| Diarrhea of piglets is frequently caused by porcine enterotoxigenic Escherichia coli (ETEC) including two types of virulence factors:fimbrial adhesins and enterotoxins. The adhesins of ETEC discovered from different animals maybe have F4 (K88), F5 (K99), F6 (987P), F41 and so on.Therefore, detection of fimbriae on animal yields can not only to diagnose the diarrhea, but also to provide the scientific experimental basis on treatment methods.F5+ETEC is the main pathogen which caused piglet yellow dysentery and white dysentery disease.It is important to establish a sensitive, convenient assay to detect F5 fimbriae. With the development and application of the immune colloidal gold chromatography, this assay provides new ideas and technology base for the establishment of F5 fimbriae detection.Polyclonal antibodies of F5 fimbriae and monoclonal antibody were purified by caprylic acid ammonium sulphate method and affinity chromatography. The concentration of purified monoclonal antibody and polyclonal antibody are 1.745mg/ml and 2.056mg/ml respectively. Through the polyacrylamide gel electrophoresis (SDS-PAGE) analyzing, they all have H line and L line obviously. And the potency of monoclonal antibody increase from 1.6×105 to 6.4×105,while the blood serum is from 3.2×104 to 6.4×104. Purified polyclonal antibody and monoclonal antibody can be used for detecting and tagging respectively.Colloidal gold which was prepared through deoxidization of trisodium citrate was uniformity under the electron microscope scanning so that they are sutiable to be used for tagging.The best combination of antibodies with colloidal gole is 54μg/ml, the optimum pH value is 7.2.Anti-F5 fimbriae monoclonal antibody labeled with colloidal gold was used as a detector.Anti-F5 fimbriae polyclonal antibody and sheep anti-mouse IgG were blotted on a nitrocellulose membrane for test line and control line, respectively.The various factors and conditions of the immunochromatographic lateral-flow assay were explored, and the optimal reaction conditions of assay were ascertained. Dilution of antibody tagged by gold particle(0.01M pH 7.2 PB,0.5% BSA,1% Trehalose),Optimal investing solution of polyclonal antibody(0.01M, pH 7.2 Tris-cl),confining liquid(0.01M pH 7.2 Tris-cl,2% BSA, 0.2%tween-20),nitrocellulose filter(Sartorius CN140), Combine pad (SB 06),samplepad(SB 06),and absorption pad(S X 27)were chosen. And to determine the NC membrane test line and control line sprayed speed 1.2μg/ml.It was confirmed by experiment that the colloidal gold immunochromatographic strip of F5 fimbriae had good sensitivity (no response to F4,F6,F41,F18ab) and stability. The strip conserved at 4℃for 120 days could still detect the sample. The strip could be handled easily by untrained person and special instrument, and test result could be obtained in 5-10min, ideally suited for primary level and clinical diagnosis.
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