Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarumin sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout ( O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measure- ments of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration ( r 2 5 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r 2 5 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing , 3g . Regular monitoring of the health of fish reared in intensive aquaculture is an important step toward re- ducing the introduction and spread of infectious dis- eases. 32 The immune system of fish may be adversely affected by changes in physiologic processes resulting from an inattention to water quality or fish rearing den-