Development Of Double-Antibodies Sandwich ELISA And Colloidal Gold Immunochromatographic Strips For PPRV Detection

Peste des petits ruminants(PPR) is an acute, spirited, contagious disease mainly in goats, sheep and wild small ruminants animals,caused by Peste des petits ruminants virus. The OIE has regulated it as a disease must be reported, China has classified it as the type I disease. At present, PPR was widespreaded in most countries of Africa,the Middle East,the Near East,the Central Asian and the South Asian subcontinent except the south Africa. It has also spreaded throughout many countries and regions surrounded China. In July,2007, PPR has the first broken-out in Tibet province of China.Later,there has broken-out PPR several times again.Meanwhile,PPR as a major animal epidemics has caused great losses on the animal husbandry economy to the local coutries and regions where it outbroken. PPR has seriously affected on production and trade of the small ruminants in our country and the world.PPR has become a key monitoring objects in animal disease prevention and control, as well as in the international trade of animal products.In this study,we has prepared and purified the ascites based on single hybridoma against the N protein of PPRV, which has been established by our lab din the early. The titer of the purified ascites is1:106. Then, Prepare and purify the polyclonal antibody serum against Peste des petits ruminants virus through a goat. Establish and optimize a double-antibodies sandwich ELISA based on the well-disposed mAb, pAb, and the cultures being confirmed PPRV positive or negative. The results of the optimization were:coating the pAb in a concentration of0.64ng/well, at4℃overnight, or at37℃1h; Wash the plates3times with the0.05%PBST; Add50uL the mAb0.385ug/well after50uL sample was added; Dilute the HRP-goat anti-mice IgG in1:5000,react in dark,at37℃,1h; Then,react with the substrate TMB15min, at37℃. Read the ODs on the ELISA microplate reader at450nm.In this study,colloidal gold of30nm has also prepared by trisodium citrate reduction method. Detect the quality of colloidal gold by Uv spectrophotometry, it has an single,narrow,maximum absorption peak at524nm.That has proved that the colloidal gold has a good quality.Establish and optimize a colloidal gold immuno-chromatography test strip method for PPRV detection,with the well-disposed mAb,pAb,the colloidal gold and the cultures proved to be PPRV positive or negative. The results of the optimization were:ajust the pH of the colloidal gold to8.2;Label the colloidal gold with the mAb at12μg/ml; purify the gold-labelled antibody by12000r/min centrifugal at4℃,45min;Dispose the sample pad,the gold-protein complex pad in the0.1M pH8.2Tris-HCl which contains1%NaC1,0.5%Tween-20,1%BSA,2%sucrose.Resuspend the precipitation in the resuspension buffer:0.1M pH8.2Tris-HCl which contains1%BSA,10%sucrose,0.5%Tween-20by1/10(v/v),then spray it on the glass fiber membrane; Dilute the pAb to3mg/ml, spray it on the T line of the NCM; Dilute the SPA with double distiled water to lmg/ml, spray it on the C line of the NCM. Then,assembly the strips in order.This study successfully established two kinds of detection methods for Peste des petits ruminants virus:a double-antibodies sandwich ELISA and a immuno-colloidal gold chromatography test strip. This has very important practical significance for the early diagnosis,the prevention and control of PPR in our country.