Binding properties of broad-specific monoclonal antibodies against three organophosphorus pesticides by a direct surface plasmon resonance immunosensor

Abstract

In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC 50 ranging from 2.93 to 19.71 ng mL −1 . Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (K D ) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10 −9  M and 4.26 × 10 −9  M for parathion, 6.75 × 10 −9  M and 4.17 × 10 −9  M for methyl-parathion, and 2.44 × 10 −8  M and 1.19 × 10 −8  M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. [Figure not available: see fulltext.].