| To develop a rapid,simple gold immunochromatographic assay(GIA)for the detection of TGEV.Polyclonal anti-Nucleocapsid protein of TGEV antibodies and monoclonal antibody were purified by caprylic acid ammonium sulphate method and affinity chromatography.The concentration of purified polyclonal antibody is 1.692mg/ml.Through the polyacrylamide gel electrophoresis (SDS-PAGE) analyzing, there were H line and L line obviously.The purity of antibody was 100%. Potency of serum fell from 2.56×105 to 6.4×104. The concentration of purified monoclonal antibody is 1.933mg/ml. Through the polyacrylamide gel electrophoresis (SDS-PAGE) analyzing, there were H line and L line obviously.The purity of antibody was 100%. Potency of ascites fell from 6.4×104to 1.6×104。Purified polyclonal antibody and monoclonal antibody were used for detecting and tagging respectively.Tri-sodium citrate with aqueous gold chloride were mixed to make 20nm gold particle, Through macroscopic observation,the gold solution was wine red, transparence and uniformity. Through electron microscope scanning ,we observed that the disposition of gold particle was uniformity and the size was uniform. Through ultraviolet scanning, we observed that the wavelength of maximum absorption peak was 520nm, the width of peak was narrow.That meant the disposition of gold particle was uniformity. The result indicated that the gold particle could be used for tagging monoclonal antibody.The gold particle was used for tagging monoclonal antibody,we determined the minimum amount of protein which could stabilize 1ml gold colloid was 0.2mg. Optimal pH was 7.6. The conjugation solidified to glass fibers, connecting to the sample pad is a nitrocellulose membrane,which contains one line of immobilized polyclonal anti-Nucleocapsid protein of TGEV antibodies,serving as the test line,and one line of immobilized goat anti-mouse IgG antibody,serving as the control line. Attaching to the other end of the membrane is an absorption paper pad,serving as the sample liquid reservoir. After the addition of a sample to the sample pad,the sample liquid will flow from the sample pad to the conjugate pad,then to the membrane,and finally reach the absorption pad.Initially,a reddish test line on the membrane will become visible if the sample contains TGEV. If the sample contains no TGEV,no conjugate can be immobilized on the membrane and no visible test line will show.The various factors and conditions of the immunochromatographic lateral-flow assay were explored,and the optimal reaction conditions of assay were ascertained. Optimal investing solution of polyclonal antibody(0.01M pH 7.4 PBS), confining liquid (0.01M pH 7.4 PBS contained 2% BSA), dilution of antibody tagged by gold particle (0.01M pH 7.4 PBS contained 0.1 % BSA and 0.5% saccharobiose), nitrocellulose filter(HF135), fiberglass membrane(PR-NJ25) , sample pad(PR-VL78),and absorption pad (CH-270K) were chosen.. Optimal condition of block was 10min in ordinary temperature. Investting concentration of polyclonal antibody on the nitrocellulose filter was0.0169mg/ml ,and the painting amount of antibody tagged by gold particle on the fiberglass membrane was0.037mg/ml.A GICA for rapid detection of TGEV was explored.The GICA device had a cut-off value 0f 100TCID50 .There was no cross-reaction.The device had a high quality in repetition. At the temperature of 4 degree,the strip test packaged for 90 days also could detect sample.The major advantages of GICA were that results could be obtained within 5min without any detecting instrument and that all needed reagents were included in the device. The method is fit for spot fast diagnosis of TGEV ,and could be used for refernce in the development of the detection of other disease.
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