Ultrasensitive Time-resolved Fluoroimmunoassay For Rapid Detection Of Chloramphenicol

Chloramphenicol(CAP) residues in human food have led to a lot of side effects include severe or fatal illnesses. It is stipulated in many countries, such as USA, Canada, Australia, EU member states and China that CAP not be detected in animals used as food. Therefore,it is important to develop a sensitive and simple method for CAP detection.Time-resolved fluoroimmunoassay (TRFIA) developed in recent ten years is a kind of infant nonradiolabeled immunoassay, which provides high specificity, high sensitivity, good repetition and can deal with a large number of samples.A rapid and sensitive method of indirect competitive time-resolved fluoroimmunoassay (TRFIA) for CAP determination was established in this paper. CAP was coupled with carrier protein bovine serum albumin (BSA) and Ovalbumin (OVA) to prepare immune antigen CAP-BSA and coating antigen CAP-OVA. Anti-CAP polyclonal antibody was raised by immunization in rabbits. CAP-OVA was coated onto the microtitre plate. CAP or sample was added into the microtitre plate as a competitor, and incubated with limited anti-CAP antibody. A goat anti rabbit IgG-Eu3+ conjugate was used to enable detection. Results showed that the detection limit of the assay was 0.008 ng/ml (8ppt) for indirect competitive TRFIA formats. The assay range was 0.008 ng/ml -100 ng/ml. The concentration of CAP for 20%, 50% and 80% binding inhibition (ED20, ED50, ED80) were (25.75±0.442) ng/ml, (0.917±0.081) ng/ml and (0.033±0.0016) ng/ml. The inter- and intra-assay variabilities of the CAP-TRFIA were 6.8% and 13.5%. The cross reactivity of the CAP-TRFIA with Chloramphenicol succinate was 19.28%, while that with Thiamphenicol was less than 0.1%, that with Penicillin and Norfloxacin was less than 0.01%. The mean recovery of CAP from milk, honey, prawn muscle tissues and chicken muscle tissues samples was 79.7%, 112.6%, 92.1% and 97.1%. The thermal stability of CAP-TRFIA format was more than 6 months. The detection limit of CAP-TRFIA was signicantly enhanced compared with that of Enzyme-Linked Immuno Sorbent Assay(ELISA) kits in markets which is 0.05ng/ml . The ELISA kits have a high rate of false positive and the stability was not very good, because of the enzyme activity was much affected by temperature, pH and other factors. It was shown that the CAP-TRFIA with high stability and optimal range is the most sensitive assays reported and will be useful to screen CAP residuals simply and economically.