猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒（SFV）抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。     

应用明胶颗粒凝集试验检测人免疫缺陷病病毒(HIV-1)抗体

明胶颗粒凝集试验是测定HIV-1抗体的新方法。本研究将明胶颗粒凝集试验与ELISA法、蛋白印迹法和间接免疫荧光试验做了比较,观察本方法的敏感性和特异性。共检测了195份来自法国和非洲象牙海岸的血清,凡是蛋白印迹法阳性的血清,明胶颗粒凝试验都是阳性。这表明本方法是特异和敏感的,方法简便,不需特殊仪器,省时,可用于HIV-1抗体的筛选,但多数蛋白印迹法可疑的血清,明胶颗粒试验均阴性。因此,对蛋白印迹法测出的可疑者应该用数种方法进行追踪检测。     

不同载体表达的人类细小病毒B19壳蛋白VP2的抗原性比较研究

构建表达质粒pcDNA3 VP2,将其转染CHO细胞建立了稳定表达的细胞系;用间接免疫荧光法和Western印迹证明了表达的VP2蛋白的特异性。对昆虫杆状病毒系统表达的VP2蛋白作初步纯化。分别用由大肠杆菌、CHO细胞和昆虫杆状病毒表达系统表达的VP2蛋白,以间接免疫荧光法和ELISA法检测人群血清中的VP2抗体,结果表明,间接免疫荧光法的敏感性高于ELISA法。     

合肥市蜀山区206例艾滋病高危人群HIV、HCV感染情况调查

目的了解合肥市蜀山区艾滋病高危人群中人类免疫缺陷病毒(HIV)、丙型肝炎病毒(HCV)感染情况,为艾滋病高危人群疾病防治工作提供依据。方法采集合肥市蜀山区206例艾滋病高危人群血清样本,分别运用金标法和酶联免疫吸附法(ELISA)进行HIV抗体、HCV抗体检测。HIV抗体筛查结果阳性或一阴一阳标本再经蛋白印迹法(WB)确证阳性为最终结果,并对结果和不同人群、年龄、性别的感染情况进行统计分析。结果在206份血清样本中检出HIV抗体阳性17例,阳性率为8.73%;检出HCV抗体阳性21例,阳性率10.19%。合并感染病例2例,占0.97%。结论检测结果提示,在高危人群中存在HIV/HCV流行和传播的风险,因此应持续加强高危人群的监测,有效阻止艾滋病和丙型肝炎的流行和蔓延。     

人冠状病毒NL63核壳蛋白和棘突蛋白的表达及其在血清学检测中的初步应用

目的:重组表达人冠状病毒NL63(HCoV-NL63)的核壳蛋白(N蛋白)及棘突蛋白(S蛋白),用于检测血清中的相应抗体。方法:用原核表达系统表达HCoV-NL63的N蛋白,建立检测N抗体的Werstern印迹法;用真核表达系统表达HCoV-NL63的S蛋白,建立检测S抗体的间接免疫荧光(IFA)法。结果:经Werstern印迹检测,重组S蛋白和N蛋白表达正确;初步建立了N蛋白纯化方法。利用建立的检测方法,检测了100份正常成人血清,总阳性率为81%。其中S抗体阳性率为66%,N抗体阳性率为38%,S抗体和N抗体均为阳性的占总数的22%,双抗体均为阴性的占总数的19%;S抗体的检出率明显高于N抗体。结论:重组HCoV-NL63N蛋白及S蛋白表达成功;S抗体和N抗体共同检测可获得较好的检测结果,减少漏检。     

肺癌病人血清中U1-A snRNP自身抗体的发现与鉴定

许多研究表明, 肺癌病人存在自身免疫现象. 为了寻找肺癌病人血清中具有潜在诊断价值的自身抗体, 采用免疫荧光染色, 免疫印迹和蛋白质芯片技术对10例肺癌病人和10例正常人血清进行了检测. 筛选中发现1例来自64岁男性肺鳞癌(Ⅲb期)病人的血清呈现特异性的细胞核染色, 并在31 kD处有一个明显的细胞核蛋白免疫印迹条带. 进而采用该病人的血清进行免疫沉淀, 对所捕获的蛋白组分进行质谱分析和数据库查询后确认31 kD条带为核内小核糖体蛋白U1-A(small nuclear ribonucleoprotein U1-A, U1-A snRNP). 进一步对36例鳞癌、26例腺癌、31例小细胞肺癌和20例健康对照血清样本进行了免疫荧光染色和免疫印迹验证, 结果显示50%的鳞癌、26.9%的腺癌、54.8%的小细胞肺癌病人血清中有抗U1-A snRNP抗体. 上述结果报道了在肺癌病人血清中出现Anti-U1-A snRNP自身抗体.     

