The Study On The Establishment Of Ci-ELISA For The Detection Of Florfenicol And Apramycin Residue

Florfenicol (FFC) is a synthetic antibacterialt that belongs to the chloramphenicols group and Apramycin (Apra) is one of the most common semi-synthes penicillin that belongs to the Aminoglycoside group. They have been increasingly used in veterinary medicine to treat microbial infections. However, the FFC and Apra residues in animal edible tissues could cause serious public health problems. Enzyme-linked immunosorbent assay (ELISA) is a convenient and fast method to screen large samples. Being most of the kits depend on foreign countries, so it is urgent to develop ELISA fast kit for veterinary drugs with own independent property right. It's very meaningful to establish a fast detection method to detect the two antibiotics residue to control animal food safety and safeguard consumers' health.The purpose of the study here is to develop an indirect competitive enzyme-linked immunosorbent assays (ci-ELISA) to monitor FFC and Apra residues in animal food. The main contents and results were as followed:1.Synthesis and Identification of Florfenicol and Apramycin Artificial Antigen.Florfenicol was coupled respectively to bovine serum albumin (BSA) and ovalbumin (OVA) by mixed anhydride method; Apramycin was coupled to BSA by carbodiimide method and was coupled to OVA by giutaraldehyde method. Four artifical antigens FFC-HS-BSA, FFC-HS-OVA, Apra-BSA and Apra-OVA were prepared. The complete antigens were identified by SDS-PAGE, UV spectrometric scanning, infrared spectrometry and immune BALB/c mice test. The results demonstrated that the artificial antigens were synthesized successfully.2. Synthesis and Identification of Florfenicol and Apramycin polyclonal antibody.The antiserum titre and specificity obtained using an indirect ELISA and Ci-ELISA respectively. The results showed that the antiserum titre of FFC was about 1:51200 and the antiserum titre of Apra was about 1:25600 .Both of the specificity were good.3. Establishment and Application of Ci-PAGE methods for Florfenicol.The Ci-ELISA method was established to detect Florfenicol residues. The optimal test conditions was as followed: the optimal working concentration of antiserum, blocking reagent, coating antigen and horseradish peroxidase marked to the goat IgG of anti-rabbit IgG were 1:6400, 5% of separated milk, 0.5 ug/mL and 1:20000 respectively. Its standard curve is in 0.18ng/ml-500 ng/ml, the curvy equation is Y=-0.1516X + 0.788, coefficient correlation R=0.9859, with a limit of detection to 0.18 ng/ml, IC50 = 79.3 ng/ml. The coefficients of variation of intra-assay and inter-assay were respectively 4.86%, 7.77%. The antiserum had cross reactivity of 0.094% and 0.098% with chloramphenicol and thiamphenicol, respectively, but had cross reactivity of less than 0.01% with others. The recoveries in chicken ranged from 84.14% to 106.1%. Accuracy of the method denoted by mean recoveries of chicken and recoveries in chicken ranged from 84.14% to 106.1%.4. Establishment and Application of Ci-PAGE methods for Apramycin.The curvy equation is y= 12.666x +28.22, coefficient correlation R=0.9724, with a limit of detection to 0.1ng/ml, IC₅₀ =52.4 ng/ml. The linear range was from 0.1～100ng/mL. The coefficients of variation of intra-assay and inter-assay were 4.15% and 6.59% respectively. The recoveries rate of chicken muscle by ELISA ranged from 89.45% to 108%. The cross-reactivities of other structure-related drugs including gentamicin streptomycin kanamycin were lower than 0.01%.