Evaluation Of ELISA Kits For Detection Of Chloramphenicol Residues And Initial Improvements

In order to detect chloramphenicol (CAP) residues by immunoassay methods, We developed the corresponding ELISA kit, But the CAP maximum residue limit(MRL) defined by many countries in edible animal products became more and more lower, accordingly the sensitivity of the ELISA kit was demanded more and more higher. Then in the research we optimized the main reagents of the kit. The results of this study are as following.1. CAP-holoantigen and enzyme-labelled antigen were synthesizedCAP-midbody was synthesized by esterification reaction, then the immunizing antigen and coating antigen were synthesized by conjugating CAP-midbody to HSA,BSA and OVA by the method of diazotization and coupling. At the same time, an enzyme-labelled conjugate of CAP—OVA and horseradish peroxidase (HRP) was synthesized by the method of periodate oxidation, and CAP—BSA—HRP was synthesized by the same method. Verification results by ultraviolet spectrophotometer demonstrated that all the CAP-holoantigens and enzyme-labelled antigens were successfully synthesized.2. Preparation of anti-CAP polyclonal (PcAb)Twenty healthy large-eared Japanese rabbits were divided randomly into 7 groups, the former 6 groups were immunized with different CAP-holoantigens, and the last group was blank. After 4 times immunization, antiserum was prepared by bloodletting from carotid. Antiserum was purified by saturated ammonium sulfate and Sephadex G200. Among 6 immunity classes, the CAP—HSA immunity class had the highest titer and ensitivity which could reach 5x10⁵ and 4.5μg/L (represent by IC₅₀) respectively.3. Preparation of anti-CAP monoclonal antibody(McAb) Balb/c mice were immunized with different CAp-holoantigens. After 4 times immunization, the stimulated splenocytes were routinely fused with SP2/0 myeloma cells to produce hybridoma cells. Through screening to hybridoma supernatants by indirect ELISA and competitive direct ELISA (CdELISA), 4 hybridoma cell lines were successfully isolated. In the procedure a new gradient screening method was established to gain cell lines that can excrete higher affinity anti-hapten antibody.4.Checking of the ELISA methodThe sensitivity of ELISA method: the fluctuation scope of IC₅₀ was from 7.9μg/L to 15.6μg/L and the mean was ll.6μg/L; the lowest detectable limit (LDL) of the ELISA method: the LDL for standard solution of CAP was 0.3μg/L and for muscle tissues from swine and chicken were 2.540μg/kg,1.896μg/kg respectively; the accuracy rating of the ELISA method: when the admoveatur concentration of CAP was 3.0μg/kg～10.0μg/kg, the recovery rate in pig muscle tissues was 60%～110%, the interassay coefficient of variation was lower than 20 %; the precision of ELISA method: the intraassay coefficient of variation was under 30ng/mL, according with residual detection demands; the comparison results between ELISA and GC method: the recovery by ELISA method was 71.2%lower than by GC method which was 87.6%, the variation coefficient by ELISA method was 6.7% higher than by GC method which was 6.6%, but allthe indexes by two methods were in normal fluctuation range.5. Development of ELISA kits for CAP detectionThe stability of ELISA kits: stability test of 2℃～8℃and accelerated stability test of 37℃showed thatthe kit could be conserved at 4℃for 6 monthes; self-made kits were compared to import kits about the sensitivity, accuracy rating and precision of ELISA method, and it indicated that all indexes of self-made kits were lower than Holland kits; the residual experiment in animals showed that self-made kits had the detectivity for CAP residues in sample. Recheck and application results showed that self-made kits had the following good qualities: simple sample pre-processing, high sensitivity, accuratissime and reliability, volant and convenient manipulation, and the self-made kit could be widely used in coherent production for CAP residues detection.