A new type sandwich immunoassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microcystin and an anti-microcystin monoclonal antibody.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.