Preparation Of Chloramphenicol Rabbit Monoclonal Antibody And Establishment Of The ELISA Procedure Basing It

Chloramphenicol (CAP) is an effective broad spectrum antibiotic widely used in veterinary practices. However, due to its side effects in humans, especially fatal bone marrow depression and aplastic anemia, CAP has been banned for use in food producing animals in many countries including China. In order to prevent the illegal use of chloramphenicol, it is necessary and urgent to develop sensitive methods for detecting CAP residues in animal tissues. Enzyme-linked immunosorbent assay is gradually applied in CAP residues assay as a rapid, highly sensitive and special method. This research aims to establish an indirect competitive enzyme-linked immunosorbent assay to detect CAP residues in animal food. The main content and results were as follows:1. Synthsis and identification of artificial antigensAn immunizing antigen CAP-BS and coating antigen CAP-BSA were synthesized by the method of diazotization. The conjugates were confirmed by UV spectrophotometer, SDS-PAGE electrophoresis and MALDI-TOFMS. The coupling ratios of the hapten and the carrier protein were 22:1. The protein concentration of CAP-BSA and CAP-OVA were determined to be 3.8 mg/mL and 2.0 mg/mL through BCA protein assay.2. Immunization and antisera screeningFour NZW rabbits with serial NO.76,77,78 and 79 were immunized in order to prepare corresponding anti-CAP antisera. The titer of the antisera was tested by ELISA. The result showed No.76 rabbit has the highest titers and it was selected to be spleen donors. The titer of No.76 rabbit anti-sera were 1:1.28×105.3. Production and purification of monoclonal antibody9-3 clones with high affinity were selected by indirect ELISA and competitive indirect ELISA. CAP 9-3H1/L2 was chosen to be taken into the second transfection in order to obtain plenty of RMcA-CAP for the further research by using competitive indirect ELISA and ELISA sandwich method were used to determine. Monoclonal antibody was purified by Protein-A Sepharose affinity chromatography method. The concentration of lgG was 1.23 mg/mL.4. Optimization of ELISA procedureThe optimal ELISA procedures for analyzing CAP were run as follows:the CAP-OVA was diluted with carbonate buffer for 10000 time and added to a 96-well plate for 50 uL well-1. After incubation at 4℃for 12h, the plate was washed for 4 times by TBST with 0.05% Tween-20. Then the binding sites which were not occupied by the coating antigen were blocked by 3% skim milk for 1 hours at 37℃. After the plate was washed as before, CAP standard solutions (50μL well-1) and primary antibody (50μL well-1) were added and incubated for 1 h at 37℃. The primary antibody was diluted 16000 time.All the reagent were diluted with TBS buffer, pH 8.0. After washing, HRP-IgG was added(100μL well-1) and the plate incubated for 45 min at 37℃. Then the plate was washed four times and the substrate solution(50 uL well-1) added. After reaction with shaking for 15 min, sulfuric acid (50μL well-1)was added. Finally, the absorbance was measured at 450 nm using a microplate reader.5. Characterization of monoclonal antibodyThe RabMab had good affinities to CAP. The competitive inhibition curves showed that the IC50 and IC10 was 1.06 ng/mL and 0.1 ng/mL respectively. The competitive inhibition enzyme-linked immunosorbent assay for CAP was established. The regression equation of this curve is y=38.447x+49.082, R2=0.9924. the curve has a favorable linearity relation within the concentration range of 0.18-6.37 ng/mL. Meanwhile, the RabMab had a good specificity with CAP. There was no cross reaction with fluoroquinolones and sulfonamides compounds. And the cross reactivity with Chloramphenicol, thiamphenicol. florfenicol were 2.09%,18.10%,12.45% respectively.6. Recovery of CAP in samplesRecovery rates of swine urine samples were between 73.31%-109.62% and RSD were between 6.98%-17.94%. Recovery rates of fresh milk samples were between 71.03%-95.01% and RSD were between 8.16%-16.93%. Recovery rates of honey samples were between 71.10%-87.96% and RSD were between 7.52%-14.74%.All these results showed the ELISA assay can meet the test need and have potential applications.