三种方法检查食蟹猴血清STLV-1抗体的研究

本文用乳胶凝集试验（ＰＡ）、免疫荧光试验（ＩＦＡ）和蛋白印迹试验（ＷＢ）对１０３份食蟹猴血清作了ＳＴＬＶ－１抗体检查。结果表明,１３份ＷＢ检查为ＳＴＬＶ－１抗体阳性的血清,ＰＡ和ＩＦＡ检查均为阳性,而９０份ＷＢ检查为ＳＴＬＶ－１抗体阴性的血清,ＰＡ检查有３份为阳性,ＩＦＡ检查有１份为阳性结果。     

制备肺炎衣原体抗原片检测血清抗体

目的:探索肺炎衣原体抗原片检测血清抗体法在诊断Cpn感染中的实际应用前景。方法:应用进口肺炎衣原体(Cpn)毒株感染Hep-2细胞,分别以瑞氏-姬母萨染色、吖啶橙染色和直接免疫荧光染色等3种方法鉴定Cpn感染细胞。纯化获取大量Cpn抗原,用于制备斑点抗原片。建立微量免疫荧光染色法(MIF)检测血清抗体,诊断Cpn感染。结果:Cpn感染Hep-2细胞的最适条件是用含1μg/mL放线菌酮的维持液,在35℃、5%CO2孵箱中培养7d,并在培养的第0、3、4、5天以2600r/min离心1h,感染成功率极高。染色反应显示,瑞氏-姬母萨染色可将Cpn包涵体染成蓝紫色或红紫色;吖啶橙染色则使Cpn感染的Hep-2细胞呈现鲜明的橘红色;免疫荧光抗体染色后,在Cpn感染细胞内可见亮苹果绿色包涵体。通过斑点抗原荧光抗体染色的方法抽样检测了100份病人血清中的Cpn抗体,其中抗Cpn-IgG抗体的阳性血清共61份,阳性率为61%。与Cpn-外周血单核细胞(Cpn-PBMC)抗原片比较,阳性检出率无明显差别。结论:用Cpn感染细胞制作的Cpn斑点抗原片可用于临床检测血清Cpn-IgG抗体,且具有特异性、敏感性高的特点,但要求检测人员有一定的经验。     

在昆虫细胞中表达EB病毒壳抗原及其在诊断中的应用

本研究的目的是以昆虫杆病毒为感染为载体表达ＥＢ病毒壳蛋白的主要多肽ｇｐ１２５,用间接免疫荧光和免疫的印迹技术证明表达产物的特异性,表达产物位于感染细胞的胞闪内,分子量约１００ｋＤ,用免疫Ｄｏｔ法检查感染细胞裂解物中存在特异性表达产物的量,重组病毒裂解产物免疫小鼠后,能产生与ＶＣＡ反应的抗体,重组病毒感染的细胞为靶细胞,与Ｂ９５－８细胞片平行检查人血清中ＶＣ／ＩｇＧ和ＶＣＡ／ＩｇＡ抗体,确定表达产物     

HIV—1 P24截短体与gp41截短体的融合蛋白在大肠杆菌中的表达、纯化和鉴定

用基因重组技术将截短的HIV－1 p24基因和gp41基因连接成嵌合基因,插入质粒pGEX-4T3,构建成重组表达质粒pGEX-F。将pGEX-F转化大肠杆菌BL21。经IPTG诱导表达,pGEX-F在大肠杆菌BL21中获得了高效表达。融合蛋白P24－gp41经Glutathione-Sepharose4B亲和层析纯化后,用间接ELISA和免疫印迹检测HIV抗体阳性血清和正常人血清,P24－gp41只与HIV抗体阳性血清反应,证明获得的融合蛋白P24－gp41有很强的抗原特异性和免疫反应性,具有较高的应用价值。     

Characterization of GP120 binding to CD4 and an assay that measures ability of sera to inhibit this binding

