Prevalence in myositis of antibodies recognizing anti-U3 RNA probably in a novel complex with 22/25 kD protein and not fibrillarin.

New antibodies against a U3 snRNP, which were named anti-Myo 22/25 antibodies, were detected in four (8%) of 53 serum samples from patients with polymyositis/dermatomyositis (PM/DM) by RNA immunoprecipitation. In the protein immunoprecipitation analysis, all four serum samples precipitated 22 kDa and 25 kDa proteins, which were not precipitated by normal serum or serum positive for antifibrillarin antibodies. Three of the four PM/DM patients had other identified autoantibodies including anti-PL-12 antibodies, antihistone antibodies (AHA), anti-SS-A antibodies and anti-SS-B antibodies defined by double immunodiffusion, ELISA or RNA immunoprecipitation, although there were no significant correlations between anti-Myo 22/25 antibodies and clinical or laboratory findings. There may be a subgroup of PM/DM patients whose sera are positive for anti-Myo 22/25 antibodies.     

Diagnostic tests for antiribosomal p protein antibodies: a comparative evaluation of immunoblotting and ELISA assays

We compared the clinical sensitivity and specificity of three different methods for the detection of serum antiribosomal P protein (anti-P) antibodies in systemic lupus erythematosus (SLE).Sera from 60 unselected SLE patients, 100 healthy subjects and 100 patients with other rheumatic inflammatory diseases were screened for anti-P antibodies by immunoblotting (IB) on P proteins from Raji cells and by two ELISA assays, one using the C-terminal 22 aminoacid long synthetic peptide (C-22) of P proteins, the other using a multiple antigen peptide (MAP) carrying four copies of the C-terminal 13 aminoacid long P peptide.Anti-P antibodies were found in 20% lupus sera by IB, 16.7% by MAP ELISA and 11.7% by C-22 ELISA. The specificity for SLE diagnosis of the three tests in healthy subjects and other rheumatic diseases was: 100% by IB, 100% (vs healthy subjects) and 97% (vs rheumatic diseases) by C-22 ELISA, 100% by MAP ELISA. The agreement between methods was good; differences in concordance rates were restricted to weak positivities. We observed a high concordance in the results of IB and ELISA methods for anti-P antibody detection. IB on P proteins extracted from human lymphoid cells is more sensitive than both ELISAs; IB and MAP ELISA perform better than the C-22 ELISA in determining weakly positive sera.     

Prevalence of hepatitis C virus infections in dialysis patients and their contacts using a second generation enzymed-linked immunosorbent assay

The prevalence of antibodies to the hepatitis C virus (HCV) was determined in 498 hemodialysis patients from three german dialysis units, 121 staff members and 42 family members using an enzyme-linked immunosorbent assay (ELISA) of the second generation which detects antibodies to a structural (C22) and to non-structural (C33c, C100, 5-1-1) recombinant antigens to HCV. Using the second generation ELISA 115 patients (23.1 %) were anti-HCV positive versus 77 (15.5%) when sera were tested by an ELISA of the first generation containing only a non-structural antigen (C100). In 34 of these 40 discordant sera antibodies against at least one viral protein was found by a recombinant immunoblot assay. Of 5 sera containing antibodies to only one viral protein (C22) 3 were HCV RNA positive by polymerase chain reaction. Epidemiological evaluation of the patients revealed that the prevalence of anti-HCV was correlated to the duration of dialysis but not to the number of blood transfusions. Of 121 staff members 2 (1.6%) and 2 of 42 family members (4.7%) were positive indicating a low risk of the patients' contacts of acquiring HCV infection.Supported by the Kuratorium für Dialyse und Nierentransplantation e.V., Emil-von-Behring Passage, W-6078 Neu Isenburg, FRG     

Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis.     

Expression of the capsid protein of Chikungunya virus in a baculovirus for serodiagnosis of Chikungunya disease

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIK fever, which typically manifests as a febrile illness. To develop a CHIKV-specific diagnostic test, CHIKV capsid protein was expressed using a baculovirus expression system. The seroreactvity of the recombinant CHIKV capsid protein was evaluated by ELISA and immuochromatographic assay (ICA), using 40 anti-CHIKV-positive and 20 anti-CHIKV-negative sera, an additional 20 normal sera samples from healthy Koreans, and 20 anti-Dengue virus sera samples. The sensitivity of the recombinant CHIKV capsid protein was 85% and 87.5% as measured by ELISA and ICA, respectively. The specificity of the recombinant CHIKV capsid protein was 100% both by ELISA and by ICA. No cross-reactivity of the capsid protein was seen with anti-Dengue virus sera samples. There was a significant correlation between the ELISA- and ICA-measured seroreactivities of the recombinant CHIKV capsid protein for anti-CHIKV IgM-positive sera samples. These results suggest that the recombinant CHIKV capsid protein could be used in a diagnostic test for identifying CHIKV disease.     

