基于量子点及免疫磁珠技术快速检测弓形虫抗体的实验研究

目的 应用量子点及免疫磁珠技术,建立一种简便、快速的弓形虫IgG抗体检测方法.方法 采用碳二亚胺交联法,将弓形虫抗原包被住磁性微球表面作为固相载体,量子点标记二抗作为检测抗体.检测待检血清中的弓形虫特异性抗体,并对检测条件进行优化.结果 量子点与二抗的最佳偶联条件:pH6.0、反应时间为2 h、二抗浓度为20 μg/mL.确立检测的最佳反应条件:抗原包被浓度50μg/mL,量子点标记二抗工作浓度为1:200.用本方法检测弓形虫抗体阳件血清的最大稀释度为1:1 000,而EHSA为1:500.结论 成功建立了弓形虫IgG抗体快速检测方法,该方法具有较好的特异性和灵敏度,且操作简便快速.     

快速胶体染料试纸条法检测弓形虫病IgG抗体的研究

目的研制一种检测弓形虫病IgG抗体的快速胶体染料试纸条法(DDIA),与国外进口试剂盒进行比较,以评价其临床应用价值。方法以本实验室筛选的胶体染料D1标记羊抗人IgG,以此标记物与弓形虫病人血清中的抗弓形虫IgG抗体结合,用点有弓形虫可溶性抗原的硝酸纤维膜通过层析捕获染料标记羊抗人IgG与弓形虫IgG抗体的复合物,采用正交试验确定DDIA的最佳检测条件;同时对弓形虫病流行病学筛查时的88份血清进行检测并与进口试剂盒的检测结果进行比较。结果DDIA的最佳检测条件为弓形虫可溶性抗原的点膜浓度为1mgml,人IgG点膜浓度为2mgml,血清用量为10μl和羊抗人IgG胶体染料检测液作1∶4稀释时的检测带清晰且背景较浅;DDIA与进口试剂盒的总符合率为97.7%,其中阳性和阴性符合率分别为100%(5252)和94.4%(3436)。结论DDIA与进口试剂盒有较高的符合率,表明该法具有较好的弓形虫病诊断价值,可进一步推广应用。     

重组主要表面抗原1用于弓形体病IgG免疫诊断的研究

目的　用纯化的重组刚地弓形虫RH株主要表面抗原 1(SAG1)作为检测抗原 ,建立间接法rSAG1 ELISA ,检测弓形虫IgG抗体并与国外进口试剂盒进行比较 ,以观察其阳性和阴性符合率 ;并对rSAG1 ELISA检测的精确度、灵敏度和特异性进行评价。方法　接种重组菌至LB肉汤中 ,IPTG诱导表达后 ,用Ni2 螯合柱进行亲和纯化 ;分别用不同浓度的重组抗原rSAG1包被聚苯乙烯酶标条 ,检测不同稀释度的阳性和阴性血清 ,以辣根过氧化物酶标记的羊抗人IgG为二抗 ,采用正交试验确定rSAG1 ELISA的最佳检测条件 ;按rSAG1 ELISA法的最佳检测条件对混合弓形虫IgG阳性和阴性血清重复测 2 0次进行精确度的检测 ;采用抗体抑制试验检测其特异性 ;同时对用进口试剂盒筛得的 5 2份弓形虫IgG阳性血清和 4 0份弓形虫IgG阴性血清进行检测。结果　制备的rSAG1重组蛋白纯度在90 %以上 ;以该重组抗原建立的rSAG1 ELISA的最佳检测条件为 :重组抗原rSAG1的包被浓度为 5 μg/ml,血清稀释度 1∶10 0 ,酶标记的羊抗人IgG 1∶2 0 0 0稀释 ;用rSAG1 ELISA对混合弓形虫IgG阳性和阴性血清的重复检测表明 :IgG阳性血清的检测值的变异系数 (CV值 )为 10 .9% ,IgG阴性血清的CV值为 10 .7% ;灵敏度检测表明血清稀释度在 1∶5 0～ 1∶2 0 0均可检出阳性 ;特异性     

点免疫金渗滤法筛选胶体金标记抗人IgG 的研究

目的:应用点免疫金渗滤法筛选适宜胶体金标记的抗人IgG(以下简称二抗),并运用多个评价指标进行比较和分析。方法:选择三个不同公司的二抗用胶体金标记,根据棋盘滴定法确定最佳标记条件;运用点免疫金渗滤法对三种二抗的胶体金标记后检测效能进行比较,采用包虫病人和健康对照血清,用包虫病特异性抗原检测包虫病患者血清中的特异性抗体,评价最优的二抗,并与酶联免疫吸附测定法进行比较。结果:A、B、C 三种二抗标记的最佳标记条件为:pH 均为8.5,加入量分别为38.4、24、19.2 g/ ml,综合评价二抗B 检测效能最优。将其与酶联免疫吸附测定法检测效能进行比较,两种方法检测结果的Kappa=0.895(P<0.05),吻合度较强。结论:应用点免疫金渗滤法,以及本文提出的评价指标,能够筛选出最佳二抗,对检测试剂盒中二抗的选择具有重要的参考价值。     

