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Relative allergenicity of modified bovine milk proteins Jang, Colin B. 1993

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RELATIVE ALLERGENICITY OF MODIFIED BOVINE MILK PROTEINS by COLIN BARRY JANG B.Sc., The University of British Columbia, 1990 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August 1993 © Colin Barry Jang, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of Food Science The University of British Columbia Vancouver, Canada  Date October 12, 1993  DE-6 (2/88)  ii ABSTRACT  Cow's milk contains a variety of proteins which are capable of eliciting an allergic reaction. The casein fraction and S-lactoglobulin of the whey fraction are recognized as the most potent allergens in cow's milk. It is not known, however, whether the individual sub-fractions of casein possess equal allergenicity. In this study, the relative allergenicities of ce81  and S casein were determined. The relative allergenicities  of both caseins after enzymic dephosphorylation were also investigated. In addition, the relative allergenicity of whey after the partial removal of S-lactoglobulin by FeCl3 precipitation was studied. The relative allergenicity of the milk proteins was determined by the passive cutaneous anaphylaxis (PCA) assay, which utilized antisera obtained from mice that were exposed to the test proteins using separate oral and intraperitoneal experimental protocols. In addition, an enzyme linked immunosorbent assay (ELISA) was used to determine the relative concentrations of antigen specific immunoglobulins G and E in the antisera. Mice which received the test proteins by oral administration were not sensitized against the proteins. In contrast, results obtained from mice exposed to the test proteins by intraperitoneal injection, revealed that dephosphorylated native  a81  ces,  casein and S casein were allergenic while  casein was not. The dephosphorylation of S-casein did  iii not significantly affect allergenicity. The partial removal of g-lactoglobulin did not significantly reduce the allergenicity of whey. The proteins which remain in the whey after treatment with FeCl3 (primarily u-lactalbumin and a residual amount of 0lactoglobulin) are equal to untreated whey in their ability to produce an allergic reaction.  iv  TABLE OF CONTENTS  PAGE Abstract^  ii  Table of Contents^  iv  List of Tables^  vii  List of Figures^ List of Appendices^  xiii xviii  Acknowledgement^  xx  Introduction^  1  Objectives^  3  Literature Review^  4  A. Immune Response to Antigens ^ A.1 Cells of the Immune Response ^  4 4  A.2 Thymus Dependent/Thymus Independent Antigens^ A.3 The Humoral Immune Response ^ B. Allergic Reactions^  4 5 9  C. Physical and Chemical Properties of Food Allergens^  11  C.1 Host and Environmental Factors Affecting Sensitization to Allergens^  12  C.2 Antigenic Determinants/Epitopes^  14  C.3 Molecular Size^  15  C.4 Stability^  16  C.5 Foreigness^  16  V D. Cow's Milk Allergens^  18  E. Assays of Relative Allergenicity^  21  E.1 Animal Models^  21  E.2 Passive Cutaneous Anaphylaxis^  23  Materials and Methods^  A. Modification of Proteins ^  27 27  A.1 Dephosphorylation of Caseins ^  27  A.2 Preparation of Acid Whey^  27  A.3 Ferric Chloride Precipitation of S-lactoglobulin^  28  B. Protein Determination^  28  C. Phosphorus Determination^  29  D. E-lactoglobulin Determination by SDS-PAGE ^30 E. Immunisation Protocol^ E.1 Oral Administration of Proteins ^  30 31  E.2 Intraperitoneal Injection of Proteins ^31 F. Preparation of Antisera ^  32  G. Determination of Relative Antigenicity^32 H. Determination of Relative Allergenicity^34 H.1 Relative IgE Concentration by ELISA^34 H.2 Passive Cutaneous Anaphylaxis ^ I. Statistical Analysis^ Results/Discussion^  34 35 36  A. Experiment 1: Orally Administered Proteins^36 A.1 Relative Antigenicity^  36  A.2 Relative Allergenicity^  39  vi B. Experiment 2: Intraperitoneally Injected Proteins^  43  B.1 Relative Antigenicity^  43  B.2 Relative Allergenicity^  46  Conclusions^  57  References^  58  vi i  LIST OF TABLES  PAGE Table 1.  Comparison of the Mean Relative IgG Values for Mice Orally Administered Casein Proteins.  Table 2.  37  Comparison of the Mean Relative IgG Values for Mice Orally Administered Whey Proteins.  Table 3.  38  Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Casein Proteins.  Table 4.  40  Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase.  Table 5.  41  Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Whey Proteins.  Table 6.  41  Comparison of the Mean Relative IgG Values for Mice Intraperitoneally Injected with Casein Proteins.  Table 7.  44  Comparison of the Mean Relative IgG Values for Mice Intraperitoneally Injected with Whey Proteins.  47  viii  Table 8.  Comparison of the Mean Relative IgE Values for Mice Intraperitoneally Injected with Casein Proteins.  Table 9.  48  Comparison of the Mean Relative IgE Values for Mice Intraperitoneally Injected with Whey Proteins.  Table 10.  51  Mean Passive Cutaneous Anaphylaxis Titres for Mice Intraperitoneally Injected with Casein Proteins.  Table 11.  52  Mean Passive Cutaneous Anaphylaxis Titres for Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase.  Table 12.  54  Mean Passive Cutaneous Anaphylaxis Titres for Mice Intraperitoneally Injected with Whey Proteins.  Table 13.  Protein Concentration of Ovalbumin Standard.  Table 14.  66  Protein Concentration of Stock Whey Solutions.  Table 16.  66  Protein Concentration of Stock Casein Solutions.  Table 15.  56  67  Phosphorus Concentration of Casein Samples.  69  ix  Table 17.  g-Lactoglobulin Concentration of Wheys.  71  Table 18.  Composition of Milk-Free Rodent Diet.  76  Table 19.  IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Native Casein Proteins.  Table 20.  96  IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Dephosphorylated Casein Proteins.  Table 21.  97  IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Whey Proteins.  Table 22.  98  IgG Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  Table 23.  99  IgG Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.  Table 24.  100  IgG Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  Table 25.  101  IgE Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  102  x  Table 26.  IgE Equations of the Lines and r 2 Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.  Table 27.  103  IgE Equations of the Lines and r 2 Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  Table 28.  104  Relative IgG Values for Individual Mice Orally Administered Native Casein Proteins.  Table 29.  105  Relative IgG Values for Individual Mice Orally Administered Dephosphorylated Casein Proteins.  Table 30.  106  Relative IgG Values for Individual Mice Orally Administered Whey Proteins.  Table 31.  107  Relative IgG Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  Table 32.  108  Relative IgG Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.  Table 33.  109  Relative IgG Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  110  xi  Table 34.  Relative IgE Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  Table 35.  111  Relative IgE Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.  Table 36.  112  Relative IgE Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  Table 37.  113  Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Native Casein Proteins.  Table 38.  114  Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Dephosphorylated Casein Proteins.  Table 39.  115  Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase.  Table 40.  116  Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Whey Proteins.  Table 41.  117  Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  118  xii  Table 42. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.^  119  Table 43. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase. ^  120  Table 44. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Whey Proteins. ^  121  LIST OF FIGURES  PAGE Figure 1.  The Humoral Antibody Response.  Figure 2.  Ovalbumin Standard Curve.  68  Figure 3.  Phosphorus Standard Curve.  71  Figure 4.  S-lactoglobulin Standard Curve.  74  Figure 5.  SDS-PAGE of Wheys and g-lactoglobulin Standards.  Figure 6.  7  75  Determination of us, Casein Specific IgG in Mice Orally Administered usi Casein.  Figure 7.  78  Determination of us, Casein Specific IgG in Control Mice (Oral Administration).  Figure 8.  78  Determination of g Casein Specific IgG in Mice Orally Administered g Casein.  Figure 9.  79  Determination of g Casein Specific IgG in Control Mice (Oral Administration).  Figure 10.  79  Determination of Dephosphorylated c  ^Specific IgG in Mice  Orally Administered Dephosphorylated us/ Casein.^  80  xiv Figure 11. Determination of Dephosphorylated ^Specific IgG in Control  c  Mice (Oral Administration).  80  Figure 12. Determination of Dephosphorylated 0 Casein Specific IgG in Mice Orally Administered Dephosphorylated g Casein. Figure 13.  81  Determination of Dephosphorylated  fl  Casein Specific IgG in Control Mice  (Oral Administration). Figure 14.  81  Determination of Whey Specific IgG in Mice Orally Administered Whey.  Figure 15.  Determination of Whey Specific IgG in Control Mice^(Oral Administration).  Figure 16.  82  82  Determination of FeC13 Treated Whey Specific IgG in Mice Orally Administered FeC13 Treated Whey.  Figure 17.  83  Determination of FeCl3 Treated Whey Specific IgG in Control Mice (Oral Administration).  Figure 18.  Determination of  c  83 ^Specific  IgG in Mice Intraperitoneally Injected with us, Casein. Figure 19.  Determination of o  84 ^Specific  IgG in Control Mice (Intraperitoneal Injection).  84  XV  Figure 20.  Determination of g Casein Specific IgG in Mice Intraperitoneally Injected with 0 Casein.  Figure 21.  85  Determination of g Casein Specific IgG in Control Mice (Intraperitoneal Injection).  Figure 22.  85  Determination of Dephosphorylated us, Casein Specific IgG in Mice Intraperitoneally Injected with Dephosphorylated us„ Casein. ^  86  Figure 23. Determination of Dephosphorylated us, Casein Specific IgG in Control Mice (Intraperitoneal Injection). 86 Figure 24. Determination of Dephosphorylated  g Casein Specific IgG in Mice Intraperitoneally Injected with Dephosphorylated g Casein. Figure 25.  87  Determination of Dephosphorylated  g Casein Specific IgG in Control Mice (Intraperitoneal Injection). Figure 26.  87  Determination of Whey specific IgG in Mice Intraperitoneally Injected with Whey.  Figure 27.  88  Determination of Whey Specific IgG in Control Mice^(Intraperitoneal Injection).  88  xvi  Figure 28.  Determination of FeC13 Treated Whey Specific IgG in Mice Intraperitoneally injected with FeC13 Treated Whey.  Figure 29.  89  Determination of FeC13 Treated Whey Specific IgG in Control Mice (Intraperitoneal Injection).  Figure 30.  89  Determination of us, Casein Specific IgE in Mice Intraperitoneally Injected with us, Casein.  Figure 31.  90  Determination of as, Casein Specific IgE in Control Mice (Intraperitoneal Injection).  Figure 32.  90  Determination of g Casein Specific IgE in Mice Intraperitoneally Injected with g Casein.  Figure 33.  91  Determination for g Casein Specific IgE in Control Mice (Intraperitoneal Injection).  Figure 34.  91  Determination of Dephosphorylated a  ^Specific IgE in Mice  Intraperitoneally Injected with ^ a  92  Figure 35. Determination of Dephosphorylated as, Casein Specific IgE in Control Mice (Intraperitoneal Injection). ^92  xvii  Figure 36.  Determination of Dephosphorylated  g Casein Specific IgE in Mice Intraperitoneally Injected with  g Casein. Figure 37.  93  Determination of Dephosphorylated ie Casein Specific IgE in Control Mice^(Intraperitoneal Injection).  Figure 38.  93  Determination of Whey Specific IgE in Mice Intraperitoneally Injected with Whey.  Figure 39.  94  Determination of Whey Specific IgE in Control Mice (Intraperitoneal Injection).  Figure 40.  94  Determination of FeC13 Treated Whey Specific IgE in Mice Intraperitoneally Injected with FeC13 Treated Whey.  Figure 41.  95  Determination of FeC13 Treated Whey Specific IgE in Control Mice (Intraperitoneal Injection).  95  LIST OF APPENDICES  PAGE Appendix 1. Protein Determination.^  66  Appendix 2. Phosphorus Determination.^  69  Appendix 3. g-lactoglobulin Determination. ^  72  Appendix 4. Rodent Diet.^  76  Appendix 5. ELISA Buffer Compositions. ^  77  Appendix 6. ELISA Absorbance vs. Time Graphs. ^78 A. ELISA IgG Determinations^  78  A.1 Experiment 1: Orally Administered Proteins^  78  A.2 Experiment 2: Intraperitoneally Injected Proteins^ 84 B. ELISA IgE Determinations^  90  Appendix 7. Equations of the Lines and r2 Values for Individual Mice.^ A. ELISA IgG Determinations ^  96 96  A.1 Experiment 1: Orally Administered Proteins^  96  A.2 Experiment 2: Intraperitoneally Injected Proteins^ B. ELISA IgE Determinations^  99 102  xix Appendix 8. IgG Values for Individual Mice. ^105 A. Experiment 1: Orally Administered Proteins ^105 B. Experiment 2: Intraperitoneally Injected Proteins^  108  Appendix 9. IgE Values for Individual Mice Intraperitoneally Injected with Proteins.^  111  Appendix 10. PCA Titres for Individual Mice. ^114 A. Experiment 1: Orally Administered Proteins ^114 B. Experiment 2: Intraperitoneally Injected Proteins^  118  XX  ACKNOWLEDGEMENT  I would like to thank all the members of my research committee for their help and comments: Dr. D. Kitts, Dr. E. Li-Chan, Dr. B. Skura and Dr. J. Vanderstoep. I would also like to thank Sherman Yee, Val Skura, Angela Kummer, Donna Smith, and Dr. L. Kastrukoff and his staff for their assistance during the course of my thesis. A very special thank you goes to Emmanuel Akita for his help above and beyond the call of duty.  1  INTRODUCTION  Food provides the human body with nutrients which are essential for maintaining life. However, foods can also have harmful effects on the human body. Under certain circumstances, certain individuals may develop adverse reactions to particular foods. Adverse reactions to foods can occur by several different mechanisms; reactions that are immunologically based are categorized as food allergies. Food allergies are defined as food induced immune reactions that are harmful to the tissues or disruptive of the physiology of the host. A food allergen is that specific component of food which takes part in the immune reaction that results in allergy. In the majority of clinical cases, protein is the food component responsible for eliciting an allergic response. The incidence of food allergies in the overall population is estimated at less than ]A (Taylor, 1985). Only a few individuals will develop the allergen specific immunoglobulin isotype (IgE) and an allergic response after exposure to a potentially allergenic food (Aas, 1978). Genetic and environmental factors, as well as the physical and chemical properties of the protein itself will affect the likelihood of allergic sensitization to the allergen. Cow's milk is a common source of food related allergies, particularly among children and infants. Due to the complex mixture of proteins in cow's milk, a number of different proteins may act as allergens. However, those individuals  2 allergic to cow's milk often show sensitivity to more than one protein (Baldo, 1984). The proteins which are the most commonly implicated in individuals allergic to cow's milk are S-lactoglobulin and the casein fraction. Modification or removal of these proteins may reduce the likelihood of allergic sensitization to milk. The purpose of the thesis, was to determine if an infant formula could be produced with reduced allergenicity. Cow's milk can be made similiar to human milk by manipulating its protein composition and acid clotting properties. Human milk does not contain the whey protein P-lactoglobulin. Since P-lactoglobulin is recognized as one of the most potent allergens in cow's milk, its removal may reduce allergenicity. In addition, human milk does not contain us, casein. Although the casein fraction is also considered to be one of the most potent allergens in cow's milk, the relative allergenicities of the individual sub-fractions have not been determined. Human and bovine milks clot differently in the stomachs of infants (Nakai and Li-Chan, 1987; Pildes et al.,  1980;  Cavell, 1979).  