温州市区育龄妇女孕前巨细胞病毒感染现状调查

目的了解温州地区育龄妇女孕前人巨细胞病毒（HCMV）感染的状况。方法收集2008年10月至2010年6日参加温州市龙湾区免费孕前优生筛查的妇女血标本2869份,采用酶联免疫吸附试验（ELISA）检测血清HCMV IgG/IgM抗体;HCMV IgM抗体阳性标本,采用实时荧光定量聚合酶链反应（FQ-PCR）检测血HCMV DNA载量;HCMV IgG/IgM抗体双阳性标本,采用尿素变性结合ELISA技术检测IgG抗体亲和力指数（AI）。结果 2869份孕前妇女血清中HC-MV IgG抗体阳性检出率为97.77%（2805/2869）,HCMV IgM抗体阳性检出率为0.77%（22/2 869）,IgG/IgM抗体均阳性检出率占0.17%（5/2 869）;22份HCMV IgM阳性标本中,血HCMV DNA阳性检出率为68.18%（15/22）;5份HCMVIgG/IgM双阳性标本中,检出低亲和力IgG抗体1份,中等亲和力IgG抗体2份,高亲和力IgG抗体2份。结论温州市区育龄妇女孕前HCMV IgG抗体阳性率高;对HCMV IgM抗体阳性孕前妇女应进行多指标检测以判断HCMV感染的状态,为减少出生缺陷、做好优生优育服务提供依据。     

EB病毒胸腺嘧啶激酶抗体在鼻咽癌血清学诊断中的作用

目的 建立EB病毒感染和鼻咽癌血清学检测的敏感,特异和有效的方法.方法 以EB病毒早期蛋白胸腺嘧啶激酶为检测抗原,建立ELISA和免疫纤维膜条方法,检测血清中的特异性的IgG抗体.结果 用ELISA和诊断条同时检测了401份血清,其中鼻咽癌患者血清127份.两种方法检测鼻咽癌患者血清抗体均阳性,274份门诊患者血清中,55份ELISA和诊断条检测抗体均阳性,3份诊断条方法检测阳性的血清,ELISA检测A值接近临界值.对胸腺嘧啶激酶的抗体阳性率明显地高于早期蛋白P54.结论 以胸腺嘧啶激酶为检测抗原,为鼻咽癌血清学诊断和高危人群的筛查提供更有效的手段.     

TTV酶联免疫方法的建立及其初步应用

目的 建立检测TTV的酶免疫技术,了解不同型肝炎患者血清中抗－TTV抗体的分布情况,并结合TTV DNA的检测分析两者的关系。方法 以原核表达的TTV ORF1蛋白为抗原建立检测抗－TTV的酶联免疫吸附试验（ELISA）方法,采用该方法检测不同肝炎患者中抗－TTV抗体;在套式聚合酶链反应（PCR）方法检测血清标本中TTV DNA。结果 所建立的检测TTV的ELISA方法具有较好的特异性,不同别肝炎患者中抗－TTV抗体阳性率分别为：甲型肝炎患者10.5%（4／38）,乙型肝炎患者12.5%（16／128）,丙型肝炎患者8.3%(7/84),丁型肝炎患者7.7%(30/93),健康人群1.3%(1/78)。统计学分析表明,非甲－庚型肝炎患者抗－TTV阳性率显著高于其他型肝炎患者（P＜0.01）,而正常人群则显著低于肝炎患者（P＜0.05）。抗－TTV阳性率与TTV DNA阳性率存在相关性（P＜0.05）。结论 在不同型肝炎患者中均可检出抗-TTV抗体,但以非甲－庚型肝炎患者阳性率高;TTV抗体可与基因同时存在于患者血清中,抗－TTV抗体可能类似抗－HCV, 是一传染性标志。     