乙型肝炎患者抗微粒体抗体检测及临床意义

<正> 国外报道部分慢性肝炎患者存在一种自身抗体—抗微粒体抗体。本文报道乙型肝炎患者抗微粒体抗体检测结果。 对象和方法 一、病例:共检测86例住院肝炎患者血清,其中HBsAg阳性39例;HBsAg阴性/抗-HBc阳性30例;非乙肝感染11例。 二、方法:抗微粒体抗体检测:采用间接免疫荧光方法。抗微粒体抗体阳性对照血清系Dr.Rizze-tto实验室提供;阴性对照包括用正常人血清取代病人血清及用荧光标记羊抗兔IgG取代荧光标记羊抗入IgG。肝内HBsAg、HBcAg检查采用ABC     

化学发光法检测抗-HEV IgG方法的建立

目的 建立化学发光检测抗HEV IgG方法,用于HEV感染的实验室诊断及流行病学调查,为试剂盒研制打下基础.方法 以HEV重组抗原包被微孑孔板,辣根过氧化物酶标记的单克隆抗人IgG抗体为第二抗体,建立抗HEV IgG的化学发光检测方法,评价其灵敏度、特异性、精密性等指标.检测患者血清中抗HEV IgG抗体并与第三方试剂比较.结果 建立了检测抗HEV IgG的化学发光方法,检测500例临床患者标本并与对照试剂盒对比,阳性符合率为99.32％,阴性符合率为98.58％,总符合率98.80％,其灵敏度、特异性、重复性均达到设计要求.结论 建立了化学发光检测血清中抗-HEV IgG的方法,具有敏感性高、特异性强、操作简便等特点,适用于戊型肝炎的临床诊断和流行病学调查.     

量子点流式微球技术检测抗胰岛素抗体方法学建立

目的将量子点荧光探针应用于流式微球技术,创建出量子点流式微球技术检测人抗胰岛素抗体的方法并对其偶联率、精密度、线性范围、灵敏度进行初步评价。方法将胰岛素与羧基化微球偶联制备免疫诊断微球,与血清或标准品中人抗胰岛素抗体结合后,加入兔抗人IgG抗体,最后加入生物素化的羊抗兔IgG抗体和链霉亲和素化的量子点,使微球表面呈现荧光,并通过流式细胞仪检测平均荧光强度(mean fluorescent intensity,MFI)并记录。结果羧基化微球偶联胰岛素的性能显著强于空白微球,偶联0.5～3 h为最佳偶联反应时间,偶联成功的免疫诊断微球放置4℃冰箱至少可稳定50 d。微球偶联微球表面所携带荧光MFI与所测抗胰岛素抗体浓度成正比,量子点流式微球技术检测同一标本的灵敏度(3.74 pg/ml)、精密度(8.24%)、线性范围(3.74～5 000 pg/ml)都优于酶联免疫吸附试验所测得灵敏度(24.53 pg/ml)、精密度(12.31%)、线性范围(24.53～2 500 pg/ml)。结论本实验创建的量子点流式微球技术可用于人血清抗胰岛素抗体测定,其检测性能良好。     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

检测人博卡病毒抗体间接酶联免疫吸附法的建立及临床应用

目的 建立间接酶联免疫吸附法(ELISA)检测人博卡病毒(HBoV)抗体,探讨其临床应用的可行性.方法 以重组表达的HBoV融合蛋白VP2作为包被抗原,建立检测HBoV抗体的间接ELISA,优化该检测方法的最佳条件,观察其精密度、敏感度和特异性,并与荧光定量PCR方法对比检测40份临床样本,同时采用免疫印迹法(Western blot)确认其符合率.结果 所建立的间接ELISA最佳抗原包被浓度为2 mg/L,酶标二抗工作浓度为1∶5000,血清标本最佳稀释度为1∶200.该方法平均批内变异系数为6.87%,平均批间变异系数为4.67%.40份临床样本间接ELISA检测结果与荧光定量PCR及免疫印迹结果一致,符合率100%.结论 利用VP2融合蛋白作为抗原,建立的检测血清HBoV抗体间接ELISA方法特异性强、重复性好,可用于HBoV抗体的定量和定性检测.     

