Study Of Benzopyrene Immunological Rapid Detection Method

Benzopyrene (3,4-Benzopyrene, BaP) is a kind of polycyclic aromatic compounds whichhas strong carcinogenic. It was produced from the exhaust gas produced by incompletecombustion of fuel, such as coal, oil and gas, and the smoking, baking and frying food. BaPhas the characteristic of long-term and hidden feature, when the body contact or intake, evenif there were no reaction, but also it can accumulate in the body, has longer incubation periodbefore showing symptoms, generally for20to25years, at the same time it also can make thefuture generations. BaP has teratogenetic and mutagenic, and it can affect the children throughmatrix via the placenta, causing deformity or embryos and larvae immune function decline,etc. BaP can cause lung cancer, gastric cancer, bladder cancer, and gastrointestinal cancer andother cancers. As the sources and harmfulness of BaP was widely, so it is very necessary tostudy out BaP indirect competitive ELISA fast detection kit. The study were prepared for BaPof immune antigen BSA-oABaP and detection antigen OVA-oABaP, and BALB/c mice wereimmunized with BSA-oABaP. Then we abtained BaP-hybridoma cell, prepared monoclonalantibody with hybridoma technology, assemble BaP rapid detection ELISA kit and evaluatedthe performance. The main contents and results of this study were as follows：1、With BaP as raw materials, the hapten was synthesised by nitration and reduction ofthe two step chemical reaction. The immune antigen (BSA-oABaP) and the detection antigen(OVA-oABaP) were prepared through conjugation of the hapten with BSA and OVA by usingEDC method. By UV and SDS-PAGE to identify coupling was successful. Then immuned theBALB/c mice, the polyclonal antiserum titer was detected by indirect ELISA, sensibility wasidentified by blocking ELISA. The results show that the BSA-oABaP wave moves to the rightwhen compared with oABaP, and the moving speed of BSA faster than BSA-oABaP in theSDS-PAGE. That is to say coupling is successful. Indirect ELISA showed a high titer above1:103of the antiserum of all the six BALB/c mice. The titer of pAb for No.6mouse is thehighest, its IC50was22.14μg/L. The rate of cross reaction of BaP pAb toBenzo(b)fluoranthene was18%, and no cross reaction to other antibiotics. The experimentshows that we obtained the BaP pAb with good sensibility and specificity. 2、Six BALB/c mice were immunized with BSA-oABaP, the titre and sensitivity ofpolyclonal antibody was detected by indirect ELISA and blocking ELISA, so as to select themouse used in cell fusion. BaP mAb was prepared by hybridoma technology. The titer、affinity、sensitivity、specificity and subtype of the mAb were characterized. Massive BaP mAbwere induced from in vivo method. There hybridoma cell lines of1D3、2A9、2E12werescreened for specificity to BaP, all the isotypes of the mAb were IgG1. The indirect ELISAtiters of them were1:3.2×102、1:5.12×102、1:6.4×102in supernatant,1:2.56×105、1:5.0×105、1:5.12×105in ascites respectively, the mAb of them showed good sensitivity with IC50of1.413μg/L、1.156μg/L、0.923μg/L to BaP, the affinity constant (Ka) was2.36×109L/mol、3.84×109L/mol、1.81×1010L/mol respectively, and all the isotypes of the mAb were IgG1.The rate of cross reaction of BaP mAb to Benzo[b]fluoranthene was3.3%, and no crossreaction to other antibiotics. The experiment shows that we obtained the BaP mAb with hightiter、high sensitivity、high affinity and high specificity.3、A rapid detection ELISA kit for determination of BaP (BaP-Kit) were developed withBaP mAb of2E12. The calibration curve of BaP-Kit with standard BaP inhibitor was typicalsigmoid curve fitted to the four parameters logistic equation with R2=0.9902and linearregression equation y=-28.662x+132.87. According to regression equation, IC50is found0.779μg/L. The sensitivity of0.054μg/L, the recoveries of BaP spiked in beef were83.85%,in pork were95.53%. The precision and accuracy of the assay as determined by inter-assayand intra-assay coefficient variation were below6%. The rate of cross reaction of BaP-Kit toBenzo[b]fluoranthene was2.7%, and no cross reaction to other antibiotics. The validity ofBaP-Kit in4℃was above four months.