Preparation And Identification Of A Broad-specificity Monoclonal Antibody Against O, O-diethyl Organophosphorus Pesticides

The invention and broad implementation of pesticides is a milestone during the agricultural modernization, and have made great contributions to increase yield and revenue. However, unscientific and chronical use of pestcides also resulted in over standard pestcide residue and jeopardize human health. Batches of hypertoxic and persistent pestcides have been replaced by organophosphate pesticide which is relatively safer and easier to degrade. However, problems resulted by the illegal use of these kinds of pesticides remain unresolved, thousands of people get sick or even lose their lives because of organophosphorous intoxication which account for more than 50% of the pesticide intoxication. In practical production, organophosphorous pesticides usually are mixed to reinforce the pesticidical effect, thus, a method for determination of multi-residues of organophosphorous pesticides is really in need.Current organophosphorous multi-residue methods such as instrumental analysis, which possess advantages of sensitivity, accuracy, and speed, but it's complicated pre-processing and expensive instruments, restrains it implement with in professional testing organizations. enzyme-linked immunosorbent assay, based on the specific binding reaction between antigen and antibody, which not only meet the state testing standards of sensitivity and accuracy, but also maintains advantages such as efficiency, easy sample handling, real time detection with low-cost, etc, are getting more and more public attention.Additionally, previous studies have verified the feasibility of broad-spectrum antibody which is able to detect a serial of pesticides with common functional group through generic hapten.The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) with specific monoclonal antibodies (McAbs) for quick detection and screening of a broad spectrum 0f O,O-diethyl organophosphorous pesticides. Diethylphosphono acetic acid was used as a generic hapten, and conjugated to carrier protein by active ester method, carbodiimide method and mixed-anhydride method to produce generic antigen which were then synthesized successfully. Balb/C mice were then respectively immunized with three immunogen using different methods for the preparation of antiserum. Antiserum was determined by indirect ELISA 6 week later after the first immunolization, and mice that produced polyclonal antibodies with the titer above 2×10⁴were chosen for cell fusion to prepare monoclonal antibodies. After selective cultivation by HAT medium and several times of screening by indirect ELISA, positive hybridoma cells were obtained. Two hybridoma cell strains that can stably secret monoclonal antibodies were obtained after three time of limited dilution clone and its supernatants have reaction neither with BSA nor with OVA, but with artificial coating antigen.The immunoglobulin class was analyzed by double immuno diffusion and the affinity and specificity of McAbs were determinated with indirect enzyme-linked immunosorbent assay. Results indicated that the immunoglobulin class of McAbs was IgG1. ELISA based on MOZ-1 showed that its IC₅₀ was 0.22μg/mL, limit of detection was 0.38ng/ml,and the linear detection range was 28.07μg/mL-1.9ng/ml. McAbs have a high affinity for diethylphosphono acetic acid (1.4×10⁷M^-1) and are able to cross-react to Chlorpyifos, Parathion, Profenofos, Omethoate, Dichlofenthion, Diazinon, Bromophos, Phoxim in the range between 7%–82%. This immunoassay is therefore capable of detecting 8 different organophosphorous pesticides. It is concluded that this assay is a qualified screening tool for quantitative or semi-quantitative detection of the 8 organophosphorous pesticides.