Study On Anenzyme-Linked Immunosorbent Assay For The Detection Of Tetracycline

Aindirect competive enzyme-linked immunosorbent assay(ic-ELISA) for the determination ofTC in animal foods was developed.Tetracycline connected with keyhole limpet hemocyanin using formaldehyde,which was used as immunogen. Aantiserum was obtained after immunization of New Zealand rabbits,thenit was purified using Protein A Sepharose-4B to obain a polyclonal antibody with high affinity and specificity.Tetracyclinewas connected with ovalbuminusing formaldehyde, which was used as enzyme coating antigen. We established indirect competition ELISA detection method, the influence of experimental factors (including antigen incubation concentration,dilution ratio of antibody, different buffer,etc.) were studied, the standard curve obtained the highest sensitivity and stability when antigen incubation concentration was 0.05 μg/well, dilution ratio of antibodywas 1:10000, the buffer was 0.1%BSA/PBS,the blocking buffer was 0.5% skim milk powder/PBS. The performance of the developed method had also been evaluated systemically.The concentration of gentamycin causing 15% inhibition is 0.01±0.004μg/L,50% inhibition is 0.33±0.09μg/L. The antibody prepared in this experiment had high sensitivity,the cross-reactivities against oxytetracycline and chlorotetracycline were 0.39%, 4.42%respectively, no cross reaction was observed with penicillin, streptomycin, chloramphenicol.Milk, honey, pork, beef, chicken, squid, bigeye,flatfish and shrimpwere chosen to spike and recovery.The recovery rate were between85.88%-109.17%,94.52%-107.59%, 84.19%-98.96%,75.91%-105.58%,84.40%-109.64%,87.40%-120.45%,64.45%-104.76%, 74.55%-112.46%,82.84%-111.85%, respectively.Limit of detection in milk and squid was 5μg/kg, in honey, pork, beef, chicken, bigeye, flatfishand shrimpwas 10μg/kg.Inter- and Intra-variation were less than 20%.The correlation of the ic-ELISA with commercial kit was investigated.The consistency of the two detection methods was very high, the linear regression equation was:y=0.8542x+2.2848, R2=0.9954, and the results showed that their relativity was very good.It showed that the method had high accuracy and precision.In this experiment we established an enzyme-linked immunosorbent assay to detect tetracyclinein food,which has high sensitivity, high specificity,highaccuracy and a simple sample preparation procedure.It meet the requirements of rapid detection.