Bovine casein which has been dephosphorylated with potato acid phosphatase, forms a fine dispersion with a microstructure similiar to that of human casein when acidified to pH 4 (Li-Chan and Nakai, 1989). However, the effect of dephosphorylation on allergenicity is unknown.  3 OBJECTIVES  There were four objectives of the thesis: 1.) To determine the relative allergenicities of native as„ and 8 casein. 2.) To determine the relative allergenicity of us, casein following enzymatic dephosphorylation. 3.) To determine the relative allergenicity of 8 casein following enzymatic dephosphorylation. 4.) To determine the relative allergenicity of whey after the removal of g-lactoglobulin.  4 LITERATURE REVIEW  A. Immune Response to Antigens A.1 Cells of the Immune Response All the cells of the blood, including those that govern immune reactions, are derived from a common precursor called a stem cell. Stem cells are generated in the bone marrow, and undergo a process known as hematopoeisis to differentiate into the various cell types (Pestka and Witt, 1985). The cell line responsible for specific immunity are the lymphocytes. The lymphocytes can be divided into two types: T-cells and B-cells, and are named after the organ in which they differentiate. Thus, stem cells which migrate to and mature in the thymus become T-cells. Stem cells which mature in the bursal equivalent (bone marrow in humans) become B-cells. T-cells are further subdivided into various subpopulations, depending on the function that each T-cell performs. B-cells can be divided into plasma and memory cells. T-cells regulate the immune response and are also involved in cell mediated immunity. B-cells are responsible for humoral or antibody mediated immunity. A.2 Thymus Dependent/Thymus Independent Antigens Antigens can be of two types: thymus dependent or thymus independent. Thymus dependent antigens require a synergistic cooperation between both B and T-cells in order to initiate a humoral response (Kimball, 1983). Thymus independent antigens require only B-cells for the production of antibodies. In humans, T-cells are required in order for the antigen to provoke  5 an immune response (Kimball, 1983). Therefore, all antigens in humans are probably thymus dependent. Thymus dependent antigens induce the production of antibodies of the IgM, IgG, IgA and IgE class, while thymus independent antigens initiate an IgM response (Sweeney and Klotz, 1987). A.3 The Humoral Immune Response Immune responses to food antigens occur in the gastrointestinal tract. The gastrointestinal tract contains lymphoid tissue capable of mounting an immune response to antigens which cross the mucosal epithelial barrier. Gut associated lymphoid tissue is found primarily in "Peyer's patches", a group of nodules found in the ileum of the small intestine (Anderson and Sogn, 1984). Overlying the Peyer's patches is a single layer of epithelium containing M-cells, which regulate antigen uptake (Owen and Nemanic, 1978). Antigen absorption in the gut by M-cells is an important access route for ingested antigens to reach lymphoid tissues and thereby stimulate the local and distant immune system (Walker, 1987). The remainder of the intestine is also a site for antigen sampling, and is probably of even more significance than the M-cell, since the surface area of the M-cell is small compared to the total surface area of the intestine (Kleinman, 1992). When the antigen penetrates the epithelial mucosa, the first structure to respond immunologically is a macrophage (Breneman, 1987) (See Figure 1). The macrophage engulfs the antigen into phagocytic vacuoles, which fuse with lysosomes and digest the  6 antigen with proteases and other degradative enzymes. However, not all the antigen is degraded; some of it is retained in immunogenic form and localized on the surface membrane of the macrophage in association with an antigen encoded by the major histocompatability complex (MHC) (Kimball, 1983). The macrophage presents the antigen fragment/MHC antigen to a particular subset of T-cell known as a T-helper (TH) cell. All TH  cells express a single type of receptor specific for a  particular antigen fragment/MHC encoded antigen. The specificity of the receptor is determined prior to contact with the antigen. Once the TH cell binds to the antigen fragment/MHC encoded antigen, the macrophage secretes interleukin-1 which activates the TH cell. The activated T-helper cell synthesizes and secretes interleukin-2 and produces its own receptors for this chemical. The binding of interleukin-2 with its receptors causes the T-helper cell to undergo mitosis and proliferate into clones with the same antigen fragment/MHC encoded antigen receptor specificity. The clones produced do not express the interleukin-2 receptor, therefore, the clones must interact with the appropriate macrophages in order for clonal proliferation to continue. B-cells have membrane bound immunoglobulins on their surface which act as antigen receptors. The receptors consist of IgD and monomers of IgM. Both isotypes exhibit the same antigen binding specificity. The specificity of the receptors is determined prior to contact with the antigen. When antigen  Figure 1. The Humoral Antibody Response.  (1) Macrophage engulfs and digests the food antigen. (2) Food antigen is presented on the surface of the macrophage in association with MHC encoded antigen. (3) T. cell with receptor for food/MHC antigens binds to macrophage. (4) Macrophage secretes IL-1. (5)T. cell secretes IL-2. (6) T. cell undergoes mitosis. (7) B-cell binds to food antigen. (8) Food antigen endocytosed by B-cell. (9) Food antigen presented on the surface of the B-cell in association with MHC encoded antigen. (10) T. cell blnds to B-cell. (11) T. cell secretes BCGF and BCDF. (12) B-cells proliferate and differentiate. (13) Plasma cells secrete antigen specific antibodies.  8 binds to the immunoglobulins on the surface of the B-cell, they are moved to one pole of the cell where they form a cap. The antigen is then taken into the cell by endocytosis. Fragments of the antigen are returned to the B-cell surface in association with the same MHC encoded antigen which was found on the surface of the macrophages. The antigen fragment/MHC encoded antigen on the surface of the B-cell serves as a bridge to physically link the B-cell with T, cells that have receptors of the same specificity. Interleukin-1 secreted by the macrophages and B-cell growth factors secreted by TH cells cause the B-cell to undergo mitosis and generate a clone with the same antigen binding specificity. The ability of the B-cell to respond to interleukin-1 and the B-cell growth factors is believed to be mediated by the production of specific receptors for each of these factors, once the B-cell has bound antigen and linked to a T„ cell. The TH cell also secretes B-cell differentiation factors which transform the proliferating B-cells into plasma cells. The ability of the proliferating cells to respond to the B-cell differentiation factors is also probably governed by the production of receptors specific for these factors. Plasma cells are capable of synthesizing and secreting antibodies. The secreted immunoglobulins may be of any isotype (IgM, IgG, IgA, or IgE), and will have the same antigen binding specificity as the original B-cell receptor. Usually, plasma cells can synthesize only one isotype at any one time. Clones of the B-cell may also become memory cells. Memory cells  9 possess antibodies on their surface which function as receptors. These antibodies have the same antigen binding specificity as the original B-cell, however, the receptors may be of other isotypes (IgG, IgA or IgE). Memory cells allow for an enhanced immune response upon subsequent exposure to the antigen. Upon reexposure, the magnitude of the humoral response is greater, the lag time between exposure to the antigen and the production of antibodies is reduced, and antibody production is extended for a greater length of time. Isotype switching also occurs; initial contact with the antigen produces IgM while subsequent exposure to the antigen produces IgG, IgA, and IgE. In addition, affinity maturation occurs. Over the course of the immune response, B-cell clones with higher affinity receptors will dominate the response and secrete antibodies with increased affinity for the antigen (Kimball, 1983). B. Allergic Reactions All allergic reactions can be classified on the basis of four different immunological mechanisms, as first described by Coombs and Gell (1975). However, the four mechanisms are not mutually exclusive; more than one mechanism may operate at the same time (Bahna, 1987). The Type I reaction is also known as immediate or acute hypersensitivity and is characterized by a rapid onset of symptoms (Taylor, 1985). Type I reactions to foods are widely documented and accepted, and it is the only allergic reaction that is relatively well understood (Kniker, 1987). The Type I  10 reaction involves antigen specific IgE. IgE is capable of binding to the surface of mast cells and basophils via the Fc region of the antibody. Both mast cells and basophils possess surface receptors specific for IgE. Each basophil can carry up to a few thousand to one million receptors for IgE on its membrane (Aas, 1978). When antigen cross links two IgE molecules on the surface of the mast cell or basophil, the cell degranulates and releases vasoactive mediators. These mediators are capable of causing smooth muscle contraction and increased vascular permeability, which results in the clinical symptoms of allergy (Pestka and Witt, 1985). While over 40 substances have been identified as mediators, histamine is responsible for the majority of the immediate effects (Taylor et al., 1989). For a Type II allergic reaction to occur, the antigen must passively attach to the cells of the host. The Fc region of a cell surface antigen-antibody complex (with IgM or IgG) is capable of activating the complement cascade via the classical pathway (Pestka and Witt, 1985). When antigen specific IgM or IgG binds to the antigen, complement is activated and the antigen along with the host cells that the antibody has attached to are destroyed. Type II reactions have not been shown to occur with foods (Taylor and Cumming, 1985). Type III allergic reactions occur when antigen binds to specific IgG or IgM and forms an immune complex. Immune complexes which circulate in the fluid of the vascular system can become trapped within tight vascular tufts and settle  11 (Breneman, 1987). The antigen-antibody complex can activate complement, and the complex along with the host cells in which it has become trapped are destroyed. The circulation of immune complexes containing food antigen and specific antibodies is a common and normal phenomenon, and whether such circulation and deposition of complexes contributes much to food-related disease is difficult to prove (Kniker, 1987). Both uncertainty and controversy exist regarding the importance of Type III reactions to food (Haddad et al., 1983). Type IV allergic reactions are also known as delayed type hypersensitivity, and are characterized by a delayed onset (> 6 hr. after ingestion of the offending food) of symptoms (Taylor, 1985). Type IV reactions are mediated by a subset of sensitized T-cells known as T-Delayed Type Hypersensitivity (T  DTH  ) cells.  In order for a Type IV reaction to occur, the antigen must be bound to a cell surface (Pestka and Witt, 1985). When a T DTH cell with specific receptors binds to the antigen, it releases lymphokines which cause non-specific cell killing. Type IV reactions can involve food, but the precise mechanism of these reactions is poorly understood at the molecular level (Taylor and Cumming, 1985). C. Physical and Chemical Properties of Food Allergens In food allergy, the role of antibodies other than those of IgE is poorly understood (Aas, 1989). Type I or IgE mediated food reactions are the only type of responses for which it is possible to demonstrate a clear relationship with the symptoms  12 of food allergies (Pastorello et al., 1987). All known food allergens have been implicated in IgE mediated reactions (Taylor et al., 1987a). This review of the physical and chemical properties of food allergens will be restricted to antigens capable of eliciting a Type I response. In order to be an allergen, the offending food constituent must be capable of stimulating the production of IgE. All food allergens capable of initiating an IgE mediated allergic response are naturally occurring proteins or glycoproteins (Aas, 1989). Very little is known about the characteristics of those food proteins which can function as allergens (Taylor, 1992). Proteins that can function as allergens are found predominantly in foods of plant and marine origin; with the exception of cow's milk and chicken egg, proteins of animal origin are rarely implicated in food allergies (Taylor, 1992). C.1 Host and Environmental Factors Affecting Sensitization to Allergens Host factors will contribute to, or determine, whether a certain molecule will act as an allergen (Aas, 1978). Host factors are equally as important as the molecular structure of the allergen (Aas, 1976). Studies have indicated that while the disposition to become allergic is inherited, the type of disease manifestation and the sensitization to a particular allergen usually are not, although they can be (Marsh, 1975). The likelihood of allergic sensitization to the proteins in food is heavily dependent on the immune responsiveness of the individual  13 (Taylor et al., 1987b). Only those individuals whose genetic code is strongly programmed to produce IgE under the normal conditions of exposure to food, are likely to become sensitized to food proteins (Aas, 1978). Several environmental factors will influence the likelihood of allergic sensitization to particular food proteins (Taylor et al., 1987b). The degree and frequency of exposure to the antigen is critical (Taylor et al., 1987b). Some experiments with animals have suggested that low dose stimulation with antigen favours the production of IgE, whereas high dose stimulation favours the production of IgG (Ishizaka, 1976). The age of the individual at the time of exposure to the antigen is also critical (Taylor et al., 1987b). Food allergy is much more common among children than adults (Fries, 1959). Although the potential exists to develop allergies at any age, infants are the most susceptible (Taylor and Cumming, 1985), since the gut of the infant is more permeable to dietary antigens than the gut of adults (Udall et al., 1981a; Walker and Bloch, 1983). Therefore, if exposure to an antigen occurs at an early age, the likelihood of allergic sensitization to that protein is increased (Taylor et al., 1987b). Age is a less important determinant of intestinal permeability than the state of maturation of the intestinal barrier (Udall et al., 