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A study of auto-anti-idiotypes to BSA Forsyth, Robert Bruce 1989

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A S T U D Y O F AUTO-ANTI-IDIOTYPES  TO BSA  By ROBERT BRUCE FORSYTH B.Sc,  The University of British Columbia,  1985  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E O F M A S T E R O F SCIENCE  in T H E F A C U L T Y O F G R A D U A T E STUDIES (Microbiology)  We  accept this thesis as to  the  required  conforming  standard  T H E UNIVERSITY O F BRITISH C O L U M B I A March  1989  © Robert Bruce Forsyth,  1989  In presenting this thesis in partial fulfilment of the degree at the  study. I further agree that permission for extensive  copying of this thesis for scholarly purposes may by  his or  her  representatives.  be  permission.  Department The University of British Columbia Vancouver, Canada  (2/88)  granted by the head of  It is understood  publication of this thesis for financial gain shall not be  DE-6  advanced  University of British Columbia, I agree that the Library shall make it  freely available for reference and department or  requirements for an  that  copying  allowed without my  my or  written  ii  Abstract  In  order  antibody  responses  sera in mice albumin  (BSA),  antigen and  specific  antibody. anti-BSA binding the  limpet  serum  contained  between  idiotypes  with  those  levels  of  Mouse the  antibodies  anti-BSA  in  to  serum  populations in  the  or anti-idiotypic reagents,  this  are internal image  of  completely  auto-anti-idiotypes  of  any  against  same  serum  the  chicken anti-BSA  inhibited  similarity produced  the  between in  manipulation  It indicates  to be internal  found  with Ids in mouse  suggests that the  antibodies.  (AAI)  of  antibodies  absence  We  response  that  This  idiotypic  antigen-sepharose  secondary  and A A I .  idiotypic  for such  species,  Id  and diphtheria  the  on  normal  Bovine serum  sera.  in  the  immune  used to measure  present  comparable  chicken  whole  raised  (KLH)  from these  present  related  selection  was  were  distantly  way  we  The chicken A A I also react specifically serum.  during  auto-anti-idiotypes  which  A A I were at  antigens,  hemocyanin  populations  antibodies  These  species  between  using three protein antigens:  antibody sera  relationships  different  ordinary protein  co-purified  columns. anti-BSA  to  in  A n avidin-biotin E L I S A  chicken  which  idiotypic  produced  keyhole  between  the  the  and chickens  toxoid ( D T ) .  that  study  populations  immune  binding  to  two with  A A I detected in there images.  is  positive  iii  Table  of  Contents  Abstract  ii.  Table of Contents  iii.  List of Figures  iv.  List of Abbreviations  v.  Acknowledgement  vii.  Introduction Materials  and  Results Discussion Bibliography  1. Methods  4. 9. 36. 42.  i v  List  Figure  1.  of Figures  Title  Specific  self  binding properties  chicken  antibodies  Page  of affinity  purified 11.  2.  Purity of the affinity  3.  Molecular size of  the  purified chicken a n t i - B S A chicken  auto-anti-idiotypic  antibodies 4.  5.  6.  7.  8.  9.  10.  13.  Idiotypic binding is due mainly to between IgG species  16. interactions 19.  Specific binding of mouse serum antibodies to affinity purified chicken a n t i - B S A measured in an E L I S A assay  21.  Molecular size of the mouse antibodies bind to the chicken auto-anti-idiotype  24.  which  The effect of removing a n t i - B S A antibody from mouse a n t i - B S A serum on binding to chicken a n t i - B S A auto-anti-idiotype  26.  T i m e course of induction of anti-idiotype and a n t i - B S A antibody in the immune response to B S A  29.  Inhibition by antigen of idiotypic binding to affinity purified chicken antibodies  33.  Inhibition of binding between chicken idiotype and auto-anti-idiotype using mouse and chicken a n t i - B S A serum antibodies  35.  V  List  of  Abbreviations  Abbreviation  A  405  Meaning.  nm  absorbence at 405 nm.  AAI  auto-anti-idiotype(s).  B6  C 5 7 B L / 6 mouse strain.  Bis  N,N'-methy lene-bis -acrylamide.  BSA  bovine serum albumin.  CFA  complete Freund's adjuvant.  DBA  D B A / 2 mouse strain.  DT  diphtheria toxoid.  EDTA ELISA  ethylenediamine-tetraacetic  enzyme linked immunosorbent assay.  h  hour(s).  Id(s) IF A  acid.  idiotype(s). ,  incomplete Freund's adjuvant.  Ig  immunoglobuUn.  KD  kilodalton.  KLH Lf PAGE PBS  keyhole limpet hemocyanin. limit of floculation unit. polyacrylamide gel electrophoresis. phosphate buffered saline.  vi  List  Abbreviation  of  Abbreviations  (continued)  Meaning.  SAS  saturated ammonium sulphate.  SDS  sodium dodecyl-sulphate.  TNP  trinitrophenyl.  Tris  tris(hydroxymethyl)aminomethane.  w/v  weight to volume.  vii  Acknowledgements  I owe a debt of gratitude to you my instructors; Hoffmann, Julia Levy and Hung-Sia Teh; doorway  before  me and introduced me  Drs. Geoffrey  who have illuminated the to a world  of mysteries.  I would especially like to thank Dr Geoffrey Hoffmann for giving me the opportunity to explore.  Thank you for your patience and your  passionate excitement for simple essence behind intricacies. that my work may honor all of you.  I hope  Here is my drop, a libation to  the sea. "I devoted myself to study and to explore by wisdom all that is done under heaven. has laid on men!"  What a heavy burden God  Proverbs 1:13  1 Introduction  The  overall  relatively  simple.  functions  which  why  how  and  effect of  behaviour  of  remove  the  the  immune  investigation  which  system  manages  interests  us  is  molecules  with use  Ig of  usual approach to studying that  simple  dominant  antigens  idiotypes.  