The Study Of Elisa Method For Fluoranthene And Ketoprofen

In this study, Fluoranthene was acted as the representative of the polycyclic aromatic hydrocarbons, derivatized and synthesized hapten. Ketoprofen was represented of the Non-steroidal anti-inflammatory drugs and acted as the hapten directly. They were linked respectively to two kinds of carrier protein, by which the artificial antigens were synthesized successfully. Using the immunogens, rabbits were immunized to obtain the antibody against Fluoranthene and Ketoprofen. The ic-ELISA methods for the detection of FL and KET were established with the antiserum against FL and KET, respectively.Main results are as follows:1. Synthesis and Identification of the Artifical Antigens for Fluoranthene and Ketoprofen.Fluoranthene and Ketoprofen were coupled to bovine serum albumin (BSA), by Activation of Lipid Method to prepare coating antigen. They were also coupled to ovalbumin (OVA) by Mixed Anhydride Method to prepare immunogen. After dialysis, The Conjugates were identified by UV Spectrum. The results demonstrated that the artificial antigens were synthesized successfully.The concentration of the FL-BSA, KET-BSA, FL-OVA and KET-OVA were 3.95 mg/mL, 5.25 mg/mL, 5.95 mg/mL, 4.28 mg/mL, respectively. The conjugating ratios of the conjugates were 16.0, 9.93, 13.1 and 8.65, respectively. 2.Preparation of Polyclonal Antibody and Determination of titer. FL-BSA and KET-BSA were used as immunogens to immunise rabbits,by which the antiserum against FL and the antiserum against KET were obtained. The titers of the antibody against FL and the antibody against KET were 1: 6.4×104, 1: 1.28×105, respectively.3. Establishment of ic-ELISA methods. Through the optimization of the factors including detection process and detection condition, sensitivity, linear range and the equation of the calibration curve of the indirect competitive ELISA was decided. The optimization tests about the detection showed as follows: The dilution ratio of the antigens and antibodies were 1: 2000 and 1: 8000 respectively for both of FL and KET; the antigens were placed 4℃about 12 hours to be enveloped; the sealed liquid was 0.5% gelatin for both of FL and KET; the closure time of the two substances was both 1.0 h; the antibodies were incubated at 37℃for 60 min to be confined for the two substances; the enzyme-antibodies were incubated at 37℃for 60 min for both of the FL and KET. One standard curve of FL was established with the antibodies against FL: the regression equation, the 50 % inhibition concentration(IC50), the limits of detect(LOD) and the linear range about the standard curve were y=-21.64x+96.55 (R2=0.960), 0.142μg/mL, 5.82ng/mL and 10 ng/L~10μg/L, respectively. Another standard curve of KET was established with the antibodies against KET: the regression equation, the 50%inhibition concentration(IC50), the limits of detect(LOD) and the linear range about the standard curve were y=-22.97x+104.46(R2=0.980), 0.235μg/L, 4.26 ng/L and 10 ng/L~10μg/L, respectively. The coefficients of variation intra-assay and inter-assay were both less than 10% for both of FL and KET,which accord with the requirements of methodology.4. The detection of the samples The standard curve of FL and KET was established by HPLC: the standard curve of FL was y=35126.00x+5363.46(R2=0.999)and the standard curve of KET was y=41440.05x+3563.48(R2=0.998).Using the ic-ELISA and HPLC to detect the recovery of FL and KET, recoveries of FL and KET in water samples ranged from 88%~110% with C.V. % of 2.00~8.00.Testing the concentration of FL and KET in the water samples and compared with the method of HPLC, there were FL and KET in the water.The results above showed that the success of this study lied in the successfully prepared artificial antigen of FL,KET, getting the antibody after animal immune. The high titer serum was obtained to establish indirect ELISA. After optimization, standard curve of FL,KET was made and the cross reaction was not obvious. This indirect ELISA detection method of FL,KET residue could satisfy the primary analysis, and provide technical basis for the establishment of ELISA kit.