HIV-1/2抗体和P24抗原联合检测试剂盒的研制与评价

目的研制HIV-1／2抗体和P24抗原联合检测酶免疫试剂盒并评价其实用性。方法联合使用基因工程HIVI／2型抗原和抗HIVP24单克隆抗体包被酶联反应板，以辣根过氧化物酶标记的HIVI／2型抗原和生物素化的兔抗HIVP24抗体作为标记物，研制了联合检测HIVI／2抗体和P24抗原的ELISA诊断试剂，并对其特异性、敏感性、稳定性等进行评价和临床考评。结果检测P24抗原质控品的灵敏度可达0．2ng／ml；与雅培公司试剂比较检测78份AIDS患者血清和85份正常人血清、对照检测中国药品生物制品检定所研制的HIV参比血清，特异度和灵敏度均为100％。临床考核检测12051份各种血清，灵敏度为100％（543／543），特异度为99．48％（11448／11508）。试剂在37℃放置6d后，试验结果无明显差异。结论本试剂盒具备特异度强、敏感度高、稳定性好、操作简便等优点，可以一步检出HIV特异性抗体和HIVP24抗原，缩短了HIV感染的检测窗口期，适用于HIV感染的实验室诊断和流行病学调查。     

白喉和破伤风类毒素抗原ELISA检测方法的建立

目的建立定量检测白喉和破伤风类毒素抗原的酶联免疫吸附试验(ELISA)方法。方法分别采用白喉、破伤风单抗包被酶标板,兔抗白喉、破伤风多抗作为二抗,辣根过氧化物酶标记的羊抗兔IgG抗体作为酶标抗体,以平行线法建立定量检测白喉和破伤风类毒素抗原含量的夹心ELISA法,并进行方法学验证。结果两种定量检测ELISA方法的验证结果均符合规定。检测白喉类毒素抗原ELISA方法的定量限为8.90×10-4Lf/ml,回收率为98.35%,批内变异系数(CV)≤10.59%,批间CV≤13.51%;检测破伤风类毒素抗原ELISA方法的定量限为2.13×10~(-3)Lf/ml,回收率为107.28%,批内CV≤13.96%,批间CV≤10.06%。结论建立了白喉、破伤风类毒素抗原ELISA检测方法,该法特异性强、准确度高、重复性好,可用于白喉破伤风疫苗产品的质量控制和生产过程控制。     

检测抗HIV-1/2型抗体的双抗原夹心方法的建立及其试剂盒的研制

目的 建立更敏感的检测人免疫缺陷病毒（HIV）抗体的方法,并研制检测试剂盒。方法 根据HIV－1／2型的基因序列及其所编码氨基酸结构,采用固相法合成了HIV－1型的gp41.1、gp41.2、gp120、p24和HIV－2型的gp36五条多肽,混合包被酶标板作为固相抗原。用辣根过氧化物酶标记以上多肽抗原作为标记物,建立检测血清中抗HIV－1／2抗体的双抗原夹心ELISA法。同时,应用该方法制备检测HIV抗体的试剂盒,并检测三批中国卫生中药品和生物制品检定所HIV诊断试剂国家参比品。结果 建立了检测HIV－1／2抗体的双抗原夹心法。用检定所参比品检测,该方法特异性、灵敏度均为100％,变异系数小于10％。与间接法相比较其灵敏度、特异性均高于间接法（P＜0．05）。检测210份其他病种患者血清均为阴性。与GBI公司的HIV抗体诊断试剂比较,检测40份卫生部药品和生物制品检定所提供的质控参比品（阳性20份,阴性20份）,GBI试剂阴、阳性符合率及总符合率分别为100%(20/20)、85％（17／20）及92．5％（37／40）,而应用该方法所研制的诊断试剂盒、阳性符合率及总符合率为100％。该试剂已通过国家卫生部质检。与雅培公司HIV诊断试剂比较检测90份献血员血清和88份HIV－1／2型感染者血清,符合率为100％。试剂盒于37℃放置4d后的检测结果的阴、阳性判定不受影响。结论 本法特异性强、灵敏度高、稳定性好,适用于献血员的筛选和临床HIV感染的检测。     

