Development of an ELISA for detection of an organophosphorus compound using monoclonal antibodies

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Abstract
This paper describes a specific and highly sensitive ELISA system using monoclonal antibodies in order to assay an organophosphorus compound. The soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2,-trimethyl-propyl diester (MATP) served as model substance. In order to obtain antibody-producing hybridomas BALB/c mice were immunized with MATP linked onto human serum albumin (HSA). The spleen cells of immunized mice were fused with syngenic plasmacytomas of the non-producer-line X63Ag8.653 with the aid of polyethylene glycol. To eliminate undesirable cross-reaction, common screening procedures were modified by directly coating the ELISA plates with hapten. Five out of 15 positive cell-lines were cloned by limiting dilution and further propagated. The respective immunoglobulin class and subclass of the obtained monoclonal antibodies was determined. Four of which were identified as IgG1, the other as IgG2a. After enrichment of antibodies in ascites and their isolation by protein A-sepharose, the affinity of various monoclonal antibodies was estimated in competitive inhibition enzyme immunoassay (CIEIA) by measuring the IC50 rates of free MATP. The rates were found to lie between 2.5 × 10−6 mol/l and 4.3 × 10−4 mol/l MATP. The IC10 rate for detectable MATP concentration was 5.4 × 10−7 mol/l MATP. Test duration was 280 min. The reactivity of the monoclonal antibodies with structurally related substances was used to check their specificity. Cross-reaction turned out to be negative. In order to develop a direct competitive ELISA, MATP was linked to horse radish peroxidase (HRPO) by adding a spacer. This helped to reduce total duration to 40 min. The detection level was further reduced to 1.3 × 10−7 mol/l MATP (corresponding to 975 pg/25 μl test-buffer) using the monoclonal antibody F71D7. Likewise, MATP was detected in goat serum, chicken serum, rabbit serum, milk and company's water in concentrations between 2.1 × 10−7 mol/l (IC10, company's water) and 4.9 × 10−8 mol/l (IC10, milk).