斑点金免疫渗滤法检测肿瘤患者血清弓形虫IgG抗体的应用研究

为建立简便、快速的斑点金免疫渗滤法 (DIGFA)检测肿瘤患者血清弓形虫IgG抗体。本文将弓形虫抗原包被于硝酸纤维素薄膜上 ,用来检测肿瘤患者血清中的弓形虫IgG抗体 ,通过胶体金———SPA直接显色 ,阳性者出现红色斑点。用该法对 2 32名肿瘤患者血清和 2 6 0名正常体检者血清进行检测 ,阳性率分别为 11. 2 1%和 1. 92 %。与IHA法对比检测 ,其结果具有良好的一致性 ,总符合率为 99. 39%。结果显示 ,斑点金免疫渗滤法检测肿瘤患者血清中弓形虫抗体简便、快速 ,适于向基层推广。     

Seroprevalence of torch infection in bad obstetric history

Primary infection with TORCH complex [Toxoplasma, Rubella, Cytomegalovirus (CMV), and Herpes simplex virus II (HSV-II)] in pregnant women can lead to adverse outcome which are initially inapparent or asymptomatic and thus difficult to diagnose on clinical grounds. Over a one-year period 380 serum samples were collected from pregnant women having bad obstetric history, attending antenatal clinic. In the present study we have shown the prevalence of Toxoplasma, Rubella, CMV, HSV-II infection in pregnant women by demonstrating the presence of IgM and IgG antibodies by ELISA test. It was found that, IgM antibodies were positive in 40 (10.52%) for Toxoplasma, 102 (26.8%) for Rubella, 32 (8.42%) for CMV and 14 (3.6%) for HSV-II. IgG antibodies were positive in 160 (42.10%) for Toxoplasma, 233 (61.3%) for Rubella, 346 (91.05%) for CMV 145 (33.58%) for HSV-II. Hence all antenatal cases with bad obstetric history should be routinely screened for TORCH as early diagnosis and appropriate intervention, will help in proper management of these cases.     

Evaluation of microparticle enzyme immunoassays for immunoglobulins G and M to rubella virus and Toxoplasma gondii on the Abbott IMx automated analyzer.

The ability of the Abbott IMx automated analyzer to detect immunoglobulin G (IgG) and IgM antibodies to rubella virus and to Toxoplasma gondii was compared with the abilities of RUBAZYME, RUBAZYME-M, ABBOTT TOXO-G enzyme immunoassay, and ABBOTT TOXO-M enzyme immunoassay, respectively. Specimens that produced discordant results were evaluated by RUBACELL II, Behring Enzygnost-Rubella enzyme-linked immunosorbent assay, Behring Enzygnost Toxoplasmosis/IgG, and bioMerieux Toxo-ISAGA (immunosorbent agglutination assay), respectively. After resolution of discordant results, IMx Rubella IgG, IMx Rubella IgM, IMx Toxo IgG, and IMx Toxo IgM antibody assays had sensitivities of 99.9, 100, 98.0, and 100%; specificities of 98.9, 99.0, 97.5, and 98.7%; and accuracies of 99.8, 99.3, 97.8, and 98.8%, respectively.     

Enzyme-immunoassays for antibodies in measles, cytomegalovirus infections and after rubella vaccination.

Enzyme-immunoassays using an indirect method with alkaline phosphatase conjugated antiglobulins were satisfactory for detection of antibody to Measles and Cytomegalovirus. The antigen was passively adsorbed to polystyrene micro-harmagglutination plates for the assays. IgM antibody to Rubella was also detected by enzyme-immunoassay at 7 and 28 days after vaccination in a person who had negative Rubella serology before vaccination. IgG antibody was detected at those times in another patient who had positive Rubella serology prior to vaccination. The enzyme immunoassays appear to have the potential for routine laboratory use for virological diagnosis.     

Performance of the Elecsys Rubella IgG Assay in the Diagnostic Laboratory Setting for Assessment of Immune Status

Rubella in early pregnancy bears a high risk for congenital defects (e.g., cataracts, hearing loss, and heart disease) and for long-term sequelae in the newborn. Despite implementation of vaccination programs in many regions, the threat of devastating consequences from congenital rubella virus infection remains and careful screening of maternal immune status before and during pregnancy helps to reduce the risk. This study compared the performance of the Elecsys Rubella IgG assay with that of other assays routinely used for screening. Samples from 1,090 women undergoing routine antenatal care were tested using the Elecsys and Enzygnost Rubella IgG assays and the hemagglutination inhibition test. Samples with hemagglutination inhibition titers of <32 (n = 148) were additionally tested using the Vidas, AxSYM, Liaison, and Architect Rubella IgG assays. Agreement of qualitative results from the Elecsys, Enzygnost, and hemagglutination inhibition assays was good in all samples. All assays showed 100.0% specificity. In samples with hemagglutination inhibition titers of <32, the Elecsys, AxSYM, and Enzygnost assays showed higher sensitivity (>90.0%) than the other immunoassays (78.6 to 82.4%). The Elecsys assay reported significantly higher rubella virus IgG levels than the other immunoassays across the whole set of 1,090 samples, with the largest bias and deviation from limits of agreement in Bland-Altman analysis. In conclusion, the Elecsys assay is highly sensitive and specific with regard to qualitative results and suitable for routine automated screening. However, given the considerable variation between quantitative results from different immunoassays, testing methods should be documented and the same assay used throughout an individual''s antenatal follow-up wherever possible.     

