重组冠状病毒核衣壳蛋白血清抗体检测

目的 SARS冠状病毒的核衣壳(Nucleocapsid)蛋白(N蛋白)是病毒的主要结构抗原，重组N蛋白可用作抗原检测患者血清中相应抗体。方法 以纯化的目的蛋白N-1，N-2分别包被96孔板，检测正常人群及患者血清中抗SAKS病毒抗体。结果 检测SAKS感染患者血清，N-1阳性检出率为55．68％，N-2阳性检出率为56．82％，与华大基因试剂盒相比符合率分别为90．12％和87．65％。结论 重组核衣壳蛋白可做为检测SARS抗体的抗原蛋白。     

Development and evaluation of serological assays for detection of human hantavirus infections caused by Sin Nombre virus.

BACKGROUND: The hantavirus cardiopulmonary syndrome (HCPS) was first recognized in 1993 after a cluster of acute respiratory distress syndrome deaths in the southwestern of the United States. The major causative agent of HCPS in North America is the Sin Nombre virus (SNV) carried by the deer mouse Peromyscus maniculatus. The first HCPS case imported to Europe was reported in 2002. OBJECTIVES: The objective of the study was to develop and evaluate ELISA and Western blot tests for the serological detection of human infections caused by SNV including those imported to Europe. STUDY DESIGN: A polyhistidine (His)-tagged recombinant nucleocapsid (rN) protein of SNV was expressed in Saccharomyces cerevisiae and purified by nickel chelation chromatography. On the basis of the purified SNV rN protein mu-capture and indirect IgM and IgG ELISAs and an IgG Western blot were developed. The evaluation of the tests was performed using a negative serum panel and a blinded serum panel from the US containing acute-phase sera from HCPS patients. RESULTS: Based upon the results obtained using a panel of negative control sera the specificity for SNV mu-capture and indirect IgM and IgG ELISAs were found to be 100%. All 33 sera from SNV-infected HCPS patients included in the blinded panel were detected by the SNV mu-capture and indirect IgM ELISAs. Twenty-nine out of the 33 SNV-IgM positive sera reacted also in the SNV-IgG ELISA. An SNV-IgG Western blot confirmed the data of the SNV-IgG ELISA. Although the majority of anti-SNV positive sera cross-reacted with rN proteins of Puumala virus and Dobrava virus, the lacking reactivity of a few sera with these heterologous rN antigens in the corresponding IgM and IgG ELISAs demonstrates the value of virus-specific test formats for acute-phase sera. CONCLUSIONS: The novel SNV ELISA and Western blot tests represent a useful tool for the serological detection of SNV infections.     

Development and evaluation of a new ELISA for the detection and quantification of antierythropoietin antibodies in human sera

Assays for the analysis of antierythropoietin antibodies (anti-EPO Abs) currently suffer from a high degree of nonspecificity or are cumbersome and time consuming to perform. They are therefore not well suited for the analysis of large numbers of human sera samples, a task that has become increasingly important due to an increase in the number of patients developing anti-EPO Abs. The objective of this study was to develop and validate a sensitive and specific ELISA for the determination of anti-EPO Abs that would suit these purposes. In this new double antigen bridging ELISA, anti-EPO Abs bind via one site to recombinant human erythropoietin (rhEPO)-biotin immobilized to streptavidin-coated microtiter plates (MTPs) and by a second site to rhEPO labelled with digoxigenin (DIG). The amount of bound antibody is determined using an anti-DIG antibody coupled to peroxidase. A rabbit polyclonal anti-EPO Ab purified by immunoadsorption is used as reference antibody preparation. The dynamic range of this ELISA was 1-75 ng/ml per assay calibrated with the reference antibody preparation. The assay was specific for anti-EPO Abs and did not react with other immunoglobulins (Ig) present in human serum. The lower limit of detection (LLD) of the assay was 0.5 ng/ml, and the lower limit of quantitation (LLQ) was 1.0 ng/ml. Anti-EPO Abs could be detected in the sera of pure red cell aplasia (PRCA) patients. In contrast to previous reports, no anti-EPO Abs could be detected in the sera of patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjogren's syndrome (SS), or in the sera of dialysis patients.     

