对串联风疹病毒(Rubella virus,RV) 3个结构蛋白6个免疫显性区域进行制备(命名为B103),并将其应用于血清学诊断中。选取RV C aa 1–30 & aa 96–123、E2 aa 31–105和E1 aa 11–39 & aa 154–277 & aa 389–412等6个免疫显...对串联风疹病毒(Rubella virus,RV) 3个结构蛋白6个免疫显性区域进行制备(命名为B103),并将其应用于血清学诊断中。选取RV C aa 1–30 & aa 96–123、E2 aa 31–105和E1 aa 11–39 & aa 154–277 & aa 389–412等6个免疫显性区域,将其串联并基因合成;将此基因片段插入TRX和His标签之间构建表达质粒;蛋白B103在大肠杆菌BL21(DE3)中诱导表达,并利用Streamline Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白,Sephadex G-25分子筛分析其折叠情况及均一性;利用免疫印迹技术对蛋白B103的抗原性进行鉴定,并建立RV-IgM抗体捕获法ELISA检测技术,初步评价此方法对阴阳血清样本的鉴别能力。蛋白B103以可溶性形式表达,其表达量约占菌体总蛋白的18.57%,经纯化后蛋白B103浓度为3.026 mg/mL,纯度为95.35%;免疫印迹实验表明蛋白B103能与RV急性期血清发生反应;对40份RV急性期血清及40份RV阴性血清进行检测发现可以很好地鉴别阴阳性血清标本;其灵敏度为92.50%,特异性为95.00%,阳性预测值为94.87%,阴性预测值为92.68%,符合率为93.75%,McNemer检验的结果提示与“金标准”诊断结果无差异,kappa=0.900,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的RV血清学诊断抗原,可以应用于RV早期诊断中。展开更多
OBJECTIVES: To investigate the sensitivity of immunoblotting and enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis and to corr...OBJECTIVES: To investigate the sensitivity of immunoblotting and enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis and to correlate autoantibody serum levels with disease activity. METHODS: In serum samples obtained from 44 pregnant patients before initiation of therapy and from the same number of healthy blood donors, the autoantibody reactivity was assayed by immunofluorescence microscopy on human skin sections as well as Western blot analysis and 2 different ELISAs by using recombinant forms of the immunodominant domain of BP180. In addition, ELISA reactivity with this autoantigen was assayed in 6 patients during the course of the disease, and its correlation with the clinical disease activity was estimated by applying the Spearman rank correlation test. RESULTS: By indirect immunofluorescence microscopy, complement-fixing autoantibodies to the dermal-epidermal junction were found in 93%of patientssera. By immunoblotting and ELISA, autoantibodies to bullions pemphigoid antigen 180 were detected in 93%and 86.3%of pemphigoid gestationis patients, respectively, but in none of the healthy controls. Serum levels of autoantibodies as detected by ELISA paralleled the patients’disease activity. CONCLUSIONS: Our study shows that immunoblotting and ELISA are sensitive tools for the detection of autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis. In addition, the ELISA is useful to monitor autoantibody serum levels.展开更多
文摘对串联风疹病毒(Rubella virus,RV) 3个结构蛋白6个免疫显性区域进行制备(命名为B103),并将其应用于血清学诊断中。选取RV C aa 1–30 & aa 96–123、E2 aa 31–105和E1 aa 11–39 & aa 154–277 & aa 389–412等6个免疫显性区域,将其串联并基因合成;将此基因片段插入TRX和His标签之间构建表达质粒;蛋白B103在大肠杆菌BL21(DE3)中诱导表达,并利用Streamline Chelating亲和层析和DEAE阴离子交换层析纯化目的蛋白,Sephadex G-25分子筛分析其折叠情况及均一性;利用免疫印迹技术对蛋白B103的抗原性进行鉴定,并建立RV-IgM抗体捕获法ELISA检测技术,初步评价此方法对阴阳血清样本的鉴别能力。蛋白B103以可溶性形式表达,其表达量约占菌体总蛋白的18.57%,经纯化后蛋白B103浓度为3.026 mg/mL,纯度为95.35%;免疫印迹实验表明蛋白B103能与RV急性期血清发生反应;对40份RV急性期血清及40份RV阴性血清进行检测发现可以很好地鉴别阴阳性血清标本;其灵敏度为92.50%,特异性为95.00%,阳性预测值为94.87%,阴性预测值为92.68%,符合率为93.75%,McNemer检验的结果提示与“金标准”诊断结果无差异,kappa=0.900,提示两种方法诊断结果一致性优异。原核表达与层析纯化可以获取抗原性优异的RV血清学诊断抗原,可以应用于RV早期诊断中。
文摘OBJECTIVES: To investigate the sensitivity of immunoblotting and enzyme-linked immunosorbent assay (ELISA) to detect autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis and to correlate autoantibody serum levels with disease activity. METHODS: In serum samples obtained from 44 pregnant patients before initiation of therapy and from the same number of healthy blood donors, the autoantibody reactivity was assayed by immunofluorescence microscopy on human skin sections as well as Western blot analysis and 2 different ELISAs by using recombinant forms of the immunodominant domain of BP180. In addition, ELISA reactivity with this autoantigen was assayed in 6 patients during the course of the disease, and its correlation with the clinical disease activity was estimated by applying the Spearman rank correlation test. RESULTS: By indirect immunofluorescence microscopy, complement-fixing autoantibodies to the dermal-epidermal junction were found in 93%of patientssera. By immunoblotting and ELISA, autoantibodies to bullions pemphigoid antigen 180 were detected in 93%and 86.3%of pemphigoid gestationis patients, respectively, but in none of the healthy controls. Serum levels of autoantibodies as detected by ELISA paralleled the patients’disease activity. CONCLUSIONS: Our study shows that immunoblotting and ELISA are sensitive tools for the detection of autoantibodies to bullous pemphigoid antigen 180 in patients with pemphigoid gestationis. In addition, the ELISA is useful to monitor autoantibody serum levels.
