| Chloramphenicol(CAP), which is considered as a broad-spectrum antibiotic, has serious adverse effect against human bodies, leading to aplastic anemia and granulocytopenia. Due to low price and stable character, Chloramphenicol is still used in aquaculture illegally. Chemiluminescent Enzyme Immunoassay (CLEIA) has the advantage of high specificity, broad linear range, etc. In the study, anti-Chloramphenicol monoclonal antibody was prepared and a method of CLEIA for determination of CAP was developed.Immunogen was prepared by mixed anhydride method and diazotization method under 3 different molar ratios from 50:1-10:1(hapten to carrier). Both Chloramphenicol Sod Succinate(HAP) and CAP were used as hapten, Bovine Serum Albumin (BSA) was used the carrier. Active ester was also applied under 7 different molar ratios from 40:1-1:1 in the preparation of coating antigen, when Ovalbumin (OVA) as the carrier. UV and IR spectrum and SDS-PAGE was used to assure the conjugation of hapten to carrier. The conjugate ration was 27:1-8:1 for HAP-BAS, 21:1-5:1 for CAP-BAS, and 26:1-1:3 for HAP-OVA through UV detection.6 Balb/c mice were immunized using 18:1 HAP-BSA and 21:1 CAP-BSA. The immunizing dose was 25,75, 100μg per mouse, respectively. One parallel group was set. After third immunization, the serum titer and sensitivity to Chloramphenicol, which can be reflected by 50% inhibiting concentration (IC50), was detected by ELISA using 4#HAP-OVA as coating antigen. Mouse 6 was chosen for boosted immune. Hybridoma technique was applied for fusing Sp2/0 mouse myeloma cells with splenocytes from Mouse 6. And the fusion rate was 84.6%. The supernatant was detected by ELISA for its titer and sentitive. After subcloning,2 hybridoma cell lines(Ⅳ1B3,Ⅳ2E7), which could secrete the specific anti-CAP antibodies, were established by limiting dilution assay (LDA). After selection by ELISA and culture expansion, cellⅣ1B3 clones, which is more sensitive, and with higher titer and lower IC50, were attained and injected into mice abdomen for anti-CAP monoclonal antibody production, The ascites of the mice was collected and extracted through caprylic acid and equilibrium ammonium sulfate.After antibodies purification, The conjugation ratio of the coating antigen was optimized at the index of IC50. The molar ratio of HAP:OVA was 3:1. Using the HAP-OVA as coating antigen, the antibody titer was 2×105 and the IC50 was 15ng/mL。The cross reaction rate of with penicillin, gentamicin, streptomycin, and tetracycline was barely zero, and with Chloramphenicol succinate was 110.3%.Enzyme-labelled antigen was synthesized by conjugating Hap with HRP according the method of mixed anhydride. The best enzyme-labelled antigen was screening by ELISA. Optimum consistency for each ingredient in CLEIA substrate was obtained by single factor experiment. The luminophore was luminal at the concentration of 5×10-4mol/L, enhancer was 4×10-4mol/L 4-iodophenol, and oxidant was 3×10-3 mol/L H2O2 solution. Direct competitive ELISA was developed and optimized. The optimum concentration of coating antigen was 1.5μg/mL, diluted rate of HAP-HRP was 1:1000, reaction time of standard (CAP) or samples with HAP-HRP was 30 min.The equation of calibration curve was Y=-13080lgX+35091 with the r=0.997 and IC50=2.3ng/mL in the concentration range from 0.067ng/mL to 36.7ng/mL. The coefficients of variation for within and between assays below 7%and 10%,respectively. The average recovery of shrimp was 96%-112% The method showed good repetition and precision and can be the foundation of CLEIS test kits.
|
PDF Full Text Request