Anti-idiotype and Anti-anti-idiotype Antibodies Generated from Polyclonal Antibodies against Microcystin-LR

Polyclonal anti-idiotype antibodies (pAb2/m) for microcystin leucine-arginine variant (MCYSTLR) were generated from ascites after immunization with affinity-purified Fab fragment of rabbit polyclonal antibodies (pAb1/r) against MCYST-LR to BALB/c mice. Competitive direct enzyme-linked immunosorbent assay (cd-ELISA) revealed that the binding of free MCYST-LR to solid-phase pAb1/r was inhibited by affinity-purified pAb2/m. pAb2/m can be used as MCYST-LR surrogate in the ELISA. Using the purified pAb2/m as immunogen, polyclonal anti-anti-idiotype antibodies (pAb3/ r), which have characteristics similar to those of original pAb1/r, were generated in rabbits. In the pAb3/r-based ELISA, the concentrations causing 50% inhibition (IC50) of binding of MCYST-LR-horseradish peroxidase (MCYST-HRP) to the solid-phase pAb3 by MCYST-LR, MCYST-arginine-arginine variant (MCYST-RR), MCYST-tyrosine-arginine variant (MCYST-YR), and nodularin (NODLN) were 0.7, 24.4, 19.2, and 24.3 ng/mL, respectively. pAb3/r showed a dose-dependent neutralization of the inhibitory effect of MCYST-LR to protein phosphatase 1 (PP1) in an in vitro assay system. pAb2/m had no effect on PP1 activities.