基于重组外膜蛋白研制检测副鸡禽杆菌抗体金标试纸

Construction of a Gold Colloidal Strip for the Antibody Detection of Avibacterium Paragallinarum Based on Recombinant Outer Membrane Protein

为研制一种采用胶体金间接法检测副鸡禽杆菌抗体的试纸条,本试验设计试纸条胶体金标记的蛋白为兔抗鸡IgG蛋白,检测线(T)为副鸡禽杆菌外膜蛋白P9,质控线(C)为羊抗兔IgG蛋白。检测试纸条的特异性、灵敏度及稳定性,并与间接ELISA法检测结果比较2种方法符合率。结果显示,当副鸡禽杆菌标准阳性血清稀释1000倍时,用该试纸条仍可检测出阳性结果,表明该检测试纸条敏感性良好;试纸条检测鸡新城疫病毒阳性血清、禽流感病毒阳性血清、鸡支原体阳性血清、鸡大肠埃希菌阳性血清等均为阴性,无交叉反应,说明试纸条具有很好的特异性;试纸条在4℃冰箱存放28 d,仍可成功检测出副鸡禽杆菌阳性血清,灵敏度下降不明显,说明该试纸条具有很好的稳定性。用此试纸条检测200份鸡传染性鼻炎疫苗免疫后的血清样品,阳性符合率为98%。此外,针对已知阴性、阳性血清样品,采用该试纸条与采用ELISA法检测,2种试验方法的检测总符合率高达99.5%。综上所述,此试纸条特异性、灵敏性及稳定性均很好,是一种理想的检测副鸡禽杆菌抗体的方法。

This study aimed to construct a gold colloidal immunochromatographic strip based on Avian paragallinarum recombinant outer membrane protein for specific,sensitive,and stable antibody detection.Nitrocellulose strip was first coated with A.paragallinarum recombinant outer membrane protein P9(detection line T)and goat anti-rabbit IgG(quality control line C),followed by rabbit anti-chicken IgG labeled with colloidal gold.The specificity,sensitivity and stability of the strip were assessed by using sera from chicken infected with A.paragallinarum or other common chicken pathogens.The coincidence rate of the test strip was compared with indirect ELISA for A.paragallinarum antibody.The results showed the detection line in the strip was still visible when A.paragallinarum standard positive serum was diluted at 1∶1000,indicating its excellent sensitivity;sera from chicken infccted with Newcastle disease virus(NDV),avian influenza virus(AI),Mycoplasma gallisepticum(MG)and Escherichia coli(E.coli)exhibited no cross reaction,indicating its high specificity;and the strip gave consistent result even after storage at 4℃for 28 days,indicating its good stability.The strip detected 98%of 200 sera from chicken with A.paragallinarum infection and had a total coincidence rate of 99.5%in comparison to indirect ELISA.Thus,the colloidal gold strip provides an ideal diagnostic reagent for specific,sensitive,and stable detection of A.paragallinarum antibodies in infected chicken.