UBC Theses and Dissertations
Separation of immunoglobulins from egg yolk using metal chelate interaction chromatography and ion exchange chromatography McCannel, Anne Marie
The immune response of chickens immunized with β-N-acetylglucosaminidase was monitored in the egg yolks of the birds using an enzyme-linked immunosorbent assay. Significantly higher levels of specific antibodies were detected in the yolks of the birds immunized with the enzyme when compared with the yolks of a control bird collected over the same period significant differences also were found in the response within the immunized group of birds, indicating individual variability to the injections. Immunoglobulins were isolated from egg yolk after a preliminary purification using alginate to precipitate lipoproteins. A ten millilitre DEAE-Sephacel ion exchange chromatography column resulted in a final product containing 16 mg of IgG with a purity of 60% when 50 mL of an egg yolk supernatant was applied. Specific antibody activity toward the antigens β-lactoglobulin and E. coli lipopolysaccharide was higher in both cases in the isolated immunoglobulin fractions which contained lower purity (40%). Lower antibody activity was observed in the 60% purified fractions. Separation of specific antibodies from non-specific antibodies appeared to occur, possibly due to the given characteristics of the specific antibodies, or due to the differences exhibited by chicken IgG subpopulations. Using metal chelate interaction chromatography, a 10 mL copper-loaded column was able to yield 104 mg of IgG with a purity of 75% when 200 mL egg yolk supernatant was applied. Again, the heterogeneous nature of chicken IgG was illustrated. A comparison of the two chromatographic techniques indicated the advantages of metal chelate interaction chromatography over ion exchange chromatography under the conditions examined. Applications of the chicken IgG isolated by metal chelate interaction chromatography to an enzyme-linked immunosorbent assay for the detection of β-N-acetylglucosaminidase was attempted. Linear relationships were obtained when standard solutions of the enzyme were used in the assay. The results indicate that MCIC-isolated chicken immunoglobulins have excellent potential for use in analytical tests.
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