Development of an indirect ELISA for detection of the adaptive immune response of black carp (Mylopharyngodon piceus)

Abstract

Black carp (Mylopharyngodon piceus) is an important fishery resource and the main breeding target in China. Due to the lack of an assay of immunoglobulin M (IgM) antibodies in black carp, there is no effective method to evaluate adaptive immune response, which limits immunological studies and vaccine development. The present study used mAbs (monoclonal antibodies) against serum IgM of grass carp as capture antibodies. The results of Western blot analysis indicated that these antibodies had strong affinity and specificity to IgM heavy chain in black carp serum and were used to detect the antibody titer, optimize the conditions, perform a sensitivity test, and develop an indirect ELISA (enzyme-linked immunosorbent assay) to detect specific IgM antibodies in the serum. This detection method has good specificity and is effective only for grass carp (Ctenopharyngodon idella) and black carp and not for crucian carp (Carassius aumtus), silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), mandarin fish (Siniperca chuatsi), black bream (Megalobrama skolkovii), or yellow catfish (Pseudobagrus fulvidraco). The lowest antigen detection level was 0.05 μg/ml. The error of experimental repetition in the same sample was 1.61–4.61%. The levels of specific IgM in black carp serum were steadily increased after immunization, peaked on day 28, and then slowly decreased. Indirect ELISA can be applied to detect the changes in specific antibodies in black carp serum. Moreover, indirect ELISA provides a convenient and reliable serological detection method for immunological research and evaluation of immune effects of a vaccine in black carp.