A Novel Antigen Detection Immunoassay for Field Diagnosis of Hepatitis C Virus Infection

Abstract

The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (?5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.     

鼠抗丙型肝炎病毒（HCV）NS3区单克隆抗体的研制及特性分析

用淋巴细胞杂交瘤技术，制备出５株抗ＨＣＶ－ＮＳ３区单克隆抗体（ＭｃＡｂ）分别命名为Ｃ７－１，３，６，８，９。单抗特性测定结果显示５株单抗均为ＨＣＶ－ＮＳ３区特异性，与ＨＣＶ其它区域的抗原及宿主菌成份均无交叉反应，此５株单抗识别ＮＳ３区上两个不完全相关的抗原位点，其中Ｃ７－１，３，６株识别同一抗原决定簇，Ｓ７－９的识别位点与此３株有一定的相关，Ｃ７－８则识别与此４株不完全相关的位点。ＨＣＶ－ＮＳ３抗     

丙型肝炎病毒抗原检测试剂在临床诊断中的初步应用

本文利用重组丙型肝炎病毒(HCV)多表位复合抗原免疫动物制备抗HCV多克隆抗体,建立HCAgELISA检测方法,对不同的临床样品进行检测,分析不同临床样品HCAg检出率,并与荧光定量PCR结果进行对比。95份HCAb阳性的样品中,51份样品HCAg阳性,阳性率为53.7%;88份重症肝病患者血清中检出HCAg阳性33份,阳性率为37.5%,均明显高于其它样品(P<0.05);73份肝功异常但HCAb阴性血清样品,检出HCAg阳性8份,阳性率为11.0%;HCAg与HCAb之间存在相关性(r=0.5076,P<0.01),HCAg与HCV-RNA符合率为85.7%。HCAg ELISA检测可作为临床HCV诊断的检测指标,为临床早期诊断及治疗预后提供依据。     

Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus

We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.     

Hepatitis C virus infection in dialysis and chronic liver patients: Viraemia dependent anti-E2-antibody response

Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.     

Evaluation of a total hepatitis C virus (HCV) core antigen assay for the detection of antigenaemia in anti-HCV positive individuals

A new, sensitive enzyme immunoassay has been developed for detecting and quantifying total hepatitis C virus (HCV) core antigen in anti-HCV positive or negative sera ("trak-C", Ortho Clinical Diagnostics, Raritan, NJ). The purpose of this study was to evaluate the performance of trak-C as an additional laboratory diagnostic marker of viraemia. The performance was compared to HCV-RNA detection in the "screening" of sera from a large heterogeneous population of hospitalised patients and outpatients. Six hundred and eighteen anti-HCV negative sera, 405 anti-HCV positive/HCV-RNA negative sera, 604 anti-HCV positive/HCV-RNA positive sera and 67 anti-HCV negative sera containing antigens or antibodies potentially interfering with the performance of the assay were analysed. Supplemental HCV antibody testing was performed using a commercial strip immunoblot assay. HCV-RNA was investigated using a qualitative commercial assay. A quantitative commercial RT-PCR was used for the analysis of selected samples. Sensitivity and specificity values were 94.7 and 100%, respectively. The latter was also confirmed when anti-HCV negative samples containing potentially interfering antigens/antibodies were examined. Sensitivity below 100% was probably due to an antigenaemia below the detection limit of trak-C. Besides, because 65.6% of HCV-RNA positive/trak-C negative samples presented specific antibodies against all four RIBA antigens, the hypothesis was raised that, in some cases, the dissociation step efficiency could be sub-optimal. In conclusion, trak-C seems suitable for identifying HCV infection on large based populations. It is a rapid to perform, reliable and specific assay that can be adapted to any laboratory setting.     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

Development of a multiplex bead-based assay for detection of hepatitis C virus

Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.     

Rapid and simple detection of a Mycobacterium tuberculosis circulating antigen in serum using dot-ELISA for field diagnosis of pulmonary tuberculosis

Tuberculosis (TB) has re-emerged as a major health problem worldwide. Developing an easy, inexpensive immunodiagnostic test is extremely important for TB diagnosis, especially in developing countries. A target mycobacterial circulating antigen of 55-kDa molecular weight was identified in sera from confirmed Mycobacterium tuberculosis infected individuals by using Western blotting based on a specific mouse IgG anti-M. tuberculosis monoclonal antibody (TB-55 mAb). No bands were identified in sera of healthy individuals. The target TB antigen was isolated and characterized as a protein. It consists of 15 amino acids; 24.6% of the amino acids are hydrophobic and 46.4% are hydrophilic. A dot-ELISA format, based on TB-55 mAb, was developed for the direct demonstration of the 55-kDa TB antigen in serum samples of pulmonary TB patients. The technical aspects of the developed dot-ELISA are simple, rapid (5 min), and reproducible, as well as sensitive (87%) and specific (93%). Using the more sensitive immunoassay; Western blot, the 55-kDa TB antigen was detected in all (100%) sera that have been shown false negative by dot-ELISA, as well as in true positive sera. In conclusion, we have developed a simple and rapid immunoassay for the direct detection of a circulating mycobacterial antigen in sera of TB infected individuals and, therefore, the developed assay can be applied for laboratory and field diagnosis of TB infection in developing countries.     