1981b). Infants with food allergies tend to lose their sensitivity as they grow older (Taylor, 1985) as a result of gut closure. Other factors which affect gut permeability, such as intestinal  14 injury, will also enhance the likelihood of allergic sensitization to food proteins (Taylor et al., 1987b). C.2 Antigenic Determinants/Epitopes An antigenic determinant or epitope is that specific area or site on a protein molecule to which an antibody or receptor of an immunocompetent lymphocyte can bind (Aas, 1989). Since all naturally occurring allergens are proteins, epitopes can be of two types; these being sequential or conformational. Sequential epitopes consist of a number of amino acid residues as found in the primary sequence of the protein. Conformational epitopes result from the steric folding of the polypeptide backbone chain, which brings together amino acid residues that were originally found at different sites on the primary sequence (Aas, 1989). In proteins, antigenic determinants are usually composed of between five and ten amino acid residues (Sweeney and Klotz, 1987). Most epitopes are conformational (Aas, 1989). To be an allergen, the protein must have at least two epitopes in order to be able to crosslink the IgE molecules once the antibodies have bound to the surface of the mast cell or basophil (Aas, 1978). Although two epitopes are required for bridging to occur, there is no evidence to conclude that those epitopes must be identical (Aas, 1978). In addition, epitopes must be on the surface of the antigen in order for IgE producing cells and antibodies to react with it (Atassi, 1975; Crumpton, 1974). Therefore, the factors that may determine the potency of a particular allergen are: (1) the number of epitopes that are  15 accessible for specific antibodies, and (2) the binding dynamics between these epitopes and antibodies (Aas, 1989). C.3 Molecular Size The ideal size (molecular weight) of food allergens ranges between 10,000 to 70,000 daltons (Taylor, 1992). The size of food allergens appears to be dictated by at least three factors: (1) the bridging requirement, (2) ability of the protein to initiate an immune response, and (3) intestinal permeability (Taylor, 1992). In order for the basophil or mast cell to degranulate, the allergen must cross link two IgE molecules on the cell membrane surface. Therefore, the allergen must have the appropriate molecular size or dimensions to allow bridging to occur. More correctly, there must be two or more epitopes located along the protein molecule at distances equivalent to those between IgE molecules on the surface of the basophils or mast cells (Aas, 1978). Thus, bridging may be less dependent on molecular size than on molecular shape (Taylor et al., 1987a). The allergen must also be capable of initiating the proliferation of immunocompetent lymphocytes and trigger the synthesis of specific antibodies. Small proteins are less likely to be antigenic (Taylor et al., 1987b). However, small peptides can act as haptens and become allergenic if they aggregate with larger proteins (Taylor et al., 1987b). A hapten is a molecule that by itself will not initiate an immune response, but may serve as an antigenic determinant when coupled to another antigenic molecule (Kimball, 1983). Proteins with  16 molecular weights in excess of 70,000 daltons are less likely to be efficiently absorbed through intestinal mucosal membranes and obtain access to IgE producing cells (Taylor, 1987a). However, larger molecules may induce IgE production following parenteral administration (Aas, 1978). C.4 Stability Allergenic food proteins are capable of inducing an allergic reaction after various food processing treatments (Taylor, 1986). Therefore, it is reasonable to assume that these food proteins are comparatively stable to heat and acid treatments (Taylor, 1992). Food allergens must also survive the digestive process, and thus, are resistant to pH mediated changes and peptic-tryptic digestion (Taylor et al., 1987a). Denaturation of food proteins with heat or chemical treatments can substantially reduce the allergenicity of proteins with conformational allergenic determinants, since such treatments could irreversibly alter the three-dimensional structure of the epitope (Taylor et al., 1987b). However, protein denaturation may have no effect on allergenicity if the allergenic determinant resides on the primary sequence of the protein molecule (Taylor et al., 1987b). C.5 Foreigness Proteins must be able to stimulate the production of specific IgE in order to be an allergen. The ability of a particular protein to induce the production of antibodies is related to the protein's perceived degree of foreigness to the  17 host (Crumpton, 1974). However, this concept of foreigness in not well understood, especially as related to the production of IgE (Taylor, 1992). In general, the more foreign or chemically different an antigen is from the normal tissues of the host, the greater its antigenicity (Sweeney and Klotz, 1987). Proteins that are similiar in nature to proteins existing in the host often have little or no antigenicity within that host (Sweeney and Klotz, 1987). An example is an experiment that was performed with the pituitary hormone adrenocorticotrophin (ACTH). Dayhoff and Eck (1969) studied ACTH in various animal species. ACTH consists of 39 amino acid residues, 24 of which were shared by the animals in their study. Species specificity to ACTH occurred in the amino acid regions between 25 and 33. When antibodies were raised against ACTH, the antibodies were found to react only with the areas of variability, not with those areas that were common to the various species. Thus, antibodies are formed only against particular areas of a foreign protein that are not shared by the host (Crumpton, 1974). It has not been possible to determine any physicochemical feature that is characteristic for allergens, apart from being proteins with a molecular weight between 10,00 to 70,000 daltons (Aas, 1989). Although the amino acid composition or sequence and the three dimensional structure of most food allergens are unknown, they do not appear to be unusual proteins in any way other than their allergenicity (Taylor et al., 1987a). The allergenicity of these proteins is unlikely to be due to the  18 existence of unique compositional features (Taylor et al., 1987a). In addition, the general structure and chemical properties of an antigen alone cannot be used to predict the type of reaction one might observe in populations (Sweeney and Klotz, 1987). D. Cow's Milk Allergens The most common allergenic food among children and infants is cow's milk (Bock and Martin, 1983; Bock et al., 1978). Cow's milk allergy (CMA) is a hypersensitivity reaction that is the result of an abnormal immunologic response to one or more proteins found in cow's milk (Taylor, 1986). Manifestations of CMA can result in a number of gastrointestinal, respiratory and dermatologic symptoms. The least commonly observed (Lebenthal, 1975), but most serious manifestation of CMA is anaphylactic shock, which can result in death (Taylor, 1986). The occurrence of Type I or IgE mediated responses has been confirmed in CMA (Taylor, 1986). Type III and Type IV allergic reactions may play a role in some forms of CMA, but further evidence is required to establish this possibility (Taylor, 1986). Cow's milk contains between 28 to 41 grams protein per litre (Bahna and Heiner, 1980) and can be divided into two main protein components: casein and whey. Milk also contains numerous minor protein fractions as well as several enzymes (Taylor, 1986). Casein (csn) constitutes approximately 809s of the proteins in milk, while the whey fraction makes up the remaining 20-96- (Swaisgood, 1985). Based on their order of  19 decreasing electrophoretic mobility, the casein fraction can be further subdivided into the ce (1-csn and us2-csn), S, 1, and  K  caseins (Bahna and Heiner, 1980). Of the caseins, u81-csn is found in the largest concentration, and constitutes approximately 34% of the total proteins in milk and 42.5% of the proteins in the casein fraction (Swaisgood, 1985). The second largest casein fraction in terms of concentration is S-csn, which constitutes approximately 25% of the total proteins in milk and 31.3% of the proteins in casein (Swaisgood, 1985). The whey fraction is composed of S-lactoglobulin, a-lactalbumin and the blood proteins (serum albumin and immunoglobulins) (Swaisgood, 1985). Of the proteins in the whey fraction, S-lactoglobulin is found in the largest concentration, and constitutes approximately 9% of the total proteins in milk and 45% of the proteins in whey (Swaisgood, 1985). Individual protein constituents will vary quantitatively with the stage of lactation, the breed of cattle and the feed used, but with little qualitative variation (McMeekin, 1954). Cow's milk contains between 18 to 25 proteins which can act as antigens (Bahna and Gandhi, 1983; Baldo, Mansson,  1961;  1984;  Hanson and  Savilahti, 1981). However, not all antigens in  milk are allergenic. In addition, those proteins which are allergenic do not possess the same allergenicity (Bahna and Heiner, 1980). The allergenicity of individual milk proteins has been evaluated by skin tests and oral challenges. S-lactoglobulin was found to produce the strongest response when  20 skin tests were performed with persons known to be allergic to cow's milk (Bleumink and Young, 1968; Goldman et al., 1963b). Oral challenge studies with isolated protein fractions showed S-lactoglobulin and casein to be the two most common allergens in cow's milk (Davidson et al., 1965; Goldman et al., 1963b; Kuitunen et al., 1975; Liu et al., 1967; Visakorpi and Immonen, 1967). u-lactalbumin was less frequently incriminated as an allergen in oral challenges, and reactions to bovine serum albumin and immunoglobulins were rare (Taylor, 1986). No challenge studies have been performed using the individual sub-fractions of casein (Taylor, 1986). IgE antibodies to several minor proteins such as lactoferrin, lactoperoxidase, alkaline phosphatase and catalase have been identified in a few patients with cow's milk allergy (Baldo, 1984), however, the significance of these proteins as allergens has yet to be determined (Taylor, 1992). It should also be noted, that individuals with cow's milk allergy may react to more than one protein in cow's milk (Taylor, 1992). The enzymatic digestion of milk proteins may also affect allergenicity. Epitopes must be expressed on the surface of the protein in order to interact with immunocompetent lymphocytes. When a protein is digested, epitopes which were hidden within the molecule can become exposed. Ishizaka et al. (1960) presented evidence that new antigenic determinants were exposed when bovine serum albumin was partly degraded with pepsin. This phenomenon has also been reported with casein, S-lactoglobulin  21 and a-lactalbumin when treated in vitro with pepsin and trypsin (Spies et al., 1970). Neither milk fat nor lactose has been shown to be antigenic (Bahna and Heiner, 1980), however, both components may affect the allergenicity of milk. Poulsen et al. (1987) demonstrated that the homogenization of milk increases the ability of the milk to induce a systemic anaphylactic reaction in sensitized mice when challenged intravenously. This phenomenon has also been confirmed in children allergic to cow's milk upon oral challenge (Host and Samuelsson, 1988). The allergenicity of cow's milk proteins might also be enhanced by the non-enzymatic or Maillard browning reaction with lactose (Smith, 1976; Bleumink and Berrens,  1966;  Bleumink and Young,  1968;  Lietze,  1969). When the Maillard browning reaction occurred between g-lactoglobulin and lactose, the amount of antigen required to elicit a positive intradermal reaction in subjects allergic to cow's milk was decreased by a factor of 100 (Bleumink,  1970;  Bleumink and Berrens, 1966). E. Assays of Relative Allergenicity E.1  Animal Models  Animal models provide comparative indications on the allergenicity of proteins from various sources and on the impact of different technologies affecting immunoreactivity in vivo (Pahud et al., 1988). However, the extent to which allergenicity in any laboratory animal model correlates with human allergenicity is unknown (Kleinman, 1992). The validity  22 of these model systems is restricted by the limited information concerning the prevailing epitopes in humans and animals (Aas, 1978). Laboratory animals may be sensitized by parenteral immunization using adjuvant (Williams and Chase, 1967; Hum n and Chantler, 1980). Two species which have been used to assess the allergenicity of milk by this method are guinea pigs and mice (Ratner et al., 1958; Poulsen et al., 1990). When mice are used in allergenicity studies, a critical factor to consider is the antigen dose. Vaz et al. (1971) found that immunizing mice with minute doses of 0.1 Ag of ovalbumin in aluminum hydroxide gel induced high titres and the persistent synthesis of IgE antibodies. Immunization with high doses of 100 Ag of antigen induced weak and transient IgE formation. The route of immunization affects the concentration of antigen and the types of cells with which the antigen will interact (Sweeney and Klotz, 1987). When a protein is ingested, it is exposed to digestive enzymes and the acid conditions of the stomach. The protein must filter through the mucous layer and intestinal cell wall before encountering immunocompetent cells (Guesry et al., 1989). These processes dramatically reduce the amount of foreign protein that reaches the immunological system (Guesry et al., 1989). Therefore, animal models which utilize oral sensitization are more desirable, since they more accurately represent the normal route of sensitization to food proteins. Anderson et al. (1979)  23 demonstrated that guinea pigs could be sensitized to cow's milk proteins by gavage via the oral route. Use of the oral guinea pig model for assessing the allergenicity of milk proteins after various treatments is well documented (Heppel et al., et al.,  1987;  1984;  Jost  Kilshaw et al., 1982; McLaughlan et al., 1981;  Pahud et al., 1985), and is an accepted in vivo experimental approach for comparing the effect of different treatments on protein allergenicity (Jost et al., 1987). Mice can also be sensitized to milk proteins by the oral route (Poulsen et al., 1990; Poulsen et al.,  1987;  Nielsen et al., 1989). A study  conducted by Paulsen and Hau (1987) indicated that guinea pigs could be replaced by mice when assessing the allergenicity of various compounds by PCA. The sensitivity of the two species is similiar, but the husbandry and handling of mice is more convenient and they are less expensive (Poulsen and Hau, 1987). E.2 Passive Cutaneous Anaphylaxis In vivo assays such as anaphylactic shock models and passive cutaneous anaphylaxis are widely employed and recommended when assessing the allergenicity of various compounds (Poulsen and Hau, 1988). Anaphylactic shock models employ test animals which have been immunized against the proteins. Following intravenous challenge with the antigen, the animals are observed to see if fatal anaphylaxis occurs. Anaphylactic shock scores have also been used to evaluate the degree of anaphylaxis. The scores are assigned according to the severity of the anaphylactic reaction observed after injection  24 of the antigen (Paulsen et al., 1987). An alternative in vivo approach is passive cutaneous anaphylaxis. PCA corresponds in every respect to systemic anaphylaxis (Ovary et al., 1963), however, it is