Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids

Background: Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-alpha has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-alpha detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-alpha antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-alpha of salmonid fish. TNF-alpha epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-alpha, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-alpha positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-alpha function.

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

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.