人冠状病毒HKU1血清学检测方法的建立及其初步应用

目的 表达HCoV-HKU1的壳蛋白（N蛋白）及棘突蛋白（S蛋白）,建立检测血清中相应抗体的方法.方法 使用原核表达系统表达纯化HCoV-HKU1的N蛋白,转膜制条建立基于蛋白印迹的N抗体检测方法.构建HCoV-HKU1 S蛋白的真核表达质粒,转染细胞后,用间接免疫荧光（IFA）建立S抗体检测方法.利用已建立的N抗体和S抗体检测方法,检测了100份正常成人血清.结果 经Western Blot检测,S和N蛋白表达正确;利用已建立的血清学检测方法分析100份正常成人血清,其中HCoV-HKU1 S抗体的阳性率为47%,N抗体的阳性率为48哂,S抗体和N抗体均阳性的占总数的21%,双抗体均阴性的占总数的22%.S抗体和N抗体共同检测可获得74%的阳性检出率.结论 所建立的方法可用于HCoV-HKU1血清学分析,共同检测HCoV-HKU1 S抗体和N抗体可获得较好的检测结果.在正常成年中HCoV-HKU1抗体阳性检出率较高.     

Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay

The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.     

Development of a recombinant truncated nucleocapsid protein based immunoassay for detection of antibodies against human coronavirus OC43

Human coronaviruses are one of the main causes of upper respiratory tract infections in humans. While more often responsible for mild illness, they have been associated with illnesses that require hospitalization. In this study, an assay for one of the human coronaviruses, OC43, was developed using a truncated recombinant nucleocapsid (N) protein antigen in an enzyme immunosorbent assay (ELISA) and evaluated using serum collected from HCoV-OC43-infected patients, healthy adults, and patients with other respiratory virus infections. Results showed that the diagnostic sensitivity and specificity of the assay were 90.9% (10/11) and 82.9% (39/47), respectively. To evaluate the clinical utility of the ELISA, serum samples collected from patients during an outbreak of HCoV-OC43 infection and previously identified as positive by HCoV-OC43 whole N ELISA were screened resulting in 100% diagnosis agreement between the testing methods. These results suggest that this assay offers a reliable method to detect HCoV-OC43 infection and may be a useful tool in coronavirus seroepidemiological studies.     

Comparative evaluation of 15 serological assays for the detection of syphilis infection

Fifteen commercial syphilis kits were assessed against the same moderately sized specimen panel that included 114 serum and plasma specimens from syphilis cases and 249 specimens from unselected blood donors. The 114 specimens from syphilis cases comprised 40 from cases of primary syphilis, 43 from cases of secondary syphilis, 19 from cases of early latent syphilis, and 12 from cases of late latent syphilis. Of the 15 kits, ten were enzyme immunoassays, four were Treponema pallidum haemagglutination assays, and one was a T. pallidum particle agglutination assay. Thirteen of the 15 kits gave final specificities of 100%; the other two kits were repeatedly reactive with one to two specimens. Initial sensitivities ranged from 93.9 to 99.1%. Most variation between kits was observed in results for the groups with untreated primary and treated late latent disease, although the differences were not statistically significant. The comparative data on kit performance derived from this study is useful for examining syphilis testing guidelines and for making informed purchasing decisions.     

Evaluation of diagnostic accuracy of 10 serological assays for detection of SARS-CoV-2 antibodies

European Journal of Clinical Microbiology & Infectious Diseases - Antibody detection is essential to establish exposure, infection, and immunity to SARS-CoV-2, as well as to perform...     

Recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins

Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.     