information  about  that  idiotypic  these  overall regulation contain  have  bearing the idiotype  many  immunized  antigens: B S A ,  auto-anti-idiotypic  they  are  antigens  are  of  idiotypic  and  between  has been to use direct  This  approach  often  has  involves  artificial  and  a  (mice  and  chickens)  different  or D T (diphtheria toxoid) in  the  resulting  (AAI)  are  so  as  a  natural  and there  is  evidence  the  bearing  yielded  much  (1,2), it has been suggested  took  same individual as  anti-  immunization  monoclonals  we  antibodies  produced (4)  this  model  One popular line  study  produce  interactions  Ids,  Auto-anti-idiotypes produced by the  to  of  desired  unimportant  Since immune responses to complex  species KLH,  idiotypes  this  accepted  molecules  of interest.  phenomena  different  two  (Id)  Though  (3).  the  effector  questions  produce  been produced by  (haptens)  idiotypic  the  has been formulated.  immune cells via their receptor  antibodies  to  in principle,  of specific  and so far no widely  receptor  idiotypic  is,  Answering  specific  The  between  system  the expression  antigen.  is far more complicated,  interactions  immune  Antigen induces  overall immune regulation  of  the  immune  called  that  of  the  We  conventional  and studied polyclonal  the idiotype.  part  antigens  approach. with  to  sera.  because  they  In many immune  systems  response  they are involved  in  are  B  to cell  2  tolerance.  AAI  immunized positive AAI be  can  mice.  B-cells  be  These  detected  bound  to  antibodies  inhibit  some  from secreting  are produced in normal  involved  in the  of  a  prone  role to  in  preventing  auto-immunity  trinitrophenyl-Ficoll normal AAI  strains  have  directed  humans  AAI  systemic  lupus  remission (13).  directed  (11,12) Thus  and  regulation  themselves  roles  to  be  view  of  or  are are  known  mice  (10).  A A I to  at  all produce  Furthermore,  in are  generally  involved of  levels  healthy  antibodies  their properties  of  during  individuals  proving  the  in  (anti-DNA)  increased  present  symmetry  an  idiotypic  between  reasoned  that in such a battle it is quite  Ids  immune  in  "battle"  and  response  anti-Ids  some survivors on "both sides" (14). clones Ids  Fi  auto-antibody  that  suppressed.  strains  the F i of crosses with  These  imagine  complementary  of  in their production of  by-products  about  Mice  that A A I  to  in  be  very  mediating  regulatory  process.  and their regulatory  system.  the  when  disease.  present  normally  they  in the immune  In  are  auto-anti-idiotypic  whether  needs  are  the  but  There is evidence  auto-antibody  against  In humans  such  symptoms.  erythematosus  interesting  More  the  (6,7)  idiotype  immunological disorders  In N Z B strain mice  against  the  may  auto-immune  milder  of  from  they  (8).  are deficient  (9).  responses  some  as acquired C l inhibitor deficiency play  cells  Ig in a plaque assay (5). immune  pathology  spleen  should that  are  have  interactions  occurs,  specific  for  there the  likely that  one could  produced  more in  there  or less large  be  antigen.  In order for a clone been  might a  We  should  be  to survive  eliminated  amounts  or  would  3  effectively  eliminate  clones  managed  that  survive.  since  However,  in both  they  complementary  to overcome  In different  survivors  would  their  species  different species  bear internal images  of  we  would should  would  sites  idiotypic  for  antibodies  those  epitopes  to  bind to Ids in mouse this expectation In this  epitopes.  expect have  expect  were all selected by stimulation  binding  Anti-idiotypic  any complementary  species we  anti-Ids.  Ids  different  different  that  the  on the  would Ids  anti-Ids  antigen  follows  a particular antigen  that  to  be  repertoires. present  (15)  to be complementary It  also  because to Ids  of  surviving  anti-  antisera  might  in chicken  antisera raised against the same antigen.  It was  that we set out to test.  study  we  in the same serum.  found chicken A A I against  antibodies  present  These chicken antibodies  (in animals immunized  with B S A ) also bind to Id produced by mice.  Antigen inhibits both  forms  of  idiotypic  binding  and  mouse  anti-BSA  serum  inhibits the binding between chicken Id and A A I . from  one  species  recognize  the  idiotypes  Thus specific A A I  from  though neither has been immunized with the other's interpret this selective preference Id.  cross  pressures to  species recognition favouring  anti-Ids  which  anti-Ids merely  of  Ids  which  as are  recognize  specifically  another  antibodies.  being  the  internal  one  species,  or  We  result  of  images,  in  other  private  4  Materials  Animals. obtained our  Inbred  and  DBA/2  Methods  and  C57BL/6  from Jackson Laboratories (Bar Harbour,  breeding  colony  between  8  and  obtained  from  12 the  from  stocks  weeks Poultry  of  obtained  age.  from  Dekalb  Division  of  the  strain  mice  were  M E ) or raised in Jackson  breed  and  chickens  Department  of  used were  Animal  Science at the University of British Columbia.  Preparation  of Immune Sera.  Chickens  were immunized  B S A (Sigma Chemical Company, St. Louis, M O . , or K L H (Calbiochem, L a Jolla, C A . ,  25 mg per injection)  5 mg per injection): week 1 in a  50% (v/v) emulsion with C F A s.c, week 2 in P B S i.v., s.c,  and week 8 in C F A s.c.  with  week 3  in I F A  Serum was obtained 12 days after the  last injection.  Another group of chickens were injected  week intervals  with diphtheria toxoid ( D T ) (Connaught Labs Toronto,  Canada).  Injections  aluminum  hydroxide gel  20 L f / m l  1 and 2 were 20 L f and 10 L f (precipitated form Y , see  at three  respectively  containing  and 1 mg/ml A l u m i n u m (as A l u m i n u m Chloride).  The third  Serum was obtained 11 days after the last  injection.  In addition small  all  chickens  the  at  ref  in  16)  injection was 5 L f in P B S .  of  s.c.  samples  different  of  times  serum were obtained during  the  from  immunization  schedule.  In all cases chicken blood was clotted 2h at 3 7 ° C in glass  tubes  then  and  refrigerated  overnight  at  4°  C  to  allow  for  clot  retraction. Groups  of  5  DBA/2  and  C57BL/6  mice  intraperitoneally with B S A (7 mg per injection) injection).  