双抗夹心化学发光免疫分析定量检测Ⅳ型胶原方法的建立

目的 建立基于双抗夹心微孔板化学发光酶免疫分析的人血清Ⅳ型胶原定量检测方法.方法 应用Ⅳ型胶原多抗进行包被,辣根过氧化物酶标记Ⅳ型胶原单抗,以鲁米诺化学发光体系检测,调整优化各种反应液的工作浓度和各类反应条件后建立双抗体夹心的检测方法;评价所建立方法的线性范围、特异性、灵敏度、稳定性等性能指标;应用肝纤维化患者血清与进口试剂进行比对实验.结果 所建立方法的灵敏度为15.5 ng/ml,线性范围25 ～ 850 ng/ml,批内、批间变异均小于8％.检测Ⅳ型胶原的临床高、中、低值血清回收率分别为98.5％、94.3％和102.6％;在4℃和37℃条件下分别进行了3、5、7d的稳定性考察,线性相关系数均＞0.99,标准偏差＜7％;比对实验分析显示与进口试剂相关性具有统计学意义.结论 成功建立了定量Ⅳ型胶原化学发光酶免疫分析方法,该方法有较好的准确性、灵敏度、重复性,与进口试剂检测结果等效.     

检测单克隆抗体不同ELISA方法的敏感性比较(文摘)

本文比较7种不同的EL1SA方法在检测9株单克隆抗体中的敏感性。7种ELSA按试剂加入程序不间分别为①抗原、鼠抗体、酶标兔抗鼠②抗原、鼠抗体、兔抗鼠、酶标羊抗兔③羊抗体、抗原、鼠抗体、酶标兔抗鼠④鸡抗体、抗原,、鼠抗体、免机鼠、酶标羊抗兔⑤鼠抗体、抗原、兔抗体、酶标羊抗兔⑥兔抗鼠、鼠抗体、抗原、酶标兔抗体⑦羊抗鼠、鼠抗体、抗原、鸡抗体、酶标兔抗鸡。抗原为烟草花叶病毒(TMV)及其衣壳蛋白(TMVP)。用碱     

水痘-带状疱疹病毒抗原的ELISA定量检测方法的建立

目的:建立水痘-带状疱疹病毒(VZV)抗原的双抗体夹心ELISA定量检测方法,用于质控VZV灭活疫苗研发和生产中抗原含量.方法:以VZV中和单抗5F6C8为包被抗体、8H5D1为酶标抗体,构建定量检测VZV抗原的双抗体夹心ELISA方法,并对本方法的特异性、灵敏度、准确性、线性和稳定性等性能进行分析.结果:建立的双抗体夹心定量检测VZV抗原的ELISA方法,线性范围为0.4 μg～13 μg/ml,相关系数为R2=0.994,定量限度为0.4.μg/ml;变异系数CV＜ 15％、准确性回收率介于87.5％ ～ 111.6％之间,稳定性37℃6天的回收率＞80％.与VZV以外的相关病毒样本没有交叉反应.结论:构建的VZV抗原ELISA定量检测方法的各项性能符合定量检测需要,可用于VZV灭活疫苗的研发和生产过程的抗原含量检测.     

