The Purification of Fish B - like Cells and Their Functional Properties

Choi, Sang Hoon

Abstract

Serum immunoglobulins from carp Cyprinus carpio were purified using affinity chromatography methods. Fish were immunized with bovine serum albumin (BSA) and the specific fish antibodies purified from the immune serum on a BSA-irnmobilized Sepharose 4B gel. The analysis of the immunoglobulins by reducing SDS-PAGE showed them to be composed of a single p,-like heavy chain of 76 kd and light chain of 28 kd. Polyclonal rabbit antibodies against the fish IgM were produced to further analyze IgM' B-like cells from carp. Irrespective of a BSA immunization, the distribution rates of IgM' B-like cells in the head kidney and spleen were about 49% and 24%, respectively. The IgM' cells were magnetically purified by using Mini-Macs column. To study whether the purified IgM' cells are B-like lymphocytes, those cells were cultured with hrIL-4 (50 U/ml) for 48 hr at 25C in 5% CO, incubator. And the titer of antibodies secreted from IgM' and IgM cells was analyzed by an enzyme-linked immunosorbent assay (ELISA). We found the IgM' cells produced a greater amount of antibodies to BSA than both IgM cells and negative control. Unexpectedly, however, moderate amount of antibodies were also detected in the supernatant of IgM cell population, indicating the difference of humoral immune responses between a fish and mammalian.