Evaluation of three serological tests for detection of antibody toPseudomonas aeruginosa in human sera

Four hundred and ninety-five sera from 325 patients from whomPseudomonas aeruginosa had been isolated and 86 control sera were tested for antibody by indirect haemagglutination tests (HAT) and complement fixation tests (CFT) using a polyvalent pseudomonas serotype-specific vaccine antigen, PEV-02. Sera were also tested by countercurrent immunoelectrophoresis (CIE) for precipitins to a species-specific protein antigen. Control sera gave titres of 160 or less by HAT and 20 or less by CFT. 2-Mercaptoethanol resistant antibody titres (immunoglobulin G) were below 40 for all control sera and none of the latter contained precipitins to common antigen. Of 325 patients, 156 (48%) gave titres of 320 or greater by HAT and of these, 114 (73%) showed elevated immunoglobulin G titres. Less patients with positive blood cutures than expected were positive by HAT and more patients with bone infections gave raised immunoglobulin G titres than expected. Cystic fibrosis patients were invariably seropositive by all tests. There was a correlation between positive CIE and CFT tests, especially in patients who were positive by HAT. Approximately half of 83 patients tested gave a serotype-specific antibody response. The tests were of little value in confirming clinically evident acute infections, but in cases of doubtful infection they did provide confirmatory evidence of an antibody response in approximately one-third of patients culture-positive forPseudomonas aeruginosa.     

Comparison of serological assays for detection of Chlamydia trachomatis antibodies in different groups of obstetrical and gynecological patients

New serological enzyme immunoassays (EIAs) were compared with microimmunofluorescence (MIF) as a "gold standard" to detect Chlamydia trachomatis antibodies in different groups of obstetrical, gynecological, and subfertile patients. There were no significant differences in seroprevalence rates, except for the group of C. trachomatis-positive patients (P < 0.01). Test characteristics were calculated for Chlamydia-EIA (Biologische Analysensystem GmbH, Lich, Germany) and pELISA (Medac, Wedel, Germany). pELISA seems to be a good alternative to MIF. It has high specificity and is easier to perform.     

Development of a human-murine chimeric immunoglobulin M for use in the serological detection of human alphavirus antibodies

Diagnosis of human alphaviral infections relies on serological techniques, such as the immunoglobulin M antibody capture-enzyme-linked immunosorbent assay (MAC-ELISA). We have humanized the alphavirus broadly cross-reactive murine monoclonal antibody 1A4B-6 to create a reagent capable of replacing human positive sera in the MAC-ELISA for diagnosis of human alphaviral infections.     

Comparison of six serological assays for human immunodeficiency virus antibody detection in developing countries.

Three commercially available assays for the detection of human immunodeficiency virus (HIV) antibodies-Vironostika enzyme immunoassay (EIA), Wellcozyme competitive EIA, and JLC Allaman indirect immunofluorescence assay--were tested on 300 serum samples from African subjects with and without HIV-related conditions. Two experimental assays both rapid and simple to perform (Biotech dip stick and Cambridge Bioscience latex agglutination) were also evaluated on the same serum samples. The results were compared with those of a commercial Western blot (WB) (immunoblot) assay from Biotech, used as the reference technique. All assays were tested in the laboratory of the AIDS Project in Kigali, Rwanda. Calculated specificity ranged from 90.8% (dip stick) to 98.6% (Vironostika EIA, Wellcozyme competitive EIA, and Cambridge Bioscience latex agglutination). Sensitivity ranged from 95.2% (Cambridge Bioscience latex agglutination) to 98.0% (Vironstika EIA) and JLC indirect immunofluorescence assay). However, the sensitivity of the latex agglutination test improved to 98.6% after the prozone effect was controlled for by serial twofold dilution of latex agglutination-negative, WB-positive samples. In situations with a high prevalence of HIV infection, any one of these tests can be regarded as an alternative to the more expensive, time-consuming, and difficult WB assay.     

