Study On Enzyme-Linked Immunosorbent Assay For The Detection Of Streptomycin

In this paper, a reliable indirect competition enzyme-linked immunosorbent assay method was developed for detecting of streptomycin in animal-derived foods and feeds.Streptomycin connected with bovine serum albumin using 1,4-butanediol diglycidyl ether, which was used as immunogen. A polyclonal antibody was obtained after immunization of rabbits. STR-CMO (streptomycin conjugated with carboxymethoxylamine hemihydrochloride) connected with ovalbumin using carbodiimide, which was used as coating antigen.We established an indirect competition ELISA detection method, and the influence of experimental factors (including coating antigen incubation concentration, dilution of antibody, ionic strength, pH, etc.) were studied. The standard curve obtained the highest sensitivity and stability when dilution ratio of antibody was 1:1000, coating antigen incubation concentration was 0.5 μg/well, the blocking buffer was 0.5% skim milk powder/PBS, ionic strength was 0.01 mol/L PBS, pH 7.4, the buffer of antibody was 0.1%BSA/PBS. The concentration of streptomycin causing 50%inhibition was 2.00±0.24 ng/mL,15% inhibition was 0.24±0.08 ng/mL. The cross-reactivity against other amino-glycoside antibiotics was all low except for dihydrostreptomycin, which had 95.50% corss-reactivity.Chicken, pork, chicken liver, pig liver, pig kidney, fish, shrimp, milk, chicken feed and pig feed were chosen for the detection of streptomycin. All samples were spiked with streptomycin at five levels (15 ng/g,30 ng/g,120 ng/g,300 ng/g,600 ng/g). The recoveries were in ranges of 71.32% to 106.94%, and the coefficients of variation of intra- and inter-were less than 18%. In order to appraise the stability of the assay, analysis of variance of the recovery was done. The experimental results had good stability with little variation within the different samples and spiked levels except for livers samples, which showed this ELISA was significantly accurate and had extensive utility value. Compared with commercial ELISA kit, the quantitative results were in good agreement (R2=0.9854), and the sensitivity of this ELISA in tissue was superior to commercial ELISA kit.