Synthesis and Transfer of Key Reagents for an ¹²⁵Iodine Chloramphenicol Radioimmunoassay

In recent years, several reports of Chloramphenicol (CAP) detected in poultry, aquaculture products or honey revealed that abuse of this antibiotic is still a serious problem, especially in developing countries. To identify and curb veterinary drug misuse, national food surveillance authorities have a great need for sensitive and efficient but non–sophisticated and low–cost screening methods. In this context, a ¹²⁵I solid–phase radioimmunoassay (RIA) for CAP was developed. ¹²⁵I as a γ–emmitter has the advantage of being directly detectable and the short half–life reduces problems with disposal of radioactive waste. The competitive assay was conceived as a double antibody tube test with anti-rabbit coated tubes, specific anti- CAP antibody from rabbit and biotinylated CAP plus ¹²⁵I–labelled streptavidin as tracer. As ¹²⁵I–labelled CAP is not commercially available and direct labelling of CAP is rather labour–intensive and needs experience, aspects which limit the application in standard surveillance labs for which the test should be fit, ¹²⁵I–labelled streptavidin, which is commercially available and is easier to label was selected as tracer. The carboxyl group of the CAP metabolite, CAP succinate, provided a functional group for forming a peptide bond with 5– biotinamidopentylamine by carbodiimide coupling. Biotinylated CAP succinate was chromatographically purified and checked for reactivity to streptavidin in a modified enzyme linked version of the original RIA with streptavidin–horse radish peroxidise (HRP) as the label. The synthesized product appeared to be biotinylated CAP succinate, judging by its UV absorption at 278 nm and binding both to anti–CAP antibody and streptavidin–HRP. The sensitivity of the tested standard curves showed that the tracer was fit for purpose. A new concept of ¹²⁵I solid–phase RIA was developed. It is applicable to a broad array of analytes and after adopting this technique to all relevant tests one lab needs only one radioactive label for all tests, making analytical procedures much easier. All stages for the development of a practical and sensitive ¹²⁵I solid–phase RIA for CAP namely assay design, synthesis of biotinylated CAP and the first test on a modified enzyme–linked base, were elaborated,. The antibodies were stabilized and all reagents safely transferred to the project partners, who were additionally provided with assay protocols for the first tests to ensure establishment of the assay. (author)