犬蠕形螨病诊断抗原制备研究

PREPARATION OF DIAGNOSTIC ANTIGEN OF CANINE DEMODICOSIS

通过刮取重症病犬蠕形螨发病部位的皮屑 ,用 5 %NaOH消化 2h后进行虫体浓集 ,将浓集的虫体冻融、研磨、超声破碎 ,经 80 0 0r min离心 2 0min ,取上清为蠕形螨盐溶性粗抗原 ;将沉渣用尿素混匀 ,再次超声 ,经 80 0 0r min离心 2 0min ,取上清为蠕形螨尿素溶性粗抗原。所得抗原用Bradford法进行蛋白浓度检测 ,浓度分别为 1 4mg mL和 1 1mg mL。经过葡聚糖凝胶层析 (G 10 0 ) ,分别得到两个层析峰。经SDS 聚丙烯酰胺凝胶电泳 ,盐溶性粗抗原显示 2条主蛋白带 ,分子量约为 4 5 7kDa和 2 7 5kDa ;尿素溶性粗抗原显示2条主蛋白带 ,分子量约为 4 6 8kDa和 2 7 5kDa ,用正常犬皮处理的对照抗原却出现至少 9条蛋白带 ,分子量范围为 :2 7kDa～ 110kDa。将抗原稀释至 1∶30 0 ,血清按 1∶30 0稀释 ,酶标SPA (葡萄球菌A蛋白 )作为二抗 ,通过ELISA检测 ,尿素溶性粗抗原层析峰第一峰具有较好抗原性 ,实际检测 12例临床检查发现蠕形螨的临床病犬和 19例未发现蠕形螨的临床健康犬 ,阳性符合率和阴性符合率分别为 91 6 7%和 78 95 %。经与其他皮肤病犬进行检测发现该抗原能较好地与其他皮肤病进行鉴别 ,与猪蠕形螨阳性血清有一定的交叉反应。

The skin scrapings from serious skin lesion of dogs infecting from demodictic unites were collected and macerated in 5% sodium hydroxide for 2h, then the parasites were concentrated by glycerol gradient centrifuge.By freezing and thawing the concentrated unites and subsequently grinding, ultrasonication, and centrifugation at 8 000r/min for 20min, the supernatant was collected as crude sodium-soluble antigen, and the pellet were mixed with urea solution and then ultrasonicated again, and with subsequently centrifugation at 8 000r/min for 20 min, the supernatant was harvested as crude urea-soluble antigen. By Bradford assay determination, the harvested protein concentration were 1.4mg/mL for sodium-soluble antigen and 1.1mg/mL for urea-soluble antigen. By separating the two kinds of crude antigens with Sephadex G-100, 2 main proteins with molecular weight of 45.7kDa and 27.5kDa from sodium-soluble antigen, and 2 main proteins with molecular weights of 46.8kDa and 27.5kDa from urea-soluble antigen were obtained. The 46.8kDa protein has strong reactivity with positive canine serum, and has some reactivity with pig positive serum. By testing 12 clinical cases of canine demodicosis and 19 clinical healthy cases, the obtained antigen was found had potential application in diagnosis of demodicosis.