Research On ELISA Methods For Octachlorostyrene

Octachlorostyrene (OCS), a toxic halogenated aromatic compound, is a persistentand bioaccumulative toxicant and belongs to potential persistent organic pollutants. Inthe last decade, established methods for detection of OCS are mainly based on gas liquidchromatography (GC), high-performance liquid chromatography (HPLC), gas liquidchromatography (GC)-tandem mass spectrometry (MS) and gel permeationchromatography(GPC). Although OCS urgently need a new, simple, sensitive andlow-cost detection method. New, fast, low-cost and simple determination method ofOCS provides a convenient conditions for the research and control of OCS.In1971, Ercegovich firstly applied immunochemical methods to environmentmonitoring, in recent years, immunoassay has been widely recognized and applied,emerging new methods and new means have laid the solid foundation for vigorouspromotion of the development of the environment POPs immunoassay. Theimmunoassay method have the advantages of specificity, high sensitivity, easy andsimple analysis capacity, the detection of low cost, does not require expensiveequipment, pre-treatment steps to simplify, which conventional instrumental analysiscan not be compared, in addition it has the selective strong and high sensitivity makeit very suitable for the analysis of detection of trace components in complexmatrices.At present, the studys of environmental POPs immunoassay, are reportedboth at home and abroad, mainly focuses on the method of ELISA. According to theliterature survey, at present no reports about the immunoassay of OCS.In this thesis, Octachlorostyrene (OCS) was derivatized and synthesized hapten,then it was linked respectively to two kinds of carrier protein, by which theimmunogen and coating antigen were synthesized successfully.Using the immunogens,rabbits were immunized to obtain the antibody against Octachlorostyrene. Theic-ELISA method for the detection of OCS was established with the antiserum againstOCS. The specific contents are as follows:(1) The Synthesis and Identification of artificial antigens for OCSIn order to establish the immune detection method of OCS, we synthesis thehapten of OCS with carboxyl reactive groups setp by step based onpentachloronitrobenzene. OCS was coupled to bovine serum albumin(BSA), by Activationof Lipid Method to prepare immunogen. It was also coupled to ovalbumin (OVA) by Activation of Lipid Method to prepare coating antigen. After dialysis, The Conjugateswere identified by UV Spectrum.The results demonstrated that the artificial antigenswere synthesized successfully. The conjugating ratios of the immunogen and coatingantigen are25:1,22:1respectively.(2) Preparation of anti-Octachlorostyrene Polyclonal Antibody and Determination of their titerOCS1-BSA, OCS2-BSA, OCS3-BSA were used as immunogens to immuniserabbits, by which the antiserum against OCS was obtained. The titers of the antibodyagainst OCS were1：128000，1：256000and1:64000.(3) Establishment of ic-ELISA methods for detection of OCSThrough the optimization of the factors including the type and concentration ofthe coating antigen and antiserum, the pH, the content of Tween-20, the type andconcentration of organic solvent of the buffer solution, the equation of the calibrationcurve of the indirect competitive ELISA was decided. The optimization tests about thedetection showed as follows: the combination of the coating antigen haptenOCS1-OVA at a concentration of10μg/mL and the antisera against immunizingantigen OCS2-BSA at a dilution of1/128000in microtiter plates. The immunoassaybuffer is15%CH3CN in0.15M PBS at pH7.4. Under the optimized conditions, thisindirect ELISA shows a linear detection range from1.4to86.3ng/mL, with an IC50value of4.46ng/mL and a detection limit of0.1ng/mL. Twelve kinds of compoundswere tested for calculating cross-reactivities, and almost all of them showed littlecross-reactivity (<5%). Water and sera samples spiked with OCS were analyzed byELISA and the achieved recoveries were satisfied with a mean recovery of91.6%.