Development Of Colloidal Gold Immunochromatographic Strip For Detection Of E.coli O157:H7and Bovine Protein

Colloidal gold immunochromatographic strip for the detection ofE.coli O₁₅₇:H₇and milk derived protein has many advantages, like rapid,sensitive, can be used for field testing, and so on. It has greatimprotance to the milk adulteration and safety testing. In this study,the conditions for preparing colloidal gold of20nm by sodium citrateand ascorbic acid reduction were optimized, and the labelingefficiency between the two methods to obtain colloidal gold werecompared, then the best conditions and prepare method were achieved.Choosing the bovine β-CN colloidal gold immunochromatographic stripas the representative, the composition materials of the strip werescreening and determined through experiments. Finally, the E.coliO₁₅₇:H₇and bovine β-CN colloidal gold immunochromatographic stripwere prepared and measured, and the strip were proved to be usable forthe detection of E.coli O₁₅₇:H₇and milk-derived protein in skim milkanalysis.The preparation of specific polyclonal antibody: E.coli O₁₅₇:H₇,bovine β-CN and α-LA were used as antigen to immunize New Zealandwhite rabbits for antiserum respectively. The antiserum were dealedwith graded salting （50%、40%、33%） and protein A affinitychromatography, the purity of the antibody were more than90%,analysed by SDS-PAGE. The determination of the colloidal gold preparation method: thecolloidal gold of20nm were prepared by sodium citrate and ascorbicacid reduction methods, and the amount of reductant, the heating timewere optimized, then get the best conditions are as follows:100ml0.01%HAuCl4solution, adding sodium citrate （1%）1.5ml, with aheating time of10min; adding the ascorbic acid （1%）2.0ml, with aheating time of5min. The colloidal gold prepared by the two methodswere then used to study the antibody label condition: they have thesame best labeling pH condition,8.0, whereas for the optimal amount ofantibody, the former（26μl/ml） is lower than the latter（65μl/ml）, and thelabeled antibody activity, the latter is higher too, which is tested byELISA. However, the ascorbic acid reduction method are notreproduceable due to the ascorbic acid can be easily oxidized. Finally,the sodium citrate reduction method was choosed as the best preparationmethod in the experiment.Development of colloidal gold immunochromatographic strip fordetection of E.coli O₁₅₇:H₇and bovine protein:1)the determination of the strip constituent materials: choosing theβ-CN colloidal gold immunochromatographic strip as the representativeto perform tests, the strip solid phase materials are determined asfollows: the Millipore135s, glass fiber SB06, polyester cellulosemembrane VL98, absorbent paper S*27.2)The development of colloidal gold immunochromatographic stripfor detection of E.coli O₁₅₇:H₇: Conjugate solution was prepared bydiluting of anti O₁₅₇:H₇IgG-colloidal gold complexes with the samevolume of0.01M PBS（pH7.4）. Then the conjugate pad （glass fiberSB06）were immersed in the conjugate solution and dried at37℃; rabbitanti-O₁₅₇:H₇IgG（500μg/ml）and goat anti-rabbit IgG（100μg/ml）wereseparately packaged onto a NC membrane（the Millipore135s） to form atest region （T） and a control region （C）, dried and block any remainingactive sites on the membrane by incubation with0.01M PBS （pH7.4）containing1%BSA, later, washed twice with0.01M PBS（pH7.4）containing1%（w/v） BSA,0.1%（v/v） Tween20, dried. The NC membrane, conjugate pad, sample pad, absorbent pad were then adheredto an adhesive PVC plate orderly to form the strip. It has the samesensitivity,105cfu/ml, to detect pure bacteria and artificialcontamination samples, which can be used for the actual sample testing,with good specific and reproducibility, can be placed90days at4℃.3)The development of colloidal gold immunochromatographic stripfor detection of bovine protein: similarly, the conjugate pad wereimmersed in the conjugate solution, which is diluted by half volume of0.01M PBS（pH7.4）, rabbit anti-β-CN IgG （200μg/ml） and goatanti-rabbit IgG（200μg/ml）were separately fixed onto a NC membraneto form the test line and the control line. The strip has a sensitivity of31.25μg/ml to the β-CN standard solution, to the diluted skim milk is37.5μg/ml, can be used in the determination of β-CN in skim milk. Also,with a good repetitive, usable for90days if keep at4℃. The detectionof α-CN was weakly positive, which suggests a fit of defect.The strip for detection E.coli O₁₅₇:H₇developed in the subjectpave the way for the bacteria’s semi-quantitative and quantitativedetection. The strip for detection β-CN has opened up a new way for thedetermination of protein content in milk, and laid the foundation for thefurther dairy adulteration detection research based on the milk derivedprotein content.