were  immunized  or K L H (100  The mice were injected with antigen in 1:1  jig per  C F A on days 0  5  and 14, rested for 1 month, injected with antigen in P B S , rested for 3 months, with  injected  antigen  with  in  antigen  PBS.  second  otherwise  soluble  stated.  1:1  C F A and again  Serum was  injection of antigen in saline. the  in  boost  obtained  7-10  14  days  days  later  after  each  T h e pooled mouse sera obtained after were  used  for  all  experiments  unless  In all cases mouse blood was clotted l h at 3 7 ° C  and refrigerated overnight at 4 ° C .  Affinity purification were  made  using  of Chicken Antibodies.  antigen  coupled to  CNBr  Affinity  activated  columns  (17)  sepharose  C L - 4 B (5 mg B S A , 5 mg K L H or 5000 L f D T per ml of beads).  In each  case 70-90% of the protein was coupled and residual reactive sites on the beads  were  blocked with 0.16  M ethanolamine.  Immune serum  diluted  1 in 3 with P B S and adjusted to 25 m M in E D T A  passed  one  4° C.  The column was then washed with P B S - E D T A followed by P B S  or  more  and eluted with 0.1 washed  times  over  the  N H C l in 0.15  appropriate antigen  M NaCl.  and neutralized and the depleted  was  column  T h e column was  serum was  passed  over  column a second time. T h e column was washed and eluted as The  fractions  were  spectrophotometer,  monitored by Varian).  absorbance  T h e peak  were neutralized with 1 M Tris Base. runs was  pooled,  at 280  fractions  at  then the  before.  nm ( D M S 90 eluted material  The eluted material from both  concentrated by dialysis on a bed of  glycol, dialysed against P B S , adjusted to p H 5-6 stored at - 2 0 ° C .  of  then  with  polyethylene  1 N H C l and  6  Adsorption  of mouse  serum  on antigen  Samples  (100  over 0.5  ml columns of B S A or K L H sepharose.  washed  of biotinylated  antibodies  through over  serum diluted  1 in 20  columns.  were passed  T h e samples were  1 h at 4 ° C with P B S (10  ml)  and eluted  with  0.1 N H C l in 0.15 M N a C l (8 ml) while collecting 2 ml fractions. acid fractions  were neutralized  with  1 M Tris-Base and all  The  fractions  were adjusted to 2.5 ml with P B S .  Preparation precipitated (SAS),  of  twice  gamma  (18)  resuspending  with  with  Protein estimated (18)  that  BSA,  45%  0.85%  precipitated with 50% S A S . 45% S A S , resuspended  globulin.  The by  nm = 0.89  and 1 A 280  PBS  were  reagent  IgG)]  (Sigma)  mM in  10-20  mg/ml)  antibodies was  were  stopped  in 25  and  (19)  was  by  with  to  of  protein  [assuming  nm = 1.5 or  was  mg/ml  0.70  mg/ml  Sigma)  adding  diluted  biotinylated  diluted  0.1  0.2  ( p H 5.0).  volume  and  1 in  for  4  mg/ml  Protein of  solutions  10  incubating (protein  hours. and  in  biotinylation  N-hydroxysuccinimide  after 2 hours by adding 0.4  m M succinate  were  or by using the bicinchoninic acid protein  biotinamidocaproyl  Serum  sera  were washed  mg/ml fowl y-globulin  dimethyl-formamide]  temperature.  Mouse  were  sulphate  U . V . absorbance  of Proteins and Antibodies.  biotinylated  [5  NaCl.  mg/ml K L H , 1 A 279  assay at 6 0 ° for 15 minutes (kit B C A - 1  Biotinylation  ammonium  concentration  measuring  nm = 0.74  mouse y-globulin (as  (w/v)  sera  and dialysed against P B S .  necessary  1 A 278  saturated  The final precipitates  determination.  where  Chicken  at  ester room  concentration  of  Antigen  and  purified  biotinylated;  the  reaction  volume  of  100  The dimethyl-formamide  mM  glycine  and  unused  7  reagent  were then removed by dialysis  Avidin-biotin idiotypes.  The E L I S A  micro E L I S A convenience stock  to specific  antigen or anti-  assay used was a modification of the standard  contained II,  whole  fractionation) otherwise  binding  the P B S - T w e e n was prepared from a 5 fold  (Immulon  antibodies,  for  using the reagents as described by V o l l e r et al (20). F o r  which  plates  ELISA  against P B S .  0.4  g/1 NaN3  Dynatech)  were  y-globulin  or the  as a preservative. coated  (prepared  protein  concentrated  antigens  with  by  (100  Microtitre  affinity  ammonium  purified sulphate  u.1 at 10 ( i g / m l  unless  stated) in carbonate buffer p H 9.6 for 3 hours at 3 7 ° C . T h e  plates were washed with P B S - T w e e n , blocked with 5% casein PBS-Tween) primary bind  for 1 hour  reagent  for 2  alkaline  (diluted  hours  at 3 7 ° C and washed. i n 5%  at 3 7 ° C .  phosphatase  casein)  were  (Sigma,  1/400  conjugate  T h e biotinylated  was added  T h e plates  and allowed  washed  washed  and  p-nitrophenylphosphate The  plates  development  were  had occurred.  Inhibition  an  antigen)  at  concentrations affinity  an E L I S A  of binding  volume twice  at  of  37°  dilution  of  in  104: p H 9.8)  until at 405  ELISA.  reagent  1  mg/ml  was added.  sufficient  colour  n m ( A 405 nm)  final  anti-BSA  were  (biotinylated  inhibitors mixed  at 200 ng/ml,  with  antibody  concentration.  of the biotinylated reagents were:  purified chicken  0.5  T h e plates  Competitive  diluted in 5% casein  desired  a  plate reader (Titertec Multiskan).  the primary  the  C  Absorbances  or serum antibodies)  equal  (Sigma  in 10% diethanolamine  incubated  were measured using  (antigen  substrate  to  and avidin-  mg/ml stock) in 5% casein was added for 1 hour at 3 7 ° C . were  (w/v in  or  T h e final  B S A at 50 ng/ml, chicken  anti-BSA  8  serum The  at a  1/4,000 dilution and mouse a n t i - B S A  mixtures  were then used in the E L I S A  calculated from the A 405  A - M e a n background n  405  nm with uncoated wells (blocked with 5% casein).  samples without  (pH  10 minutes  6.8),  inhibitor. T h e background is  polyacrylamide  preparative fractionation for  )  nm for samples with inhibitor and A Q is the A  n m for the  C  \ 100  405  reducing  gel  electrophoresis:  of antibodies.  Samples  in non-reducing sample  were heated  The  % T is  total monomer contributed by the crosslinking monomer  gels  were  then  cut  into  blue.  BSA  to be isolated  antibodies  were  individually  polypropylene  pieces  and some  changed fresh  pooled  buffer.  