EB病毒潜伏膜蛋白2A抗原表位的融合表达及其意义

为了探讨含有EB病毒潜伏膜蛋白2A(LMP2A)抗原表位片断表达的融合蛋白在鼻咽癌血清学检测中的应用意义,通过重叠延伸PCR方法,合成了3对相互重叠的寡核苷酸引物,涵盖LMP2A的主要的4个抗原表位,将它们拼接在一起构建一个多肽融合基因,克隆到PGEX-4T-2载体中表达融合蛋白,以GST亲和层析柱法纯化融合蛋白,鉴定后以此为抗原检测鼻咽癌患者的血清。结果表明,获得了含EB病毒LMP2A主要的4个抗原表位的融合蛋白(EC2A),蛋白纯度达90%以上,ELISA结果显示鼻咽癌患者血清的检出率为77.9%,正常人群血清为阴性,与常规的VCA-IgA法进行比较,有9份(13.3%)血清VCA-IgA为阴性而EC2A-IgG检出阳性,为鼻咽癌的临床检测提供了新思路,也为后续的单克隆抗体制备奠定了基础。     

Application of recombinant Echinococcus granulosus antigen B to ELISA kits for diagnosing hydatidosis

Echinococcus granulosus causes human cystic echinococcosis as an important public health problem in many regions of the world. There are some problems in primary diagnosis such as cross-reaction with sera from patients with other parasitic disease in serological tests. The use of an appropriate source of antigenic material is a very important and crucial point in the improvement of the serodiagnostic features such as enzyme-linked immunosorbent assay (ELISA) method. We expressed and purified recombinant AgB of Echinococcus granulosus and used as antigen in ELISA method. Serum samples were given from 36 cystic hydatid disease patients that have been confirmed by surgical operation as well as 36 healthy individuals sera were tested by ELISA method using recombinant AgB and compared with commercial kit (Euroimmun) for specificity and sensitivities value. The sensitivity of 91.66% and specificity of 97.22% were determined by homemade kit.     

鹦鹉热嗜衣原体蛋白酶样活性因子免疫优势区基因重组蛋白的诊断应用研究

目的 检测鹦鹉热嗜衣原体(Chlamydophila psittaci,Cps)蛋白酶样活性因子(chlamydial protease-like activity factor,CPAF)免疫优势区基因在E.coli BL21中高效表达的产物在Cps感染诊断中的应用,为建立Cps感染的快速诊断奠定实验基础.方法 利用生物信息学软件筛选出Cps CPAF免疫优势区基因序列(CPAFm,A196～A450),设计特异性引物,PCR扩增目的基因,将其克隆入pGEX6p-2载体后转化E.coli BL21,利用IPTG诱导表达重组蛋白,Western blot鉴定重组蛋白.以纯化的重组蛋白作为包被抗原,建立血清学诊断的间接ELISA法;同时用商品ELISA试剂盒与建立的间接ELISA法检测180份可疑Cps感染的病鸭血清标本,将检测结果进一步用Western blot方法验证.结果 构建原核重组质粒pGEX6p-2/CpsCPAFm,表达相对分子质量约为54×103的目的蛋白产物.以纯化的重组蛋白为包被抗原建立间接ELISA法检测Cps参考血清,其阴、阳性符合率均为100％;与肺炎嗜农原体(C.pneumoniae,Cpn)、沙眼衣原体(C.trachomatis,Ct)无交叉反应.对180份可疑病鸭血清标本进行检测,与商品Birds Chlamydia Psittaci IgG ELISA试剂盒比较,以Western blot方法作对照,自建的ELISA法符合率均为100％,Birds Chlamydia Psittaci IgG ELISA Kit符合率为77.5％～95.0％.结论 以Cps CPAF免疫优势区(A196～A450)作为诊断抗原,建立血清学诊断的间接ELISA法具有较好的临床诊断价值.     