Recombinant 56-kilodalton major outer membrane protein antigen of Orientia tsutsugamushi Shanxi and its antigenicity

The gene encoding the 56-kDa protein of Orientia tsutsugamushi Shanxi was amplified by a nested PCR and cloned into the expression vector pQE30. The 56-kDa protein of O. tsutsugamushi Shanxi (Sxh56) was expressed as a fusion protein with the His(6)-binding protein of Escherichia coli by deleting the signal peptide-encoding sequence from the 5' end of the open reading frame. The recombinant protein formed inclusion bodies when expressed in E. coli M15. The recombinant protein was examined for reactivity with mouse sera against three antigenic prototypes of O. tsutsugamushi by an immunoblot assay. The recombinant Sxh56 reacted only to polyclonal antiserum to O. tsutsugamushi Gilliam in an enzyme-linked immunosorbent assay (ELISA) and in an immunoblot assay. Recombinant Sxh56 was purified by Ni-nitrilotriacetic acid affinity chromatography and injected into mice to evaluate its ability to stimulate immune responses. High levels of immunoglobulin G and T-cell proliferation appeared in mice immunized with the recombinant protein. The recombinant Sxh56 was used in an ELISA to evaluate the ability of the method to detect antibodies to O. tsutsugamushi in human and animal sera. Thirty sera from mice infected with O. tsutsugamushi Gilliam or Shanxi and 55 sera from normal mice were detected in the ELISA with recombinant Sxh56, and the sensitivity and specificity were 96.67 and 100%, respectively. One hundred fifty-one positive sera and 412 negative sera to O. tsutsugamushi Gilliam were detected in an indirect immunofluorescence assay with the recombinant protein, and the sensitivity and specificity were 96.36 and 88.08%, respectively. These results strongly suggest that the recombinant Sxh56 is a suitable type-specific immunodiagnostic antigen and vaccine candidate.     

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.     

Detection of anti-LKM-1(anti-CYP2D6) by an enzyme-linked immunosorbent assay in adult patients with chronic liver diseases.

Anti-liver kidney microsome-1 (LKM-1) autoantibody, which is a serological marker for autoimmune hepatitis type II, recognizes Cytochrome P450 IID6 (CYP2D6). This autoantibody is also detected in a portion of patients with chronic hepatitis C. Anti-LKM-1 has been measured by indirect immunofluorescence (IF) using rat liver and kidney sections. However, this method has some problems in specificity and is so laborious to handle with many samples. In this study, in order to determine anti-LKM-1, we established an enzyme-linked immunosorbent assay (ELISA) for anti-CYP2D6 using a recombinant CYP2D6 fusion protein. We studied sera from 29 patients positive for anti-LKM-1 by the new ELISA. We further studied sera from a total of 301 patients with various liver diseases and 100 sera from normal controls negative for anti-LKM-1 by the new ELISA. The specificity of the ELISA was ascertained by absorption tests using sera positive for anti-LKM-1. In 29 sera from patients positive for anti-LKM-1 by IF, we found a good correlation between the logarithms of the antibody titers determined by IF and ELISA indexes obtained by our new method. Anti-CYP2D6 was positive in 12 of 12 (100%) patient with autoimmune hepatitis type II and 16 of 17(94.1%) with chronic hepatitis C positive for anti-LKM-1 by IF. In other 401 sera negative for anti-LKM-1 by IF, anti-CYP2D6 was all negative except a few sera. We established a new ELISA for anti-LKM-1 (anti-CYP2D6). This ELISA system is sensitive, antigen-specific and easy to be done. Therefore, this assay allows a routine test of many serum samples, especially for diagnosing autoimmune hepatitis type II.     

