Anti-idiotype monoclonal antibodies against anti-microcystin antibody and their use in enzyme immunoassay.
Microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, are suspected as tumor-promoter contaminants of environmental water. We have previously developed an enzyme-linked immunosorbent assay (ELISA) for MCs based on an anti-MC MAb (MAb-mc). We describe here the production of anti-idiotype MAbs (MAb-ids) which react with MAb-mc and their use in a new ELISA for MCs. For the production of MAb-id, hybridoma cells were generated from mice immunized with MAb-mc. Two MAbs were selected for their ability to inhibit the binding of MAb-mc to microcystin-LR (MCLR)-bovine serum albumin conjugate in ELISA. The one with the higher inhibitory activity, designated Id7 (IgG1, kappa), was further characterized. ELISA and immunoprecipitation analysis revealed that Id7 specifically bound to MAb-mc but not to control IgG1, and the binding was inhibited by free MCLR. Therefore, Id7 is a MAb-id to MAb-mc and potentially possesses the structural image of MCLR. To establish MAb-id based ELISA, Id7 was tested for use in three types of competitive ELISA for MCs. The best format enabled reliable measurements of MCLR in the range of 100-1000 pg/ml with a coefficient of variation of less than 3%. In addition, microcystin-RR and microcystin-YR, principal MCs found in environmental water, were cross-reacted well (67-111% of MCLR) in the ELISA although 6(Z)-MCLR, a minor component, was less reactive (7% of MCLR). A comparative study of the MAb-id based ELISA with previously established MAb-mc based ELISA revealed good correlation (n = 14, r = 0.97) between the two methods for measurements of MCs content in freshwater samples. Thus, developed MAb-id based ELISA is an useful alternative for environmental monitoring of MCs.
Tsutsumi
T
T
Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Japan.
Nagata
S
S
Yoshida
F
F
Ueno
Y
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism*; D000458:Cyanobacteria; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D052998:Microcystins; D010456:Peptides, Cyclic / Q000276:immunology* / Q000378:metabolism; D014871:Water Microbiology
D000911:Antibodies, Monoclonal; D002273:Carcinogens; D052998:Microcystins; D010456:Peptides, Cyclic; C078588:microcystin