gel The  were  (Bis)]. stained  the chicken anti-  were sliced into horizontal strips  chopped  tubes with  and the  sections  Unstained sections containing  about  into  5  1 gel  mm  squares,  volume  of  bicarbonate and rolled end over end for 3 days  with  (21,22)  of monomer (acrylamide and Bis) and % C is the %  with Coomassie  was  to 6 0 °  acrylamide (4%  C ) with stacking gels containing 3% T (5% C ) acrylamide [(24)  the  A  buffer (0.0625 M Tris H C l  on linear gradients (23) of 3% to 10% or 3% to 20% T  of  the  separation,  2% S D S and 10% glycerol) and subjected to P A G E  the total % (w/v)  was  equation:  A j - M e a n background  V  Non  1/30,000.  1-  % Inhibition=  is the A 405  at  assay. The inhibition  nm values according to the  (  Aj  serum  0.05  which  placed M  at 4 ° C .  in  ammonium The buffer  slices were rolled for an additional 3  days  two  were  buffer  and this eluted material was  samples  stored at  from -20°C.  each  slice  9  Results  Auto-anti-idiotypic antibody  populations.  populations  specific  avidin-biotin coated in  preparation  each  the  is,  specific  (Fig.  1).  measure  purified  antibodies.  contain  binding  antibodies  reacts with  specific  biotinylated  in each  anti-BSA  Antibodies  antibody  purified antibody  with  reacts  each  for other  plates  purified  the  same  specifically  with  and a n t i - D T reacts  preparation  react  This  by the V - r e g i o n . (AAI)  to  affinity  antigens.  auto-anti-ids  and an  preparations  within  anti-KLH  in  chicken  chicken  their  antibodies  binding is mediated  preparations  strongest effect,  The  anti-KLH  that the  affinity  purified  affinity  chicken  with antibodies  purified  same  to  preferentially  anti-BSA,  indicates  used  form.  That  anti-DT  weakly  was  of  react  preparation. chicken  Affinity  ELISA  with  in  for B S A , K L H or D T were  non-biotinylated  with  antibodies  only  specificity  That is,  against  the  the  antigen  Since the chicken a n t i - B S A preparation gave the it was  Characterization  used to further characterize  of  chicken  anti-BSA  that the  specific  not due  to residual antigen  the  antibodies  to chicken anti-BSA.  the  involved  phenomenon.  in  binding  W e did experiments  binding of chicken a n t i - B S A  to chicken  in the purified antibodies.  B S A was separated by non-reducing S D S P A G E .  of  to show  anti-BSA  is  C h i c k e n anti-  The major bands in  the chicken a n t i - B S A migrate at the same positions  as a purified yolk  IgG  was  standard  overloaded albumin)  ( F i g . 2).  no  albumin  bands  could  Although band  be  nor  detected  the  lower in  gel  molecular  the  chicken  intentionally  weight  (degraded  anti-BSA,  either  10  Fig. 1.  Specific  chicken antibodies.  self  binding properties  BSA  (b)  (•,  anti KLH. :  • ,  of  anti-BSA.  to plates coated with purified chicken a n t i - K L H (a), anti-  and anti-DT (c).  duplicates.  purified  T h e results represent the binding in an E L I S A  biotin labeled, purified chicken antibodies • , anti-DT)  of affinity  The error bar is the standard deviation of  In panel (b) the two control curves (those for  anti-KLH  and  anti-DT) overlap, and in some cases the error bar is smaller than  the  symbol.  Biotinylated  antibody  (jig/mL)  12  Fig. 2.  Purity of the affinity purified chicken a n t i - B S A .  of proteins  were separated  electrophoresis  30 ng  by non reducing S D S polyacrylamide gel  on a linear 3 to 20% gradient gel and stained  Coomassie Blue.  Samples  Lane 3 is purified chicken a n t i - B S A ,  and lane 4 is B S A ,  with  approximately  approximately 0.3 (ig. Lane 2 is chicken  egg yolk I g G (25) as a standard. Lane 1 is molecular weight standards (Sigma S D S - 6 H : myosin (band not visible) phosphorylase anhydrase)  b, bovine  with  P-galactosidase,  serum albumin, ovalbumin and  molecular  weights  as indicated.  carbonic  Molecular ro co oi  weight cn cn  co  o>  (KD)  14  visually  or  by  scanning  densitometer.  1% B S A contamination  toas  little  The  m i n i m u m B S A contamination  as  account for the E L I S A to  be  about  irrelevant protein  5%.  chicken  antibody  is  that  would  amounts  the  so  visible  have  equivalent ( F i g . 2, 3a).  been  needed  from mixing  of  mixtures  standard  clearly  estimated  (anti-KLH)  and  BSA  BSA  as  to  were  were  mixed  maintain a  used  10% B S A .  to  coat  Plates coated at mixtures  with purified chicken a n t i - B S A .  less sensitive to the effect reagent. varying BSA  The binding quantities  was  binding  of  compared.  curves  0.06  biotinylated In  the  ng/ml  equal to that of about 0.4 Thus the  plates  coated  with  of  biotinylated  |ig/ml  of the  increases containing  of  linear  anti-BSA chicken  portion  B S A produces  biotinylated  biotinylated chicken a n t i - B S A  anti-BSA  biotinylated  chicken  B S A or biotinylated  approximately  ELISA  The assay is even  of B S A contamination  to  with  constant  of the protein as B S A bind the same amount of chicken  as plates coated  to  experiments  The binding of chicken anti B S A to these plates  linearly up to about 5%  signal was Varying  concentration  plates.  A  chicken  would have  anti-  of a  of  the  signal  anti-BSA.  to contain  15 %  contamination with B S A to account for the binding observed. Fractionation demonstrate  gel  electrophoresis  was  binding is due to molecules  The affinity purified chicken a n t i - B S A was  ammonium  unstained  preparative  that the specific  chicken I g G . 0.05M  with  portion  of  bicarbonate  from  the non-reducing  transverse  SDS  PAGE  used  the size of eluted  slices  gel.  to  of  Each  with the eluted  fraction was used to coat wells of an E L I S A plate to yield a profile of the gel from top to bottom. the E L I S A  Biotinylated chicken a n t i - B S A  bound in  (Fig. 3b) mainly to material from gel slices corresponding  15  Fig. 3.  