Compared accuracy of two enzyme linked immunosorbent assay (ELISA) methods performed with polyclonal antibodies to measure immunoglobulin levels inPlatichthys flesusL. 1758 (Flounder)

Abstract

Polyclonal antibodies (PAbs) directed against flatfish (Platichthys flesusL.) immunoglobulins (IgM) were prepared and characterised for use in immuno-assays. IgG antibodies produced in two rabbits reacted specifically with flatfish IgM. A double-antibody sandwich enzyme-linked immunosorbent assay (DS-ELISA) and a competition ELISA (C-ELISA) have been developed to measure flatfish immunoglobulin concentration in serum. The DS-ELISA employs biotinylated and non-biotinylated Ig, while C-ELISA uses only one biotinylated Ig. The C-ELISA method gave an average value of 2·630±0·067mg ml−1for flounder IgM and the DS-ELISA 4·010±0·072mg ml−1. Total serum protein titration was determined by the Bradford method and values of 20% and 30·4% IgM in the total serum protein were calculated using the C-ELISA and DS-ELISA, respectively. A comparative study of these two methods reveals that both can be used. However, the C-ELISA seems to be more accurate and easier to use. It is concluded that the ELISA technique using PAbs is a potential method to measure flounder IgM and could be helpful in the assessment of the immune status in flatfish.