重组抗原在HSV-Ⅰ感染ELISA检测中的应用

目的　用单纯疱疹病毒(HSV)重组蛋白作为包被抗原建立一种特异性和灵敏性较高的ELISA方法。方法　将重组糖蛋白D(gD)和HSV Ⅰ分别作为包被抗原,检测5 7份临床标本,同时用国产和德国试剂盒进行检测;将德国试剂盒作为金标准,另外三者检测结果在特异性、灵敏性和符合率等方面与其进行比较。结果　与德国试剂盒相比,在特异度、敏感度、符合率方面,重组抗原分别为5 7 1%、82 0 %、78 9%。病毒抗原分别为5 7 1%、78 0 %、75 4 % ;国产试剂盒分别为10 0 0 %、4 8 0 %、5 4 4 %。重组蛋白重复性实验结果经统计学处理差异无统计学意义(P >0 0 5 )。结论　用酵母菌表达的HSV Ⅰ重组gD蛋白作为包被抗原进行ELISA检测是一种敏感、特异的方法,具有较大的应用价值。     

A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease

Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.     

Segmentation expression of capsid protein as an antigen for the detection of avian nephritis virus infection in chicken flocks

Subclinical pathological changes in the kidneys of broiler chickens and suppression of growth caused by the avian nephritis virus (ANV) affect poultry flocks worldwide. A test for detection of virus-specific antibodies in serum would be useful for epidemiological investigations, however the poor propagation in cell cultures has restricted the development of serological tests based on the use of ANV particles as antigens. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of ANV-specific antibodies in chicken serum, using a recombinant protein antigen prepared by segmentation expression of the capsid protein antigen epitope of ANV (HM029238) transfected into Escherichia coli. The expressed fusion protein was detected by Western blotting with ANV-positive serum, and the optimal immunoreactive fusion P1 protein was determined. Using the optimized P1-ELISA, ANV-specific antibodies were detected in commercial chicken flocks aged 10-25 days obtained from the Liaoning Province, China. Out of 960 serum samples, 459 (47.8%) were positive for infection with ANV. These results indicate that the P1-ELISA is helpful for preliminary serological diagnosis of ANV infection, and could be used to for screening in ANV infection and for determining antibodies against ANV.     

Establishment of an indirect ELISA method for antibody detection of porcine pseudorabies by recombinant gB,gC, and gD proteins

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide. This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET-28a⁺ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichia coli. The results of the polymerase chain reaction and enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme-linked immunosorbent assay (ELISA) analysis of 184 PRV-positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D-ELISA has the highest specificity and sensitivity among the ELISA methods developed in this study. We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein-based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.     

Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis

Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3 mg refolded protein per 200 mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.     

Development and application of a double-antigen sandwich enzyme-linked immunosorbent assay for detection of antibodies to porcine circovirus 2

A double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) is described for detection of porcine circovirus 2 (PCV2) antibodies using the well-characterized recombinant PCV2 capsid protein. In a comparative test of 394 pig sera against an indirect immunofluorescence (IIF) test and a commercial ELISA kit (also based on the recombinant PCV2 capsid protein), the results showed that the diagnostic sensitivity, specificity, and accuracy of the assay were, respectively, 90.61, 94.02, and 91.62% compared with IIF and 94.38, 95.28, and 94.67% compared with the commercial ELISA kit. Assay of 12 PCV-free pigs over a 5-week period produced only PCV2-negative titers by all 3 methods. These results and the seroprofiles of 4 pig farms obtained by both the commercial ELISA kit and the double-antigen sandwich ELISA indicate that the sandwich ELISA is a reliable method for detection of antibodies to PCV2. Additionally, the method described here permits the use of undiluted test serum samples simultaneously loaded with horseradish peroxidase (HRP)-conjugated antigen into the test well, and the complete test procedure can be performed in less than 90 min. This double-antigen sandwich ELISA should be a useful tool to aid swine industry professionals in deciding the intervention strategies for the control of PCV2-associated diseases.     