easier to quantitate than systemic anaphylaxis (Watanabe and Ovary, 1977) and there is less suffering on the part of the animals. To perform the PCA test, serum collected from an immunized animal is injected intracutaneously in a suitable recipient. The IgE in the sera binds to mast cells in the skin via its Fc fragment. Fc fragments vary according to antibody class, and it is for this reason that not all isotypes are capable of binding to the mast cells in the skin (Watanabe and Ovary, 1977). After a specific time period to allow binding to occur, the allergen and a non-toxic dye are intravenously injected into the recipient animal. When the allergen crosslinks two IgE molecules on the surface of the mast cell, the mast cell degranulates and releases vasoactive substances. The release of the vasoactive substances increases the permeability of the capillaries, which allows the dye to diffuse into the surrounding tissue and cause the skin around the reaction sites to change colour. For precise readings, the animal should be killed, skinned and the reaction measured on the inside of the skin (Watanabe and Ovary, 1977). PCA reactions can be quantitated by the size of the reaction, or by the endpoint technique (Watanabe and Ovary, 1977). The size of the reaction is roughly proportional to the antibody content (within certain limits), and can be quantitated by measuring the  25 diameter of the reaction (Watanabe and Ovary, 1977). This technique is seldom used since it is much more demanding technically than the endpoint technique. The endpoint technique employs a series of diluted antisera. The reciprocal of the highest dilution to give a positive reaction is used as the endpoint or titre. The standard assay for quantitating antigen specific mouse IgE has been the PCA reaction in rats (Watanabe and Ovary, 1977). The antisera from mice can be titred in both homologous and heterologous species. In homologous species (eg. micemice), both IgE and IgG, participate in the PCA reaction. In heterologous species (eg. mouse-rat) only IgE is involved in the reaction (Ovary et al., 1975). Only in vivo studies on animal models or human beings can give precise information on the relative allergenicity of a given protein after processing (Guesry et al., 1989). In vitro tests should only be used when the results have been confirmed by in vivo studies (Guesry et al., 1989). The radioallergosorbent test (PAST) and enzyme linked immunosorbent assay (ELISA) are two in vitro techniques which have been used to measure the levels of a particular immunoglobulin class to specific antigens in the serum. To perform a RAST, the antigen is bound to a solid phase. The test serum is added to the antigen, and incubated to allow binding to occur between the antigen and antibodies. A radiolabeled anti-Ig antibody specific for the isotype under study is added (Chandra and  26 Jeevanandam, 1984) and the amount of bound radioactivity is measured, giving an estimate of the antigen specific Ig in the serum (Aas, 1978). A similiar principle is used for the ELISA, however, instead of using a radiolabeled anti-Ig antibody, an enzyme coupled to the anti-Ig antibody is substituted and a substrate is added. The enzyme acts on the substrate which causes it to change colour or become fluorescent (Anderson, 1990). The amount of colour or fluorescence is measured, which provides an estimate of the antigen specific Ig in the serum. In vitro methods such as RAST and ELISA are routinely used to measure antigen specific IgE. Alone, these tests cannot be used to assess relative allergenicity, but must be employed in conjunction with an in vivo method. The RAST and ELISA methods measure free IgE in the serum, not cell-bound IgE (Chandra and Jeevanadam, 1984). In addition, they are unable to evaluate the extent of antigen induced mediator release (Conroy and Adkinson, 1977). The drawback with in vitro tests such as the RAST and ELISA, is that they are designed to measure a single isolated event (eg. antigen-IgE binding), and fail to take into account other physiological events in the allergic reaction (Poulsen and Hau, 1987).  27 MATERIALS AND METHODS  A. Modification of Proteins A.1 Dephosphorylation of Caseins a81-csn (donated by Guillermo Arteaga, PhD candidate, Department of Food Science, The University of British Columbia, and prepared by the method of Zittle and Custer (1963), minimum 90% us,-csn), and S-csn (Sigma, St. Louis, MO, USA, minimum 90% g-csn) were enzymatically dephosphorylated according to the procedure of Li-Chan and Nakai (1989). a51-csn or g-csn solutions (0.5%) at pH 7.0 were treated with 50 mg acid phosphatase (Sigma, St. Louis, MO, USA)/g protein and held at 37°C for 2 hours. Following incubation, the samples were dialysed (regenerated cellulose membrane, MWCO 6,000 - 8,000) against distilled, deionized water adjusted to pH 7 at 4°C for 65 hours. Native casein samples were dialyzed against distilled, deionized water at 4°C for 48 hours. A.2 Preparation of Acid Whey Unpasteurized milk was obtained from The University of British Columbia Dairy Unit. Butterfat was removed from the milk by centrifugation at 5,000 x g for 30 minutes at 4°C. The casein fraction was separated from the whey by isoelectric precipitation; the milk was heated to 37°C and the pH adjusted to 4.6 with 1N HC1 (Li-Chan and Nakai, 1988). The precipitated casein was removed by centrifugation at 10,000 x g for 20 minutes at 4°C. The whey was recentrifuged at 15,000 x g for 10  28 minutes at 4°C to remove residual casein. The acid whey was dialyzed against distilled, deionized water for 48 hours at 4°C. A.3 Ferric Chloride Precipitation of S-lactoglobulin S-lactoglobulin was removed from the acid whey by the method of Kaneko et al. (1985). An aliquot of 1M FeC13 (BDH Inc., Toronto, ON, Canada) was added to undialyzed acid whey which had been adjusted to pH 4.2, to give a final concentration of 7.5 mM FeCl3. The pH was maintained at 4.2 throughout the procedure by the simultaneous dropwise addition of a 3N NaOH solution. The whey was incubated in an ice water bath for 2 hours, and the precipitated S-lactoglobulin was removed by centrifugation at 10,000 x g for 15 minutes at 4°C. Excess Fe3' was removed by adjusting the pH to 9.0 and adding 2 mg/ml Na2HPO4. The pH was readjusted back to 9.0 and the whey allowed to stand for 1 hour. The precipitated iron salts were removed by centrifugation at 15,000 x g for 10 minutes at 4°C. The whey was dialyzed against distilled, deionized water at 4°C for 48 hours. B. Protein Determination The protein concentration of the sample stock solutions were determined by the Biuret method using Sigma Diagnostics (St. Louis, MO, USA) procedure number 541. The procedure was modified for use as a microassay using microtitre plates. Solutions (60 Al) of the samples or a diluted chicken egg albumin (Sigma, St. Louis, MO, USA) standard were mixed with 250 Al of total protein reagent in the wells of an ELISA microtitre  29 plate (Dynatech Laboratories Inc., Chantilly, VA, USA), and the absorbance read immediately at 550 nm using a SLT Labinstruments EAR 400 plate reader (Salzburg, Austria). A standard curve was constructed using the SlideWrite Plus program (Advanced Graphics Software Inc., Sunnyvale, CA, USA) and the protein concentrations of the samples determined from the standard curve (See Appendix 1). The standards were assayed in quadruplicate and the samples in triplicate. No blank wells were included in the assay to correct for defects in the plate which might contribute absorbance. C. Phosphorus Determination Determination of the phosphorus content of a1-csn and g-csn before and after dephosphorylation were performed by a modified procedure of Morrison (1964). The casein samples or standard (NaH2PO4 x 2H20 solution containing 200 Ag phosphorus/ml) were dried in glass test tubes calibrated at 5 ml. Sulfuric acid (0.03 ml) was added to each tube, and heated in a boiling hot water bath for approximately 15 seconds. Aliquots of hydrogen peroxide (10 pl, 30% w/v) were added until the acid became clear. The tubes were heated in a boiling hot water bath for 1 minute, then allowed to cool. Water (3 ml) was used to wash down the inside walls of each tube, and 0.1 ml of a 33% (w/v) sodium sulfite solution was added. An ammonium paramolybdate solution (1.0 ml, 2% w/v) was added to each tube, followed by 0.5 ml of a 2% (w/v) ascorbic acid solution which was prepared immediately before use. The tubes were heated at 100°C for 10  30 minutes, then cooled. The volume was adjusted to 5.0 ml with distilled deionized water, and the absorbance measured at 822 nm using a Shimadzu UV-160 spectrophotometer (Kyoto, Japan). A standard curve was constructed using the SlideWrite Plus program (Advanced Graphics Software Inc., Sunnyvale, CA, USA) and the phosphorus concentrations of the samples determined from the standard curve (See Appendix 2). All samples and standards were done in triplicate. D. Z-lactoglobulin Determination by SDS-PAGE The S-lactoglobulin concentration of the whey before and after precipitation with FeC13 was determined using the Pharmacia PhastSystem (Pharmacia LKB Biotechnology, Uppsala, Sweden) on a 12.5% (w/v) gel with Coomassie Brilliant Blue staining. The samples or a diluted g-lactoglobulin (Sigma, St. Louis, MO, USA) standard were adjusted to approximately pH 8 with 1.5M Tris-HC1. An aliquot of a 25% w/v sodium dodecyl sulfate solution and 2-mercaptoethanol was added to give a final concentration of 2% for each reagent respectively. An aliquot of a 1% w/v chicken egg albumin (Sigma, St. Louis, MO, USA) internal standard was also added to each sample and standard to give a final concentration of 0.1%. The protein samples were heated in a boiling hot water bath for 5 min. and allowed to cool before 0.5 ill of each sample was applied to the gel. The proteins were quantified using a Pharmacia PhastImage Gel Analyzer (Pharmacia LKB Biotechnology, Uppsala, Sweden). A standard curve was constructed using the SlideWrite Plus program (Advanced Graphics  31 Software Inc., Sunnyvale, CA, USA) and the concentrations of S-lactoglobulin in the samples were determined from the standard curve (See Appendix 3). The g-lactoglobulin concentrations of the samples and standard were determined from a single gel. The protein concentration of the g-lactoglobulin standard was determined by the Biuret method as described in section B. E. Immunisation Protocol Two separate methods were used to expose Balb/C mice to the proteins; the proteins were administered by oral gavage or by intraperitoneal injection. Mice were obtained either from Charles River Canada Inc. (St.-Constant, PQ, Canada), or from a mating with mice from The University Of British Columbia Animal Unit. Mice of both sexes, aged 6-8 weeks old, and maintained on a milk free diet (See Appendix 4) were used in all experiments. E.1 Oral Administration of Proteins Mice were orally administered the test proteins according to the method of Poulsen et al. (1990). Protein (10 Ag) in 100 Al of sterile diluent (H20) was administered on day 1. Oral booster doses of 1 Ag of protein in 100 Al of sterile diluent were administered on days 11 and 22. E.2 Intraperitoneal Injection of Proteins The mice were intraperitoneally injected with the test proteins using a modified procedure of Poulsen et al. (1990). Test protein (10 gig) mixed with 1 mg of aluminum hydroxide adjuvant (Pierce, Rockford, IL, USA) in 200 Al of sterile diluent^was^intraperitoneally^injected^on^day^1.  32 Intraperitoneal booster doses of 1 Ag of protein mixed with 1 mg aluminum hydroxide adjuvant in 200 pl of sterile diluent were administered on days 11 and 22. F. Preparation of Antisera The mice were bled 29 days after initial exposure to the test proteins. Antiserum was prepared using a modified procedure of Garvey et al. (1977). Whole blood was kept at room temperature for 2 hours to induce clot formation, then stored at 4°C for 24 hours to allow the clot to contract. Antiserum was separated from the clot by centrifugation at 2,000 x g for 30 minutes at 4°C. The antiserum was diluted 5x with PBS and stored at -70°C. G. Determination of Relative Antigenicity The relative concentrations of antigen specific IgG in murine serum were determined by an indirect ELISA and used as a measure of antigenicity. Immulon 2 microtitre ELISA plates (Dynatech Laboratories Inc., Chantilly, VA, USA) were coated with 50 pl of 0.01% w/v antigen in carbonate buffer (see Appendix 5) overnight at 4°C. After removing the excess antigen, the plates were washed 3x with PBS/ TWEEN (see Appendix 5) and 200 pl of a 0.25% ovalbumin (Canadian Lysozyme, Aldergrove, BC, Canada) blocking buffer (see appendix 5) was applied. The microtitre plates were incubated at 35°C for 30 minutes, and the blocking removed. The wells of the plates were coated with 50 pl of diluted antisera and incubated at 35°C for 1 hour. After the antisera was removed, the plates were washed 3x with  33 PBS/TWEEN and coated with 50 Al of 0.001% w/v anti-mouse IgG alkaline phosphatase conjugate (Sigma, St. Louis, MO, USA) for 1 hr. at 35°C. The conjugate was removed, and the plates washed 3x with PBS/TWEEN, and once with distilled deionized water. P-nitrophenyl phosphate substrate (50 Al) (Sigma, St. Louis, MO, USA) in diethanolamine buffer (see Appendix 5) was added and the plates incubated at 35°C. ^All samples were analyzed in triplicate. The absorbance at 405 nm was measured (using a reference filter at 620 nm) using a SLT Labinstruments EAR 400 plate reader (Salzburg, Austria) over time and subtracted from blanks. Absorbance values above 1.50 were excluded from the plot. The rate of colour development is proportional to the amount of conjugate which has reacted. This in turn is related to the amount of antibody being assayed (Voller, 1980). The absorbances were plotted over time and the slopes of the graphs (multiplied by 1.0 x 10') were calculated using the SlideWrite Plus program (Advanced Graphics Software Inc., Sunnyvale, CA, USA).^The slopes were used as estimates of the relative concentrations of antigen specific IgG in the sera and expressed as a relative IgG values. Absorbance points measured after a single designated time were not used to determine the relative concentrations of antigen specific IgG, because the ELISA plate reader was incapable of reading all the wells of the plate simultaneously. Therefore, wells containing identical amounts of antibody could have different absorbances. By using the slope of the graph, this error was eliminated. ^Also, any  34 defects in the wells of the plate which might contribute to the absorbance were also eliminated. In addition, plotting the absorbance values over time allows for the identification of outlying groups of measurements, while measuring the absorbance at a single designated time does not. H. Determination of Relative Allergenicity H.1 Relative IgE Concentrations The relative concentrations of antigen specific IgE in the sera were determined by an indirect ELISA. The same procedure for determining the relative concentration of IgG (section G) was used for determining IgE, except that an anti-mouse IgE alkaline phosphatase conjugate (Pharmingen, San Diego, CA, USA) was substituted to detect the appropriate isotype. H.2 Passive Cutaneous Anaphylaxis Heterologous passive cutaneous anaphylaxis (PCA) was performed according to the procedure of Akita and Nakai (1990). Male CD rats (Charles River Canada Inc., St.-Constant, PQ), 9-11 weeks old, were intradermally injected with 100 Al of doubling dilutions of antisera. After 4 hours, 1.0 mg of antigen (protein) diluted in 1.0 ml of 2.