There is evidence that the initial interaction between HIV-1 and the host that is essential for infection is the specific binding of the viral envelope glycoprotein, gp120, to the CD4 molecule found on certain T cells and monocytes. Most individuals infected with HIV develop antibodies against the gp120 protein. Although in vitro treatment of CD4+ T cells with mAb to a specific epitope of the CD4 molecule (T4a) blocks virus binding, syncytia formation, and infectivity, it is unclear if antibodies to gp120 from an infected individual that can inhibit the binding of gp120 to CD4 is in any way related to the clinical course of disease. Our present study characterizes the binding of 125I-labeled rgp120 to CD4+ cells, and describes an assay system that measures a potentially relevant form of immunity to HIV infection, i.e., the blocking of HIV binding to CD4+ cells. Optimal binding conditions included a 2-h incubation at 22 degrees C, 4 x 10(6) CD4+ cells, and 1 nM gp120. The dissociation constant (KD) for gp120 binding to cell surface CD4 was 5 nM, and was inhibited by soluble CD4 and by mAb to T4a but not to T3 or T4. For the binding inhibition assay, negative controls included healthy seronegatives, seronegatives with connective tissue diseases, patients with HTLV-1 disease, and patients infected with HIV-2. In studying over 100 sera, the assay was highly sensitive (98%) and specific (100%). The majority of HIV+ sera could inhibit binding at dilutions of 1/100 to 1/1000. No correlation was noted between binding inhibition (BI) titer in this assay and clinical stage of HIV infection. In addition, there was no correlation between BI titer and HIV neutralizing activity. The BI titer was correlated with the titer of anti-gp160 (r = 0.63) and the titer of anti-gp120 (r = 0.52) antibodies determined by Western blot dilution. As with neutralizing antibodies and other forms of immune response to HIV, it is unclear what role antibody blocking of HIV binding to CD4+ cells may play in active immunity to HIV in infected individuals. This activity may prove to have some value in protection against initial HIV infection and, thus, the assay may be of use in monitoring vaccine trials.     

HIV-1 Gag p17-p24在痘苗病毒中的表达及性质研究

研究了重组痘苗病毒表达的HIV1核心蛋白(Gag)p17p24蛋白的一些生物学及免疫学特点。间接免疫荧光、DotELISA及Westernblot结果表明,构建的两株重组病毒分别表达了HIV1Gagp24及p17p24融合蛋白。电镜观察证实,Gagp24及p1724重组蛋白均可形成病毒样粒子。重组病毒可诱导小鼠产生抗HIV1Gagp24抗体。重组病毒感染BHK21细胞后,可见由于细胞凋亡而致的染色体DNA断裂“梯子”电泳图。     

两种HSV-1及猴B病毒相关抗体测定方法的比较

目的以人单纯疱疹病毒（HSV-1）做为抗原,利用空斑法和IFA法比较猴BV和人HSV-1阳性血清两种不同血清的中和能力的差异,建立一种实用、准确、可靠的病毒毒力的检测方法。方法首先,将HSV-1病毒悬液作连续的10倍稀释,取1 mL接种于已经长成单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数,算出病毒悬液中每毫升所含蚀斑单位,即滴定出HSV-1的TC ID50。同时,用免疫荧光方法（IFA）对猴和人疱疹阳性血清进行滴定,得到其血清的效价。其次,用滴定出的病毒液分别与两种阳性血清体外中和后,接种到单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数。最后,计算出其蚀斑减少率。结果用1%甲基纤维素作覆盖层的蚀斑数量平均为10-5PFU,能形成115-116个/mL蚀斑,形状呈黍米大小的规则圆形,其蚀斑边缘清晰。IFA滴定的人HSV-1阳性血清与猴BV阳性血清的中和抗体均为1∶80。人HSV-1和猴BV两种阳性血清的空斑减少率均为100%。结论确定了利用1%甲基纤维素做为覆盖层可得到清晰可靠的蚀斑,由此方法检测到用人HSV-1可以代替猴B病毒,筛查猴B病毒抗体。且为将来进行药物筛选和中和实验中利用病毒空斑法建立方便、可靠的方法。     

Detection of Piscirickettsia salmonis in fish tissues by an enzyme-linked immunosorbent assay using specific monoclonal antibodies

