UBC Theses and Dissertations
Chicken egg yolk immunoglobulin Y and fragments : purification, allergenicity and their neutralization of Escherichia coli heat labile toxin Akita, Emmanuel M.
The importance of egg yolk as a convenient and inexpensive source of antibodies (IgY) is well recognized. The use of IgY in immunotherapy and immunodiagnostics will be facilitated by developing more efficient ways of purifying IgY from the yolk. In im- munotherapy, additional problems which should be addressed in order to facilitate the use of IgY include stability against acidity of the stomach and proteolytic enzymes as well as its potential allergenicity. In this research, a method was developed using sim ple water dilution at pH of 5.0 - 5.2, to separate water-soluble plasma proteins (WSF) from egg yolk granules. IgY was purified from the WSF using a combination of several techniques including salt precipitation, alcohol precipitation, ultrafiltration, gel filtration and ion exchange chromatography. Salt precipitation, ultrafiltration, followed by gel fil tration is the recommended sequence. Over 100 mg of electrophoretically pure IgY was routinely obtained from one egg. Comparison of the water dilution (WD) method with three published methods of purifying IgY, showed that the WD method was superior in terms of yield, ease of use and potential for large scale production of IgY. A method also was developed using peptic digestion for a large scale preparation and purification of immunologically active Fab’. Optimum yield of Fab’ was obtained after peptic digestion of IgY at pH 4.2 for 9 h at low NaCl concentration. These conditions led to the complete digestion of pFc’ fragment leaving only the Fab’ fragment. By combination of ultrafiltration and anion exchange, and conditions which allowed binding of the small amount of contaminants in the digest to the anion exchange column, pure Fab’ fragments were easily obtained in the eluent. This approach allowed purification of large amounts of protein on a small column, therefore, improving the efficiency of purification. The allergenicity of IgY was studied using passive cutaneous anaphylaxis. It was observed that whether in the form of the yolk, WSF, the whole IgY molecule or its Fab’ fragments, IgY was less allergenic compared to egg white, a known allergen. Under immunization conditions where the IgY was found to be allergenic, the Fab’ fragment was found to be less allergenic than the whole IgY molecule. Consequently, Fab’ might provide an alternate form of administering IgY. The protective effect of IgY and Fab’ as measured by their neutralization of entero toxin produced by enterotoxigenic E. coli was also studied. The neutralization activity of IgY was not reduced by removal of the pFc’ fragment. Heat pasteurization at 65 °C for 15 min did not adversely affect the neutralization activities of IgY or Fab’. IgY was found to be fairly stable at lower pH. This was demonstrated by the fact that some residual neutralization activity was maintained for IgY and Fab’ when incubated at pH 2.0 for up to 4 hr. However, presence of high salt which precipitated the IgY and not the Fab’, led to complete destruction of the neutralization activity of IgY but not Fab’.
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