Molecular  antibodies. non  size of the chicken auto-anti-idiotypic  Affinity purified chicken a n t i - B S A was  reducing S D S polyacrylamide gel electrophoresis  separated  by  and eluted  from  gel slices as described in the Materials and Methods section. Separate sets of wells on E L I S A  plates were coated with the material  from each of these gel fractions diluted 1/5 The  top panel (a) shows densitometer  BSA)  scans of lanes 3 (chicken anti-  and 4 ( B S A ) of the gel (which is shown in F i g . 2).  panels  are E L I S A  a n t i - B S A (b) fraction. stained  The bottom  binding of biotinylated affinity purified chicken  and mouse anti-(chicken y globulin) (c) to each gel  T h e arrows indicate the approximate positions molecular weight  kinase Sigma F-0387; 9400).  with carbonate buffer.  markers (84  KD,  of the pre-  fructose-6-phosphate  27 K D , triosphosphate isomerase  Sigma T -  The asterisks in panel (c) indicate the fractions which were  biotinylated and assayed  (Fig.4)  to determine  species bind to the chicken IgG on the E L I S A  which biotinylated Ig plate.  Migration mm  17  to the was  position  (Fig. 3a  no binding to  position  of  the  - lane 3)  of  the  main protein band.  any material from the  B S A band (Fig. 3a  There  slices corresponding  - lane  4).  There was,  to  the  however,  some binding to higher molecular weight material which may be I g M or  dimeric  y-globulin  IgA  (23)  binds  to  since some  mouse higher  anti-serum molecular  against weight  whole  chicken  material  in  that  region of the profile (Fig. 3c). Material  from  the  marked  with  asterisks  chicken  a n t i - B S A was  gel  in  fractions  Fig.  3c  measured  corresponding  was  biotinylated  ( F i g . 4).  These  to  the  and  binding  fractions  the higher molecular weight ( I g M or IgA) material and the peak. for  peaks to  represent main IgG  Binding of material in the peak corresponding to I g G accounts the  vast  members  majority  of  mainly due  the  of  affinity  the  signal.  purified  to binding among  Thus  chicken  members  the  binding  anti-BSA  of the  between  population  IgG populations  than binding between IgG and some minor component  is  rather  such as I g M or  IgA.  Idiotypic  complementarity  between antibodies from  chicken immune responses to BSA. sera  bind  in  antibodies  but  Furthermore anti-KLH  the not  neither  sera  ELISA to  to  affinity  chicken  normal  Biotinylated  anti-KLH  mouse  bind to chicken  purified  serum  mouse  anti-BSA  chicken  anti-BSA  antibodies nor  (Fig  5d-i).  hyperimmune  mouse  a n t i - B S A even though  high titers of a n t i - K L H antibody (Fig. 5a- c).  mouse and  the  latter  have  18  F i g . 4. IgG species.  Idiotypic  binding is due  Samples  between  (200 |il) of the Ig containing gel fraction  extracts marked with asterisks  in F i g . 3c were dialyzed against P B S  and biotinylated by adding 20  of  2 hours and dialyzed against P B S . adjusted to 300 jil  mainly to interactions  biotinylation reagent,  The biotinylated samples were  and used at 1/50.  purified chicken a n t i - B S A was standard deviation of duplicates.  Binding to plates coated with  measured. Slices  signal and are IgG (compare F i g . 3c).  incubated  The error bar is  the  8 and 9 give the strongest  Relative binding (A 405 nm)  20  F i g . 5.  Specific binding of mouse  serum antibodies to affinity  purified chicken a n t i - B S A measured in an E L I S A show  assay.  The results  the binding of various biotin labeled normal or immune mouse  sera to plates coated with affinity purified chicken a n t i - B S A ( # ) , affinity purified chicken a n t i - K L H (O), sera used in the various panels  were  B S A (•),  and K L H ( • ) .  The  obtained from: unimmunized  mice (a); groups of mice after the second boost with soluble antigen (b, c, f, and i);  two individual mice after the first boost with soluble  antigen (d and e);  a group of mice seven days and ten days  respectively after the first boost with soluble antigen (g and h). data points  are means  of duplicates.  The  Biotinylated  mouse  serum  (log  of  di l u t i o n  factor)  22  In pooled sera of both D B A / 2 individual mice (Fig 5d,e)  a large proportion of the mouse  able to bind the chicken anti-Id. mouse BSA  anti-BSA  equal  sera tested the titration curves  or less.  amounts  antibody  at  Thus,  of  that equal  bound antibody, half  of  the  or  sera  more  the  for binding to excess  are separated by about a two  assuming  approximately  antibody is  In all except one (Fig. 5f)  and excess chicken a n t i - B S A  dilution  and B 6 mice and in the sera of  fold  signals  are produced by  contain  idiotype  of  the  level  of  binding anti-BSA  antibody. T o rule out antigen as the explanation as  well,  biotinylated  preparative binds  to  gel  anti-BSA  electrophoresis  chicken  same position  mouse  anti-BSA  as  the  serum  (Fig 6).  was  A l l of  antibodies  antibodies  for this  (top  which  specific  fractionated the  to  using  material  panels)  bind  binding  which  migrates  B S A (centre  at  the  panels)  and the main peak of the immunoglobulins as detected by binding to rabbit anti-mouse  Ig antibodies  (bottom  panels).  This peak is  well  resolved from the position of the B S A band (66 K D ) where no binding was  observed. Biotinylated  antigen  binding  sepharose bind  chicken  sepharose  from  anti-BSA  non-binding  anti-BSA  anti-BSA  are  were recovered  B S A column.  antibodies  against  chicken  sera  BSA  The bind  anti-BSA  to  were  fractions  M o r e than 90%  but not K L H sepharose  the  purified  and  columns.  to  chicken  mouse  by  (Fig 7). in the simplest the  antibodies.  by  of the  removed  also  fractionated  adsorbing  mouse  with  antibodies  adsorbtion  with  into BSA which BSA  Antibodies which bind  B S A binding fraction explanation  chicken  is  that  A A I contained  to  eluted mouse in  the  23  F i g . 6.  Molecular size of the mouse antibodies  chicken  auto-anti-idiotype.  