Immunoassay Methods Development For The Determination Of Camptothecin And Organoph Osphate Pesticides Based On Monoclonal Antibody

Agric-chemicals(including biological and chemical pesticide)play a critical rule for agricultural production,meanwhile,its environmental effects and human health risk has been concerned world-wildly.In this study,immunoassay methods have been developed for the determination of camptothecin and organophosphate pesticides based on monoclonal antibody.Camptothecin is the only one natural plant ingredient which has specifical inhibition activity to topoisomerase-I by far,and also is effective on solid tumours and malignant tumors as well as its derivatives.Therefore,it would be more interesting to study the antitumor mechanisms of camptothecin for drug development,using antibody technology and proteomics for cells or molecular interaction analysis.Organophosphate pesticides are high efficiency,broad-spectrum insecticides which have been widely used as a class of chemicals.However,the accumulate toxicity of organophosphorus pesticides could caused serious health effect to human.So it certainly needs to develop a detecting method that can detect the residues of organophosphate pesticide sensitively and broadly to ensure food safety.Based on previous research,in this research,we studied the relationship between structure of hapten and the specificity of antibody with different immunogen.For camptothecin hapten,two different sites have been changed and transformed into carboxyl group,and then conjugated with carrier protein KLH.After Balb/c mice immunization,hybridoma cell has been selected.Same procedure has been conducted for the development of monoclonal antibody against organophosphate pesticides,while,with only one organophosphate general hapten conjugated with two different carrier protein KLH and BSA as immunogen for broad-specificity monoclonal antibody production.Furthermore,the variable region gene of antibody was amplified from genomic DNA of hybridoma cells by PCR reaction,and single-chain variable fragment antibody(scFv)was produced.The main results are as follows:(1)For camptothecin monoclonal antibody producing,two different types of hapten(7-CPT and 20-CPT)have been achieved by camptothecin structure modification,and conjugated with carrier protein KLH for immunization of mice.The efficiency of antiserum raised by 7-CPT-KLH is about 800 times dilution,however only hapten can be recognised,the I50 of 7-CPT is 12.193?g/mL;the efficiency of anti-serum raised by 20-CPT-KLH is about 250000-500000 times dilution,and both hapten and camptothecin can be recognised,the I50 of 20-CPT is 8.100?g/mL,the I50 of camptothecin is 235.020?g/mL;there after,hybridoma cell 1G9 has been successfully selected,which can specifically bind camptothecin,with I10 about 0.894?g/mL,and I50 about 338.844?g/mL.(2)Two different hybridoma cell lines(BPB-BSA-3D2B3,BPB-KLH-3H6G3)have been successfully selected,which can secret monocolonal antibody for hapten recognization;After cross reactivity analysis,8 pesticides have been confirmed that can be specifictal recognized by monoclonal antibody(BPB-BSA-3D2B3)with I50 below 30 ?g/mL,and 13 pesticides can be specifically recognized by when it is detected by monoclonal antibodies(BPB-KLH-3H6G3)with I50 below 30 ?g/mL,repectively.(3)Furthermore,mRNA have been extracted and tranformed to cDNA from hybridoma cells to amplificate variable genes of heavy and light chain of antibody,and analysised;Single-chain antibody gene is constructed by using SOE-PCR method,and KLH-scFv gene and BSA-scFv gene were obtainde successfully,then compaired with amuni acid modeling.The results showed that two different type genom have been conformed and maitained.