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Studies on structural stability of chicken’s egg yolk immunoglobulin (IgY):

University of British Columbia

Immunoglobulins play a critical role in therapy, diagnostic assays and purification of important compounds. Hen egg yolk immunoglobulins, or IgY, have been studied intensively by researchers. Many techniques for isolation of IgY have been established. IgY has many advantages over the mammalian antibodies. It does not bind with mammalian complements or rheumatoid factor. Low cost and convenient production make it an attractive antibody for human use. To obtain specific antibodies, elution protocols often expose IgY to harsh conditions such as low pH, which may lead to denaturation. This research aims to study the effects of acidic pH, temperature, the denaturant guanidine-hydrochloride on IgY and the reversibility of those effects. Structural properties were studied by ultraviolet spectroscopy, intrinsic fluorescence, extrinsic fluorescence with hydrophobic probes, and enzyme linked immunosorbent assay. IgY was found to be sensitive to acidic pH (2.8) with irreversible changes when treated at 37°C. IgY denatured with > 3 M guanidine hydrochloride concentration showed a significant drop in antigen binding activity determined by enzyme linked immunosorbent assay suggesting that the changes resulting in inactivation are only partly reversible. These results should be considered in establishing processes for the isolation or utilization of IgY antibodies. For practical use of IgY in food or therapeutic formulations it is important that it is stable to processing conditions. Stability of IgY to freezing and freeze drying was investigated by monitoring its solubility, intrinsic fluorescence, extrinsic fluorescence and enzyme linked immunosorbent assay activity. IgY was found to be stable to freezing at -8°C but showed a significant drop in solubility and enzyme linked immunosorbent assay activity after freeze drying. Immunoaffinity chromatography has been used in the past to isolate IgY and other antigens. Structural conformation of eluted IgY was investigated in the present study. Surface hydrophobicity of IgY antibodies eluted at pH 2.8 was significantly higher than the crude IgY isolate before affinity purification. Trehalose in the eluting medium had a protective effect on IgY as it decreased surface hydrophobicity of pH eluted IgY.

Thesis/Dissertation

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2009-05-20

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http://hdl.handle.net/2429/8008

Forestry

University of British Columbia

UBCV

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