游离丙型肝炎病毒核心抗原检测的临床价值探讨

目的:探讨游离丙型肝炎病毒(HCV)核心抗原检测在HCV感染诊断中的价值。方法:采用荧光定量PCR法检测HCV-RNA、ELISA法同步检测抗-HCV和游离HCV核心抗原。结果:191例HCV感染者HCV-RNA的检出率为71·2%(136/191);抗-HCV的检出率为97·4%(186/191);游离HCV核心抗原的检出率为33·0%(63/191)。其中有2例经抗病毒治疗的患者HCV-RNA和抗-HCV检测均阴性,但游离HCV核心抗原检测阳性;另有1例患者抗-HCV阴性,而游离HCV核心抗原阳性,经HCV-RNA证实为HCV感染。27例非HCV感染者HCV-RNA、抗-HCV和游离HCV核心抗原检测结果均为阴性。结论:HCV核心抗原检测作为抗-HCV检验的补充试验对HCV感染的诊断具有重要价值。     

Expression of immunoreactive forms of the hepatitis C NS5A protein in E. coli and their use for diagnostic assays

Summary.  In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV. Received December 26, 2001; accepted April 15, 2002 Published online July 10, 2002     

检测抗丙型肝炎病毒总抗体的双抗原夹心法的建立

以丙型肝炎病毒（ＨＣＶ）结构区和非结构区基因工程表达抗原及人工合成多肽包被酶标板，用辣根过氧化物酶标记抗原，建立了检测血清中抗－ＨＣＶ总抗体的双抗原夹心法。与普遍采用的检测抗－ＨＣＶＩｇＧ的间接酶联免疫吸附试验（ＥＬＩＳＡ）比较，其灵敏度（１∶１２８）稍高于间接法（１∶６４），特异性相同，均为１００％。该法可同时检测抗－ＨＣＶ各类抗体，使检出率提高１０％。     

Antigenic variation of core, NS3, and NS5 proteins among genotypes of hepatitis C virus.

Assays that detect antibody to hepatitis C virus (HCV) are used to screen blood donors and patients with hepatitis. Current enzyme-linked immunosorbent assay (ELISA)-based methods are invariably based upon antigens from expressed recombinant proteins or oligopeptides from HCV type 1. Some HCV antigens used in screening assays are coded by regions of the HCV genome that show extensive variability; therefore, HCV type 1-based assays may be less effective for the detection of antibody elicited by infection with other genotypes. In this study, we have measured antibody reactivity of sera from 110 hepatitis C patients infected with type 1b, 3a, or 4a to genotype-specific and cross-reactive epitopes present in recombinant proteins from HCV genotypes 1b (core, NS3, and NS5), 3a (NS3, NS5), and 4a (core, NS3), corresponding to those used in current third-generation screening ELISAs. By comparing the serological reactivities of sera to type-homologous and type-heterologous antigens, we detected a significant type-specific component to the reactivity to NS3 (61 to 77% of the total reactivity) and NS5 (60% of the total reactivity). Furthermore, despite the similarities in the amino acid sequences of the core antigens of type 1b and type 4a, we also found significantly greater reactivity to type-homologous antigens, with approximately 25% of reactivity being type specific. These findings are consistent with previous findings of fivefold weaker reactivity of sera from HCV type 2- and HCV type 3-infected blood donors in the currently used third-generation ELISAs and suggest that these assays are suboptimal for screening populations in which the predominant genotype is not type 1.     

血清丙型肝炎病毒抗原的免疫酶斑点法检测

目的建立一种可筛查早期HCV感染者的简单快捷方法。方法制备特异性的抗HCV核心蛋白和抗HCVNS3的单克隆抗体(mAb)酶标记物,应用免疫酶斑点法检测各种类型肝炎患者和正常人血清中的HCV-Ag,并与双抗体夹心ELISA检测HCV-Ag及RT-PCR检测HCVRNA的结果进行比较。结果免疫酶斑点法检测表明,HCV-Ag的检出率在抗HCV抗体阳性、乙肝和非甲非戊肝炎患者中,分别为15.4%(8/52)、1.73%(19/1099)和1.03%(1/97);在81例甲肝和67例戊肝患者及50例正常人中均未检出。免疫酶斑点法与双抗体夹心ELISA及RT-PCR的检测的结果没有显著性差异,但其与RT-PCR检测结果有一定的相关性。结论免疫酶斑点法特异性强,操作简便快捷,不需要特殊仪器设备,为HCV的早期诊断和常规筛查提供了有效的方法。     