%, (w/v) Evans Blue dye (Sigma, St. Louis, MO, USA) was administered intravenously via the penile vein. PCA reactions were evaluated 45 minutes after injection of the antigen/dye. The titre value equalled the reciprocal of the highest dilution of antiserum to give a positive reaction. A positive reaction was considered a circular blue spot larger than 5 mm in diameter. All antisera  35 were tested in duplicate. I. Statistical Analysis Differences between the protein treatments and the negative controls were determined using t-tests at a significance level of p < 0.05 as described by Ott (1988).  36 RESULTS/DISCUSSION  Six protein samples and a negative control (water) were used in each experiment. The casein samples consisted of: usicsn, dephosphorylated a81-csn, S-csn, and dephosphorylated S-csn. The results of the phosphorus determination showed 66.5% and 84.1% dephosphorylation for cesi-csn and S-csn respectively (See Appendix 2). These results are lower than those obtained by Li-Chan and Nakai (1989). Li-Chan and Nakai (1989) obtained 70% and 99% dephosphorylation for usl-csn and S-csn respectively. The remaining two protein samples consisted of whey and FeCl3 treated whey. The results of the S-lactoglobulin determination indicated that at least 85.5% of the S-lactoglobulin had been removed from the FeCl3 treated whey (See Appendix 3). The concentration of g-lactoglobulin in the FeC13 treated whey was below the minimum detection limit of the PhastImage program. Therefore, the lowest concentration used for constructing the glactoglobulin standard curve was substituted to determine the minimum amount of g-lactoglobulin that had been removed from the whey. The actual amount of g-lactoglobulin removed from the FeC13 treated whey exceeded this value. A. Experiment 1: Orally Administered Proteins A.1 Relative Antigenicity The relative IgG values for mice orally administered the test proteins are given in Tables 1 and 2. T-tests were performed on the data to detect significant differences (p < 0.05) between the samples. The results indicate that the mice  37  Table 1. Comparison of the Mean Relative IgG Values for Mice Orally Administered Casein Proteins. ANTIGEN  ANTISERA  as1 CASEIN  CASEIN  MEAN ABSORBANCE +^S.E.M.b  MEAN IgG VALUE ± S.E.M. (abs.c/min.)x103  0.110^+^0.005e  0.63^+^0.04  NEGATIVE CONTROL  0.130^+^0.007e  0.76^+^0.05  S CASEIN  0.018^+^0.003f  0.13^+^0.02  NEGATIVE CONTROL  0.023^+^0.002f  0.16^+^0.01  DEPHOS  DEPHOS  0.281^+^0.050e  1.42^+^0.35  CASEIN  CASEIN NEGATIVE CONTROL  0.134^+^0.019e  0.76^+^0.06  DEPHOS  DEPHOS  0.045^+^0.006f  0.28^+^0.03  CASEIN  CASEIN 0.041^+^0.003f  0.23^+^0.02  S CASEIN  gd  d  gd  NEGATIVE CONTROL  'Antisera diluted 1/25. bS.E.M. = Standard Error of the Mean. 'Absorbance daephosphorylated eAbsorbance measured after 150.41 minutes of incubation at 37°C. fAbsorbance measured after 149.05 minutes of incubation at 37°C.  38  Table 2. Comparison of the Mean Relative IgG Values for Mice Orally Administered Whey Proteins. MEAN ABSORBANCE^MEAN IgG VALUE ± S.E.M. + S.E.M.b^(abs.c/min.)x103  ANTIGEN  ANTISERAa  WHEY  WHEY  0.065^+^0.003d  0.26^+^0.01  NEGATIVE CONTROL  0.062^+^0.003d  0.26^+^0.02  FeC13 TREATED WHEY  0.070^+^0•003d  0.29^+^0.02  NEGATIVE CONTROL  0.091^+^0.011d  0.35^+^0.07  FeC13 TREATED WHEY  'Antisera diluted 1/25. bS.E.M. = Standard Error of the Mean. cAbsorbance dAbsorbance measured after 205.91 minutes of incubation at 37°C.  39  were not sensitized against any of the test proteins. A.2 Relative Allergenicity The relative IgE concentrations were not measured, since the IgG data suggested that the mice were not sensitized against the proteins, ie. there was no antigen specific IgG in the sera. It should be noted, that the antigenic epitopes which give rise to IgG may not be the same epitopes which produce IgE (Taylor, 1989 et al.). A food protein that induces an IgG response in animal models, even humans, will not necessarily induce an IgE response (Taylor, 1989 et al.). IgE is much harder to detect than IgG, because it is normally found in extremely low concentrations in the serum. IgE is the least prevalent of the five antibody classes (Metcalfe et al., 1991). In the normal human subject, IgE comprises approximately 0.002 96 of the total serum immunogobulins (Metcalfe et al., 1991). The concentration of IgE in normal human serum is approximately 1/40,000 that of IgG (Kimball, 1983). Since the results indicated that no antigen specific IgG was in the antisera, it is improbable that IgE would be detected. The IgG data indicated that the test proteins were not antigenic when administered orally. Since a protein must be antigenic in order to be allergenic, it would be reasonable to assume that the mice which had been orally administered the test proteins, would exhibit no allergenicity towards the proteins. Tables 3, 4, and 5 show the PCA titres for the mice orally administered the proteins. There was no response at the lowest  40  Table 3. Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Casein Proteins. ^ ANTIGEN^ANTISERA MEAN TITRE + S.E.M.a N Rb  CASEIN^CASEIN NEGATIVEc CONTROL  ^  NR  ^ S CASEIN^S CASEIN NR ^ NEGATIVE NR CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED^NR us, CASEIN^CASEIN ^ NEGATIVE NR CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED^NR S CASEIN^S CASEIN NEGATIVE^NR CONTROL aS.E.M. = Standard Error of the Mean. bAll negative controls consisted of a pooled antiserum sample. cNR = No Response at the lowest dilution tested (1/5).  41  Table 4. Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase. ^ ANTIGEN ANTISERA^MEAN TITRE + S.E.M.a POTATO ACIDb DEPHOSPHORYLATED^NRc PHOSPHATASE CASEIN DEPHOSPHORYLATED^NR S CASEIN ^ NEGATIVEd NR CONTROL aS.E.M. = Standard Error of the Mean. bMice challenged with 0.00005 g enzyme. cITR = No Response at the lowest dilution tested (1/5). dUegative control consisted of a pooled antiserum sample.  Table 5. Mean Passive Cutaneous Anaphylaxis Titres for Mice Orally Administered Whey Proteins. ANTIGEN  ANTISERA  MEAN TITRE + S.E.M.a  WHEY  WHEY  NRb  NEGATIVEc CONTROL  NR  FeCl3^FeC13^ NR TREATED WHEY^TREATED WHEY NEGATIVE^NR CONTROL aS.E.M. = Standard Error of the Mean. bINTR = No Response at the lowest dilution tested (1/5). cAll negative controls consisted of a pooled antiserum sample.  42 dilution for all the protein samples tested. ^This result supported the assumption that the mice were not sensitized against the proteins. It is not known why the mice were not sensitized against the test proteins, since other researchers have been successful in obtaining sensitization following oral exposure. A possible explanation may be the strain of mice used. ^The oral immunisation procedure and schedule of Poulsen et al. (1990) was followed, however, Poulsen et al. (1990) used inbred NMRI mice in their experiments. NMRI mice are not available in North America, so Balb/C mice were substituted in this study. However, Poulsen et al. (1987) have shown that Balb/C mice can be orally sensitized to milk proteins.^Although in their experiment, Poulsen et al. (1987) fed the mice ad libitum over several generations. Balb/C mice may not have been suitable with the immunisation protocol used for this experiment. It should also be noted that Poulsen et al. (1990) performed homologous PCA in their experiment, therefore, both IgE and IgG, would participate in the PCA reaction. It is possible that the titre values obtained in their experiment were due solely to the influence of IgG, alone. Nielsen et al. (1989) compared the relative allergenicity of homogenized and non-homogenized milk using mice orally exposed to whole milk. However, in their experiments, the effect of IgG, on the PCA reaction was determined.^A positive IgE mediated PCA response could be obtained with homogenized milk, but not with non-homogenized  43 milk. The results of their experiments indicated that the physical state of the milk fat affected the allergenicity of the proteins. The protein preparations used in our experiments were pure and devoid of fat, which may have had the same effect as using non-homogenized milk. After experiment 1 was completed, it was not clear why the mice were not sensitized against the proteins; either the proteins were not antigenic and/or the route of exposure was inappropriate for sensitizing the mice. To resolve this question, a second experiment was performed using a different route of exposure for the test proteins. Instead of orally exposing the mice to the antigens, the proteins were injected intraperitoneally in experiment 2. B. Experiment 2: Intraperitoneally Injected Proteins B.1 Relative Antigenicity Table 6 shows the relative IgG values for the mice intraperitoneally injected with the casein proteins. T-tests were performed to determine if significant differences existed between the treatments. No significant difference (p < 0.05) was found between usl_csn and the negative control. This result indicated that the mice intraperitoneally injected with a1-csn were not sensitized against the protein. It should be noted that the IgG response of mouse number 5 was abnormally high in comparison to the other mice administered the same protein. For this reason, mouse number 5 was treated as an outlier and removed from the statistical analyses. All other casein  44  Table 6.  Comparison of the Mean Relative IgG Values for Mice Intraperitoneally Injected with Casein Proteins.  ANTIGEN  ANTISERAa  CASEIN  CASEIN  MEAN ABSORBANCE^MEAN IgG VALUE + S.E.M. +^S.E.M.b^(abs.'/min.)x103 0.023^+^0•003d'f  0.38d +^0.08  NEGATIVE CONTROL  0.019^+^0•006g  0.26^±^0.02  S CASEIN  0.607^+^0.058f  7.31^+^1.33  NEGATIVE CONTROL  0.001^+^0.000g  0.07^+^0.01  DEPHOS  DEPHOS  0.597^+^0.063f  7.71^+^1.43  CASEIN  CASEIN NEGATIVE CONTROL  0.011^+^0•001g  0.25^+^0.02  DEPHOS se CASEIN  0.221^+^0.030f  2.86^+^0.56  NEGATIVE CONTROL  0.005^+^0.001g  0.14^+^0.02  S CASEIN  usl e  DEPHOS se CASEIN  usle  aAntisera diluted 1/1000. bS.E.M. - Standard Error of the Mean. 'Absorbance dMause #5 was treated as an outlier and not included in the average. eDephosphorylated fAbsorbance measured after 59.83 minutes of incubation at 37°C. °Absorbance measured after 59.90 minutes of incubation at 37°C.  45  proteins (dephosphorylated a1-csn, S-csn and dephosphorylated S-csn) were significantly different (p < 0.05), from the negative control, which indicated that the mice were sensitized against these proteins by this route of exposure. A significant difference (p < 0.05) was found between untreated a1-csn and dephosphorylated usl-csn. The mean IgG value for the dephosphorylated ors,-csn was approximately 20 times greater than the mean for untreated usl-csn. Dephosphorylation of usl-csn significantly increased the antigenicity of the protein. Dephosphorylation may have exposed new antigenic determinants that were previously hidden, or changed the conformation of the protein to form new conformational epitopes, although, it is not known what effect dephosphorylation has on the tertiary structure of the molecule. It should be noted, that the increase in antigenicity may not be completely attributable to the exposure or formation of new antigenic determinants on the protein. The enzyme which was used to dephosphorylate the proteins may have also contributed to the antigenicity. Therefore, the increase in antigenicity as a result of dephosphorylation was probably lower than what the results have indicated. A significant difference (p < 0.05) was found between us,csn and g-csn. The mean IgG value for g-csn was approximately 19 times greater than the mean IgG value for a1-csn. ^This result suggests that S-csn is more antigenic than usl-csn. A significant difference (p < 0.05) was found between  46 untreated S-csn and dephosphorylated S-csn. The mean IgG value for dephosphorylated S was approximately 2.5 times lower than the mean for the untreated protein. Dephosphorylation of S-csn significantly decreased the antigenicity of the protein. Dephosphorylation may have destroyed conformational epitopes or changed the conformation of the protein so that epitopes became inaccessible to immunocompetent lymphocytes. It is not known what effect dephosphorylation had on the tertiary structure of the molecule. It should be noted, that the enzyme used to dephosphorylate the protein could also contribute to antigenicity. Therefore, the reduction in antigenicity as a result of dephosphorylation was probably greater than what the results have indicated. Table 7 shows the relative IgG values for the mice intraperitoneally injected with the whey proteins. A significant difference (p < 0.05) was found between each of the treatments and the negative control, which indicated that the mice were sensitized against the proteins. A significant difference (p < 0.05) was found between the whey and the FeC13 treated whey. The mean IgG value of the FeCl3 treated whey was approximately 1.7 times lower than the mean for the untreated whey. Thus, the removal of S-lactoglobulin decreased the antigenicity of the whey fraction. B.2 Relative Allergenicity Table 8 shows the relative IgE values for the mice intraperitoneally injected with the casein proteins. No  47  Table 7.  ANTIGEN WHEY  FeC13 TREATED  Comparison of the Mean Relative IgG Values for Mice Intraperitoneally Injected with Whey Proteins. ANTISERAa  MEAN ABSORBANCE^MEAN IgG VALUE + S.E.M. +^S.E.M.b^(abs.c/min.)x103  WHEY  0.505^+^0•023d  6.49^+^0.33  NEGATIVE CONTROL  0.009^+^0.007d  0.22^+^0.01  FeC13 TREATED  0.245^+^0.012d  3.81^+^0.26  NEGATIVE CONTROL  0.020^+0.001d  0.38^+^0.02  'Antisera diluted 1/1000. bS.E.M. = Standard Error of the Mean. 'Absorbance dAbsorbance measured 50.85 minutes after incubation at 37°C.  48  Table 8. Comparison of the Mean Relative IgE Values for Mice Intraperitoneally Injected with Casein Proteins. ANTIGEN CASEIN  g CASEIN  DEPHOS asi e CASEIN  DEPHOS se CASEIN  ANTISERAa as1 CASEIN  MEAN ABSORBANCE^MEAN IgG VALUE + S.E.M. +^S.E.M.13^(abs.c/min.)x103 0.016^+^0•004d'E  3.80d +^1.10  NEGATIVE CONTROL  0.004^+^0.001g  1.78^+^0.70  S CASEIN  0.100^+^0.009E  20.26^+^2.67  NEGATIVE CONTROL  0.027^+^0•009g  5.74^+^1.95  DEPHOS asie CASEIN  0.061^+^0.004E  14.52^+^1.75  NEGATIVE CONTROL  0.015^+^0.004g  3.92^+^1.18  DEPHOS se CASEIN  0.089^+^0.011E  19.24^+^3.02  NEGATIVE CONTROL  0.031^+^0.004g  6.71^+^0.55  akntisera diluted 1/20. = Standard Error of the Mean. cAbsorbance dMouse #5 was treated as an outlier and not included in the average. eDephosphorylated E.Absorbance measured after 3.93 hours of incubation at 37°C. °Absorbance measured after 3.94 hours of incubation at 37°C.  49 significant difference (p < 0.05) was found between a81-csn and the negative control. Since the IgG values indicated that the mice injected with u81-csn were not sensitized against the protein, this result was expected. As observed with the IgG data, mouse number 5 showed an abnormally high IgE response in comparison to the mice injected with the same protein. Therefore, mouse number 5 was treated as an outlier and removed from the statistical analyses.^All other proteins (dephosphorylated ces1-csn, S-csn, dephosphorylated S-csn) were significantly different (p < 0.05) than the negative control. A significant difference (p < 0.05) was found between dephosphorylated a1-csn and untreated us,-csn. ^The mean IgE value for dephosphorylated as, was approximately 3.8 times greater than the mean^for the untreated protein. Dephosphorylation^increased the ability of the protein to produce antigen specific IgE.