A RAPID AND EFFICIENT PURIFICATION METHOD FOR HORSE IGG(T) USING A RAT MONOCLONAL-ANTIBODY

1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized an imals were fused to a non-secreting LOU/C immunocytoma (IR983F). The h ybridomas were cultured in HAT-containing medium and cloned under limi ting dilution conditions. Supernatants from the growing hybrids were s creened by ELISA using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG( T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 10(10) M(-1). 3. Ascites was i nduced in LOU (IgK-1b) rats by injecting the hybridoma cells and incom plete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1a chain column. 4. The purifi ed mAb was then coupled to Sepharose. Immunoelectrophoretically pure I gG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purifi ed IgG(T).