which bind to  The protein components of  biotinylated mouse antisera (B6 a n t i - B S A on the left,  the  whole  D B A anti-BSA  in the centre and D B A a n t i - K L H  on the right) were separated by non-  reducing  electrophoresis  S D S polyacrylamide gel  and eluted  slices as described in the Materials and Methods section. these fractions coated KLH  gel  Each of  was assayed in the E L I S A for binding to a plate  with purified chicken (•),  from  B S A (•),  against mouse Ig ( • ) .  a n t i - B S A (O),  purified chicken anti-  and the y globulin fraction of rabbit antisera The arrows indicate  the approximate  positions  in the stained portion of the gel of the molecular weight markers (97 K D , phosphorylase b Sigma S D S - 6 H ;  66 K D , bovine  albumin and 14 K D , a-lactalbumin Sigma S D S - 7 ) .  serum  D B A anti B S A  B6 anti B S A  D B A anti K L H  j  o  I  I  I  l  I  .tt  I  ha  a  / \ t~-  to to  H*  I  I  t  1  *  o-  a  i  |  i  i  i  tt t  :  a  *  j 1 <J>  1  I  I  1  "  T-  i  |  t  |v,  IO  O)  <o  •  i  i  •  i  i  i  t t  1  <tf  Q  Q  o>  to to  i  i  t Q *t T-  1  ||  i -  i  H i JD JL'  0  I  I  I  10  I  I  I  I  I  20  I 0  Migratio n  I  I  in  I  I  I  10  PAGE  I  I  I  (gel  I  I  20  I  0  I  I  I  I  slice number)  I  10  I  I  20  25  F i g . 7.  T h e effect of removing a n t i - B S A  anti-BSA  serum on binding to chicken  antibody from mouse  anti-BSA  auto-anti-idiotype.  Samples of biotinylated D B A (panel a) and B 6 (panel b) mouse antiB S A sera were passed over small columns of K L H or B S A sepharose. T h e data shown represents antibodies eluate.  the binding in the E L I S A  in the first fraction (~2  of  the  ml) of the runthrough and of  The error bar is the standard deviation of duplicates  negligible  in some cases.  Practically all of the anti-idiotypic  is adsorbed to the B S A column.  the  and is activity  26  0.60.50.40.3-  E c  0.2-  m o  0.1 0.0-  1 ||| 0  r  KLH column  Run-through. O)  c c  0.6-  0>  0.4-  •5  Eluate  0.5-  0.3-  KLH column  rr  BSA column  0.2-  0.1 0.0 Run-through  Eluate  Column fraction  27  The time course of the level of anti-BSA after immunization prepared  by  with BSA.  ammonium  and AAI in chickens  C h i c k e n y-globulin  sulphate  precipitation  of  fractions  small  serum obtained throughout the immunization period. samples  were  biotinylated measured  used  BSA,  (Fig.  to  coat  mouse  8).  Some  present  by  immunization.  7  but  the  onto  produces  1.3  equal  this  the  binding  adjusted  so  ELISA ± 0.2  molar quantity  plates  little  Id  of  biotinylated  each  the  the  and anti-Id  BSA.  activity  proteins  the  made  coated  antibody  obtained  Since  is  biotinylated  anti-BSA  signal  the  first  A n estimate was  chicken  with  antibody  after  F o r the biotinylated  (mean ± S E ) times  that  to coat  binding  response  response.  biotinylated  was  signal varied linearly  primary  secondary  of  anti-BSA  this assay of the ratio of molar signals for binding of  directly  and  and  T h e main peak of both a n t i - B S A  antibody as compared to B S A .  (Fig.  very  of  The y - g l o b u l i n  chicken  were  samples  antibody in the y-globulin used  in  is produced during the for  and  conditions  B S A binding  day  plates  in excess and the  the proportion of specific plate.  anti-BSA  The  biotinylated reagent was  ELISA  were  with  peak  an  signals  8a and c) are roughly the same for binding of biotinylated B S A for  binding  ratio  antibodies  of  indicates  biotinylated that  the  affinity  purified  concentration  of  chicken  anti-BSA,  idiotype  is about 50-100% of that of the a n t i - B S A antibodies  point in the time  course.  binding at this  28  F i g . 8. BSA  Time  course  antibody in the immune response  prepared  from  small  samples  times through the course  (b),  binding  of  excess  and affinity  gamma  globulin  of  anti-idiotype  to B S A .  chicken  wells of E L I S A  biotinylated  serum  purified chicken fraction  plates.  B S A (a), anti-BSA  taken  at each  time  at  various  or B S A  (•)  The data represent  mouse (c)  and anti-  G a m m a globulin was  of immunization with K L H ( • )  and used to coat separate the  of induction of  to  during the  anti-BSA  serum  antibodies immune  in  the  response.  The error bar (in most cases smaller than the symbol) is the standard deviation of duplicates. from  two  indicated Freund's  chickens. by  the  F o r B S A the values Antigen  arrows  adjuvant  or  (in as  injections complete a  were obtained using  were  given  ( C F A ) or  solution  the  times  incomplete  (IFA)  (Sol)  at  sera  in  saline).  Relative binding  (A 405  nm  units per hour)  to vo  30  Antigen  (BSA) as an inhibitor  anti-BSA.  BSA  concentrations  was  with  included  constant  reagents binding in the E L I S A chicken BSA  antibodies.  anti-BSA  B S A (Fig. 9). binding  of  needed  is  an  inhibitor  amounts  of  various  to plates coated of  both  completely  inhibited  of  to block  the reagents.  to chicken at  biotinylated  biotinylated  by  the  purified  mouse  to affinity  high  anti-  purified  concentrations  of  since K L H does not block the  Higher concentrations  the binding of  various  with affinity  purified chicken a n t i - B S A  This inhibition is specific  any  binding  as  T h e binding  serum and affinity  chicken  of idiotypic  various  anti-BSA  of  B S A are  antibodies  than  to block the binding of biotinylated B S A to the purified chicken antiBSA.  This may reflect  a greater intrinsic affinity of the Ids and anti-  Ids for each other than for B S A or a greater effective affinity due the  multivalence  Specific  of  antibodies.  antiserum  chicken anti-BSA. same  assay  affinity BSA  as  can  be  chicken  immune  chicken  serum (Fig.  another  antisera  serum  anti-BSA  can  10a).  or y-globulin  proportion  of  the  (Fig.  Normal  chicken  binding  to  inhibitors in  the  affinity  purified  high  the  of  biotinylated  chicken  concentrations  same  chicken  chicken serum  and  much  less  and anti-Ids  in the  affinity  and c).  anti-BSA  blocked  by  antibodies  involved  that in  antithe  another immune  inhibition.  