139名跨境工作人员淋巴脉络丛脑膜炎病毒和汉坦病毒感染调查分析

目的调查跨境务工人员经鼠传播淋巴脉络丛脑膜炎病毒(lymphocytic choriomeningitis virus, LCMV )和汉坦病毒(hantavirus, HV)的感染状况, 初步评估鼠传病毒感染风险。方法本研究于2019—2020年间, 针对常年在境外从事户外工程工作的务工人员开展调查, 采集血清样本, 通过基于LCMV重组核蛋白的间接ELISA方法、免疫印迹法和基于LCMV重组糖蛋白的间接免疫荧光法检测样品血清中LCMV特异性IgG抗体, 采用检测汉坦病毒IgG抗体的商业化ELISA试剂盒和汉坦病毒感染抗原片检测血清中HV IgG抗体情况。结果共调查了139名跨境工作者, 年龄在25～57岁之间, 64%(89/139)有多个国家工作经历, 涉及26个国家, 包括14个亚洲国家和12个非洲国家, LCMV IgG抗体检出率11.51%(16/139), 阳性样本经免疫印迹法和间接免疫荧光法核实, 抗体检出率略高于类似调查中发现的人群感染率;HV抗体检出率0.72%(1/139), 但尚不能确定获得感染的时间和地区。结论本研究揭示了境外务工人员中LCMV和HV病毒感染状...     

Enhanced antiglobulin-mediated neutralization of herpes simplex virus-IgG complexes by complement and heterologous anti-immunoglobulin

Summary The ability of IgG anti-Fc and anti-Fab to neutralize infectious herpes simplex virus-IgG (HSV-IgG) complexes was determined. When limiting amounts of antiglobulin were used, antibody directed against the Fab portion of human IgG was significantly more effective than anti-Fc antibodies in neutralizing the HSV-IgG complexes. The detection of viral bound antibody was enhanced by the incorporation of heterologous antiglobulin or complement in the antiglobulin neutralization test. Specifically, HSV-IgG which had been incubated with rabbit antihuman globulin was further neutralized by goat antirabbit IgG or guinea pig serum complement. This augmented neutralization test could prove useful in detecting small amounts of antibody bound to virus in infectious isolates from patients or experimental animals with viral diseases.With 4 Figures     

MPT64抗体的适体在结核病血清学检测中的应用

目的 利用SELEX技术筛选MPT64抗体的适体建立混合夹心ELISA检测体系,应用于临床血清标本的检测,探讨该方法 潜在的实验室诊断价值.方法 利用竞争ELISA检测方法 ,测定不同浓度的MPT64抗原对最后一轮ssDNA文库与靶物质亲和力的抑制率,选取优势序列且亲和性值较高的适体用于检测,优化混合夹心ELISA检测方法 ,确定MPT64抗体的检测下限和线性范围,同时检测230例临床血清标本.结果 在竞争ELISA结果 中,总反应体系为100μl,MPT64抗原浓度由2μg/ml增加到256 μg/ml时,对ssDNA文库的抑制率也由0.25%增加到80%.优化的混合夹心ELISA的检测体系:ssDNA包被浓度为0.1μg/孔、血清稀释度为1/200、辣根过氧化物酶标记的羊抗人IgG抗体浓度为1/40 000.MPT64抗体的检测下限为3 mg/L,线性范围为10～1000 mg/L.利用该体系检测100例结核病患者、100例健康体检者和30例非结核病患者血清,检测结果 用GraphpadPrism软件进行分析,结核病组与健康体检组、结核病组与非结核疾病对照组差异均具有统计学意义(P<0.001).统计学分析得到检测的特异性与敏感性分别为96.1%和31.0%.结论 利用适体建立的混合夹心ELISA用于结核病的血清学诊断具有一定的诊断价值.     

Identification of Human Sperm Antigens to Antisperm Antibodies*

ABSTRACT: We have successfully applied SDS (sodium dodecyl sulfate) gel/protein blot radioimmunobinding method to identify the molecular size of sperm antigens that elicit antisperm antibodies from patients with unexplained infertility. Following the transfer of renatured proteins from SDS gel of human sperm extract onto nitrocellulose strips, the radioimmunobinding was performed by incubating the strips with patients' sera at 1:100 dilution and then with relabeled goat antihuman immunoglobulin G (IgG) or protein A as detecting probes. Unique sperm antigens that reacted with some patients' sera were identified following the autoradiography of the incubated paper strips. Among the fifty-nine standard serum samples from the Reference Bank of the World Health Organization, about one-fourth of them were found to react predominantly with a sperm protein band having the reference value (Rf value) of 0.2 and the approximate molecular weight of 90,000 dalton. A similar analysis was also performed with serum samples from vasectomized patients. Some of them also revealed a specific binding with the sperm antigen(s) of similar molecular weight. The results of this analysis were also compared with those of conventional tests for sperm antibodies as well as those of microplate radioimmunoassays and enzyme-linked immunoassays. This study suggests that SDS gel/protein blot radioimmunobinding method can be a useful tool for the molecular identification of unique human sperm antigen(s) that elicit naturally occurring antisperm antibodies in patients with unexplained infertility.     

Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates

Summary A 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C 2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C 2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.     

Rapid immunoenzymatic technique for titration of rabies antibodies IgG and IgM results

Techniques usually employed for the detection of rabies' antibodies are costly, time consuming, and sometimes fail to detect early antibodies. The introduction of immunoenzymatic techniques in the serology of viral disease represents a new and important advance. We therefore adapted this technique to the detection of rabies antibodies.We have found that the sera from rabies patients who had not received antirabies treatment do not show seroneutralizing antibodies until several days after the onset of symptoms. However, antibodies can be detected some days earlier by the immunoenzymatic method in the same samples.Furthermore, the immunoenzymatic test was applied to the detection of both the IgM or the IgG class of antirabies antibodies using an antihuman Ig-or antihuman IgG-peroxydase conjugate.     

Specific serum IgA, IgG and IgM antibody determination by a modified indirect ELISA-technique in primary and recurrent herpes simplex virus infection

Twenty-three patients with a herpetic infection as diagnosed by a positive culture of herpes simplex virus (HSV) were studied with respect to serological responses of IgA, IgG and IgM antibodies in paired serum samples by an indirect (sandwich) enzyme linked immunosorbent assay (ELISA). Eight of the patients had a primary infection and 15 a recurrent one. In the ELISA test a detergent treated cell lysate of HSV type 1 was used as antigen. In the IgM assay all sera were pretreated with antihuman IgG with the purpose to precipitate IgG of the samples. The conjugate was a F(ab)2-fragment of antihuman-IgM. In primary infections all patients had significant titre rises of IgG and presence of high IgM titres in the convalescent serum. IgA antibodies were found in all of them, while titre rises were detected in 5/8. In recurrent infections titre rises of IgG and IgA antibodies were found in 4 and 5, respectively. Six had detectable IgM in one or both of the paired samples. The IgG titres were higher in recurrent infections than in primary, in contrast to IgM of which much higher titres were found in primary infections. It is concluded that in primary infections a conclusive serological diagnosis was established in all patients, whereas in recurrent infections this was achieved in two of three patients. The indirect ELISA method used for IgM detection was sensitive, reliable and convenient. Interfering rheumatoid factor was effectively eliminated by treatment with antihuman IgG.     

A sensitive chemiluminescence based immunoassay for antibody to staphylococcal peptidoglycan

A sensitive chemiluminescence based immunoassay is described for measuring antibody to staphylococcal peptidoglycan in blood and dialysates from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). Peptidoglycan was isolated from a strain of S. epidermidis obtained from the dialysate of a CAPD patient with peritonitis and after sonication used to coat polystyrene beads. The coated beads were incubated with standard or sample and bound IgG was detected by the addition of affinity-purified goat anti-human IgG labelled with acridinium ester. After a wash stage 0.1 M nitric acid containing 0.1% hydrogen peroxide was added to the beads. Subsequently the chemiluminescence produced following the addition of 0.3 M sodium hydroxide was measured over a 2 s time interval with an automatic luminescence analyser. Using this technique the optimum dilution of serum for detecting antibodies to peptidoglycan was found to be 1/800 and for overnight effluent from CAPD patients the dilution was 1/8. Initial values of serum and dialysate antibody levels from 34 subjects are presented. This method has the advantage that it will detect concentrations of anti-peptidoglycan which are less than 1% of those in sera, the reagents remain stable for long periods and large numbers of samples can be processed on the same day.     

检测可溶性TREM-1的ELISA法的建立及初步应用

目的:建立定量检测可溶性TREM-1的抗体夹心ELISA法。方法:采用抗人TREM-1单克隆抗体(mAb)包被酶标板,以兔抗鼠TREM-1多克隆抗体为夹心抗体、HRP标记羊抗兔IgG为检测抗体、重组小鼠可溶性TREM-1为标准品,建立检测可溶性TREM-1的ELISA法,并对30例正常人和30例急性肺部感染患者血清样本进行了检测。结果:建立的夹心ELISA法检测TREM-1的线性范围为0.78～200μg/L,批内、批间变异系数分别为6.52%和9.46%。30例正常人和30例血清急性肺部感染患者TREM-1的含量分别为(0.69±0.18)μg/L和(1.16±0.42)μg/L,两者比较差异有统计学意义(P<0.001)。结论:成功建立了一种灵敏度高、稳定性好的检测可溶性TREM-1的抗体夹心ELISA法。     

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.