Robust and sensitive monoclonal enzyme-linked immunosorbent assay for the herbicide molinate.
This paper reports on the generation of monoclonal antibodies and the development of a new enzyme-linked immunosorbent assay (ELISA) for the detection of molinate (S-ethyl hexahydroazepine-1-carbothioate). Hybridoma cells were generated using spleen and lymph node cells from a mouse immunized with S-2-carboxyethyl hexahydroazepine-1-carbothioate conjugated to keyhole limpet hemocyanin. After screening with a competitive ELISA, two monoclonal antibodies, mAbs 16C11 and 14D7, with IC(50) values of 82 +/- 2 and 173 +/- 8 ng/mL, respectively, were selected. MAb 16C11 can detect molinate concentrations of 1 ng/mL with no cross-reactivity to any other thiocarbamate pesticides; however, it was susceptible to the presence of organic solvents and to variation in buffer ionic strength. MAb 14D7 tolerated concentrations up to 5% of propylene glycol and 12.5% of acetonitrile in the assay buffer. The sensitivity of mAb 14D7 was further improved by decreasing the amount of coating antigen in the ELISA; the final inhibition assay showed an IC(50) of 69.2 +/- 1.4 ng/mL. In summary, mAb14D7 provided a more sensitive and robust assay, as compared with previous polyclonal antibody-based assays, with the additional advantage of being based upon a consistent and unlimited source of a defined reagent.
Rufo
Caterina
C
Cátedra de Inmunología, Facultad de Química, Universidad de la República, Instituto de Higiene, Av. A. Navarro 3051, 11600 Montevideo, Uruguay.
Hammock
Bruce D
BD
Gee
Shirley J
SJ
Last
Jerold A
JA
González-Sapienza
Gualberto
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001381:Azepines / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006540:Herbicides / Q000032:analysis*; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D013859:Thiocarbamates / Q000032:analysis*
D000911:Antibodies, Monoclonal; D001381:Azepines; D006540:Herbicides; D013859:Thiocarbamates; C009821:molinate