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。     

Hepatitis C virus antibody prevalence among human immunodeficiency virus-1-infected individuals: Analysis with different test systems

Sera of 383 human immunodeficiency virus (HIV)-l-infected individuals from Frankfurt (Main)/Germany were assayed by two hepatitis C virus (HCV) screening tests (Abbott second generation, Ortho second generation). This population showed a prevalence for reactivity with both tests of 20.8% (80/383). Examination of all reactive sera (91/383) by a supplemental assay (Chiron RIBA 2) gave for 46 sera a positive, for 33 sera an indeterminate, and for 12 sera a negative result. Further analysis focussed on these RIBA 2-indeterminate and -negative samples. Analysis of the sera using an in-house Western blot with three different Escherichia coli-expressed HCV proteins revealed that none of the RIBA 2-nega-tive, but 24 of the 33 RIBA 2-indeterminate sera, including 3 of 4 c33c (NSS)-reactive samples, were reactive with a recombinant core protein. Twenty-one of 22 c22-3 (core) indeterminates stained the core antigen in the in-house Western blot and 3 of them in addition a NS5 moiety. HCV-polymerase chain reaction (PCR) was positive for 14 of the 24 RIBA 2 -indeterminate sera, but for none of the RIBA 2-negative or Western blot nonreactive samples. Discrepant results between the two screening tests could not be explained by differences in the antigen compositions (i.e., a NS3-NS4 moiety of 111 amino acids present in the Ortho enzyme-linked immunosor-bent assay (ELISA), not present in the Abbott or RIBA 2 assays). © 1994 Wiley-Liss, Inc.     

Major immunoreactive domains of human ribosomal P proteins lie N-terminal to a homologous C-22 sequence: application to a novel ELISA for systemic lupus erythematosus

The aim of this study was to identify immunoreactive domains on human ribosomal P0, P1 and P2 proteins, other than the C-22 peptide, to develop a novel ELISA using a combination of these proteins and to compare this ELISA with one using the C-22 peptide. Human recombinant P0, P1, P2 and mutant P0 lacking the homologous C-22 peptide (N-P0) were produced in bacteria and tested by ELISA and immunoblotting using sera from 48 patients with systemic lupus erythematosus (SLE), 48 with an unrelated inflammatory disorder (Crohn's disease) and 47 healthy controls. ELISA with P0, P1 and P2, premixed at equimolar concentrations, gave higher OD readings than each protein tested individually. Eighteen SLE sera tested positive by ELISA with premixed P0, P1, P2 but only 3 tested positive with the C-22 peptide. Twenty-two SLE sera reacted positively, as determined by immunoblotting, with 5 different P protein combinations: P1P2, P0P1P2, P1, P0P1, P0 and P1. Only sera reactive with all three P proteins reacted with the C-22 peptide, with absent or minimal reactivity with N-P0. Native antigens yielded sensitivity (6/48, 13%) similar to the C-22 peptide assay. An ELISA with premixed P1 and P2 gave higher OD values than the arithmetic means with P1 or P2. Fifteen SLE patients had antibodies to double stranded (ds)-DNA, of which 6 also had antibodies to P0P1P2 by ELISA but 12 reactive with P0P1P2 did not have discernable ds-DNA antibodies. Ribosomal P autoantibodies react mainly with epitopes N-terminal to a homologous C-22 peptide. An ELISA with premixed P0, P1 and P2 has 5-fold greater sensitivity (38%) for SLE than an assay with the conventional C-22 peptide (7%). The combined sensitivity for SLE for antibodies to P0P1P2 and ds-DNA is 56%, higher than C-22 and ds-DNA, 38%. Only one of the SLE patients had neuropsychiatric lupus.     

A new specific serodiagnosis system for Lyme disease: use of synthetic peptides derived from outer surface protein C of Borrelia burgdorferi