基于N-ELISA的呼吸道合胞病毒快速中和抗体检测方法的建立

目的制备针对呼吸道合胞病毒(RSV) N蛋白的兔多克隆抗体, 以其作为检测抗体建立基于ELISA的快速中和抗体检测方法。方法构建pET30a-N质粒, 表达纯化N蛋白, 免疫新西兰兔制备针对RSV N蛋白的多克隆抗体作为检测抗体。阳性血清系列稀释后与100半数组织培养感染剂量(TCID50)/孔的RSV中和, 接种Hep-2细胞培养, 80%丙酮固定细胞, ELISA方法检测感染细胞中病毒的N蛋白, 当每孔的吸光度值低于临界值时, 视为中和试验阳性孔, 阳性孔血清的最高稀释度为该血清的中和抗体滴度。优化抗体稀释度、检测时间、细胞密度及中和时间, 建立基于N-ELISA的中和抗体检测方法, 对建立的实验方法进行细胞代次、边缘孔效应、准确性、重复性以及精密度验证, 初步应用于人RSV IgG阳性血清检测, 分析与微量中和法的相关性。结果成功构建出pET30a-N质粒, 表达纯化的N蛋白纯度较高, 特异性良好;三免后兔血清效价为1∶51 200, 可特异性结合RSV;制备的抗RSV N兔多抗的效价为1∶51 200, 特异性良好, 建立的快速中和抗体检测方法在4 d就可检测, 中和时间可缩短...     

庚型肝炎病毒NS5区优势抗原表位的表达及其抗原性的初步评价

目的 表达庚型肝炎病毒（HGV）基因且NS5区部分基因重组抗原，分析其抗原性。方法 分别亚克隆了HGV NS5a、NS5b以及NS5b与C区嵌和的基因至pThoiC表达载体上，构建成3个重组表达质粒，分别大肠埃希菌JM109（DE3），用IPTG诱导表达重组蛋白。表达产物经纯化后采用Western blot和间接ELISA方法分析3个重组蛋白的抗原性。结果 经酶切和序列分析鉴定正确，3种表达蛋白均高效表达且相对分子质量与预期大小相符。Western blot分析和间接ELISA试验表明，3种表达蛋白均能与抗－HGV阳性血清发生特异性反应。将应用重组蛋白检测的抗－HGV抗体与混合重组抗原（包括C区合成肽、NS5a合成肽、NS3区基因重组抗原）的检测结果进行了比较，在混合重组抗原阳性的22份血清中，P5a检出率为68％（15／22），P5b检出率为91％（20／22），Pc-5b检出率为73％（16／22）。在70份阴性标本中，3种抗原的检出率分别为P5a:7%(5/70);P5b:1%(1/70);Pc-5b:6%(4/70)。3个重组抗原单独检出阳性的标本，其中有一部分经RT－PCR检测亦为阳性。结论 原核表达的NS5区蛋白所检测的抗－HGV抗体不能完全被其他区段的抗原所覆盖，是免疫诊断HGV感染所必需的抗原表位之一。     

以Rtac2/3为抗原用于鼻咽癌病人检测的初步研究

目的 在大肠杆菌中表达EBV的早期基因BRLF1，并纯化这个重组蛋白。用纯化的蛋白作为抗原与鼻咽癌（NPC）病人血清中的特异性抗体发生反应，以寻找新的NPC筛检或诊断标志物。方法 用表达和纯化的BRLF1基因C端2／3部分蛋白（Rtae2／3）建立间接ELISA方法，检测了59份NPC患者血清中的抗Paa IgG抗体，同时59份健康者血清作对照。结果 59份NPC患者血清中50份阳性，而59份健康者对照血清中只有7份阳性。NPC组的阳性率与健康对照组之间差异有统计学意义（P〈0．01）。此方法的灵敏度为84．7％，特异性为88．1％。结论 （1）检测人血清中的抗Rta-IgG可以作为NPC诊断的重要标志物之一。（2）如果检测抗Rta-IgG与检测Zebra IgG抗体试验联合使用，用于NPC的筛检和诊断能够进一步提高灵敏度或特异性。     

