Abstract
When mice were immunized with the conjugate EIT244-KLH containing malathion-hapten with a P-NH-C instead of P-S-C bond, two monoclonal antibodies (mabs) MLT2-23 and MLT40-4 specific to the insecticide malathion were isolated and characterized in a direct competitive enzyme-linked immunosorbent assay (dc-ELISA). Other haptens with the P-S-C bond failed to raise antibodies against malathion. Malathion was determined in the ranges of 5.3 to 75 ng/ml and of 7.0 to 190 ng/ml in a dc-ELISA based on the mabs MLT2-23 and MLT40-4, respectively. Then, cDNA clones encoding heavy chain and light chain regions of both mabs were isolated from two individual cDNA libraries constructed from mRNA fractions extracted from hybridoma cells producing the corresponding mabs. Two types of single-chain variable fragment (scFv) antibody genes with the sequences of VH-linker-VL (HL) and VL-linker-VH (LH), respectively, were constructed on the basis of the cDNA clones of each mab, inserted into the phagemid vector pCANTAB5E and expressed in Escherichia coli HB2151 cells. The IC50 in an indirect competitive ELISA (ic-ELISA) with MLT2-23/HL scFv and MLT2-23/LH scFv for malathion was 81 ng/ml and 72 ng/ml, respectively, compared to 60 ng/ml with the parent mab MLT2-23. On the other hand, MLT40-4/LH scFv showed an IC50 value of 150 ng/ml, in contrast to 75 ng/ml with the parent mab MLT40-4, while MLT40-4/HL scFv hardly reacted at all with malathion in the ic-ELISA. It was found that the order of linkage of both VL and VH allowed the scFv antibodies to alter their antigen-binding affinity or antigen-antibody reactivity in the case of MLT40-4 scFvs.