A new specific serodiagnosis system for Lyme disease was developed using the highly specific partial peptide of outer surface protein C of Borrelia burgdorferi. Finally three peptides (OspC-I, -II and -III) were selected from the outer surface protein C (OspC) amino acid sequence of Borrelia burgdorferi sensu lato and were synthesized. OspC-I is located in the region that is conserved among species of Lyme disease spirochetes, whereas OspC-II and -III are located in the variable regions of the OspC from B. garinii type strain 20047. An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against these synthetic peptides was carried out using sera from patients with Lyme disease. Furthermore, sera from patients with syphilis, tsutsugamushi disease and rheumatoid arthritis were used as control sera to demonstrate specificity of each peptide in the ELISA. The results showed that the false positive results in control sera of OspC-I, -II and -III ELISA for immunoglobulin M (IgM) antibody were 8, 22 and 16%, and those for IgG antibody were 11, 43 and 35%, respectively. These results suggested that the ELISA using OspC-I was the most specific. Therefore, sera from patients with Lyme disease were tested OspC-I ELISA. Of the 21 patients, 12 were in the acute phase and nine in the convalescent phase, 17 (81%) were positive by IgM or IgG ELISA. The sensitivities of IgM and IgG ELISA were 83 and 33% for acute-phase sera, and 22 and 78% for convalescent-phase sera, respectively, suggesting that the IgM response to OspC-I peptide was often detectable in the early stage of infection. Our data demonstrated that OspC-I was one of the common epitopes among species of Lyme disease spirochetes, and therefore this is a suitable antigen for serodiagnosis of early stage Lyme disease with high specificity.     

Performance of immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays using a West Nile virus recombinant antigen (preM/E) for detection of West Nile virus- and other flavivirus-specific antibodies

Focus Technologies developed an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a mu-capture IgM ELISA for the detection of West Nile virus (WNV)-specific antibodies based on a WNV preM/E protein recombinant antigen. Normal and disease state serum panels were used to assess the performance characteristics of the two WNV ELISA kits. Totals of 807 and 1,423 sera were used to assess the IgG ELISA and IgM ELISA kits, respectively. The Focus Technologies IgG ELISA had a sensitivity of 97.6% and a specificity of 92.1% (excluding non-WNV flavivirus sera). The comparative method for WNV IgG may lack sensitivity in detecting IgG in early WNV infection, so the specificity of the Focus IgG ELISA may be higher than 92.1%. When sera from patients either infected with or vaccinated against other flaviviruses were tested on the WNV IgG assay, 35% of the sera reacted as positive for WNV IgG. Yellow fever and Japanese encephalitis vaccinees were less reactive in the IgG ELISA than St. Louis and dengue fever patients. The Focus Technologies IgM ELISA had a sensitivity and a specificity of 99.3% (excluding the non-WNV flavivirus sera). The overall cross-reactivity for the IgM ELISA to flavivirus sera was 12%, with 31% of St. Louis encephalitis patients found to be WNV IgM positive and no yellow fever vaccinees found to be WNV IgM positive. In a selected population of 706 sera, 15 false-positive WNV IgM sera were identified. The use of a background subtraction method for the IgM ELISA eliminated all 15 false-positive results, giving a specificity of 100% for the Focus IgM ELISA.     

Antigenic diversity of Norwalk-like viruses: expression of the capsid protein of a genogroup I virus, distantly related to Norwalk virus

Summary.  The gene encoding the capsid protein of a genogroup I Norwalk-like virus (NLV) (Hu/NLV/Stav/95/Nor) was cloned and expressed in insect cells using a baculovirus vector. The His-tagged recombinant capsid protein (rStav) was antigenic and immunogenic, showed an apparent molecular weight of approximately 68 kD in protein gels, and was only soluble under denaturing conditions. The amino acid sequence of the rStav protein showed 65–88% similarity to capsid protein sequences from other genogroup I NLV and was most closely related to Desert Shield virus. Norwegian recruit sera were tested for antibodies against rStav by Western blotting (rStav WB). The sera had previously been tested for antibodies against a recombinant Norwalk virus capsid protein in an ELISA (rNV ELISA). Several rNV ELISA-negative sera showed a positive response in the rStav WB, indicating that the use of antigens representing different stains may be necessary when screening sera for antibodies against genogroup I NLV. Received July 7, 1999/Accepted November 9, 1999     

Sensitivity and specificity of an enzyme-linked immunosorbent assay using the recombinant DNA-derived Treponema pallidum protein TmpA for serodiagnosis of syphilis and the potential use of TmpA for assessing the effect of antibiotic therapy.

The recombinant DNA-derived Treponema pallidum membrane protein TmpA, purified from Escherichia coli K-12, was used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its suitability in a screening test for syphilis and to monitor the effect of antibiotic treatment. The sensitivity of the TmpA ELISA was 76% for primary syphilis, 100% for secondary syphilis, and 98% for early latent syphilis. All except 1 of 15 serum samples positive for yaws were positive in this test. A specificity of 99.6% was found by testing more than 938 donor samples. The sensitivity and specificity of the TmpA ELISA are comparable to that of the T. pallidum hemagglutination assay, and therefore the test may be useful for the diagnosis of untreated syphilis. After antibiotic treatment, the level of anti-TmpA antibodies in sera of syphilis patients dropped sharply within 1 year. Thus, TmpA might be a useful antigen for monitoring successful treatment of syphilis.     

Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening

The transmembrane envelope (TM) proteins of retroviruses are used as antigen in diagnostic immunoassays and they represent a conserved target for neutralizing antibodies. To analyze the situation in infections with the feline foamy virus (FFV), its recombinant TM protein was produced and used for ELISA and Western blot analyses. Screening sera from 404 German cats showed that 39% reacted against the TM protein, the same infection rate was determined using the Gag protein. Epitope mapping showed antibodies against the membrane proximal external region (MPER) of the TM protein in the sera from infected cats, but attempts to induce neutralizing antibodies by immunization with the recombinant TM protein failed. This is the first report demonstrating that the TM protein of the FFV is highly immunogenic and valuable for serological screening. Similar to HIV-1, but in contrast to different gammaretroviruses, immunization with the TM protein of FFV did not induce neutralizing antibodies.     

Serodiagnosis using recombinant nipah virus nucleocapsid protein expressed in Escherichia coli

Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for human and swine sera and an IgM capture ELISA for human sera were established using the recombinant NiV-N protein as an antigen. One hundred thirty-three suspected patient sera and 16 swine sera were used to evaluate the newly established ELISA systems in comparison with the CDC inactivated-virus-based ELISA systems. For the human sera, the NiV-N protein-based indirect IgG ELISA had a sensitivity of 98.6% and a specificity of 98.4%, and the NiV-N protein-based IgM capture ELISA had a sensitivity of 91.7% and a specificity of 91.8%, with reference to the CDC ELISA systems. The NiV-N-based IgM ELISA was found to be more sensitive than the inactivated-virus-based ELISA in that it captured eight additional cases. For the swine sera, the two test systems were in 100% concordance. Our data indicate that the Nipah virus nucleocapsid protein is a highly immunogenic protein in human and swine infections and a good target for serodiagnosis. Our NiV-N protein-based ELISA systems are useful, safe, and affordable tools for diagnosis of Nipah virus infection and are especially fit to be used in large-scale epidemiological investigations and to be applied in developing countries.     

用基因工程表达的抗原早期诊断鼻咽癌

目的为了建立鼻咽癌（ＮＰＣ）早期诊断方法。方法以基因工程表达的、经纯化的ＥＢ病毒（Ｅｐｓｔｅｉｎ－Ｂａｒｖｉｒｕｓ，ＥＢＶ）早期抗原（ＥＡ）成分ＥＡ－Ｄ和ＥＡ－Ｒ作为诊断抗原，建立了酶联免疫吸附试验（ＥＬＩＳＡ），检查３０例ＮＰＣ病人及４９例正常人血清中的ＥＡ／ＩｇＡ抗体。结果用ＥＬＩＳＡ检测抗体较用细胞涂片免疫酶方法（ＩＥ）敏感。ＥＬＩＳＡ检测ＮＰＣ病人血清中ＥＡ／ｌｇＡ抗体，阳性率为１００％，ＥＡ／ｌｇＡ抗体效价均≥１∶１００。而用ＩＥ法，平行检测３０例ＮＰＣ病人血清中ＥＡ／ｌｇＡ抗体效价，结果６例为阴性（＜１∶１０），抗体阳性率为７０％。ＥＬＩＳＡ明显地提高了ＮＰＣ的检出率。以ｐ１３８（ＥＡ－Ｒ）和ｐ５４（ＥＡ－Ｄ）分别或混合包被，检测对ＥＢＶ特异的ＥＡ－Ｄ和ＥＡ－Ｒ的抗体。结论表明在ＮＰＣ病人血清中存在对ＥＡ两种抗原的抗体，对ＥＡ－Ｄ的抗体滴度高于对ＥＡ－Ｒ的抗体。因此，以两种抗原混合包被作为诊断抗原建立的ＥＬＩＳＡ方法，为ＮＰＣ的早期诊断提供更敏感、特异和简便的手段。     

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.     

Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot

Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1--75%, S1-N--25%, S1-C--55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA--100% positive, S2 WB--30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases.