Colloidal Gold Immunochromatography Assay And Pilot Studies On Rapid Detection Of CAP

Based on the specific binding of antigen and antibody, immunochromatography colloidal gold strip is a sensitive method for microanalysis of antigen or antibody. In this paper, Chloramphenicol (CAP), one kind of antibiotic which is addition forbidden in the area of foodstuff, as detection object, the following work were done.Colloidal gold was prepared by reducing trisodim cicitrate. According to the different addition amount of trisodim cicitrate, the effect of different heating time to the preparation of colloid gold was studied. The quality of the product was identified by Spectrophotometry, scanning electron microscope and laser particle size method. The above three methods were compared by the diameter of colloidal gold.Monoclonal antibody was produced by immuning Asctic Fluid against CAP by SP2/0 Cells in Balb/C Mice, and purified with protein G Affinity Chromatography Column.Computed by empirical formula of protein, OD₂₈₀/1.35, the concentration of McAb was initial measured.The activity and titer of McAb was determined by indirect-competitive ELISA, and then the competition inhibition curve of CAP was established.By Spectrophotometry the optimum pH of colloidal gold conjuncted with CAP McAb and minimum dose of McAb were determined. The compound was purified by high speed centrifugation, the diameter and distribution of the compound was studied by Ultraviolet Spectrophotometry, Laser particle size analyzer and electron microscope. At last the activity of compound was determined by indirect-competitive ELISA and test paper measuring.Chloramphenicol succcinate was coupled with BSA by EDC method and the conjugation rate of the reaction was measured by Ultraviolet Spectrophotometry, the activity of product was tested by indirect-competitive ELISA.The solution and material of Test strip were carefuuly selected and then all the parts were assembled into immunochromatography colloidal gold strip for CAP detection, which could be used to test negative and positive samples (10ng/mL CAP).Based on the study above, we can draw the following conclusions: While the addition amount of trisodim cicitrate was 1.0mL, the optimum heating-time was 15min; while the addition amount of trisodim cicitrate was 1.5mL, the optimum heating-time was 12min; while the addition amount of trisodim cicitrate was 4.0mL, the optimum heating-time was 9min.The total amount of CAP-McAb was about 6.147mg, we divided the McAb into 2 tubes, the titer of the tubes with lower concentration was 1:3000, IC₅₀ was 0.368ppb and the detection limit was 0.047ppb, while those of the higher concentration were 1:5000, 0.426ppb and 0.055ppb, respectively.The optimum pH of colloid gold conjuncted with CAP McAb was 8.0, 200μL colloid gold corresponded to a minimum dose of protein of 1.92μg.The compound was active and its size was a little lager than colloid gold.CBSA/CCAP=34.4,that was to say there were 34.4 molecular of CAP conjunctied to each BSA,the product was proved to be active,and the best dilution of coating antigen was 1:13000.The result of test strips indicates that it's easy to differentiate positive samples and negative samples. The detection limit of test strips is 10ng/mL.