Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
James H. Yuan
Mark S. Elliot
Gerald J. Pepe
Lloyd Wolfinbarger, Jr.
Organophosphorus compounds (OPs) comprise one of the major classes of pesticides in use today because they are highly effective and less toxic. Because of their extensive use, there is a need for monitoring OPs in water and vegetables. Gas chromatography (GC) is the usual method for OPs screening, but it is time-consuming and has the high cost of instrumentation. The goal of this project was to find an easy method which was sensitive and had less instrumentation required to test OPs.
Microwells and microtubes were utilized as the solid-phase immobilization for the development of an enzyme immunoassay for OPs. A sensitive and simple microtube based competition enzyme immunoassay for the quantitative determination of organophosphorus compounds was developed. Malathion (phosphorodithioate), parathion (phosphorothionate), tamaron (phosphorothiolate) and mevinphos (phosphate ester) were chosen as the antigens for the antibody production. Gamma globulins were isolated from whole anti-sera by DEAE-cellulose chromatography. A carbodiimide coupling method was utilized to prepare the organophosphorus compound-peroxidase conjugates (OP-HRPO). Microtubes coated with antibody were incubated with analyte and OP-HRPO conjugate. The assay was performed within 20 minutes at room temperature, utilizing the competitive reaction of the analyte and the OP-HRPO, which produces more color, when less analyte is present.
Due to the high cross-reactivity of the different OPs to the antibodies and the low cross-reactivity of the different secondary metabolites (dimethyl phosphite, p-nitrophenol, ethyl propionate and methyl isobutyrate) to the antibodies, two of the immunoassay tests were conducted to be nonspecific which enabled them to act as a general screening method for OPs.
The assays of these four classes of OPs when compared to GC had the correlation coefficients ranging from 0.912 to 0.999 in water samples and from 0.949 to 0.999 in vegetable extract screening, which can be adapted to field testing.
The reproducibility of the immunoassay in the detection of OP residues in water and vegetable samples with the percent coefficients of variation (%CV) for the organophosphorus compounds assays ranged from 51.59% to 14.06% at 1 ng/mL and 6.03% to 4.34% at 100 ng/mL. The CVs of 1 ng/mL appeared to be high since they were below or close to the detection limit.
"Competitive Enzyme Immunoassay by Polyclonal Antibodies Against Organophosphorus Pesticides"
(1993). Doctor of Philosophy (PhD), dissertation, Chemistry and Biochemistry, Old Dominion University, DOI: 10.25777/2n72-6d44