^Presumably, dephosphorylation exposed new antigenic determinants that were previously hidden, or induced conformational changes in the protein which resulted in the formation of new conformational epitopes. However, the increase in antigen specific IgE cannot be completely attributed to dephosphorylation of the protein alone. The enzyme used for dephosphorylation could have also induced the production of specific IgE, which would inflate the results. A significant difference (p < 0.05) was found between us,csn and S-csn. The mean IgE value for S-csn was approximately 5.3 times greater than the mean for us„, which indicated that S  50 casein had a greater capacity for inducing the production of antigen specific IgE. The mean IgE value of untreated S casein was almost identical to the mean for the dephosphorylated protein. No significant difference (p < 0.05) was found between the treatments, which suggested that dephosphorylation did not affect the ability of S-csn to elicit the production of specific IgE. Although dephosphorylation might destroy or hide the antigenic determinants responsible for the production of IgG, the epitopes for IgE appeared to be unaffected. It should be noted, that the enzyme used to dephosphorylate the protein could induce the production of specific IgE. Therefore, dephosphorylation may reduce the capacity of S casein to produce IgE, but the presence of the enzyme may have masked this effect. Table 9 shows the relative IgE values for mice intraperitoneally injected with the whey proteins. A significant difference (p < 0.05) was found between each of the treatments and the negative control. Although the mean IgE value for the untreated whey was almost double the mean for the FeCl3 treated whey, no significant difference was found between the two treatments. Thus, the removal of the S-lactoglobulin did not appear to significantly reduce the ability of the whey to induce the production of specific IgE. Table 10 shows the passive cutaneous anaphylaxis titres of the mice intraperitoneally injected with the casein proteins. The t-test results of the PCA titres mirror the results obtained  51  Table 9.  ANTIGEN WHEY  FeC13 TREATED WHEY  Comparison of the Mean Relative IgE Values for Mice Intraperitoneally Injected with Whey Proteins. ANTISERAa  MEAN ABSORBANCE^MEAN IgG VALUE + S.E.M. + S.E.M.b^(abs.c/min.)x103  WHEY  0.081 +^0.012d  11.30^+^2.83  NEGATIVE CONTROL  0.001 +^0•000d  0.42^+^0.16  FeC13 TREATED WHEY  0.041 +^0.004d  6.63^+^0.39  NEGATIVE CONTROL  0.003 +^0•001d  0.61^+^0.24  akntisera diluted 1/20. bS.E.M. = Standard Error of the Mean. cAbsorbance dAbsorbance measured after 6.07 hours incubation at 37°C.  52  Table 10. Mean Passive Cutaneous Anaphylaxis Titres of Mice Intraperitoneally Injected with Casein Proteins. ANTIGEN^ANTISERA ^MEAN TITRE + S.E.M.a a s1^ CASEIN^CASEIN  NRb''  NEGATIVEd^NR CONTROL S CASEIN^S CASEIN^176 + 35 NEGATIVE^NR CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED^84 + 19 as1 CASEIN^CASEIN NEGATIVE^NR CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED^160 + 0 S CASEIN^g CASEIN ^ NEGATIVE NR CONTROL aS.E.M. = Standard Error of the Mean. IDNR = No Response at the lowest dilution tested (1/5). 94ouse #5 was treated as an outlier and not included in the average. dAll negative controls consisted of a pooled antiserum sample.  53  by measuring the concentrations of antigen specific IgE. There was no response at the lowest dilution tested (1/5) for the mice injected with a1-csn.^This indicated that a1-csn was not allergenic. Mouse number 5 produced an unusually large PCA titre in comparison to the other mice injected with the same protein, and was removed from the statistical analyses. A significant difference (p < 0.05) was found between untreated a1-csn and dephosphorylated a1-csn. This result indicated that dephosphorylation increased the allergenicity of the protein. Table 9b shows the PCA titres for the mice when challenged with the enzyme which was used to dephosphorylate the caseins. The results indicate that the enzyme contributes to, but is not wholly responsible for, allergenicity. Thus, the PCA titre reflects the increase in allergenicity due to dephosphorylation of the protein, and the inherent allergenicity of the enzyme. A significant difference (p < 0.05) was found between u81-csn and 8-csn. Thus, g-csn is more allergenic than usl-csn. The mean PCA titres for untreated g-csn and dephosphorylated 13-csn were similiar, and no significant difference (p < 0.05) was found between the two treatments. This result indicates that dephosphorylation has no significant effect on the allergenicity of this protein. The results from Table 11 shows that the enzyme used to dephosphorylate the protein contributes to, but is not completely responsible for,  54  Table 11.  Mean Passive Cutaneous Anaphylaxis Titres for Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase. ^ ANTIGEN ANTISERA^MEAN TITRE + S.E.M.a POTATO ACID b PHOSPHATASE  DEPHOSPHORYLATED^27 + 7 CASEIN DEPHOSPHORYLATED S CASEIN  ^  81 + 21  NEGATIVE^ NRc'd CONTROL aS.E.M. = Standard Error of the Mean. bMice challenged with 0.00005 g enzyme. cNR = No Response at the lowest dilution tested (1/5). dNegative control consisted of a pooled antiserum sample.  55  the observed allergenicity. Dephosphorylation may reduce the allergenicity of the protein, but the presence of the enzyme inflates the PCA titre which may have masked this effect. If the enzyme could be immobilized, its influence on allergenicity would be removed. The enzyme used in these experiments was derived from potatoes. An acid phosphatase from a source less foreign to mice and humans could be substituted, which might reduce its effects on allergenicity. Table 12 shows the PCA titres for the mice intraperitoneally injected with the native and modified whey proteins. Although the mean titre for the FeCl3 treated whey was almost half the mean titre for the untreated whey, no significant difference was found between the two treatments, due to the large range in variation of titres for the mice exposed to the untreated whey. Therefore, the removal of Z-lactoglobulin does not appear to significantly reduce the allergenicity of whey. It is important to note that whey is composed of a variety of proteins. The next largest whey fraction after g-lactoglobulin is cy-lactalbumin, which is also the second most incriminated allergenic whey protein in oral challenge studies (Lebenthal, 1975). The proteins which remained in the whey after treatment with FeC13 (primarily a-lactalbumin with a residual amount of 0-lactoglobulin) appear to equal untreated whey in their ability to elicit an allergic reaction.  56  Table 12. Mean Passive Cutaneous Anaphylaxis Titres of Mice Intraperitoneally Injected with Whey Proteins. ANTIGEN WHEY  ANTISERA^MEAN TITRE + S.E.M.a WHEY^ 608 + 171 NEGATIVE^NRb CONTROL  ^ 320 + 78 FeC13^ ^ FeC13 TREATED WHEY TREATED WHEY ^ NEGATIVEc NR CONTROL aS.E.M. = Standard Error of the Mean. IDNR = No Response at lowest dilution tested (1/5). eNegative control consisted of a pooled antiserum sample.  57 CONCLUSION  The milk proteins that were administered orally to mice failed to sensitize the animals. The mice could, however, be sensitized to the same proteins (except usi) when intraperitoneally injected. Intraperitoneal administration of the proteins removes the molecular size and stability (resistance to digestive enzymes and acid conditions) constraints on the antigens, which normally exist when a food is ingested. These factors can reduce the amount of antigen which comes into contact with the immune system. The mice were not sensitized against a51-csn by either route of exposure (oral administration or intraperitoneal injection). These results strongly indicate that as,-csn is non-allergenic. Dephosphorylation of as/-csn with acid phosphatase increased the allergenicity of the protein. S-csn was more allergenic than a81-csn. Dephosphorylation of S-csn with acid phosphatase did not significantly affect the allergenicity of the protein. The potato acid phosphatase used to dephosphorylate the caseins contributed to allergenicity. Partial removal of the S-lactoglobulin from whey did not significantly reduce the allergenicity. 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Development of the gastrointestinal mucosal barrier. I. the effect of age on intestinal permeability to macromolecules. Pediatr. Res. 15:241. Udall, J.N., Colony, P., Fritze, L., Pang, K., Trier, J.S. and Walker, W.A. 1981b. Development of gastrointestinal mucosal barrier. II. the effect of natural versus artificial feeding on intestinal permeability to macromolecules. Pediatr. Res. 15:245. Vaz, E.M., Vaz, N.M. and Levine, B.B. 1971. Persistent formation of reagins in mice injected with low doses of ovalbumin. Immunol. 21:11. Visakorpi, J.K. and Immonen, P. 1967. Intolerance to cow's milk and wheat gluten in the primary malabsorption syndrome in Infancy. Acta Paed. Scand. 56:49. Voller, A. 1980. Heterogeneous enzyme-immunoassays and their applications. Ch. 9. In "Enzyme-Immunoassay", E.T. Maggio (Ed.), P. 181. CRC Press Inc., Boca Raton, Florida. Walker, W.A. 1987. Pathophysiology of intestinal uptake and absorption of antigens in food allergy. Ann. of Allergy 59 (part II):7. Walker, W.A. and Bloch, K.J. 1983. Gastrointestinal transport of macromolecules in the pathogenesis of food allergy. Ann. of Allergy 51:240. Watanabe, N. and Ovary, Z. 1977. Antigen and antibody detection by in vivo methods; a reevaluation of passive cutaneous anaphylaxis reactions. J. Immun. Met. 14:381. Williams, C.A. and Chase, M.W. (Ed.). 1967. "Methods in Immunology and Immunochemistry", Academic Press, New York, New York. Zittle, C.A. and Custer, J.H. 1963. Purfication and some of the properties of us-casein and K-casein. J. Dairy Sci. 46:1183.  66 APPENDIX 1 PROTEIN DETERMINATION  Table 13. Protein Concentration of Ovalbumin Standard. MEAN ABSORBANCEa ABSORBANCE PROTEIN' SAMPLE^REPLICATE #^(280 nm)^+ S.E.M.b (mg/ml) ^ OVALBUMIN 1^0.69^0.69 + 0.00^9.9 2^0.69 3^0.70 aStandard diluted 1/10. bS.E.M. = Standard Error of the Mean. 'Extinction Coefficient of Ovalbumin (Kirschenbaum, 1976) = 7  Table 14.^Protein Concentration of Casein Stock Solutions.  SAMPLE^REPLICATE #  ABSORBANCE (550 nm)  MEAN ABSORBANCE PROTEIN + S.E.M.a^(mg/ml)  ces1  1 2 3  0.49 0.52 0.53  0.51 +^0.09  8.45  S  1 2 3  0.24 0.24 0.25  0.24 +^0.00  3.83  DEPHOSPHORYLATED  1 2 3  0.17 0.18 0.19  0.18 +^0.00  2.72  DEPHOSPHORYLATED S  1 2 3  0.17 0.19 0.18  0.18 +^0.01  2.73  us,  aS.E.M. = Standard Error of the Mean.  67  Table 15. Protein Concentration of Whey Stock Solutions. MEAN ABSORBANCE PROTEIN + S.E.M.a^(mg/ml)  SAMPLE  REPLICATE #  ABSORBANCE (550nm)  WHEY  1 2 3  0.16 0.17 0.17  0.17 + 0.00^2.52  FeC13 TREATED WHEY  1 2 3  0.06 0.06 0.06  0.06^+^0.00^0.76  aS.E.M. . Standard Error of the Mean.  68  0.75  0.60  Y = 0.058 X + 0.021 - r2 = 0.996  0.45 0.30 ^ 0.15 _  0.00 0.00  I^  2.20  I  4.40  6.60  8.80  11.00  PROTEIN (mg/m1) Figure 2. Ovalbumin Standard Curve.  The Biuret assay was used to determine the protein concentration of the fl-lactoglobulin standards.  69 APPENDIX 2 PHOSPHORUS DETERMINATION  Table 16.  Phosphorus Concentration of Casein Samples. REP' #  as l  0.380  1 2 3  0.84 0.90 1.01  0.92 +^0.04^13.81  DEPHOS asic CASEIN  0.999  1 2 3  0.79 0.81 0.85  0.82 +^0.01  4.62  S  0.383  1 2 3  0.45 0.47 0.47  0.46 +^0.01  6.76  1.093  1 2 3  0.22 0.21  0.22 +^0.00  1.07  SAMPLE  DEPHOS iy CASEIN  (mq)  ABSORBANCE (822 nm)  MEAN ABSORBANCE^PHOSPHORUS + S.E.M.b^(q/mg protein)  PROTEIN  'Replicate bS.E.M. = Standard Error of the Mean. cDephosphorylated  70 CALCULATIONS FOR DETERMINING % DEPHOSPHORYLATION  % Dephosphorylation = 1 -  Pf  X  Pi  100  Where: Pi = phosphorus concentration before dephosphorylation Pf =  phosphorus concentration after dephosphorylation  4.62 yg phosphorus % Dephosphorylation c = 1 - ^mg protein ^x 100 = 66.5% 13.81 aq phosphorus mg protein 1.07 yg phosphorus % Dephosphorylation S = 1 - ^mg protein ^x 100 = 84.1% 6.76 yq phosphorus mg protein  71  2.00  1.60  Y = 0.175 X + 0.011 - r2 = 0.999  1.20  _  0.80  _  0.40  0.00 0.00  2.20  4.40  6.60  8.80  11.00  PHOSPHORUS (MICROGRAMS)  Figure 3. Phosphorus Standard Curve.  Phosphorus concentrations of the standards were determined using the method of Morrison (1964).  72 APPENDIX 3 S-LACTOGLOBULIN DETERMINATION  Table 17. S-Lactoglobulin Concentration of Wheys. SAMPLE WHEY FeC13 TREATED WHEY  OPTICAL^S-LACTOGLOBULIN DENSITY^CONCENTRATION (mg/ml) 2.341^1.167 _ a^  _  aBelow the minimum detection limit of the PhastImage program.  73 CALCULATIONS FOR DETERMINING 96 S-LACTOGLOBULIN REMOVED  % S-lactoglobulin removed = 1 - ^x 100 Si Where: Si = S-lactoglobulin concentration of whey gf =  S-lactoglobulin concentration of FeCl3 treated whey  Since the concentration of S-lactoglobulin in the FeC13 treated whey was below the minimum detection limit of the PhastImage program, the lowest concentration used for constructing the S-lactoglobulin standard curve was substituted. ^This value represents the minimum % of S-lactoglobulin removed. Please note, that the actual amount of S-lactoglobulin removed exceeds this value.  Minimum % S-lactoglobulin = 1 - 0.169 mg/ml x 100 = 85.5% removed^ 1.167 mg/m1  74  4  Y = 2.156 X - 0.175 r2 = 0.999  0 0^  1  ^  BETA-LACTOGLOBULIN ClughnO  Figure 4. P-lactoglobulin Standard Curve.  2  75  12345678  Figure 5. SDS-PAGE Gel of Wheys and fl-lactoglobulin Standards.  (1) fl-lactoglobulin standard; 1.69 mg/ml and internal standard, (2) P-lactoglobulin standard; 1.27 mg/ml and internal standard, (3) P-lactoglobulin standard; 0.85 mg/ml and internal standard, (4) FeCl, Treated Whey and internal standard, (5) Untreated Whey and internal standard, (6) 0-lactoglobulin standard; 0.34 mg/ml and internal standard, (7) P-lactoglobulin standard; 0.17 mg/ml and internal standard, (8) Ovalbumin internal standard.  76 APPENDIX 4 RODENT DIET The following diet is based on the American Institute of Nutrition AIN-76 semi-purified diet' for rats and mice.  Table 18.^Composition of Milk-Free Rodent Diet INGREDIENT Sucrose' Soy Protein Isolateb Corn Starchc Canola Oild Non-Nutritive Fiber' Mineral Mix f Vitamin Mixg D-L Methioninec Choline bitartratec  % LEVEL OF INGREDIENT (w/w) 50.0 20.0 15.0 5.0 5.0 3.5 1.0 0.3 0.2  'B.C. Sugar (Vancouver, B.C., Canada.) b921 protein (ICN Nutritional Biochemicals, Cleveland, OH, USA). cICN Nutritional Biochemicals, Cleveland, OH, USA. dSunfrie (Vancouver, B.C., Canada). eAlphacel; composed of finely ground alpha-cellulose (ICN Nutritional Biochemicals, Cleveland, OH, USA). LAIN Mineral Mixture 76 (ICN Nutritional Biochemicals, Cleveland, OH, USA) gAIN Vitamin Mixture 76 (ICN Nutritional Biochemicals, Cleveland, OH, USA) 'ICN Biomedicals Catalog/Animal Research Diets. 1991/1992. p. 1036.  77 APPENDIX 5 ELISA BUFFER COMPOSITIONS The following compositions are based on the ELISA buffers described by Kummer et al. (1992). 1. Carbonate Coating Buffer (pH 9.6) 1.59 g Na2CO3 2.93 g NaHCO3 0.50 g NaN3 1.00 1 H2O 2. Phosphate Buffered Saline (PBS) (pH 7.4) 8.00 g NaCl 0.20 g KH2PO4 1.15 g Na2HPO4 0.20 g KC1 1.00 1 1120 3. PBS/TWEEN - as described for PBS (composition 2), in addition to 0.5 ml Tween 20. 4. Blocking Buffer - as described for PBS (composition 2), in addition to 2.50 g chicken egg albumin. 5. Diethanolamine Substrate Buffer (pH 9.8) 0.10 g MgC12 x 6 H2O 0.20 g NaN3 97.00 ml Diethanolamine 1.00 1 H20  78  APPENDIX 6 ELISA ABSORBANCE VS. TIME GRAPHS A. ELISA IgG Determinations A.1 Experiment 1: Orally Administered Proteins 0.35  0.28  0.21  Mouse 1 + ^ Mouse 2 A Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A ---- -Mouse 6 • ---Mouse 7 • ---  0.14  0.07  0.00 0^75^150^225  ^  300  ^  375  TIME (MINUTES)  Figure 6. Determination of as, Casein Specific IgG in Mice Orally Administered as, Casein. 