An enzyme-linked immunosorbent assay (ELISA) for the detection of Piscirickettsia salmonis in fish tissue samples was developed. The test uses a combination of different monoclonal antibodies specific against P. salmonis in the capture step of the assay. The antibodies 7G4, 6E2 and 2C1 chosen for the capture step are bound to the solid support with an adhesive protein purified from a bivalve mollusc, resulting in a high yield of adsorption and binding stability. The monoclonal antibody 7G4, used as a second antibody, is conjugated to horseradish peroxidase. The resulting ELISA test detected 7 different isolates of P. salmonis and does not cross-react with several other fish pathogens, revealing a high specificity and sensitivity. The test also detects P. salmonis in kidney tissue of infected coho salmon with 98% correlation with the immunofluorescence assay.     

Replication of human immunodeficiency virus in two T-cell lines and their application as antigens for immunofluorescence

Two T-cell lines, TALL-1 and CCRF-CEM, were infected with human immunodeficiency virus (HIV), strain LAV, to explore the time course of the appearance of various virus specific antigens, and to establish an antibody assay system by indirect immunofluorescence (IF). These cells were infected with LAV at two different input multiplicity of infection (MOI). Antigens were tested by Western blot analysis (WB) and IF. Antigens for WB were extracted from the infected cells at various times after infection, but pooled sera of American HIV carriers could not recognize gp41 or gp160. Antigen expression was highest in CCRF-CEM, but, as the antigen for IF, TALL-1 infected at the MOI of 8.0 was the most suitable 7 days after infection, because it includes a fairly large number of uninfected cells, which served as the internal control.     

不同人类免疫缺陷病毒感染途径群体中戊型肝炎病毒的流行情况

本研究旨在了解不同人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染途径群体中戊型肝炎病毒(hepatitis E virus,HEV)抗体情况,探讨HEV疫苗接种的必要性。采集HIV感染者的血清或血浆,利用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测HEV IgG抗体、IgM抗体及抗原,荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测HEV核酸,Roche高纯化HIV-1核酸定量检测试剂盒(PCR荧光法)检测HIV感染者的HIV载量。比较分析不同HIV感染途径群体中HEV流行率的差别。结果显示,HIV感染者中HEV IgG抗体的阳性率为37.4%,静脉吸毒、成分献血和传播途径不明HIV感染群体的HEV IgG抗体阳性率分别为49.3%、39.5%和30.4%。HEV核酸荧光PCR检测结果均为阴性。3种HIV感染群体之间HEV IgG抗体阳性率差异无统计学意义(χ~2=2.978,P0.05)。HEV IgG阳性与阴性感染者之间HIV载量差异无统计学意义(P0.05)。结果提示,为保护HIV感染者免受HEV感染,应考虑接种HEV疫苗。     

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.     

Multicenter evaluation of the new HIV DUO assay for simultaneous detection of HIV antibodies and p24 antigen

A multicenter survey was performed to evaluate a new semi-automated human immunodeficiency virus fourth generation antibodies and antigen simultaneous assay. This assay showed a sensitivity of 100% and specificity of 99.6% among sera obtained from hospitalized patients or blood donors. Sera obtained from commercially available as well as in-house seroconversions were tested showing that HIV DUO is able to reveal an infected state in 11 out of 14 cases earlier than conventional tests. This new assay improves old test performances in terms of sensitivity, maintaining specificity at very high levels.     

Detection of Sendai virus antibody in mouse and guinea pig sera by an enzyme-linked immunosorbent assay with protein A

An enzyme-linked immunosorbent assay using horseradish peroxidase (HRPO)-labeled protein A (P-ELISA) was established for detection of Sendai virus (SV) antibody in mouse and guinea pig sera. Sensitivity and specificity of P-ELISA were compared with those of ordinary ELISA using HRPO-labeled immunoglobulin G (IgG-ELISA) and the hemagglutination inhibition (HI) test. P-ELISA was 100 to 1,000 times more sensitive than the HI test for detection of the antibody in SV-naturally infected mice. P-ELISA and IgG-ELISA showed similar sensitivities for detection of the antibody in naturally infected mouse and guinea pig sera. A high specificity was demonstrated in P-ELISA with a cut-off optical density value of 0.2 (492 nm), while a non-specific reaction was observed when IgG-ELISA was used to both mouse and guinea pig sera at a low dilution (1:10-20). The antibody in rat sera was not detected by P-ELISA although it was realized by IgG-ELISA.