健康体检儿童肺炎支原体检出情况研究

目的了解参与健康体检儿童人群的肺炎支原体(Mycoplasma pneumoniae, Mp)检出情况。方法随机入组2023年参与健康体检的6～12岁小学生1 303人, 采集受试者口咽拭子及静脉血标本, 使用分离培养、核酸检测以及血清学抗体方法检测受试者Mp检出情况, 并进行统计学分析。结果 1 303例健康儿童中Mp培养阳性检出率为4.1%(53/1 303), 核酸阳性检出率为7.3%(95/1 303), 血清学IgM抗体阳性检出率为13.6%(177/1 303)。经统计学分析, 健康儿童人群中Mp培养与核酸检测以及血清IgM抗体检测阳性率之间的差别均具有统计学意义(P<0.05)。结论 2023年健康体检儿童中Mp检出率约为4.1%。分离培养方法对健康人群中Mp检出率检测准确性优于核酸以及血清学方法, 核酸和血清学方法会高估正常人群中Mp检出率。     

抗体膜片法检测乳腺癌患者血清C-erbB-2蛋白及其方法学评价

目的:建立血清C-erbB-2蛋白的抗体膜片检测方法,并进行方法学评价.方法:抗体膜片法检测血清C-erbB-2蛋白,免疫组化方法检测乳腺组织C-erbB-2蛋白.结果:30例乳腺癌、26例乳腺增生、30例正常对照组血清中C-erbB-2蛋白阳性率分别为40.0%、7.7%和6.7%,三组相比较有显著差异(P＜0.05);乳腺癌组织C-erbB-2蛋白阳性和阴性者其血清C-erbB-2蛋白阳性率分别为91.67%和11.11%,血清与乳腺组织C-erbB-2蛋白表达具有相关性(P＜0.01);抗体膜片法检测血清C-erbB-2蛋白的灵敏度、特异度和准确度分别为40.0%、92.8%和74.4%.结论:抗体膜片法检测血清C-erbB-2蛋白与乳腺癌病理学诊断符合率高,且方法简便易行,临床具有实用价值.     

建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒（FMDV）感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白（VP1epi）作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。     

用蛋白印迹试验检测精神分裂症患者血清中博尔纳病病毒-p24抗体的研究

目的：探讨博尔纳病病毒（BDV）感染在我国的流行情况及其与精神分裂症的相关性。方法：在对优化表达的GST－BDV－p24融合蛋白进行特异性鉴定的基础上，通过确定融合蛋白与第一抗体、第二抗体间的最佳反应条件，建立可行的检测BDV－p24特异抗体的蛋白印迹（Western-blot）方法，进而对黑龙江地区精神分裂症患者和正常人对照血清中BDV－p24特异性抗体进行了检测，并通过血清－GST蛋白吸收后W estern-blot实验对阳性血清进行了确认。结果：116例精神分裂症患者中检出BDV－p24阳性血清10例，阳性检出率为8.6%，而正常人血清标本中未检出阳性。结论：我国存在博尔纳病病毒的感染，博尔纳病病毒的感染可能与精神分裂症的发生有关。     

Antibody Responses One Year after Mild SARS-CoV-2 Infection

Understanding the long-term kinetics of antibodies in coronavirus disease 2019 (COVID-19) is essential in interpreting serosurvey data. We investigated the antibody response one year after infection in 52 mildly symptomatic patients with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, using three commercial immunoassays and a surrogate virus neutralization test (sVNT) kit. Anti-N pan-immunoglobulin (Ig), anti-S IgG, and anti-S1 IgG were detected in 43 (82.7%), 44 (84.6%), and 30 (57.7%), respectively. In 49 (94.2%), the antibody could be detected by either anti-N pan-Ig or anti-S IgG assay. In the sVNT, 30 (57.7%) had positive neutralizing activity. Despite waning immunity, SARS-CoV-2 antibodies can be detected up to one year after infection, even in mild COVID-19 patients.     

Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA

This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417-422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.     