Evaluation of a New Immunochromatographic Test Using Recombinant Antigen B8/1 for Diagnosis of Cystic Echinococcosis

Diagnosis of cystic echinococcosis (CE) is based on the identification of the cyst(s) by imaging, using immunodiagnostic tests mainly as complementary tools in clinical settings. Among the antigens used for immunodiagnosis, previous studies described a good performance of the recombinant antigen B8/1 (rAgB) in an enzyme-linked immunosorbent assay (ELISA) format; however, in remote parts of areas where the disease is endemic, the implementation of an ELISA is difficult, so a more simple, rapid, and reliable method such as the immunochromatographic test (ICT) is required. In this study, using a set of 50 serum samples from patients with surgically confirmed CE, we compared the performance of an ICT and that of an ELISA using the rAgB. The overall sensitivities of ICT and ELISA were not statistically different (78% versus 72%; P = 0.36). The overall agreement between both tests was moderate (κ = 0.41; P < 0.01). Concordance between ICT and ELISA was substantial or almost perfect for patients with liver involvement (κ = 0.65; P < 0.001) and patients with more than one hydatid cyst (κ = 0.82; P < 0.001), respectively. Moreover, specificity analysis using a total of 88 serum samples from healthy individuals (n = 20) and patients (n = 68) with other parasitic infections revealed that ICT had a specificity of 89.8%. ICT and ELISA had similar performance for the detection of specific antibodies to E. granulosus, and ICT had a high specificity, opening the possibility of using ICT as a screening tool in rural settings.     

Nipah virus glycoprotein: production in baculovirus and application in diagnosis

A method for serological diagnosis of Nipah virus (NiV) is described. DNA encoding truncated G protein of NiV was cloned into the pFastBac HT vector, and the fusion protein to His-tag was expressed in insect cells by recombinant baculovirus. The resulting His-G recombinant fusion protein was purified by affinity chromatography and used as the coating antigen for serological testing by indirect enzyme-linked immunosorbant assay (ELISA). When tested against a panel of swine serum samples, the recombinant G protein-based ELISA successfully discriminated all 40 samples previously determined to be serum neutralizing test (SNT) positive from 11 SNT negatives samples. The data show that the recombinant G protein exhibits the antigenic epitopes and conformation necessary for specific antigen-antibody recognition. The main advantage of the recombinant G protein-based NiV ELISA compared to an ELISA using whole virus antigen is the use of a single antigenic protein instead of inactivated whole virus which is required to be prepared under high risk and cost. This test is suitable for routine diagnosis of NiV and also for epidemiological surveys as it allows highly reliable testing of a large number of sera rapidly.     

Serodiagnosis of sheep hydatidosis with hydatid fluid, protoscolex, and whole body of Echinococcus granulosus antigens

Diagnosis of hydatidosis is based on immunodiagnostic methods along with radiological and ultrasound examinations. A great number of immunological assays have been developed for detection of anti-hydatid cyst antibodies. The principal intermediate host of Echinococcus granulosus in most endemic regions of the world is sheep. Antibodies to various antigens are detectable in the sera of some, but not all infected sheep. The objective of the present study was to develop a specific and simple antigen-based ELISA method for diagnosis of hydatidosis in sheep with different (hydatid fluid, protoscolices, and whole body of E. granulosus) antigens. A total of 100 sera were collected from sheep with hydatidosis proven by inspection of hydatid-infested livers and lungs of the sheep slaughtered in Mashhad abattoir. Hydatid fluid and protoscolex were isolated from livers or lungs of sheep with hydatid cyst in sterile conditions. Whole body of E. granulosus was isolated from intestine of infected dogs. Sera samples were examined by ELISA with different antigens. The results of antibody detection by indirect ELISA, using different antigens, showed that the hydatid fluid was the most effective antigen of those assessed for detection of infection with hydatidosis in sheep. Findings of this study indicated that antibody detection assay is a sensitive approach for diagnosis of hydatid cyst in sheep.     