0.35  0.28  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 -----  0.21  0.14  0.07  0.00 75^150^225  ^  300  ^  375  TIME (MINUTES)  Figure 7. Determination of us, Casein Specific IgG in Control Mice (Oral Administration).  79 0.90  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A --Mouse 6 • ----  0.72 sr) 0.54 W U Z  0.36  0 0 0^0.18 -4 0.00 0  ^  65  ^  130  ^  195  ^  260  ^  325  TIME (MINUTES)  Figure 8. Determination of 13 Casein Specific IgG in Mice Orally Administered 0 Casein. 0.90  Mouse 1 + Mouse 2 A ^ Mouse 3 0 --Mouse 4 0 -----  0.72  0.00 0  65  130  195  260  325  TIME (MINUTES)  Figure 9. Determination of 0 Casein Specific IgG in Control Mice (Oral Administration).  80  1.25  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A-----  1.00  0.75  0.50  0.25  0.00 0  75  150  225  300  375  TIME (MINUTES)  Figure 10. Determination of Dephosphorylated as1 Casein Specific IgG in Mice Orally Administered Dephosphorylated as1 Casein. 1.25  Mouse 1 + Mouse 2 A Mouse 3 0 - - - Mouse 4 0 -----  1.00  0.75  0.50  0.25  0.00  0  75  150  225  300  375  TIME (MINUTES)  Figure 11. Determination of Dephosphorylated as, Casein Specific IgG in Control Mice (Oral Administration).  81 0.30 0.24 0.18  Mouse 1 +^ Mouse 2 A ^ ^ Mouse 3 0 - - - - Mouse 4 0 ----^A Mouse 5 A --Mouse 6 0 --Mouse 7 • --- ^A  A A  0.12 0.06 0.00 ^ ^ ^ ^ ^ 0 75 150 225 300 375 TIME (MINUTES)  Figure 12. Determination of Dephosphorylated A Casein Specific IgG in Mice Orally Administered Dephosphorylated A Casein. 0.30 Mouse 1 + ^ Mouse 2 A ^ 0.24 - Mouse 3 0 - - - - Mouse 4 0 ----0.18 0.12 0.06 0.00  0  75  150  225  300  375  TIME (MINUTES)  Figure 13. Determination of Dephosphorylated g Casein Specific IgG in Control Mice (Oral Administration).  82 0.27  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A --Mouse 6 • ----  0.22  0.16  0.11  0.05  0.00 0  90  180  270  360  450  TIME (MINUTES)  Figure 14. Determination of Whey Specific IgG in Mice Orally Administered Whey. 0.27  0.22  Mouse 1 + Mouse 2 A ^ Mouse 3 0 --Mouse 4 0 -----  0.16  0.11  0.05  0.00 0  90  180  270  360  450  TIME (MINUTES)  Figure 15. Determination of Whey Specific IgG in Control Mice (Oral Administration).  83 0.27 Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 o ----Mouse 5 A ----Mouse 6 • ---Mouse 7 • ---  0.22 0.16 0.1 1  0.05 0.00  0  90  180  270  360  450  TIME (MINUTES)  Figure 16. Determination of FeC13 Treated Whey Specific IgG in Mice Orally Administered FeC13 Treated Whey. 0.27 Mouse 1 + ^ Mouse 2 A ^ 022 - Mouse 3 0 - - Mouse 4 0 ----0.16 0.1 1  0.05 0 .00  0  90  180  270  360  450  TIME (MINUTES)  Figure 17. Determination of FeC13 Treated Whey Specific IgG in Control Mice (Oral Administration).  ^ ^  84 A.2 Experiment 2: Intraperitoneally Injected Proteins 1.50 ^ Mouse 1 +^4/ Mouse 2 A ^/A --d^1.20 a^Mouse 3 0 — - - -^,/,/ 41^Mouse 4 a ----kr/ 0 Mouse 5 A ----- / I. ..,^ 0.90^ -^ t/  al^  /  Z -4 0.60^/.,1' m M A" 0^/ cn al 0.30 .  165 ....... ^..^...... ^ '2"dkifILL4L-A^ 0.00 ^0^55^110^220^275 TIME (MINUTES)  Figure 18. Determination of ce81 Casein Specific IgG in Mice Intraperitoneally Injected with asi Casein. 1.50 ^ Mouse 1 + Mouse 2 A ^ 1.20 - Mouse 3 0 — --Mouse 4 0 ----Mouse 5 A ----0.90 0.60 0.30 0.00  0^55^110^165^220^275 TIME (MINUTES)  Figure 19. Determination of ce81 Casein Specific IgG in Control Mice (Intraperitoneal Injection).  85 1.50  1.20  0.90  t/  0.60  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A ----  0.30  0.00 0  ^  55  ^  110  ^  165  ^  220  ^  275  TIME (MINUTES)  Figure 20. Determination of g Casein Specific IgG in Mice Intraperitoneally Injected with Casein. 1.50  =  Mouse 1 + Mouse 2 A Mouse 3 0 - - - Mouse 4 0 ----Mouse 5 A -----  120  0.90 0 •4^0.60  0.30  0.00 0  55  110  165  220  275  TIME (MINUTES)  Figure 21. Determination of 13 Casein Specific IgG in Control Mice (Intraperitoneal Injection).  ^  ...„, ,, ..„-. '-^-.0 ,,/^-........„7 , i ....„ . „, ,  1.20  A• /121^,^ . 7...„-^ .. ,^„^  e 0 ••- /^ 1 e.. .^...." .7 / .7 // .Bli/^ ,,,, ., -"/^ -"/ .Y  a 0.90  _  44 0.60 0 •  *7 '  ...,  ,-  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 o ----Mouse 5 A -----  -,,,;-,/K/ A %IN  0.30  ." e /  0.00  86  , ,, ,A  1.50  0  AN  55  110  165  220  275  TIME (MINUTES)  Figure 22. Determination of Dephosphorylated ces1 Casein Specific IgG in Mice Intraperitoneally Injected with Dephosphorylated as, Casein. 1.50 1.20 0.90  Mouse 1 + Mouse 2 A ^ Mouse 3 0 -- --Mouse 4 0 ----Mouse 5 A -----  .4 0.60 0 • 44  0.30 0.00 0  55  110  165  220  TIME (MINUTES)  Figure 23. Determination of Dephosphorylated us, Casein Specific IgG in Control Mice (Intraperitoneal Injection).  275  87 1.50  1.20  0.90  Mouse 1 + Mouse 2 ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A  0.60  0.30  0.00 0  55  110  165  220  275  TIME (MINUTES)  Figure 24. Determination of Dephosphorylated A Casein Specific IgG in Mice Intraperitoneally Injected with Dephosphorylated 0 Casein. 1.50  1.20  0.90  Mouse 1 + Mouse 2 Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  0.60  0.30  0.00 0  55  110  165  220  275  TIME (MINUTES)  Figure 25. Determination of Dephosphorylated A Casein Specific IgG in Control Mice (Intraperitoneal Injection).  88  1.50 =^1.20 Nt. •  0.90  0 4t 0.60 0.30 0.00 ^ ^ ^ ^ ^ 0 42 84 126 168 210 TIME (MINUTES)  Figure 26. Determination of Whey specific IgG in Mice Intraperitoneally Injected with Whey. 1.50 Mouse 1 + Mouse 2 A ^ 1.20 Mouse 3 0 - - - - Mouse 4 0 - - - - 0.90 - Mouse 5 A ----  0.60 0.30 0.00  0  ^  42  ^  84  ^  126  •^ ^  ^  168  210  TIME (MINUTES)  Figure 27. Determination of Whey Specific IgG in Control Mice (Intraperitoneal Injection).  89 1.50  1.20  Mouse 1 + Mouse 2 A ^ Mouse 3 0 -Mouse 4 0 ----Mouse 5 A ----- - -  0.90  0.60  0.30  0.00 42  ^  84  ^  126  ^  168  ^  210  TIME (MINUTES)  Figure 28. Determination of FeC13 Treated Whey Specific IgG in Mice Intraperitoneally injected with FeC13 Treated Whey. 1.50  '-do  1.20  0.90  Mouse 1 + Mouse 2 A Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  0  <  0.60  0 0.30  0.00  -  6""a6='=ill'4"^I^  0^42^84^126  ^  168^210  TIME (MINUTES)  Figure 29. Determination of FeC13 Treated Whey Specific IgG in Control Mice (Intraperitoneal Injection).  ^  90 B. ELISA IgE Determinations 0.42 Mouse 1 + 0.34 - Mouse 2 A ^ Mouse 3 0 --- --^ Mouse 4 0 -- ---^ Mouse 5 • -----^• ..,. 0.25^ .,.t. • #--' ,0.17 -^ --  •  )k-. •  ±---  .A'. .....-'A  •  o  , 0.08^,^ ^-------  ,-.----  8 ------Ar^ 0 ----9-------6 ---------0__ .-^ 5_ .-Q ---------^ ....- A ------------ cr  ^ ^ ------ ---0.00^- = t--74^ '4^ 0.00^2.50^5.00^7.50^10.00^12.50 -------  TIME (HOURS) Figure 30. Determination of us, Casein Specific IgE in Mice Orally Intraperitoneally Injected with a 0.42 0.34 0.25  Mouse 1 + Mouse 2 A Mouse 3 o -Mouse 4 0 ----Mouse 5 • -----  0.17 0.08 0.00 ^  0  W  0  0.00^2.50^5.00  ---  °  4-^--  --- ------------------ ----- 1-2.50  TIME (HOURS) Figure 31. Determination of a ^Specific IgE in Control Mice (Intraperitoneal Injection).  91 0.42 0.34 025  Mouse 1 + Mouse 2 A Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  0.17 0.08 0.00 0.00  2.50  5.00  7.50  10.00^12.50  TIME (HOURS)  Figure 32. Determination of g Casein Specific IgE in Mice Intraperitoneally Injected with g Casein. 0.42 0.34 0.25  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - Mouse 4 0 ----Mouse 5 A -----  0.17 0.08 0.00 0.00  •  • -^ 0  • ^ •  •  •  0  0 ••• ••• •••^  ••••  -------  ^  -------  -----  --------  A-  2.50 2.50^5.00^7.50^10.00^12.50 TIME (HOURS)  Figure 33. Determination of 0 Casein Specific IgE in Control Mice (Intraperitoneal Injection).  92  0.42  0.34  0.25  Mouse 1 + Mouse 2 A Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  A  A^  0  A  •4^0.17 0 0.08  TIME (HOURS)  Figure 34.  Determination of Dephosphorylated as/ Casein Specific IgE in Mice Intraperitoneally Injected with a 0.42  0.34  0.25  Mouse 1 + Mouse 2 A ^ Mouse 3 0 -- --Mouse 4 0 ----Mouse 5 A -----  0.17  0.08  TIME (HOURS)  Figure 35. Determination of Dephosphorylated as/ Casein Specific IgE in Control Mice (Intraperitoneal Injection).  93 0.42  Mouse 1 + ^ Mouse 2 A ^ 0.34 - Mouse 3 - - - - Mouse 4 0----Mouse 5 A ----025 0.17  0.08  A^ 0.00 0.00  2.50  5.00  7.50  •^•  10.00  12.50  TIME (HOURS)  Figure 36. Determination of Dephosphorylated 13 Casein Specific IgE in Mice Intraperitoneally Injected with g Casein. 0.42  Mouse 1 + Mouse 2 A 0.34 - Mouse 3 0 - Mouse 4 o ----Mouse 5 A ----0.25 0.17  0.08  0.00 0.00  2.50  5.00  7.50  10.00  12.50  TIME (HOURS)  Figure 37. Determination of Dephosphorylated g Casein Specific IgE in Control Mice (Intraperitoneal Injection).  94 0.35  0.28  0.21  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 • ------  0.14  0.07  0.00 0.00  2.50  5.00  7.50  10.00  12.50  TIME (HOURS)  Figure 38. Determination of Whey Specific IgE in Mice Intraperitoneally Injected with Whey. 0.35 Mouse 1 + Mouse 2 A ^ 028 - Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 • ----021 0.14 0.07 0.00 0.00^2.50^5.00^7.50^10.00^12.50 TIME (HOURS)  Figure 39. Determination of Whey Specific IgE in Control Mice (Intraperitoneal Injection).  95 0.35 0.28 021  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  0.14  TIME (HOURS)  Figure 40. Determination of FeCl3 Treated Whey Specific IgE in Mice Intraperitoneally Injected with FeC13 Treated Whey. 035  028  0.21  Mouse 1 + Mouse 2 A ^ Mouse 3 0 - - - - Mouse 4 0 ----Mouse 5 A -----  0.14  0.07  0.00 0.00  2.50  5.00  7.50  1000  12.50  TIME (HOURS)  Figure 41. Determination of FeC13 Treated Whey Specific IgE in Control Mice (Intraperitoneal Injection).  96 APPENDIX 7 EQUATIONS OF THE LINES AND  r2  VALUES FOR INDIVIDUAL MICE  A. ELISA IgG Determinations A.1 Experiment 1: Orally Administered Proteins  Table 19. IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Native Casein Proteins. ANTIGEN us, CASEIN  S CASEIN  ANTISERA  EQUATION OF THE LINE  MOUSE #  r2  VALUE  us, CASEIN  1 2 3 4 5 6 7  Y Y Y Y Y Y Y  = = = = = = =  6.50X 6.10X 6.20X 8.40X 5.24X 6.48X 4.97X  x x x x x x x  x x x x x x x  10-3 10-3 10-3 10-2 10-4 10-3 10-2  0.99 0.93 0.89 0.90 0.95 0.98 0.67  NEGATIVE CONTROL  1 2 3 4  Y Y Y Y  = = = =  8.23X 7.93X 5.70X 8.42X  x 10-4 +^6.89 x x 10-4 +^1.08 x x 10-4 +^3.24 x x 10-4 +^1.22 x  10-3 10-2 10-3 10-2  0.87 0.93 0.98 0.98  J3 CASEIN  1 2 3 4 5 6  Y Y Y Y Y Y  = = = = = =  1.00X 9.50X 1.09X 8.20X 2.06X 2.12X  x x x x x x  x x x x x x  10-3 10-3 10-3 10-3 10 -4 10-4  0.65 0.70 0.37 0.87 0.58 0.97  NEGATIVE CONTROL  1 2 3 4  Y Y Y Y  = = = =  1.23X 1.60X 1.64X 1.86X  x 10-4 -^7.16 x x 10-4 +^8.70 x x 10-4 +^2.99 x x 10-4 -^1.65 x  10-4 10-5 10-3 10-3  0.86 0.75 0.96 0.96  10-4 10-4 10-4 10-4 10-4 10-4 10-4  10-4 10-5 10-4 10-5  + 4.44 + 4.20 +^5.44 +^1.02 -^4.43 +^9.67 +^2.62  -^2.23 -^2.63 -^3.17 -^2.20 10-4 -^8.39 10-4 -^3.51  97  Table 20. IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Dephosphorylated Casein Proteins. ANTIGEN  ANTISERA  DEPHOS  DEPHOS  CASEIN  CASEIN  EQUATION OF THE LINE  MOUSE #  r2 VALUE  1 2 3 4 5  Y Y Y Y Y  = = = = =  7.02X 8.45X 1.06X 2.84X 1.67X  x x x x x  10-4 10-4 10-3 10-4 10-3  +^8.92 +^1.67 +^1.88 +^1.42 + 4.93  x x x x x  10-3 10-2 10-2 10-1 10-2  0.86 0.47 0.37 0.97 0.75  NEGATIVE CONTROL  1 2 3 4  Y Y Y Y  = = = =  9.27X 6.79X 6.03X 8.36X  x x x x  10-4 10-4 10-4 10-4  +^1.51 +^8.96 +^8.68 +^1.68  x x x x  10-2 10-3 10-3 10-2  0.37 0.96 0.55 0.43  DEPHOS  DEPHOS  CASEIN  CASEIN  1 2 3 4 5 6 7  Y Y Y Y Y Y Y  = = = = = = =  3.58X 4.35X 2.13X 2.59X 2.68X 2.34X 1.58X  x x x x x x x  10-4 10-4 10-4 10-4 10-4 10-4 10-4  +^3.07 +^6.45 + 2.94 +^5.48 +^7.33 -^4.54 +^2.38  x x x x x x x  10-3 10-3 10-3 10-3 10-3 10-3 10-3  0.64 0.29 0.72 0.74 0.90 0.70 0.81  1 2 3 4  Y Y Y Y  = = = =  2.22X 2.71X 2.28X 1.80X  x x x x  10-4 10-4 10-4 10-4  +^5.49 +^7.35 +^7.36 +^5.52  x x x x  10-3 10-3 10-3 10-3  0.97 0.99 0.47 0.96  ,,,^a usi  ga  asia  ga  NEGATIVE CONTROL  aDephosphorylated  98  Table 21. IgG Equations of the Lines and r2 Values for Individual Mice Orally Administered Whey Proteins. ANTIGEN WHEY  FeC13 TREATED WHEY  ANTISERA  EQUATION OF THE LINE  MOUSE #  r2 VALUE  WHEY  1 2 3 4 5 6  Y Y Y Y Y Y  = = = = = =  2.96X 2.64X 2.42X 2.55X 2.01X 2.95X  x 10 -4 x lo-4 x 10 -4 x lo-4 x 10 -4 x lo-4  +^5.14 -^4.81 +^2.75 +^3.92 +^4.08 +^7.03  x x x x x x  10-3 10-4 10-3 10-3 10-3 10-3  0.97 0.95 0.91 0.94 0.94 0.93  NEGATIVE CONTROL  1 2 3 4  Y Y Y Y  = = = =  2.58X 3.05X 2.21X 2.52X  x x x x  10-4 10-4 10-4 10-4  +^5.74 +^8.23 +^3.82 +^9.78  x x x x  10-3 10-3 10-3 10-3  0.90 0.92 0.94 0.96  FeC13 TREATED WHEY  1 2 3 4 5 6 7  Y Y Y Y Y Y Y  = = = = = = =  2.67X 3.32X 2.42X 3.49X 3.15X 2.38X 2.70X  x x x x x x x  10-4 10-4 10-4 10-4 10-4 10-4 10-4  + 4.57 +^6.36 +^8.06 +^1.23 +^9.38 +^4.85 +^8.77  x x x x x x x  lo-3 10-3 lo-3  10 -2 10-3 10 -3 10-3  0.95 0.95 0.96 0.96 0.97 0.98 0.98  NEGATIVE CONTROL  1 2 3 4  Y Y Y Y  = = = =  2.16X 4.16X 2.26X 5.28X  x x x x  10-4 10-4 10-4 10-4  +^8.36 +^1.40 +^1.15 +^2.57  x x x x  10-3 10-2 10-2 10-2  0.97 0.86 0.96 0.95  99 A.2 Experiment 2: Intraperitoneally Injected Proteins  Table 22. IgG Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins. ANTIGEN  ANTISERA  asi CASEIN  CASEIN  S CASEIN  EQUATION OF THE LINE  MOUSE #  r2  VALUE  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.06X 6.36X 2.24X 4.66X 8.59X  x x x x x  10-4 10-4 10-4 10-4 10-3  -^1.38 -^7.58 -^1.15 +^9.57 +^1.34  x x x x x  10-3 10-4 10-3 10-4 10-1  0.91 0.99 0.87 0.77 0.78  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.87X 2.71X 3.07X 2.03X 2.28X  x x x x x  10-4 10-4 10-4 10-4 10-4  -^5.70 -^3.90 -^4.53 -^4.28 -^4.17  x x x x x  10-3 10-3 10-3 10-3 10-3  0.99 0.99 0.98 0.96 0.91  S CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.24X 5.79X 5.00X 4.45X 8.96X  x x x x x  10-2 10-3 10-3 10-3 10-3  +^2.63 +^1.69 +^1.80 +^1.06 +^1.31  x x x x x  10-1 10-1 10-1 10-1 10-1  0.95 0.93 0.82 0.97 0.98  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.23X 5.50X 8.50X 3.80X 4.50X  x x x x x  10-4 10-5 10-5 10-5 10-5  -^4.79 -^1.86 -^2.71 -^2.19 -^1.18  x x x x x  10-3 10-3 10-3 10-3 10-3  0.94 0.52 0.46 0.74 0.58  ces1  100  Table 23.^IgG Equation of the Lines and r2 Values forIndividual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins. ANTIGEN^ANTISERA DEPHOS cesi a CASEIN  DEPHOS ga CASEIN  EQUATION OF THE LINE  MOUSE #  r2 VALUE  DEPHOS °Isla CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.74X 1.20X 7.95X 9.82X 6.02X  x x x x x  10-3 10-2 10-3 10-3 10-3  +^2.73 +^1.04 +^6.32 +^1.74 +^1.61  x x x x x  10 -2 10-1 10-2 10-1 10-1  0.95 0.99 0.99 0.96 0.97  NEGATIVE CONTROL  1 2 3 4 5  Y = Y= Y = Y = Y =  2.98X 2.12X 3.16X 1.92X 2.22X  x x x x x  10-4 10-4 10-4 10-4 10-4  -^3.36 -^5.59 -^2.92 -^2.56 -^1.91  x x x x x  10-3 10-3 10-3 10-3 10-3  0.99 0.90 0.93 0.96 0.73  DEPHOS ga CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.13X 3.87X 4.24X 1.58X 3.48X  x x x x x  10-3 10-3 10-3 10-3 10-3  +^3.37 +^7.28 +^9.25 +^6.59 +^6.19  x x x x x  10-3 10-2 10-2 10-3 10-2  0.96 0.98 0.99 0.99 0.98  NEGATIVE CONTROL  1 2 3 4 5  Y= Y= Y= Y= Y =  1.36X 1.91X 1.79X 1.32X 8.40X  x x x x x  10-4 10 -4 10-4 10-4 10-5  -^2.97 -^3.95 -^3.65 -^2.57 -^1.00  x x x x x  10-3 i010-3 10-3 10-3  0.95 0.73 0.98 0.91 0.77  aDephosphorylated  101  Table 24. IgG Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Whey Proteins. ANTIGEN WHEY  FeC13 TREATED WHEY  ANTISERA  EQUATION OF THE LINE  MOUSE #  r2 VALUE  WHEY  1 2 3 4 5  Y Y Y Y Y  = = = = =  6.08X 7.40X 5.51X 6.12X 7.32X  x x x x x  110-3 icr3 lo-3 icy3 lo-3  +^1.40 +^1.90 +^1.02 +^1.33 +^2.24  x x x x x  10-1 10-1 10-1 10-1 10-1  0.97 0.96 0.96 0.98 0.97  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.94X 2.15X 2.49X 2.03X 2.42X  x x x x x  10-4 10-4 10-4 10-4 10-4  +^2.11 -^1.61 -^1.46 -^1.63 -^1.59  x x x x x  10-3 10-3 10-3 10-3 10-3  0.67 0.88 0.94 0.80 0.89  FeC13 TREATED WHEY  1 2 3 4 5  Y Y Y Y Y  = = = = =  3.28X 4.42X 3.09X 4.50X 3.76X  x x x x x  10-3 10-3 10-3 10-3 10-3  +^2.88 +^6.58 +^3.58 +^6.41 + 4.00  x x x x x  10-2 10-2 10-2 10-2 10-2  0.95 0.95 0.96 0.96 0.97  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  3.49X 3.72X 4.74X 3.53X 3.27X  x x x x x  10-4 10-4 10-4 10-4 10-4  -^1.14 +^8.64 + 4.12 -^6.95 -^5.30  x x x x x  10-3 10-4 10-3 10-2 10-5  0.97 0.86 0.96 0.95 0.95  102 B. ELISA IgE Determinations  Table 25.  