单克隆与多克隆双抗体夹心法测定血清可溶性IL-2受体

建立了灵敏的酶标双抗体夹心法测定血清可溶性IL-2受体(sIL-2R)。主要步骤为以单抗包被,加入待检抗原,再加多克隆兔抗IL-2 R血清作用,最后加酶标羊抗兔示踪并放大其反应。本法简便、重复性好、灵敏度达100u/ml.可适用于临床与基础研究。     

双抗原夹心法检测抗HIV-1/2总抗体方法的建立和评价

目的 进一步提高HIv感染诊断试剂的敏感性。方法 以人工合成的HIv—1 gp41．1(sP1)、gp41．2(sP2)、gp120(sP3)、P24(sP4)和HIV—2gp36(sP5)5条多肽，采用双抗原夹心酶链免疫吸附试验(ELISA)原理，以sP1、sP3、sP4和sP5混合包被酶标板作为因相抗原，辣根过氧化物酶标记sp1、sp2、sP4和sP5多肽为标记物，建立了检测抗HIV—1／2总抗体的双抗原夹心ELISA法。结果 检测卫生部药品生物制品检定所第2代40份质控参比血清，其特异性和灵敏度均为100％，高于间接ELISA法(特异性为90％，灵敏度为65％)。检测210份其他病种患者血清均为阴性，与雅培HIVAB试剂比较检测如份健康献血员血清和88份HIv感染者血清，符合率为100％。结论 本方法特异性强、敏感性高，操作简便，适用于献血员的筛选和临床HIv感染的检测。     

重组抗原Em18双抗原夹心酶联免疫吸附试验法检测泡球蚴抗体的建立及初步评价

目的 建立双抗原夹心酶联免疫吸附试验(ELISA)检测泡状棘球蚴抗体的方法.方法 重组抗原Em18(rEm18)作为包被抗原,以辣根过氧化物酶(HRP)标记rEm18抗原为检测抗原,建立检测人血清中泡状棘球蚴抗体的双抗原夹心ELISA方法;应用方阵滴定法对包被抗原、酶标抗原等条件进行优化,并对该系统的灵敏性、特异性、重复性进行初步分析评价.结果 rEm18最佳包被浓度为2.5μg/mL,HRP标rEm18稀释度为1∶800.灵敏度为92％(46/50),特异性为94％(47/50),假阴性率为8％ (4/50),假阳性率为6％ (3/50),批内变异≤9.55％,批间变异≤14.79％,与Em2-ELISA试剂盒检测结果有良好的一致性.结论 本研究成功建立了快速检测泡球蚴特异性抗体的ELISA法,其可以用于人泡球蚴病的早期诊断.     

癌抗原15-3酶促化学发光免疫分析方法的建立

目的 建立癌抗原15-3(CA15-3)酶促化学发光免疫分析方法.方法 利用双抗体夹心法建立CA15-3化学发光检测体系,分别对该体系的最低检测限、线性、分析特异性、准确度和精密度进行评估,并与进口全自动化学发光检测结果进行对比.结果 本方法灵敏度为0.26 U/mL,线性范围为0～ 300 U/mL,与CEA和CA125无交叉反应,添加回收率在97.0％ ～105.6％之间,分析内与分析间CV均小于10％,与进口全自动化学发光试剂同时检测160份样本的CA15-3浓度,检测结果的线性相关系数为0.987.结论 成功建立了CA15-3酶促化学发光免疫分析方法.该方法灵敏度高、重复性好,在临床应用中可替代进口化学发光检测试剂.     

Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.     

甲型肝炎病毒IgM AlphaLISA检测方法的初步建立

目的 建立甲型肝炎病毒抗体IgM的AlphaLISA检测.方法 浓缩甲型肝炎病毒抗原并生物素化,通过dot-Blot法摸索出生物素化的甲型肝炎病毒的最适量为6 × 10-8ng.优化供体珠、受体微珠、血清等实验反应条件,建立了甲型肝炎病毒的AlphaLISA的IgM检测方法.并对23份感染甲型肝炎病毒的IgM阳性患者和70份健康人的血清进行检测.结果 AlphaLISA甲型肝炎病毒的IgM方法检测:灵敏度(真阳性率)为78％;特异度(真阴性率)为98.5％;假阳性率为1％;假阴性率为22％;阳性预测值为95％;阴性预测值为93％;准确度为94％.结论 建立了均相、快速检测AlphaLISA甲型肝炎病毒IgM检测方法并得到初步应用.     