Use of a recombinantDictyocaulus viviparus antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of bovine dictyocaulosis

A specific recombinant antigen was evaluated for its immunodiagnostic potential in an enzyme-linked immunosorbent assay (ELISA). The antigen was used as a glutathione S-transferase (GST) fusion protein (DvGST3-14) or as a pure recombinant parasite protein (Dv3-14). A total of 55 sera from cattle experimentally infected withDictyocaulus viviparus, 24 sera from naturally infected cattle and 25 sera from helminth-free cattle were examined. ELISA results obtained for the sera from experimental infections showed a calculated specificity of >99% for both antigen preparations had a sensitivity of 93% (DvGST3-14) and 91% (Dv3-14). For field sera from natural infections, the specificity was calculated to be 90% (DvGST3-14) and >99% (Dv3-14) and the sensitivity, 85% (DvGST3-14) and 70% (Dv3-14).Dedicated to Prof. Dr. K.T. Friedhoff on the occasion of his 60th birthdayThis work was supported by grant Az Schn 267/4-1 from the Deutsche Forschungsgemeinschaft     

The immunodiagnostic potential of Echinococcus granulosus adult-worm antigens in human cystic echinococcosis

 Echinococcus granulosus adult-worm antigens were characterised for assessment of their immunodiagnostic potential for human cystic echinococcosis (CE). The analysis of worm extracts by enzyme-linked immunosorbent assay (ELISA) showed a sensitivity of 83% for CE, which is comparable with data obtained for cyst-fluid-based serodiagnostic tests. Immunoprecipitation of in vitro-translated E. granulosus worm mRNA revealed a range of low-molecular-weight antigenic proteins (12–45 kDa) recognised by human CE sera. E. granulosus adult worms may provide an additional or alternative source to metacestode material for the isolation of both native and recombinant antigens to be considered for the serodiagnosis of human echinococcosis. Received: 15 April 1996 / Accepted: 22 July 1996     

Expression of Peste des petits ruminants virus nucleocapsid protein in prokaryotic system and its potential use as a diagnostic antigen or immunogen

In this study, both partial and full-length nucleocapsid (N) gene of Peste des petits ruminants virus (PPRV) were cloned into pET33b vector and expressed in Escherichia coli (BL21) with the objective of replacing live PPRV antigen with recombinant protein in ELISA. The expressed proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot by using a PPRV N protein specific monoclonal antibody. The expressed histidine-tagged fusion proteins were purified using affinity Ni-NTA column and were assessed for their conformation in terms of reactivity by ELISA. The immunogenicity of recombinant proteins was also assessed in rabbits and anti-N antibody response against PPRV was observed in all the immunized rabbits, when tested by competitive and indirect ELISAs. In sandwich ELISA, a mean OD_492 nm of 1.4 and 0.90 was obtained for crude lysate having expressed the N protein and the PPRV antigen, respectively. Further, the N protein was tested as a coating antigen in competitive ELISA instead of PPRV antigen for serological diagnosis of PPR infection. This indicates the diagnostic potential of the PPRV recombinant N proteins, which are safe and better alternatives to live PPRV antigen in ELISA for clinical or sero-surveillance of PPR in enzootic or non-enzootic countries.     

Combination of native and recombinant cytomegalovirus antigens in a new ELISA for detection of CMV-specific antibodies

BACKGROUND: Serological detection of cytomegalovirus (CMV)-specific antibodies varies greatly due to antigen composition and the lack of antigen standardization. OBJECTIVES: To develop and evaluate a new ELISA with native and/or recombinant cytomegalovirus antigens for the detection of anti-CMV IgG and IgM antibodies. RESULTS: The diagnostic performance of three anti-CMV ELISAs coated with different CMV antigen preparations, (i) native CMV antigen, (ii) a mixture of recombinant CMV peptides pp150, pp28, gB2 and pp52 and (iii) a combination of native CMV antigens and recombinant CMV IE1 antigen applied in the new Genzyme Virotech CMV ELISA, were compared. All tested sera were derived from patients or healthy blood donors and were predefined with the Dade Behring Enzygnost((R)) CMV ELISA as well as by CMV PCR analysis. Additionally, official well-characterized serum panels were also tested. The new Genzyme Virotech CMV ELISA IgG/IgM test applying a combination of native antigens and recombinant IE1 antigen was evaluated and the performance was compared to the Dade Behring Enzygnost((R)) CMV ELISA. The sensitivities were 98.9% (IgG) and 98.2% (IgM), the specificities were 98.8% (IgG) and 98.9% (IgM) for the Genzyme Virotech CMV ELISA. Furthermore all sera of the BBI mixed titer performance panel as well as the BBI seroconversion panel were identified 100% correctly with the new Genzyme Virotech ELISA. CONCLUSIONS: These data suggest that the new Genzyme Virotech CMV ELISA has higher sensitivity and specificity than ELISAs based on native antigens or recombinant peptides only. Specific combinations of native and recombinant antigens increase the serological detection of CMV infections and may add to further standardization of CMV serology.     

Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women

An enzyme-linked immunosorbent assay (ELISA) using two recombinant antigens of Toxoplasma gondii (GRA1 and GRA6 Nt) was developed in order to differentiate between pregnant women with a serological profile of recently acquired infection and those with chronic infection. Both proteins were expressed in Escherichia coli as glutathione S-transferase fusion proteins. Thirty-two serum samples from subjects who presented seroconversion within 3 months before sampling (group 1; acute profile), 46 serum samples from women who had a positive serology at least 1 year before sampling (group 2; chronic profile), and 100 serum samples from pregnant women who were not infected by T. gondii (group 3) were examined for immunoglobulin G (IgG) reactivity. For both antigens, the specificity reached 98%. In both groups of infected patients, the overall sensitivity scored was 60% for GRA1 and 83% for GRA6 Nt. In group 1, 34% of sera reacted with GRA1 whereas 84% of sera reacted with GRA6 Nt; in group 2, however, sensitivities were 78.2 and 82.6%, respectively. Combination of the readings obtained with both antigens yielded a sensitivity of 91%. A serological follow-up of 10 women who seroconverted during pregnancy displayed three different serological patterns: (i) a GRA profile paralleling the IgG curve, as detected by the commercial kit, (ii) a GRA1 profile, or (iii) GRA1 and GRA6 Nt profiles remaining negative for at least 8 weeks after the reference test gave positive results. Taken together, these results suggest that neither GRA1 nor GRA6 Nt is sensitive enough to be used routinely to differentiate between acute and chronic toxoplasmic infections.     

Application of a recombinant Echinococcus multilocularis antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of human alveolar echinococcosis

A highly antigenic polypeptide fragment of the recombinant Echinococcus multilocularis antigen II/3 was produced in Escherichia coli and purified for application in enzyme-linked immunosorbent assay (ELISA). The antigen II/3-encoding 1.0 kb DNA sequence was reduced by sonication into smaller DNA fragments which were subsequently cloned into lambda gt11. Three clones could be isolated from the sublibrary, all synthesizing a recombinant antigen as a stable beta-galactosidase fusion protein. In a further step, the 0.6-kb insert from one positive clone was subcloned into the plasmid pAR 3038, which directed efficient synthesis of the antigen fused to only 11 amino acids from the N-terminus of the phage T7 major capsid protein. The plasmid-encoded antigen (antigen II/3-10) was purified from a bacterial cell extract and then tested in an ELISA. Using sera from 88 patients with an E. multilocularis-infection, a high diagnostic sensitivity of 90% was demonstrated. Investigation of sera from 220 patients with various helminthic infections showed a specificity of 99%, suggesting the suitability of the antigen II/3-10 as an immunodiagnostic tool.     

重组表达的着丝粒蛋白-B N-端抗原在抗着丝粒自身免疫病血清检测中的应用

目的 分析重组表达的CENP-B抗原对抗着丝粒自身免疫病血清的鉴定结果，探讨其作为临床诊断指标的可行性。方法 通过构建原核表达载体，在大肠杆菌中高效表达CENP-B-N端300个氨基酸的1段多肽；纯化包涵体，制备重组表达的CENP-B抗原，并利用ELISA、Western blot的方法对临床诊断为阳性的ACA血清进行鉴定。结果 重组表达的CENP-B N-端抗原对ACA血清的ELISA阳性检出率为99％，Western blot阳性检出准确率为97％。结论 重组表达的CENP-B抗原可以作为抗着丝粒自身免疫病血清临床诊断的1个指标。     

Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes

Neurocysticercosis (NCC) caused by infection with the larvae of Taenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using an Escherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.