Preparation Of Monoclonal Antibody Against Phthalate Esters(2-ethylcaproic) And Establishment Of An Indirect Competition ELISA Method

Phthalate (2-ethyl caprice) phthalate (DEHP) belong to the ophthalmic acid ester compounds, from large output, increasing the material softness characteristics of good, cheap, widely used in plastic products. This kind of material is a kind of environmental hormone, are classified as suspected environmental hormones, high doses of DEHP in the human body can’t completely metabolism, causing accumulation, for a long time to stay in the human body can cause side effects, harm to health. The European Union, the United States environmental protection agency (pea) has already passed some laws and regulations to strictly control the plasticizer testing standard, and are now using citric acid ester, epoxy acid esters such as security environmental protection plasticizer do update substitute products. Some regulatory measures now being developed in China. DEHP has established a variety of detection methods at home and abroad, such as high performance liquid chromatograpHy (HPLC), gas chromatograpHy (GS), gas chromatograpHy-mass spectrometry (GC-MS);the method is accurate, sensitive and effective, but all need expensive instrument, professional operation skills and complex sample handler. And ELISA method can solve these problems, it is a simple, accurate, efficient, cheap, and specific detection method, suitable for rapid screening of bulk samples.This study with monoclonal antibody and enzyme linked immunosorbent assay technique as the foundation, through the protein synthesis of artificial antigen of DEHP connection technology, preparation of the high sensitivity, strong specificity, high titer of antibody, and establish the DEHP Celia detection method.1.DEHP synthesis and identification of artificial antigens. Through first after adding nitro DEHP, again through the diazole method coupling links with proteins, preparation of artificial antigen of DEHP-BSA and DEHP-HSA, the ultraviolet scanning and immunological method coupling successful preliminary identification. DEHP-BSA as immunogenic, DEHP-HSA as the package is original.2.DEHP single preparation. Used DEHP-BSA routine immunization of BALB/c mice, after five free serum titer1:12000above. By hybridism technology and enzyme-linked immune technology can last1strain screening for stable production of DEHP specificity monoclonal antibody hybridism, one of the4C2strain cells secrete antibodies to DEHP exhibited strong combining ability, using the in vivo induced body, the4C2hybridism injected into abdominal cavity of mice, preparation of DEHP ascots, preparation of ascots titer of1:50000, protein concentration is15.9mg/mL.3.The establishment of ELISA detection method. Using4C2hybridism preparation of DEHP ascots Celia way to detect the DEHP is established. Establish standard competition curve, concentration of DEHP in2-50ng/mL, the standard curve is linear equation for y=0.3308+0.6313x(R2=0.9935), and IC50was8.16ng/mL, LOD is less than0.5ng/mL, LOQ is2.12ng/mL. The sheet resistance of DEHP inhibition rate was100%, the sheet resistance of pHthalate, deputy pHthalate butyl ester cross reaction rate were35.4%,39.5%, and the other cross reaction rate is less than10%.4.Add recycling experiment. Build Celia method is applied to cap liners to add recycling test. When the fluid to do2times dilution of the sample, the sample matrix interference disappeared basically. Within0～20Ngami, the curve equation is:y=0.4698+0.7246x, R2=0.9959, and IC50was3.76ng/g, LOD is lower than1.563ng/g. When adding DEHP concentrations between5-25ng/mL, recovery rate was between76.67%～95.23%, the difference in difference between batch and batch is less than10%.