Acute phase serodiagnosis using Quik-Sep ion exchange chromatography

A commercial ion-exchange column method (Quik-Sep) for isolation of immunoglobulin M (IgM) free from immunoglobulin G (IgG) was evaluated. After column separation, serum IgM recovery averaged 88% (46-100%) with IgG removal averaging 95% (91-100%). Rubella and Toxoplasma gondii IgM antibodies were recovered without a change in titer, whereas IgG antibodies were removed effectively. Rheumatoid factor (RF) interference and IgG blocking antibodies were eliminated following column chromatography.     

Binding of IgG to B cell via HLA molecules

Binding of immunoglobulins to major histocompatibility complex (MHC) molecules was demonstrated by two different assays: the binding of IgG to B cells by flow cytometry, and purified MHC antigens with an Elisa assay. Fc fragment from immune-complex binds to the Fc receptor on B lymphocytes. Here, Fab was also shown to bind to B cells. This binding was inhibited by specific human allo anti-HLA Class I and II sera directed at the polymorphic sites. Thus, in addition to the Fc receptor, MHC can also serve as a binding site for IgG. In an Elisa assay using purified antigens, IgG was shown to bind to HLA Class I and II molecules. Other proteins such as transferrin, human serum albumin, gelatin, etc., did not bind to the MHC proteins. Immunoglobulins bound to MHC molecules by sites on the Fab fragment independent of the hypervariable region. This was demonstrated by the retention of antibody activity even after binding of antibody (anti-lactoferrin) to MHC. The relative avidity between Fab and HLA Class I and II was 4-8 x 10(5) M-1.     

Recombinant nucleocapsid protein-based IgG enzyme-linked immunosorbent assay for the serological diagnosis of SARS

The recombinant nucleocapsid protein (rNP) of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) was expressed in a baculovirus system. The purified SARS-CoV rNP was used as an antigen for detection of SARS-CoV antibodies in IgG enzyme-linked immunosorbent assay (ELISA). The ELISA was evaluated in comparison with neutralizing antibody assay and the authentic SARS-CoV antigen-based IgG ELISA. Two-hundred and seventy-six serum samples were collected from health care workers in a hospital in which a nosocomial SARS outbreak took place and used for evaluation. The SARS-CoV rNP-based IgG ELISA has 92% of sensitivity and specificity compared with the neutralizing antibody assay and 94% sensitivity and specificity compared with the authentic SARS-CoV antigen-based IgG ELISA. The results suggest that the newly developed SARS-CoV rNP-based IgG ELISA is a valuable tool for the diagnosis and seroepidemiological study of SARS. The SARS-CoV rNP-based IgG ELISA has an advantage over the conventional IgG ELISA in that the antigen can be prepared by laboratory workers without the risk of infection.     

Comparison of carbohydrate and peptide biotinylation on the immunological activity of IgG1 murine monoclonal antibodies

When the classical amino acid esterification procedure was used for the biotinylation of the IgG1 monoclonal antibody J28 it resulted in a loss of immunological activity. This antibody recognizes the fetoacinar pancreatic (FAP) antigen and the decrease in reactivity was directly proportional to the molar biotin/antibody ratio indicating substitutions at or near the antibody combining site. This effect was specific to J28 since the IgG1 Mab F22 which recognises the same antigen was not damaged by this procedure. Active Mab J28 conjugates were obtained using biotinylation via oligosaccharide moieties. The biotinylation efficiency using this method was dependent on the previous degree of antibody periodate oxidation and the maximal substitution was 3 mol biotin per mol of antibody. Using these conditions the sensitivity of the biotinylated J28 for the FAP antigen was similar to that obtained when using non-substituted antibody in the two antibodies technique.     