Diagnosis of HCV infection by 3rd-generation ELISA in HCV-RNA positive hypertransaminasemic patients with 2nd-generation ELISA negative results

To evaluate the improved sensitivity of 3rd-generation assays for the detection of ani-HCV antibodies in diagnosing cases of HCV infection, we have re-tested by 3rd-generation ELISA test (ELISA-3) serum samples from immunocompetent patients with chronic hypertransaminasemia who were HCV-RNA positive but tested negative with 2nd-generation ELISA (ELISA-2). Out of 21 HCV-RNA positive/ELISA-2 negative samples, 3 (14.3%) were ELISA-3 positive. Among the ELISA-3 reactive samples, two were indeterminate by RIBA-3 (one was reactive with c1 00 and the other with c22), and one was negative. These results demonstrate that even in the clinical setting ELISA-3 improves the diagnosis of HCV infection. The improvement seems to be related to a better reactivity of HCV peptides rather than to the inclusion of the new determinant NS5. However, the sensitivity of the tests for the detection of anti-HCV antibodies remains to be improved.     

E2 and NS5: New antigens for detection of hepatitis C virus antibodies

The value of two new hepatitis C virus (HCV) antigens for detection of HCV antibodies was studied. These two recombinant antigens were derived from the nonstructural-5 (NS5) and envelope -2 (E2) region of the HCV genome. In a panel of 33 HCV-RNA positive samples with indeterminate Riba-2 confirmatory test results, 29 samples (88%) showed additional antibody reactivity against E2 and 12 samples (36%) snowed additional reactivity against NS5. Among 39 HCV-RNA negative, Riba-2 indeterminate donor samples, no additional E2 or NS5 reactivity was found in 34 samples (87%); while 5 samples (13%) showed additional reactivity against NS5 and/or E2. E2 reactivity thus resolved the majority of hitherto indeterminate samples. In serial samples from nine posttransfusion hepatitis C patients, NS5 and E2 antibodies did not appear earlier than classical HCV antibodies. However, E2 antibodies eventually appeared in all nine patients. The recombinant E2 might be a candidate antigen for future HCV antibody assays. © 1994 Wiley-Liss, Inc.     

丙型肝炎病毒阳性血清体外感染人肝细胞的研究

目的 研究丙型肝炎病毒(HCV)抗体(Ab)阴性,HCV-RNA阳性血清建立体外感染肝细胞模型.方法 HCV Ab阴性,HCV-RNA阳性的窗口期血清与人肝细胞共同培养,用反转录-聚合酶链反应(RT-PCR)、免疫荧光染色、Western blot、共聚焦显微镜和透射电镜等方法检测细胞内HCV核酸复制、蛋白质表达及超微结构改变.结果 细胞与病毒共同培养7～45 d,细胞内和/或培养上清中可间断检出HCV正、负链RNA;细胞浆内有HCV 核心和NS3抗原的表达;细胞超微结构有改变,并于感染后第24天时观察到类似病毒样颗粒.结论 窗口期血清中的HCV能在人肝细胞7701中复制一段时间.     

Monoclonal antibody-based antigen capture immunoassay for detection of circulating non-structural protein NS1: implications for early diagnosis of Japanese encephalitis virus infection

An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.     

Immunochemical identification and detection of a 36-kDa Toxoplasma gondii circulating antigen in sera of infected women for laboratory diagnosis of toxoplasmosis

The detection of Toxoplasma gondii circulating antigens has been indicated to be a reliable diagnostic approach of active human toxoplasmosis. However, few reports have appeared in the literature regarding the diagnostic potential of T. gondii circulating antigens. Here, a specific antibody and western blot analyses were used to demonstrate the presence of a highly reactive antigen of 36-kDa, not only in the extract of T. gondii tachyzoites, but also in selected sera of women with confirmed laboratory and clinical signs of recent toxoplasmosis. The 36-kDa Toxoplasma antigen was purified from T. gondii tachyzoites and human serum using electroelution from preparative polyacrylamide gels. The purified polypeptides showed a single peak at 10.9 min when analyzed by capillary zone electrophoresis. Based on the above encouraging results, we have developed an ELISA format for the detection of target Toxoplasma antigen (TAg-ELISA) in human serum samples. The TAg-ELISA detected the target antigen in 88% sera of acutely infected women and showed high degree of specificity (91%) among sera from non-infected women. In conclusion, the detection of 36-kDa Toxoplasma circulating antigen in human sera appears to be a promising alternative approach for laboratory diagnosis of active T. gondii infection.