mouse  This indicates  of  or  ( K L H ) give  completely 10b  idiotypic  T h e binding  by  either  antigen  be  to  inhibited from  of  were tested as  BSA.  M o r e importantly, binding of Ids chicken  inhibitor  anti-BSA  completely  anti-BSA  against  an  for inhibition by  chicken  serum  as  Specific  purified  to  purified anti-BSA a  large  auto-anti-  31  idiotypic  binding have Ids  This albumin more  since  than  y-globulin |ig/ml  inhibition non  10  by  similar to the mouse a n t i - B S A antibodies. mouse  immune  fold  less  and  serum  inhibition (Fig. 9).  about  effective  1000  not  irrelevantly  from mouse a n t i - B S A serum  whereas  is  u,g/ml  as  due  immune  inhibitors.  gives 50% of  to  BSA  is  mouse  mouse  serum  sera  are  Furthermore,  inhibition  at  10-20  required  for  50%  32  Fig. 9.  Inhibition by antigen  purified chicken antibodies. various concentrations BSA,  BSA to  B S A (solid symbols)  purified chicken  on E L I S A plates.  the  than 2.5%  a n t i - B S A ) to  added at reagents  (•:  affinity  purified chicken  anti-  A s a control, K L H (hollow symbols) was added  of four replicates  inhibition.  affinity  A : mouse anti-BSA serum and • :  same biotinylated reagents ( L 7 J , 0 , A , 0 ) .  standard deviation  was  to block binding of biotinylated  • : chicken anti-BSA serum,  affinity  of idiotypic binding to  The error bar is the  and is plotted  where  it is  larger  33  34  F i g . 10.  Inhibition of  anti-idiotype Normal  (•),  using  binding between chicken  mouse  and  a n t i - K L H (O)  chicken  idiotype  anti-BSA  or a n t i - B S A ( • )  serum  antibodies  and  antibodies. (as  chicken  sera in panel a,  as mouse sera in panel b and as y g l o b u l i n  mouse  panel  sera  in  c)  were  added  biotinylated  affinity  purified  chicken  ELISA  chicken  anti-BSA  on the  with  standard deviation than 2.5% for  inhibition.  blocking  individual chicken  of four replicates  by  chickens,  anti-BSA.  one  various  anti-BSA plate.  an  T h e error bar is  the  curves  anti-BSA  serum  are  of  is  the  to  in  two  which  from  dilutions  antibodies  and is plotted  In panel a the  chicken  at  auto-  where [both  for  source  marked  two of  it is  sera the  larger (•)] from  purified  Gamma globulin (log Lig/mL)  36  Discussion  W e find that Id and A A I are present simultaneously in the sera of  chickens immunized with conventional protein antigens.  secondary present  response  at  a  concentration  to  one  antigen  concentration  of  the  of  antibodies  ( B S A ) the  the  order  against  the  corresponding  Ids  Furthermore,  binding  chicken with  A A I also  the  same  bear internal immune which  epitopes were  and  recognize  images the  of  the  to  an  protein  the  antigens.  show  that  confirms for a  conclusion (15)  antigen  these  with  the  the chicken A A I appears This  Furthermore,  recognize  the  produced in mice immunized  antigen. the  be  indicates  at high levels to  to of  This  experiments  all of  validity of  on that antigen. to  antibodies  50-100%  antigen.  responses  inhibition  Nearly  complementary  selected  internal  immune  antigen.  response are  in  A A I appears  of  that it may be common for A A I to coexist  In the  bear  "negative  it indicates  negative  that  images  normal  antibodies images"  that the (that  to  of  anti-Ids  is,  to  be  images).  T h e presence of A A I in chicken antisera is not very s u r p r i s i n g since  their production during  documented (4).  other normal immune responses  If the anti-Ids  are induced by Id they  be present together at some time during the response. presence their  of  ability  curious. antigen  large quantities  of  However, the  in chicken serum and  the  antigen  specific  with  They  could  perhaps as  must both  anti-Ids  copurify  antibodies  well  these  to  specific  is  be  present  soluble  in  immune  sera  antibodies together  complexes.  is  with Such  37  complexes  could bind to  antibodies  and  both  together.  Others  an antigen column via the  components  have  participate in soluble  would  reported that  elute  Id  immune complex  and  antigen  from  the  A A I can  formation (26),  specific column  coexist  albeit  and  as part  of a pathological condition ( selective I g A deficiency). A  second possible  antibodies  in  the  explanation for the presence  column  purified  material is  of anti-idiotypic  that  these  antibodies  may have dual specificity; they may bind both to the antigen and to Ids  on  specific  antigen and  specific  antibodies.  anti-idiotypic  could  Clones  be  expected  advantage in the immune response. from  both  continue  antigen  to  be  and  by  are  to  both  have  a  They would receive  antigen-specific  stimulated  that  idiotypes,  idiotypes  even  and  after  antigenselective  stimulation they  the  could  antigen  is  eliminated. T h e idea of these being dual specific antibodies may not be far-fetched, common  since  multispecific  (27,28).  Furthermore,  reactivity Bona  antibodies  et  al.  have  of  specific  for IgG) also bind to IgG F c fragments. They gave the name  monoclonal  against  rheumatoid  the  that  regions  human  A / J mice  shown  is  anti-idiotypic  (a  in  (29)  antibodies  certain  IgM K m G l  raised  between  too  V  factor  "epibody" to any antibody which binds both to an antigen and to the V  regions  of  observations a  the  Balb/c  antibodies  against  have been made by K a n g  monoclonal  antibodies  other  which  against  T15  binds  to  the  They  (a myeloma which  was found to react with both  antigen.  and Kohler (30)  itself.  anti-phosphorylcholine  same  (PC)  bears  response).  P C and T 1 5 .  who reported  raised the  Related  hybridoma  dominant Id of One  hybridoma  This monoclonal binds to  38  its own V - r e g i o n s and so they called it an "autobody".  