Humoral response to spike S1 and S2 and nucleocapsid proteins on microarray after SARS-CoV-2 infection

Many aspects of the humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as its role in protection after natural infection, are still unclear. We evaluated IgA and IgG response to spike subunits 1 and 2 (S1 and S2) and Nucleocapsid proteins of SARS-COV-2 in serum samples of 109 volunteers with viral RNA detected or seroconversion with different clinical evolution (asymptomatic, mild, moderate, and severe coronavirus disease 2019), using the ViraChip® Test Kit. We observed that the quantification of antibodies to all antigens had a positive correlation to disease severity, which was strongly associated with the presence of comorbidities. Seroreversion was not uncommon even during the short (median of 77 days) observation, occurring in 15% of mild-asymptomatic cases at a median of 55 days for IgG and 46 days for IgA. The time to reach the maximal antibody response did not differ significantly among recovered and deceased volunteers. Our study illustrated the dynamic of anti-S1, anti-N, and anti-S2 IgA and IgG antibodies, and suggests that high production of IgG and IgA does not guarantee protection to disease severity and that functional responses that have been studied by other groups, such as antibody avidity, need further attention.     

Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.     

Monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acute-phase sera of severe acute respiratory syndrome patients

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.     

Current immunoassays and detection of antibodies elicited by Omicron SARS-CoV-2 infection

The present study aimed to determine whether current commercial immunoassays are adequate for detecting anti-Omicron antibodies. We analyzed the anti-SARS-CoV-2 antibody response of 23 unvaccinated individuals 1–2 months after an Omicron infection. All blood samples were tested with a live virus neutralization assay using a clinical Omicron BA.1 strain and four commercial SARS-CoV-2 immunoassays. We assessed three anti-Spike immunoassays (SARS-CoV-2 IgG II Quant [Abbott S], Wantaï anti-SARS-CoV-2 antibody ELISA [Wantaï], Elecsys Anti-SARS-CoV-2 S assay [Roche]) and one anti-Nucleocapsid immunoassay (Abbott SARS-CoV-2 IgG assay [Abbott N]). Omicron neutralizing antibodies were detected in all samples with the live virus neutralization assay. The detection rate of the Abbott S, Wantai, Roche, and Abbott N immunoassays were 65.2%, 69.6%, 86.9%, and 91.3%, respectively. The sensitivities of Abbott S and Wantai immunoassays were significantly lower than that of the live virus neutralization assay (p = 0.004, p = 0.009; Fisher's exact test). Antibody concentrations obtained with anti-S immunoassays were correlated with Omicron neutralizing antibody concentrations. These data provide clinical evidence of the loss of performance of some commercial immunoassays to detect antibodies elicited by Omicron infections. It highlights the need to optimize these assays by adapting antigens to the circulating SARS-CoV-2 strains.     

Simultaneous detection of antibodies to mouse hepatitis virus recombinant structural proteins by a microsphere-based multiplex fluorescence immunoassay

We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera. All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681). The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay. The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity). Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%. In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity). Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera. Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.     

人冠状病毒229E、OC43与SARS-CoV血清学相关性的研究

目的 检测正常人和SARS患者血清中3种人冠状病毒（229E、OCA3和SARS-CoV）特异性抗体．分析3种冠状病毒血清学相关性。方法 采用免疫印迹、免疫荧光和ELISA方法检测100例健康献血员、34例SARS患者恢复期以及11例SARS患者双份血清中229E、OCA3和SARS-CoV3种冠状病毒核衣壳（N）蛋白抗体。结果 用免疫荧光方法检测100例健康献血员血清中229E、OCA3和SARS-CoV IgG阳性率分别为98％、100％和1％，34例SARS患者恢复期血清中3种冠状病毒IgG的阳性率均为100％；免疫印迹检测100例健康献血员血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别9r7％、99％和2％，34例SARS患者恢复期血清中229E、OCA3和SARS-CoVN蛋白IgG阳性率分别97％、100％和100％；11例SARS患者的急性期和恢复期双份血清中，免疫荧光检测有5例出现229E IgG滴度4倍或以上升高，10例出现OC43 IgG滴度4倍或以上升高，ELISA检测2例出现229EN蛋白IgG滴度4倍以上升高，没有一例出现OCA3N蛋白抗体滴度升高。结论 正常人群中普遍存在229E和OCA3两种人冠状病毒抗体，SARS-CoV感染者存在对人冠状病毒229E和OCA3血清学交叉反应，提示核衣壳蛋白不是引起血清学交叉反应的主要抗原，结果对研究SARS溯源有重要意义。     

Detection of antibodies to Shigella lipopolysaccharide in urine after natural Shigella infection or vaccination.