检测血清中抗乙型肝炎X抗体ELISA竞争法的建立及初步应用

应用基因工程表达的乙型肝炎X抗原(HBxAg)和抗-HBx单克隆抗体(抗-HBx McAb),国内首次建立了检测血清中抗-HBx抗体的ELISA竞争法。此方法敏感性高,血清抗-HBx效价达1∶10即可测出:特异性强,与抗-HBs、抗-HBc、抗-HAV和抗-HD等皆无有意义竞争;稳定性和重复性好;而且由于使用了单克隆抗体,可使方法标准化。对200份临床肝炎血清进行了检测,结果显示抗-HBx阳性率为2.5％,此方法可用于流行病学调查及临床诊断。     

A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available.     

Use of monoclonal antibody in a blocking ELISA to detect group specific antibodies to bluetongue virus

An enzyme-linked immunosorbent assay (ELISA) in the form of a blocking test is described for the detection of group specific antibodies to bluetongue virus (BTV). The test relies upon interruption of the reaction between BTV antigen and a group specific murine monoclonal antibody against BTV by addition of serial dilutions of bovine or ovine test sera containing specific antibodies to BTV which inhibit binding of the monoclonal antibody to the BTV antigen. This is detected as a reduction in the optical density (O.D.) reading obtained with the monoclonal antibody alone. The test is capable of specific detection of antibodies to all 22 serotypes of BTV but, unlike the agar gel precipitin (AGP) test, does not show cross-reactions with antibodies to epizootic haemorrhagic disease of deer (EHD) viruses. Furthermore, antibodies to cellular proteins which complicate interpretation of the AGP test and the indirect ELISA are not detected in the blocking ELISA. The high sensitivity and specificity of the blocking ELISA make it an ideal alternative to the AGP test. The use of a monoclonal antibody would facilitate standardisation of diagnostic testing between laboratories.     

Development of a peptide ELISA to discriminate vaccine-induced immunity from natural infection of hepatitis A virus in a phase IV study

Hepatitis A virus (HAV) is a highly infectious agent that causes acute liver disease. The infection can trigger the production of antibodies against the structural and non-structural proteins of HAV. Nonetheless, vaccination with an HAV vaccine leads to the production of a primary antibody against the structural proteins. Because the non-structural proteins are only produced during active virus replication, there is no or very little antibody production against the non-structural proteins. However, the current commercial immunoassay cannot distinguish between antibodies produced during natural infection and those from vaccination against HAV. In our study, six immune-dominant epitopes from the non-structural proteins were designed, synthesized, linked together and cloned into pGEX-5X-1 plasmid. The recombinant protein was expressed in E. coli and purified by Ni²⁺-coated magnetic agarose beads. Then the purified recombinant protein was used as an ELISA antigen to detect antibodies for HAV non-structural proteins in serum samples. Seventy-seven attenuated and 89 inactivated vaccinated samples collected from our previous phase IV study of HAV vaccines were detected by peptide ELISA developed in this study. The mean OD₄₅₀ value for the vaccination samples and acute infection samples were 0.529 (0.486 for the attenuated group and 0.567 for the inactivated group) and 1.187, respectively. According to the receiver operating characteristic (ROC) curve, the sensitivity and specificity of the peptide ELISA were 93.80% and 91.00%, respectively. This peptide ELISA was confirmed to discriminate vaccine-induced immunity from natural infection of HAV in a phase IV study with high sensitivity and specificity.     

