Preparation Of Anti-Thyroxine Antibody Immunoglobulin Y And Its Application In RIA

It is considered that laying hen, as the immune object, has some advantages such as need of fewer of antigens to immunize, no need of abstraction acquiring amount of antibodies which are efficiently transferred from blood to the yolk, and so on. Antibodies in the yolk are collectively named yolk immunoglobulin(IgY).T4 esterized with methanol and conjugated with bovine serum albumin was used as antigen. We adopted two different ways for immunizing two hens and observed production pattern of antibody. Hen A was primarily immunized with 20 U g antigen. After 20 days, boosted with 50 u g. The third immunization was carried out with 100 u g at 62 days after primary immunization. Specific antibody in serum appeared at day 36 following the primary immunization. In yolk of hen A's egg, it appeared at 8 days later than that in serum. At the same time, hen B primarily immunized with lOOug antigen and boostered at day 45 with 50 ug antigen. Specific antibody in serum appeared at day 35 following the primary immunization. In yolk of hen B's egg, it appeared at 6 days later than that in serum. During two months after primary immunization, the litres of serum was higher than that of the yolk. From then on, they became similar. In the end, the yolk litres exceeded. However, they were no especially different. In general, litres of hen A was superior to lhat of hen B. The highest litres was on the decline by two months after the final immunization was ended. In this study, n-Bulanol for defatted IgY was superior to water dilution(WD). After IgY was defatted wilh n-Butanol, further purification of IgY wilh sodium sulphate and ammonium sulphate sailing out were compared. As a result, the salting out procedure of ammonium sulphalewas superior to sodium sulphate. After purification, higher purity(94.1%) and higher yield(9.5mg IgY/ml yolk) of antibody were obtained. In general, n-Butanol for defatted IgY is a simple ,rapid and efficient procedure.With anti-T-JgY establishing a radioimmunoassay, the quality of anti-T4 IgY was appraised : litres was 1:25 00 , permitting rang of litres in the RIA was from 1:2500 to 1:5000 ; the degree of cross reactivities with Ta was 1.28%; affinity constants was 2.13 X 109L/mol. The methological indices for the method were listed as follows: the detection limit or sensitivity was 1.67ng/ml; intra-assay CV was 5.3%, and inter-assay CV was 7.0%; the average recovery was 96.13%; The regression analysis was performed for the same sel of specimens(20)subjecled to bolh IgG-RIA and IgY-RIA, and results were quite well positively correlated(r=0.9988).