Quantification of Antibody (IgY) Titers in Hen Eggs Following Immunization and their Use in Detecting Cell Surface Molecules on Nitrocellulose Membranes

Abstract

HLA‐A*0201 α chain and β2m were expressed from a prokaryotic system, and after refolding and purification, the α chain and β2m were used to immunize eight laying hens. The titer of egg yolk antibody against α chain increased from 10² to 10^5.3. The titer of egg yolk antibody against β2m increased from 10¹ to 10^4.7. The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.

Keywords:

• Quantification
• Titer
• HLA‐A*0201 α chain
• β2‐Microglobulin
• IgY
• Immunization
• Cell surface molecules

Acknowledgments

This work was supported by grants from NSFC & the Research Grant Council of Hong Kong Joint Research Fund (30170872, 30418003), 973 projects (2001CB510008, 2003CB514113) & 863 projects (2001AA215121, 2003AA215050).