Production and characterization of human monoclonal lymphocytotoxic autoantibodies from a renal dialysis patient

Abstract: We have produced human monoclonal lymphocytotoxic autoantibodies from a renal dialysis patient by the generation of a mouse/human heterohybridoma. The antibodies are of the IgM class and react with the patient's autologous cells, the B-Iymphoblastoid cell line producing the antibody, normal T and B lymphocytes, B cells from chronic lymphatic leukemia patients (CLL cells), and the autoantibody-sensitive cell line K562. Screening of the monoclonal antibodies (mAb) against panels of normal T and B cells and CLL cells demonstrated that different reactivity profiles could be generated at different dilutions of the mAb. These profiles were identical to those seen with autoantibodies from different renal patients and this suggests that these profiles do not imply different antibody specificities but differing target cell sensitivity. Reactivity profiles seen in the fluorescence binding assays suggest that the target cell sensitivity is dictated not by antigen density alone but also by antibody/ antigen affinity. The results from studies of enzyme treatment of target cells and lectin inhibition of the molecular specificity suggest that the autoantibodies are polyreactive, capable of binding sialic acid-dependent epitopes and other negatively-charged cell surface molecules.     

从噬菌体抗体库筛选获得中和性人源抗甲型肝炎病毒Fab抗体

目的 研究人源抗甲型肝炎病毒基因工程抗体,为预防甲型肝炎病毒感染提供有效的方法。方法 采用噬菌体表面展示技术,从一名甲型肝炎恢复期病人的抗凝血中分离淋巴细胞,提取总ＲＮＡ逆转录后,用一组人ＩｇＧ Ｆａｂ特异性引物扩增。结果 克隆和表达了８株人源抗甲型肝炎病毒抗体Ｆａｂ段基因,经ＥＬＩＳＡ检测为特异性人抗甲型肝炎病毒Ｆａｂ段抗体。结论 该８株人源抗甲型肝炎病毒Ｆａｂ抗体都能与具有中和活性的鼠抗甲型肝炎病毒单克隆抗体产生竞争抑制反应,选其中的２株做体外中和实验,证明都有中和甲型肝炎病毒的活性。     

Combined immunoaffinity cDNA-RNA hybridization assay for detection of hepatitis A virus in clinical specimens.

To apply cDNA-RNA hybridization methods to the detection of hepatitis A virus (HAV) in clinical materials, we developed a two-step method in which a microtiter-based, solid-phase immunoadsorption procedure incorporating a monoclonal anti-HAV capture antibody was followed by direct blotting of virus eluates to nitrocellulose and hybridization with 32P-labeled recombinant HAV cDNA. This immunoaffinity hybridization method is simple and involves few sample manipulations, yet it retains high sensitivity (10- to 30-fold more than radioimmunoassay) and is capable of detecting approximately 1 X 10(5) to 2 X 10(5) genome copies of virus. The inclusion of the immunoaffinity step removes most contaminating proteins and thus facilitates subsequent immobilization of the virus for hybridization. It also permits positive hybridization signals to be related to specific antigens and adds a level of specificity to the hybridization procedure. When the method was applied to 23 fecal specimens collected from individuals during week 1 of symptoms due to hepatitis A, 13 specimens were found to be reproducibly positive for HAV RNA by immunoaffinity hybridization, whereas only 11 contained viral antigen detectable by radioimmunoassay.     

一对高亲和力抗人肌钙蛋白Ⅰ单克隆抗体的制备及鉴定

目的 获取抗人肌钙蛋白Ⅰ（cTnI）的单克隆抗体（McAb）.方法 以人cTnI作为抗原,免疫Balb/c小鼠,通过杂交瘤技术制备了抗人cTnI高亲和力、高特异性单克隆抗体.随后采用间接ELISA法测定抗血清效价,用protein G亲和纯化法纯化抗体,抗原竞争ELISA法鉴定抗体亲和力,SDS-PAGE法鉴定纯度,Western blotting鉴定抗cTnI单克隆抗体的特异性,竞争ELISA法分析抗原结合位点.结果 筛选出9株稳定分泌抗cTnI的单抗杂交瘤细胞株,其中A3、A9两株免疫球蛋白亚类均为IgG2a,分泌的抗体纯度高,与CK-MB、cTnT无交叉反应,效价均为：1：1024000,亲和力分别为4.21×10^8mol/L、1.07×10^8mol/L,抗原结合位点不同.结论 成功制备出了一对高亲和力、高特异性抗人cTnI单克隆抗体.