Isolation of immunoglobulin in yolk (IgY) and rabbit serum immunoglobulin G (IgG) specific against bovine lactoferrin by immunoaffinity chromatography

Hens were intramuscularly immunized and rabbits were subcutaneously immuniz ed once every two weeks for 6 weeks using bovine lactoferrin (LF) as antige n. Antibody titers of both yolk (IgY) and rabbit serum (IgG) were as high a s 1.68 x 10(8) at the 6th and 8th weeks, respectively, after the initial im munization treatment. However, antibody titer against LF in yolk was 9.4 x 10(7) at 16 weeks. While antibody titer of rabbit serum declined sharply to 2.1 x 10(7) at the 12th week and to 2.6 x 10(6) at the 13th week after the initial immunization. The purification efficiency (specific activity of pu rified antibody against LF/specific activity of the corresponding antiserum or yolk against LF) of rabbit serum IgG purified by laboratory-prepared LF -Sepharose 4B immunoaffinity column (0.05 mg LF/ml wet gel) was about 2400, similar to that of IgY purified by LF-Sepharose 4B immunoaffinity column. Different amounts (0-15.0 mg) of IgY purified by LF-Sepharose 4B immunoaffi nity chromatography were applied to the same column to determine the bindin g capacity (q(m)) and dissociation constant (Kd) of LF-Sepharose 4B immunoa ffinity gel for IgY specific against LF. It was found that q(m) was 0.81 mg IgY/ml wet gel (1.620 mg IgY/mg LF) and K-d was 6.4 x 10(-6) M as determin ed by Langmuir-type adsorption isotherms. (C) 2001 Elsevier Science Ltd. Al l rights reserved.