Preparation Of Gold Immunochromatographic Kit For Group A Porcine Rotavirus

Infection of porcine rotavirus spread widely in China and all the other countries where pigs are raised. Quick and exact surveillance is the premise for prevention and cure of the disease.Though there are many methods to detect porcine rotavirus, the methods that are fit for spot fast diagnosis of PRV are few .On account of this, gold-immunochromotography assay are used to establish a quick,convenient and sensitive method to detect the porcine rotavirus of A group.Anti-VP6 protein polyclonal antisera were obtained by using purified recombinant PRV VP6 protein immune rabbit,and ascites monoclonal antibody against PRV VP6 protein was received by injecting hybridoma cell line C5D10 to BALB / C mice. Antiserum and ascites was purified by by caprylic acid ammonium sulphate method and protein A (and G) affinity chromatography.The purified immunoglobulinG(IgG) was analyzed by the SDS-PAGE and indirect ELISA. The SDS-PAGE showed that the purified antibodies consist ofγglobulin and a little other globulin. The ELISA titers of purified polyclonal antibodies and monoclonal antibody were 10-5and 1:1200, respectively. 20 nm colloidal gold particles which were coupled with the purified monoclonal antibody were prepared by reduction of sodium citrate.The optimized amount and pH value of labelling were 60μg/ml and 8.5, respectively.The labelled monoclonal antibody was purified on low temperature ultra centrifuging.The purified labelled antibody was diluted at ratio of 1:2.5 and adsorption on fiberglass. The test lines was purified polyclonal antibodies on the nitrocellulose filter, concentration of purified polyclonal antibodies was 0.8mg/ml;The control line was goat anti-rabbit IgG which concentration was 0.5mg/ml striked on the nitrocellulose filter. Virus culture as already identified positive and negative control tissue culture was detected by the self-produced diagnostic kits , which the result was demonstrated at least 10 minutes, in addition, there were two sharply red strips at the testline and control line,so was it recognized positive. By contrast, cell cultures reflected obviously red only at the control line, so did it mean negative. Furtherly, the experiments were performed about sensitivity,specificity and repeatabilty of kit. The results showed that the test papers detected virus culture (TCID50:0.1ml 10-6.25)had a cut-off value of 1:32,and did not react with correlated diarrhea virus such as TGEV and PEDV,and the results of different batches were the same ,which concluded the kits are sensitive,repetitive and specific.The kit prepared in the study by gold-immunochromotography assay establishes the base for quickly detecting PRV.