Development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds

Abstract

Currently, diagnosis of exposure to toxic low-molecular-weight compounds is effected by the use of chromatographic techniques. Such an approach is limited by the need for expensive equipment and sample clean-up before carrying out the analysis. To overcome those drawbacks, we have been involved in the development of an immunoassay for diagnosis of exposure to toxic organophosphorus compounds such as pinacolylmethyl phosphonofluoridate (soman), which is a chemical warfare agent. Prior estimates suggested that it is necessary to be able to detect soman at a concentration below 2.5 x 10(-7) M. Using four previously developed monoclonal antibodies, an enzyme-linked immunosorbant assay (ELISA) was used to optimize assay conditions and identify the antibody with the highest apparent affinity. The minimum required assay time was 2.0-2.5 h with no loss in sensitivity. To determine the specificity of the highest affinity antibody, a competitive inhibition enzyme immunoassay (CIEIA) was performed with six structural analogs of soman. The IC50 values for these analogues were 5 x 10(-7) M for 4-nitrophenylpinacolylmethylphosphonate, 8 x 10(-7) M for dipinacolylmethylphosphonate, 2 x 10(-6) M for diisopropylmethylphosphonate, 3 x 10(-5) M for 4-nitrophenylmethyl(phenylphosphinate) and 6.5 x 10(-5) M for 4-nitrophenylethyl(phenyl)phosphinate. 4-Nitrophenyl-di(n-butyl)phosphinate did not inhibit binding. Those inhibitors with branched alkyl side-chains, similar to the soman molecule, were effective inhibitors. Compounds, which contained predominately aromatic groups, were poor inhibitors. We are continuing to probe the binding specificity of the monoclonal antibody to determine its utility in further assay development. Our present results suggest that the antibody chosen may have the appropriate specificity and affinity for immunodiagnosis of exposure to soman.

Direct hapten coated immunoassay format for the detection of atrazine and 2,4-dichlorophenoxyacetic acid herbicides

“…The fact that antibodies could be made virtually against any substance, and its usages in developing highly sensitive assay makes this approach quite useful for the analysis of these toxic molecules [1][2][3][4]. These assays apart from being highly specific, exhibit the desired sensitivity and accuracy for the detection of low molecular weight contaminants present in our environment.…”

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

“…Therefore, there is a growing demand for more rapid and less costly methods for determining small molecular residues in the environmental sample. Enzyme-linked immunosorbent assay (ELISA) is the alternative technique widely used and accepted due to its sensitivity, fastness, and cost-effectivity for the identification of chemicals like pesticides [11][12][13][14][15] . To achieve better performance of antibody, enzyme tracer and analyte, incubation strategy has been chosen so that analyte can compete strongly even in the low concentration.…”

Design and Development of Imprinted Polymer Inclusion Membrane‐Based Field Monitoring Device for Trace Determination of Phorate (O,O‐Diethyl S‐Ethyl Thiomethyl Phophorodithioate) in Natural Waters

“…Additionally, most of these techniques require sophisticated and time consuming extraction procedures such as liquid solid extraction (LSE) or solid phase microextraction (SPME) [8 -10]. Biological methods such as immunoassay have also been reported [11]. Long analysis time and extensive sample handling with multiple washing steps limit the applications.…”