The purpose of the present study was to explore the possibility of detecting antibodies to Shigella sonnei lipopolysaccharide (LPS) in urine after infection or vaccination. Urinary immunoglobulin A (IgA) and IgG antibodies and specific IgA secretory protein against S. sonnei LPS were measured by enzyme-linked immunosorbent assay (ELISA), after adjustment for urine concentration. A significant antibody level was defined as one above a cutoff value calculated from the geometric mean + 2 standard deviations of urinary anti-S. sonnei LPS levels in 43 healthy hepatitis B vaccinees (controls). Of 11 culture-proven cases of S. sonnei shigellosis, at convalescence 9 (82%) had significantly elevated levels of urinary antibodies to the homologous LPS. The S. sonnei conjugate vaccine, composed of S. sonnei O-specific polysaccharide covalently bound to recombinant exoprotein A of Pseudomonas aeruginosa, elicited a significant urine IgA or IgG anti-LPS response in 60% (6 of 10), 56% (9 of 16) 43% (16 of 37), and 14% (3 of 21) of the volunteers at 2 weeks, 6 weeks, 6 months, and 12 months after vaccination, respectively. The specificity of the urine antibody response to S. sonnei LPS was documented by the total lack of response in subjects who received parenteral Shigella flexneri 2a-recombinant exoprotein A conjugate (69 urine samples) or meningoccal tetravalent control vaccines (4 urine samples). All the volunteers who lacked a significant response to S. sonnei LPS in serum also lacked such response in urine samples. Seventy-four percent of the volunteers with a significant IgA or IgG anti-LPS response in serum at convalescence or 14 days after vaccination showed a similar response in urine. The ratio of the titer of secretory protein bound to IgA anti-S. sonnei LPS in urine to that in serum was 303 times higher than the ratio of anti-S. sonnei LPS total IgA titer in urine to that in serum, indicating that the urine IgA is of secretory origin. These findings suggest the possible use of urinary Shigella LPS antibodies as markers of systemic and secretory immune responses after natural infection or vaccination. At this stage, because of its limited sensitivity, the detection by ELISA of Shigella LPS antibodies in urine cannot replace the same assay in serum as a definitive test in an individual with a negative result.     

Prokaryote expression of HPeV-1 VP1 protein, production of VP1 polyclonal antibody and the development of an ELISA

The VP1 protein of human parechovirus (HPeV) plays a critical role in receptor binding based on its functional arginine-glycine-aspartic acid (RGD) motif region. Currently, only the neutralisation assay is used for seroepidemiological surveys of HPeVs. In the present study, the VP1 gene of HPeV-1 was cloned into the vector pET28a(+) to express the His-tagged VP1 protein in the bacterium Escherichia coli Rosetta. The recombinant protein was purified from inclusion bodies by Ni(+)-NTA affinity chromatography under denaturing conditions, followed by a refolding process in gradient urea. The identity and antigenicity of the His-tagged protein was confirmed by Western blotting using an anti-His monoclonal antibody and human HPeV-1-positive serum respectively. Polyclonal antibodies against the His-tagged VP1 protein were raised in rabbits by standard procedures, and the reactivity and specificity were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. An indirect ELISA was developed based on the fusion protein VP1, and evaluated in order to facilitate the detection of antibodies in persons who had been infected naturally with HPeVs. A serological survey was performed using the assay amongst children in the Shanghai region of China; the seropositivity rate was found to be about 73%.