口蹄疫病毒感染与免疫鉴别诊断及免疫评价二联试纸的优化

Optimization of Dual Strip for FMDV Immunized Antibody Evaluating and Discriminating Vaccinated Animals from Infected Animals

【目的】建立口蹄疫病毒(FMDV)感染与免疫鉴别诊断及免疫评价二联试纸稳定的生产工艺,促进其产业化生产及临床应用。【方法】本研究以胶体金免疫探针结合猪IgG,FMDV结构蛋白(SP)及非结构蛋白(NSP)的表位多肽偶联载体蛋白制备人工抗原,设置两条检测线精准拦截抗体的检测模式,以试纸条特异性及敏感性为评价指标,对胶体金免疫探针用蛋白、试纸拦截用多肽抗原、检测线位置及喷膜缓冲液、金标蛋白保存液、样品垫缓冲液以及样品稀释液等参数进行优化。采用优化后的试纸检测266份田间猪血清样品,并与口蹄疫O型抗体液相阻断ELISA检测试剂盒(LPB ELISA)和3ABC阻断ELISA抗体检测试剂盒(3ABC ELISA)检测结果进行对比,计算该试纸与商品ELISA试剂盒的符合率。【结果】经优化后,口蹄疫感染与免疫鉴别诊断及免疫评价二联试纸生产工艺参数如下:以胶体金标记金黄色葡萄球菌A蛋白(SPA)为免疫探针;选择混合多肽的形式设置拦截线,以非结构蛋白多肽(BSA-NSPs)喷涂T1线,结构蛋白多肽(BSA-SPs)喷涂T2线,以0.1 mol/L Tris-HCl为喷膜缓冲液;以ddHO含15 mg/mL BSA、13.25 mg/mL NaHPO·12HO、15.9 mg/mL NaHPO·2HO、10%Trition X-100和0.3 mg/mL NaN为金标蛋白保存液;以0.02 mol/L NaBO·10HO含10 mg/mL酪蛋白、5%Trition X-100、0.3 mg/mL NaN为样品垫缓冲液;以生理盐水含1.0%Tween-20为样品稀释溶液。制备的FMDV感染与免疫鉴别诊断及免疫评价二联试纸与3ABC阻断ELISA抗体检测试剂盒的符合率为96.20%,与口蹄疫O型抗体液相阻断ELISA检测试剂盒的符合率为94.36%。【结论】通过对试纸生产工艺的优化,建立了稳定的生产工艺,制备的二联试纸检测线显色清晰可见,肉眼识别度高,试纸的检测特异性和敏感性良好。本研究为该试纸的批量生产和产业化应用奠定了基础,为基层口蹄疫的检测提供了稳定、特异、敏感、准确的检测方法。

【Objective】 The purpose of this study was to establish a stable production process of Foot-and-mouth disease virus(FMDV) infection and immune differential diagnosis and immune evaluation test paper, and promote its industrial production and clinical application.【Method】 In this study, the colloidal gold immunoprobe was combined with the epitope polypeptide coupling carrier protein of porcine IgG,FMDV structural protein(SP) and non structural protein(NSP) to prepare artificial antigen, and two detection lines were set to accurately intercept the detection mode of antibody. Taking the specificity and sensitivity of the test strip as the evaluation index, the parameters such as protein for colloidal gold immunoprobe, polypeptide antigen for test strip interception, detection line position, spray film buffer, gold standard protein preservation solution, sample pad buffer and sample diluent were optimized.The optimized test paper was used to detect 266 field pig serum samples, and the results were compared with the results of foot-and-mouth disease O antibody liquid phase blocking ELISA(LPB ELISA) and 3 ABC blocking ELISA antibody detection kit(3 ABC ELISA).The coincidence rate between the test paper and commercial ELISA kit was calculated.【Result】 After optimization, the parameters of the dual test strip for differential diagnosis of FMDV infection and immunity and immune evaluation were as follows: Staphylococcus aureus A protein(SPA) labeled with colloidal gold was used as immunoprobe, the mixed BSA-peptides were used as test lines.BSA-NSPs and BSA-SPs were dispensed on NC membrane as the test line 1 and test line 2,respectively.0.1 mol/L Tris-HCl was used as membrane spraying buffer.ddHO containing 15 mg/mL BSA,13.25 mg/mL NaHPO·12 HO,15.9 mg/mL NaHPO·2 HO,10% Trition X-100 and 0.3 mg/mL NaNwere used as gold standard protein preservation solution.0.02 mol/L NaBO·10 HO containing 10 mg/mL casein, 5% TritionX-100,0.3 mg/mL NaNwere used as sample pad buffer.Normal saline containing 1.0% Tween-20 were used as sample dilution.After optimization, the coincidence rate of the optimized strip with FMDV 3 ABC ELISA and FMDV LPB ELISA was 96.20% and 94.36%,respectively.【Conclusion】 By optimizing the production process, a stable production technology was developed.The color of the test lines of the optimized strip were clearly visible with high recognition by the naked eyes.The specificity and sensitivity of the strip were markedly improved.This study laid the basis for the mass production and industrial application of the strip and provided a stable, specific, sensitive and accurate method for FMDV detection.