IgE Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins. EQUATION OF THE LINE  MOUSE #  r2 VALUE  ANTIGEN  ANTISERA  asi CASEIN  as, CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.59X 1.15X 4.45X 7.01X 2.74X  x x x x x  10-3 10-3 10-3 10-3 10-2  -^7.78 -^1.91 +^6.38 +^1.99 +^1.80  x x x x x  10" 10-3 10" 10-3 10-2  0.36 0.50 0.83 0.70 0.97  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.39X 0.00X 4.61X 2.09X 8.23X  x x x x x  10-3 100 10-3 10-3 10"  -^2.47 +^0.00 -^4.09 -^2.46 -^1.95  x x x x x  10-3 100 10-3 10-3 10-3  0.40 0.00 0.51 0.41 0.45  8 CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.96X 2.90X 1.06X 2.30X 1.91X  x x x x x  10-2 10-2 10-2 10-2 10-2  +^1.67 +^2.43 + 4.65 +^1.52 +^1.07  x x x x x  10-2 10-2 10-3 10-2 10-2  0.97 0.98 0.92 0.98 0.96  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.48X 0.00X 1.06X 4.72X 1.09X  x x x x x  10-3 100 10-2 10-3 10-2  -^1.46 +^0.00 +^3.81 -^1.25 + 3.61  x x x x x  10" 100 10-2 10-3 10-3  0.40 0.00 0.57 0.87 0.29  g CASEIN  103  Table 26. IgE Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins. EQUATION OF THE LINE  ANTIGEN  ANTISERA  DEPHOS  DEPHOS asia CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  9.65X 2.13X 1.35X 1.58X 1.24X  x x x x x  10-3 10-2 10-2 10-2 10-2  +^1.22 -^1.01 +^4.36 +^7.39 +^3.64  x x x x x  10-3 10-2 10-3 10-3 10-3  0.91 0.91 0.95 0.74 0.96  NEGATIVE CONTROL  1 2 3 4 5  Y = Y = Y= Y = Y =  3.05X 2.95X 8.84X 3.87X 8.82X  x x x x x  10-3 10-3 10-3 10-3 10-4  -^3.44 -^3.21 -^2.44 -^5.97 -^8.76  x x x x x  10-3 10-3 10-3 10-4 10-4  0.48 0.30 0.76 0.35 0.31  DEPHOS sa. CASEIN  1 2 3 4 5  Y Y Y Y Y  = = = = =  2.60X 2.13X 2.10X 2.17X 6.23X  x x x x x  10-2 10-2 10-2 10-2 10-3  +^1.89 -^1.01 +^1.36 +^1.51 +^2.32  x x x x x  10-2 10-2 10-2 10-2 10-3  0.75 0.91 0.81 0.93 0.87  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  6.84X 8.08X 7.05X 7.19X 4.40X  x x x x x  10-3 10-3 10-3 10-3 10-3  -^1.17 -^4.53 + 4.41 -^3.56 +^3.12  x x x x x  10-4 10-4 10-4 10-4 10-3  0.95 0.73 0.98 0.91 0.77  asi  a  CASEIN  DEPHOS sa CASEIN  aDephosphorylated  MOUSE #  r2  VALUE  104  Table 27. IgE Equations of the Lines and r2 Values for Individual Mice Intraperitoneally Injected with Whey Proteins. ANTIGEN WHEY  FeCl3 TREATED WHEY  ANTISERA  EQUATION OF THE LINE  MOUSE #  r2 VALUE  WHEY  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.11X 2.26X 6.56X 4.21X 1.20X  x x x x x  10-2 10-2 10-3 10-3 10-2  -^3.49 +^1.62 +^1.18 +^9.97 +^1.07  x x x x x  10-3 10-2 10-3 10-3 10-2  0.82 0.95 0.96 0.83 0.75  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  1.38X 3.53X 1.01X 0.00X 6.21X  x x x x x  10-4 10-4 10-4 100 10-4  + 4.25 -^1.02 -^1.75 +^0.00 -^2.33  x x x x x  10-4 10-3 10-3 10° 10-3  0.05 0.21 0.64 0.00 0.49  FeCl3 TREATED WHEY  1 2 3 4 5  Y Y Y Y Y  = = = = =  6.03X 7.71X 6.75X 5.32X 7.36X  x x x x x  10-3 10-3 10-3 10-3 10-3  -^3.95 -^2.36 +^5.18 -^5.84 -^2.86  x x x x x  10-3 10-3 10-4 10-4 10-3  0.79 0.62 0.56 0.85 0.85  NEGATIVE CONTROL  1 2 3 4 5  Y Y Y Y Y  = = = = =  7.21X 1.28X 1.53X 0.00X 6.53X  x x x x x  10-4 10-4 10-3 10° 10-4  +^1.59 +^8.33 -^3.16 +^0.00 -^2.05  x x x x x  10-3 10-4 10-4 10° 10-3  0.07 0.03 0.40 0.00 0.31  105 APPENDIX 8 IgG VALUES FOR INDIVIDUAL MICE A. Experiment 1: Mice Orally Administered Proteins  Table 28. Relative IgG Values for Individual Mice Orally Administered Native Casein Proteins. MEAN ABSORBANCE^IgG VALUE ANTIGEN^ANTISERAa^MOUSE #^+ S.E.M.b^(abs./min.)x103 as,  CASEIN  S CASEIN  us, CASEIN  1 2 3 4 5 6 7  0.114^±^0.000c 0.105^+^0.006 0.121^+^0.016 0.146^+^0.011 0.084^+^0.004 0.117^+^0.002 0.098^±^0.015  0.65 0.61 0.62 0.84 0.52 0.65 0.50  NEGATIVE CONTROL  1 2 3 4  0.140^±^0.013c 0.137^+^0.008 0.097^+^0.003 0.146^+^0.003  0.82 0.79 0.57 0.84  S CASEIN  1 2 3 4 5 6  0.011^+^0.003d 0.011^+^0.003 0.012^+^0.005 0.010^+^0.000 0.029^+^0.006 0.031^+^0.003  0.10 0.10 0.11 0.08 0.21 0.21  NEGATIVE CONTROL  1 2 3 4  0.018^+^0•003d 0.022^+^0.004 0.028^+^0.001 0.025^+^0.003  0.12 0.16 0.16 0.19  'Antisera diluted 1/25. bS.E.M.^Standard Error of the Mean. 'Absorbance measured after 150.41 minutes of incubation at 37°C. dAbsorbance measured after 149.05 minutes of incubation at 37°C.  106  Table 29. Relative IgG Values for Individual Mice Orally Administered Dephosphorylated Casein Proteins. MEAN ABSORBANCE  IgG VALUE (abs./min.)x103  ANTIGEN  ANTISERAa  MOUSE #  DEPHOS usic CASEIN  DEPHOS usic CASEIN  1 2 3 4 5  0.128^+^0•011d 0.153^±^0.041 0.188^+^0.061 0.618^±^0.016 0.320^+^0.046  0.70 0.85 1.06 2.84 1.67  NEGATIVE CONTROL  1 2 3 4  0.164^+^0.054d 0.117^+^0.005 0.105^+^0.023 0.149^+^0.042  0.93 0.68 0.60 0.84  DEPHOS  DEPHOS  CASEIN  CASEIN  1 2 3 4 5 6 7  0.059^+^0.011e 0.074^+^0.031 0.035^+^0.006 0.044^+^0.007 0.047^+^0.004 0.030^+^0.004 0.025^+^0.002  0.36 0.44 0.21 0.26 0.27 0.23 0.16  1 2 3 4  0.039^+^0.001e 0.048^+^0.001 0.043^+^0.010 0.032^+^0.001  0.22 0.27 0.23 0.18  gc  gc  NEGATIVE CONTROL  'Antisera diluted 1/25. bS.E.M. = Standard Error of the Mean. cDephosphorylated dAbsorbance measured after 150.41 minutes of incubation at 37°C. eAbsorbance measured after 149.05 minutes of incubation at 37°C.  107  Table 30.  Relative IgG Values for Individual Mice Orally Administered Whey Proteins.  ANTIGEN  ANTISERA'  MOUSE #  MEAN ABSORBANCE + S.E.M.b  WHEY  1 2 3 4 5 6  0.073^±^0.009c 0.054^+^0.003 0.056^±^0.005 0.061^+^0.004 0.047^+^0.003 0.072^+^0.006  0.30 0.26 0.24 0.26 0.20 0.30  NEGATIVE CONTROL  1 2 3 4  0.060^+^0.005c 0.076^+^0.005 0.049^+^0.003 0.064^+^0.002  0.26 0.31 0.22 0.25  FeC13 TREATED WHEY  1 2 3 4 5 6 7  0.062^+^0.005c 0.077^+^0.004 0.060^+^0.002 0.090^+^0.004 0.078^+^0.003 0.055^+^0.002 0.067^+^0.001  0.27 0.33 0.24 0.35 0.32 0.24 0.27  NEGATIVE CONTROL  1 2 3 4  0.056^+^0.002c 0.105^+^0.010 0.060^+^0.003 0.142^+^0.011  0.22 0.42 0.23 0.53  WHEY  FeC13 TREATED WHEY  IgG VALUE (abs./min.)x103  'Antisera diluted 1/25. bS.E.M. = Standard Error of the Mean. cAbsorbance measured after 205.91 minutes of incubation at 37°C.  108 B. Experiment 2: Mice Intraperitoneally Injected With Proteins  Table 31.  Relative IgG Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins.  ANTIGEN  ANTISERA'  MOUSE #  MEAN ABSORBANCE +^S.E.M.b  usl CASEIN  as, CASEIN  1 2 3 4 5  0.011^+^0.061c 0.034^+^0.001 0.013^+^0.001 0.030^+^0.004 0.693^+^0.016  0.21 0.64 0.22 0.47 8.59  NEGATIVE CONTROL  1 2 3 4 5  0.014^+^0•002d 0.012^+^0.000 0.012^+^0.001 0.008^+^0.001 0.013^+^0.001  0.29 0.27 0.31 0.20 0.23  S CASEIN  1 2 3 4 5  0.712^+^0.255c 0.485^+^0.050 0.485^+^0.050 0.377^+^0.016 0.711^+^0.011  12.36 5.79 5.00 4.45 8.96  NEGATIVE CONTROL  1 2 3 4 5  0.001^+^0.000' 0.000^+^0.000 0.001^+^0.001 0.000^+^0.000 0.001^+^0.000  0.12 0.06 0.09 0.04 0.05  S CASEIN  IgG VALUE (abs./min.)x103  'Antisera diluted 1/1000. bS.E.M. = Standard Error of the Mean. cAbsorbance measured after 59.83 minutes of incubation at 37°C. dAbsorbance measured after 59.90 minutes of incubation at 37°C.  109  Table 32.  Relative IgG Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins.  ANTIGEN  ANTISERAa  MOUSE #  MEAN ABSORBANCE +^S.E.M.13  DEPHOS cesic CASEIN  1 2 3 4 5  0.190^+^0.013c 0.864^+^0.013 0.568^+^0.009 0.823^+^0.023 0.541^+^0.007  2.74 12.03 7.95 9.82 6.02  NEGATIVE CONTROL  1 2 3 4 5  0.014^+^0•001d 0.007^+^0.001 0.013^+^0.002 0.009^+^0.001 0.010^+^0.002  0.30 0.21 0.32 0.19 0.22  DEPHOS  DEPHOS  CASEIN  CASEIN  1 2 3 4 5  0.069^+^0.004c 0.306^+^0.013 0.356^+^0.005 0.103^+^0.003 0.273^+^0.005  1.13 3.87 4.24 1.58 3.48  1 2 3 4 5  0.003^+^0.001d 0.007^+^0.003 0.006^+^0.000 0.006^+^0.001 0.003^±^0.001  0.14 0.19 0.18 0.13 0.08  DEPHOS usic CASEIN  gc  gc  NEGATIVE CONTROL  IgG VALUE (abs./min.)x103  'Antisera diluted 1/1000. bS.E.M. = Standard Error of the Mean. cAbsorbance measured after 59.83 minutes of incubation at 37°C. dAbsorbance measured after 59.90 minutes of incubation at 37°C.  110  Table 33.  Relative IgG Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  ANTIGEN  ANTISERAa  MOUSE #  WHEY  1 2 3 4 5  0.327^+^0.115c 0.602^+^0.017 0.391^+^0.013 0.452^+^0.002 0.614^+^0.004  6.08 7.40 5.52 6.12 7.32  NEGATIVE CONTROL  1 2 3 4 5  0.011^+^0.003c 0.009^+^0.001 0.009^+^0.001 0.008^+^0.003 0.010^+^0.002  0.19 0.22 0.25 0.20 0.24  FeC13 TREATED WHEY  1 2 3 4 5  0.201^+^0.008c 0.297^+^0.006 0.196^+^0.001 0.301^+^0.008 0.232^+^0.006  3.28 4.42 3.09 4.50 3.76  NEGATIVE CONTROL  1 2 3 4 5  0.016^+^0.001c 0.020^+^0.002 0.028^+^0.001 0.017^+^0.002 0.017^+^0.001  0.35 0.37 0.47 0.35 0.33  WHEY  FeC13 TREATED WHEY  MEAN ABSORBANCE^IgG VALUE + S.E.M.b^(abs./min.)x103  aAntisera diluted 1/1000. bS.E.M. = Standard Error of the Mean. cAbsorbance measured after 50.85 minutes of incubation at 37°C.  111  APPENDIX 9 IgE VALUES FOR INDIVIDUAL MICE INTRAPERITONEALLY INJECTED WITH PROTEINS  Table 34. Relative IgE Values for Individual Mice Intraperitoneally Injected with Native Casein Proteins. MEAN ABSORBANCE^IgG VALUE ANTIGEN^ANTISERAa^MOUSE #^+ S.E.M.b^(abs./min.)x103 us, CASEIN  13 CASEIN  as, CASEIN  1 2 3 4 5  0.008^+^0.005' 0.002^+^0.001 0.020^+^0.003 0.032^+^0.006 0.136^+^0.003  2.59 1.15 4.45 7.01 27.41  NEGATIVE CONTROL  1 2 3 4 5  0.002^+^0•001d 0.000^+^0.000 0.012^+^0.002 0.006^+^0.003 0.001^+^0.001  1.39 0.00 4.61 2.09 0.82  S CASEIN  1 2 3 4 5  0.100^+^0.003c 0.116^+^0.022 0.050^+^0.004 0.113^+^0.004 0.089^+^0.004  19.56 28.99 10.61 23.02 19.10  NEGATIVE CONTROL  1 2 3 4 5  0.014^+^0•005 d 0.000^+^0.000 0.048^+^0.015 0.024^+^0.004 0.049^+^0.028  2.48 0.00 10.62 4.72 10.88  'Antisera diluted 1/20. bS.E.M. = Standard Error of the Mean. 'Absorbance measured after 3.93 hours of incubation at 37°C. dAbsorbance measured after 3.94 hours of incubation at 37°C.  112  Table 35. Relative IgE Values for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins. MOUSE #  MEAN ABSORBANCE +^S.E.M.b  DEPHOS usic CASEIN  1 2 3 4 5  0.043^+^0•003d 0.072^+^0.008 0.062^+^0.003 0.072^+^0.013 0.038^+^0.012  9.65 21.26 13.51 15.81 12.38  NEGATIVE CONTROL  1 2 3 4 5  0.008^+^0.004e 0.008^+^0.005 0.034^+^0.006 0.019^+^0.007 0.005^+^0.002  3.05 2.95 8.84 3.87 0.88  DEPHOS  DEPHOS  CASEIN  CASEIN  1 2 3 4 5  0.129^+^0•023d 0.072^+^0.008 0.103^+^0.015 0.110^+^0.009 0.030^+^0.003  25.97 21.26 21.03 21.73 6.23  1 2 3 4 5  0.029^+^0.011e 0.035^+^0.015 0.031^+^0.001 0.032^+^0.007 0.030^+^0.012  6.84 8.08 7.05 7.19 4.40  ANTIGEN  ANTISERAa  DEPHOS usic CASEIN  gc  gc  NEGATIVE CONTROL  IgG VALUE (abs./min.)x103  'Antisera diluted 1/20. bS.E.M. = Standard Error of the Mean. eDephosphorylated dAbsorbance measured after 3.93 hours of incubation at 37°C. eAbsorbance measured after 3.94 hours of incubation at 37°C.  113  Table 36.  Relative IgE Values for Individual Mice Intraperitoneally Injected with Whey Proteins.  ANTIGEN  ANTISERA'  MOUSE #  MEAN ABSORBANCE + S.E.M.b  WHEY  1 2 3 4 5  0.069^+^0.009 0.165^+^0.008 0.044^+^0.001 0.040^+^0.004 0.089^+^0.013  11.08 22.59 6.56 4.21 12.03  NEGATIVE CONTROL  1 2 3 4 5  0.001^+^0.001 0.000^+^0.000 0.003^+^0.000 0.001^+^0.001 0.000^+^0.000  0.14 0.35 1.01 0.00 0.62  FeC13 TREATED WHEY  1 2 3 4 5  0.035^+^0.006 0.047^+^0.011 0.044^+^0.010 0.032^+^0.003 0.045^+^0.005  6.03 7.71 6.75 5.32 7.36  NEGATIVE CONTROL  1 2 3 4 5  0.006^+^0.004 0.000^+^0.000 0.007^+^0.003 0.000^+^0.000 0.001^+^0.001  0.72 0.13 1.53 0.00 0.65  WHEY  FeC13 TREATED WHEY  IgG VALUE (abs./min.)x103  aAntisera diluted 1/20. bS.E.M. = Standard Error of the Mean. 'Absorbance measured after 6.07 hours of incubation at 37° C.  114  APPENDIX 10 PCA TITRES FOR INDIVIDUAL MICE A. Experiment 1: Orally Administered Proteins  Table 37.  ANTIGEN CASEIN  13 CASEIN  Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Native Casein Proteins. ANTISERA  MOUSE #  TITRE  1 2 3 4 5 6 7  NRa NR NR NR NR NR NR  NEGATIVE CONTROL  1-4b  NR  g CASEIN  1 2 3 4 5 6  NR NR NR NR NR NR  NEGATIVE CONTROL  1-4  NR  CASEIN  aNR = No Response at the lowest dilution tested (1/5). bPooled sample.  ^  115  Table 38. Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Dephosphorylated Casein Proteins. ANTIGEN^ANTISERA^MOUSE #^TITRE DEPHOSPHORYLATED DEPHOSPHORYLATED 1 ^NRa cis,^ as,^ 2^NR CASEIN^CASEIN^3^NR 4^NR 5^NR NEGATIVE^1-4b^NR CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED 1 ^NR S CASEIN^S CASEIN^2^NR 3^NR 4^NR 5^NR 6^NR 7^NR NEGATIVE^1-4^NR CONTROL 1TR = No Response at the lowest dilution tested (1/5). bPooled sample.  116  Table 39. Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase. ANTIGEN  ANTISERA  MOUSE #  TITRE  POTATO ACIDa PHOSPHATASE  DEPHOSPHORYLATED usi CASEIN  1 2 3 4 5  NRb NR NR NR NR  DEPHOSPHORYLATED S CASEIN  1 2 3 4 5 6 7  NR NR NR NR NR NR NR  1-4'  NR  NEGATIVE CONTROL  aMice challenged with 0.00005 g enzyme. biNTR = No Response at the lowest dilution tested (1/5). 'Pooled sample.  117  Table 40. Passive Cutaneous Anaphylaxis Titres for Individual Mice Orally Administered Whey Proteins. ANTIGEN  ANTISERA  MOUSE #  TITRE  WHEY  WHEY  1 2 3 4 5 6  NRa NR NR NR NR NR  NEGATIVE CONTROL  1-4b  NR  1 2 3 4 5 6 7  NR NR NR NR NR NR NR  1-4  NR  FeCl3 TREATED WHEY  FeCl3 TREATED WHEY  NEGATIVE CONTROL  aNR = No Response at the lowest dilution tested (1/5). bPooled Sample.  118  B. Experiment 2: Intraperitoneally Injected Proteins  Table 41.  ANTIGEN us,  CASEIN  g CASEIN  Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Native Casein Proteins. ANTISERA  MOUSE #  TITRE  usl CASEIN  1 2 3 4 5  NRa NR NR NR 320  NEGATIVE CONTROL  1-5b  NR  g CASEIN  1 2 3 4 5  160 320 80 160 160  NEGATIVE^1-5^NR CONTROL aITR. = No Response at the lowest dilution tested (1/5). bPooled Sample.  ^  119  Table 42. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins. ANTIGEN^ANTISERA^MOUSE #^TITRE DEPHOSPHORYLATED DEPHOSPHORYLATED 1 ^20 as,^a sl^ 2^80 CASEIN^CASEIN^3^160 4^80 5^80 NEGATIVE^1-5a^NRb CONTROL DEPHOSPHORYLATED DEPHOSPHORYLATED 1 ^160 S CASEIN^0 CASEIN^2^160 3^160 4^160 5^160 NEGATIVE^1-5^NR CONTROL aNR = No Response at the lowest dilution tested (1/5). bPooled Sample.  120  Table 43. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Dephosphorylated Casein Proteins and Challenged with Potato Acid Phosphatase. ANTIGEN  ANTISERA  POTATO ACIDa PHOSPHATASE  DEPHOSPHORYLATED as, CASEIN  1 2 3 4 5  5 40 40 40 10  DEPHOSPHORYLATED S CASEIN  1 2 3 4 5  80 80 80 5 160  MOUSE #  TITRE  NEGATIVE^1-4b^NRc CONTROL 'Mice challenged with 0.00005 g enzyme. bPooled Sample. cl\TR = No Response at the lowest dilution tested (1/5).  121  Table 44. Passive Cutaneous Anaphylaxis Titres for Individual Mice Intraperitoneally Injected with Whey Proteins. ANTIGEN^ANTISERA^MOUSE #^TITRE WHEY  WHEY^1^640 2^1280 3^320 4^160 5^640 NEGATIVE^1-5a^NRb CONTROL  FeCl3^FeC13^1^320 TREATED WHEY^TREATED WHEY^2^160 3^160 4^640 5^320 NEGATIVE^1-5^NR CONTROL 'Pooled sample. 13NR = No Response at lowest dilution tested (1/5).  

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