Subsequently  a V H region peptide involved in the binding has been identified ( 3 1 ) . Consistent  with  a dual  specificity  interpretation of  our results  fact that, to within the time resolution of the study, idiotype  is  the  B S A binding and  binding activity occur in the chicken immune response  with  identical kinetics (Fig. 8 ) . If  the  binding  between  antibodies  in  these  antibody preparations is indeed due to the presence "autobodies," question  posed  biological to  observation  by  Kang  relevant  explore  response  this  more  .  et  to B S A .  al.  From  closely  could  begin  ( 3 1 ) as  to to  Id  and  chicken  of "epibodies" or  address whether  this perspective  the  purified  the  autobodies  it would be  A A I clones  important  in  are  worthwhile  the  immune  Panels of a n t i - B S A monoclonal antibodies could be  raised and screened  for binding to one  another and could be used to  screen for the A A I producing clones.  time  If the population of a n t i - B S A antibodies is in fact selected  with  to be  self-  self-recognizing,  regulating. idiotypic  This  regulation,  quantitative idiotypes  analysis  contrast  produced  in  idiotypes  present  serum  is  that than  to  human at  our  be  could  a  relatively  be  more  models  in  which  results  it  has  responses  to  that  point  probably because be  antigen.  then  population may become  largely  simple  readily two  or  system  of  amenable  to  more  levels  of  are involved.  In  This  would  this  carefully The  same  tetanus in  to  would  remove also  reported  toxoid the  in that report the  absorbed  absorption  been  any  remove  do  immune assay  not  required  AAI  bind  response  antibodies any  that  to (6).  that  the  against  the  A A I of  the  kind  described  here  and could  seriously  reported simultaneous presence with  bacterial  lupus  (32)  (11,12)  affinity  see  of  chicken  direct  Others  between  to  chicken  stimulation  present  during  contain  A A I against  in  patients  IgA  the  of  and  with  systemic  deficiency  we  also  antibodies  can  be  auto-anti-idiotypic  immune mouse  see  (26)  response. Ids.  The  understood  antibodies  Mouse  However,  this  which  the they  mouse  serum  recognize  were  and vice  not  stimulated  versa.  the  which bear internal images antibodies  remarkable  anti-Ids  are  mostly  discussion  not by  most  auto-anti-ids)  Pontillon  et  formed in human responses internal  image  anti-Ids.  al.  The different  sharing species  is  of  also  would  not  by  the  A n y anti-  chicken  Ids  explanation  This  of  antigen  epitopes  against those epitopes.  described are  not  (33).  to house  general may have a tendency  Ids  is  to the production in chickens of A A I ,  are recognized by mouse that  by  could  alone  T h e simplest  process  in  terms  serum to bind to these chicken  that a selection majority of  leads  specific  (4)  serum  anti-  in  antibodies and to inhibit the binding of chicken Id to A A I . in  and  mouse  the chicken idiotypes.  explain the ability of immune mouse  Ids  have  (8).  antibodies,  binding specifically  interaction  results.  idiotypic binding of antibodies in chicken serum to the  purified  idiotypes  and  selective  acquired C - l inhibitor deficiency We  the  of Id and A A I in a rabbit immunized  polysaccharide  erythematosus  affect  in  the  internal However,  images;  dust mite allergens  suggests  that  This  literature  the  that is  (which see  the  auto-anti-ids (7)  are also  auto-anti-idiotypes  in  to bear internal images.  idiotypic  in contrast  specificity to  the  by  antibodies  behaviour of  Ids  from  two  detected  by  40  anti-Ids  raised against  studies,  Oudin  by  and M i c h e l  immunizing  antiserum  purified  from  rabbits  In their classic  pioneering  (34,35) produced anti-idiotypic with  individual  precipitated specific  antibodies.  bacterial  rabbits.  antibodies  The  from  cells  agglutinated  resulting  the  original  antibodies  anti-idiotypic  not  specificities  shared even  detected  between  immunization  with  anti-idiotypic  antibodies.  shared  with  those  an  other  which  few)  Some  of  shared  anti-idiotypic reagents  are  species.  are  against  so  because  a  Ids  individuals.  do  In general  production of  these  Ids  the  same  to  distinct)  Thus  A they  range which  of are  fraction of are internal  (15,36).  Some  of biologically active antigens  can even such as  mimic  hormones  In chickens immunized with B S A almost all (rather than only a of  the  A A I are internal image.  the A A I selected  different  immunization  with Id.  One internal  clones  human  plausible  image can  is  This  observation  in the course of an immune response  qualitatively  cell  leads  those epitopes  of the effects  (37).  of the  from  on the antigen and as such could be recognized by  antibody against  some  antibody  recognize  Oudin's  members  (genetically  images of epitopes any  by  sera  serum but not  the sera of other rabbits immunized with the same antigen. idiotypic  with  anti-Ids  speculation  suggested by be  activated  anti-idiotypic  T  cell  from  as the  by  to  those  why  lines  have  A A I might  by  tend  active  to  be  that anti-idiotypic T -  (38). been  that  to antigen are  selected  observation  antibody  suggests  In  vitro cultured  produced  which  are  activated by immune serum Ig when presented in the context  of  class  present  II  antigen  (HLA-DQ). to T-cells get  If  we  speculate  that  B-cells  help (lymphokines for example)  which  self  from those T -  41  cells  then  specifically  any  anti-idiotypic  picking  B-cell  up and presenting  clone  could  antibody  get  help  molecules to  by anti-  idiotypic T-cells in the same way that antigen specific B-cells pick up antigen at very low concentrations, antigen specific T-cells (39). internal  image  would  get  accumulate  it and present it to  In this situation B cells producing an more  stimulation  since  they  would  recognize a broad range of idiotypes and thus be able to interact with more  anti-idiotypic T-cells.  epitopes  Conversely B cells against  antigen  could possibly be stimulated by picking up internal image  antibody and presenting it to T-cells.  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