Comparison of radioimmunoprecipitation assays for the detection of human anti-tumor virus antibodies

The demonstration of human antibodies reactive in radioimmunoprecipitation assays (RIAs) with primate tumor virus (oncornavirus) antigen has implications for a possible viral etiology of certain human tumors. The positive results reported by us contradicted previously published negative findings and led to considerable scientific controversy. We feel much of the discrepancy may be of methodological origin. An attempt is therefore made in this communication to resolve these apparent discrepancies by comparing various published parameters of the RIAs used in the search for human antibodies reactive with oncornavirus antigens.     

Comparison of serologic assays for detection of antibodies against human herpesvirus 8

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.     

Enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein N

Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.     

Performance of serological assays for early detection of human immunodeficiency virus type 1 seroconversion.

Early detection of sexually transmitted human immunodeficiency virus type 1 (HIV-1) infection was investigated in newly infected persons to determine the sensitivities of currently available serologic techniques. Serial serum samples were obtained from 51 newly infected persons in a cohort of 1,153 homosexual or bisexual men participating in the Baltimore Center of the Multi-Center AIDS Cohort Study during the first 2.5 years of follow-up. Of 51 participants, 45 seroconverted between any two seminannual visits and 6 were found to have been infected just prior to study entry. Five enzyme-linked immunosorbent assays (ELISAs), two immunoblots, and an HIV-1 P24 antigen capture assay were performed on a panel of all serial serum samples from these individuals. The sensitivity of ELISAs varied between 50 and 84% in seroconverters with less-developed antibody response. In this group of seroconverters, the most sensitive antibody assay was an immunoblot from Biotech (95%) and HIV-1 P24 was found infrequently (9.5%). The sensitivities of ELISAs and immunoblot were 100% in individuals with more-developed antibody patterns, and no HIV-1 P24 was detected among them.     

Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8.

BACKGROUND: Serologic assays for the detection of antibodies to human herpesvirus type 8 (HHV-8) are important for epidemiological studies and to further investigate the proposed pathogenesis of the virus in cancer. Although a variety of assays are available, a lack of optimization and standardization makes their usefulness uncertain, and may be responsible for the controversy concerning the prevalence of infection. OBJECTIVES: To refine an indirect immunofluorescent assay (IFA) for the detection of latent antibodies and a recombinant ORF 65 ELISA for the detection of lytic antibodies in order to increase their ability to differentiate individuals at higher and lower risk for HHV-8 infection. STUDY DESIGN: Sera from Kaposi's sarcoma (KS) patients and blood donors (BDs) were used to modify assay parameters in an attempt to better discriminate between the two populations. Modifications included methods of substrate fixation, incubation times, sample dilution, and antigen/conjugate concentrations. RESULTS: Optimal modifications to the latent IFA included acetone fixation of substrate, and dilution of sera to 1:64 which enhanced detection of HHV-8 antibodies from 68 to 92% in the KS population. Similarly, successful refinement of the ORF 65 ELISA to increase the signal-to-noise ratio included the use of 88 ng of ORF 65 antigen per well and serum dilutions of 1:50. Optical density-to-cut-off ratios directly correlated with titers, thereby introducing a strategy to predict antibody concentrations. The ORF 65 ELISA and the latent IFA were both able to discriminate between the two populations but with different efficiencies. CONCLUSIONS: Although neither the latent IFA nor the ORF 65 ELISA produced perfect test indices, improvement in their performances was noted following the optimization strategies. The ELISA produced better detection of antibodies to the virus than the IFA and permitted prediction of sample titers, thus improving cost and time effectiveness.     

Anti-cholesterol antibodies in human sera

In the last 30 years many research showed that high serum cholesterol level is a great risk for the atherosclerosis. In recent years, it has become clear that the immune system has a major role in atherosclerosis development and progression, and has binding capacity to cholesterol as well. It has been demonstrated in animal experiments, that anti-cholesterol antibodies (ACHA) can prevent cholesterol diet induced atherosclerosis. Our group is looking for the answer, whether ACHA have the same function in animals and in humans, or not. In this review we summarize our studies in human sera. We measured serum ACHA levels in different groups of patients with atherosclerotic diseases in patients with viral infections and in healthy population. In the summary we write about the possible functions of ACHA in the human immune system.