European multicenter study of the LIAISON automated diagnostic system for determination of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM and the IgG avidity index

The LIAISON system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles. The system allows fast and precise measurement of Toxoplasma-specific immunoglobulin G (IgG) and IgM antibody levels and measurement of the IgG avidity index even at low levels of Toxoplasma-specific IgG antibodies in a single step without manual interference. Seven European centers participated in a multicenter evaluation of the LIAISON system. The sensitivity and specificity of the LIAISON system compared to the Sabin-Feldman dye test were 99.3 and 96.8%, respectively. In a comparison of the LIAISON Toxoplasma-specific IgM assay with an immunosorbent agglutination assay, the LIAISON assay had a sensitivity of 96.7% and a specificity of 95.4%. The LIAISON IgG assay showed agreements of 91, 100, and 100% with the AXSYM IgG (Abbott), VIDAS IgG (bioMerieux), and Platelia IgG (Bio-Rad) assays, respectively. The LIAISON IgM assay showed agreements of 95% with the AXSYM IgM and Platelia IgM assays, 96% with the ISAGA IgM assay (bioMerieux), and 97% with the VIDAS IgM assay. The coefficient of correlation between the LIAISON system and the VIDAS Toxoplasma-specific IgG avidity index was 0.81. By use of the Toxoplasma-specific IgG avidity index assay with specific IgM-positive samples, the diagnosis of infection with Toxoplasma gondii in early pregnancy has been improved significantly. The LIAISON avidity assay is a valuable assay for the exclusion of recently acquired infection with T. gondii (less than 4 months) in pregnant women, and it decreases significantly the necessity for follow-up testing.     

Distribution of virus antibody activity among human IgG subclasses.

Human IgG subclass antibody activity against viruses was studied by separating the IgG3 fractions from sera exhibiting high titres for rubella, polio types I, II and III, and herpes I viruses. The sera were fractionated on DEAE Affi-Gel Blue and protein A-Sepharose CL-4B columns using specific subclass antisera for identification. All IgG3 fractions exhibited a molecular weight of 164,000 daltons, a pI mean of 8.21 and S20,W1% = 6.2 as determined by polyacrylamide gel electrophoresis, isoelectric focusing and analytical centrifugation. Quantitative determination of the individual subclass concentrations by nephelometry showed them to be within the biological norm. The concentration and distribution of IgG in the sera and that of the IgGl, -2 and -4 and IgG3 fractions were used as a basis for studying antiviral activity. The IgG3 fractions showed a greater ratio of IgG concentration to antibody titre than the IgGl, -2 and -4 fractions as determined in neutralization and haemagglutination inhibition tests. The IgG3 fraction from anti-rubella serum bound 96.6% 125I-labelled rubella virus (HP 77/DE5). The IgG 3-125I-rubella immune complex was separated over a protein A-Sepharose CL-4B column and confirmed with subclass-specific antisera in radial immunodiffusion plates. Individual Gm allotype analyses showed markers distributed as follows: G3m(5,-6,10,11,13,-16,21,-24) for all the serum donors indicating similar genotypic expression of IgG3s.     

化学发光法检测抗-HEV IgG方法的建立

目的 建立化学发光检测抗HEV IgG方法,用于HEV感染的实验室诊断及流行病学调查,为试剂盒研制打下基础.方法 以HEV重组抗原包被微孑孔板,辣根过氧化物酶标记的单克隆抗人IgG抗体为第二抗体,建立抗HEV IgG的化学发光检测方法,评价其灵敏度、特异性、精密性等指标.检测患者血清中抗HEV IgG抗体并与第三方试剂比较.结果 建立了检测抗HEV IgG的化学发光方法,检测500例临床患者标本并与对照试剂盒对比,阳性符合率为99.32％,阴性符合率为98.58％,总符合率98.80％,其灵敏度、特异性、重复性均达到设计要求.结论 建立了化学发光检测血清中抗-HEV IgG的方法,具有敏感性高、特异性强、操作简便等特点,适用于戊型肝炎的临床诊断和流行病学调查.     

A multicenter study of IgG and IgM toxo-test enzymun

A multicenter study was performed to evaluate Boehringer Mannheim Enzymun test toxo IgG and IgM in comparison with others IgG Elisa tests (Biotrol, Biomerieux, Abbott) or IgM Elisa test (Abbott) or in comparison with other methods (direct agglutination, indirect immunofluorescence, dye test or ISAGA). The IgG study of 700 sera and IgM study of 430 sera lead to conclude to a good sensibility and specificity of IgG and a lower specificity for IgM kit to detect toxoplasmosis.     

Automated microparticle enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii.

Fully automated microparticle enzyme immunoassays (MEIA) for the IMx immunoassay analyser were developed to detect IgG and IgM antibodies to Toxoplasma gondii. The IgG MEIA results are expressed in International Units (IU) of IgG antibody interpolated from a six point calibration curve covering the range from 0 to 300 IU/ml. Reproducible results were obtained from a calibration curve stored in the instrument for at least one month. The qualitative IgM MEIA expresses results as an index using a single calibrator included in each run. The Toxo IgG MEIA and Toxo IgM MEIA were in 98% and 97% agreement, respectively, with the reference assays used. Twenty four sera can be completely processed in about 35 minutes.     

Comparison of two techniques for detection of anti-Plasmodium falciparum antibodies: Falciparum-spot IF (Biomérieux) and Malaria IgG Celisa (BMD). Preliminary results]

We compared a new Elisa assay to detect malaria antibodies: Malaria IgG Celisa (BMD) with the IFAT technique Falciparum-spot IF (Biomérieux): sensitivity, specificity, predictive positive and negative values were 81%, 99%, 95%, 95%, respectively. Eight patients had positive thick blood smear out of 23 performed. For these eight confirmed acute malaria cases, the Elisa assay was negative in five instances. For two recent malaria attacks both Elisa and IFI were negative. With blood donors, two sera were IFAT positive and Elisa negative; 16 were IFAT doubtful and Elisa negative. Doubtful results rose up to 13.5% by IFAT against 1.5% by Elisa assay. We preferred kappa coefficient instead of chi 2 test for data analysis, which measures the concordance degree between the two techniques. Here concordance is moderate. Choosing an Elisa assay to detect the transmission of malaria for at-risk blood donors collides with the method sensitivity compared with IFAT as reference.     

Analysis of IgG subclasses (IgG1 and IgG3) to recombinant SAG2A protein from Toxoplasma gondii in sequential serum samples from patients with toxoplasmosis

The kinetics of the humoral immune response was evaluated using the recombinant SAG2A protein comparatively to soluble Toxoplasma antigen (STAg) by ELISA in sequential serum samples of patients with toxoplasmosis up to 12 months of illness onset. The follow up of IgM and IgA levels to STAg showed a gradual decrease, with the majority of patients (88%) seropositive for IgM up to 12 months of infection, whereas IgA seropositivity was relatively low (78%) compared to IgM (100%) in the first 3 months of infection. The follow up of IgG and IgG1 antibodies showed a similar increasing profile for both SAG2A and STAg, with slightly higher seropositivity for STAg. The kinetics of IgG3 to STAg was similar to that of IgG1, contrasting with the kinetics of IgG3 to SAG2A that showed high levels up to 6 months of infection, with continuous decreasing over the time. Higher IgG3 seropositivity to SAG2A than STAg was also observed in the initial phases of infection. A higher IgG3/IgG1 ratio for SAG2A than STAg was detected in the first 3 months of infection, with decreasing profile over the time. The associations of IgG3/IgG1 ratio>1.0 with positive IgM or IgA antibodies were predominantly found in the first 3 months of infection, whereas associations of IgG3/IgG1 ratio<1.0 with positive IgM or negative IgA antibodies were mostly observed from 3 to 12 months of infection. In conclusion, our results demonstrate a differential kinetics of IgG3 antibodies to SAG2A and STAg in patients with toxoplasmosis up to 12 months of infection. Also, the IgG3/IgG1 ratio to SAG2A in association with classical serological markers of acute phase could be potential tools to distinguish early acute from convalescent phases of Toxoplasma gondii infection.     

Evaluation of the Immulite 2000 Toxoplasma quantitative IgG et Toxoplasma IgM for the diagnosis of human toxoplasmosis

Four hundred and ninety five human sera with clinical and biological data were tested for the evaluation of Immulite 2000 Toxoplasma Quantitative IgG and Immulite 2000 Toxoplasma IgM produced by Diagnostic Products Corporation (Los Angeles, USA) for the diagnosis of human toxoplasmosis. The results of these kits were compared to those of the University Hospital of Nancy where the reference assays were Enzygnost Toxoplasmosis IgG and Enzygnost Toxoplasmosis IgM (Berhing-Dade, Germany), Toxoscreen (bioMérieux, France), ISAgA Plus (IgM et IgA) (bioMérieux, France). The sensitivity and the specificity of IgG detection by Immulite 2000 Toxoplasma Quantitative IgG were 98% and 100%, respectively. The high sensitivity of IgM detection by Immulite 2000 Toxoplasma IgM was adapted to the early diagnosis of toxoplasmic primo-infection and to the pediatric diagnosis or follow-up of congenital toxoplasmosis but could reveal IgM a long time after primary infection.     

Evaluation of Toxoplasma gondii immunoglobulin G (IgG) and IgM assays incorporating the newVidia analyzer system

The new Vidia system is a fully automated system based on chemiluminescence and antigen bound to magnetic microparticles, which allows a fast measurement of Toxoplasma gondii-specific immunoglobulin G (IgG) and IgM levels. The analytical performances of the Vidia Toxo IgG and IgM assays were compared with those of the automated Vidas, AxSYM, and Liaison Toxo IgG and IgM assays. The comparative evaluation was performed utilizing 204 frozen sera belonging to 166 subjects and 201 fresh sera collected from 198 subjects. For the Vidia Toxo IgG system, the sensitivities were 100% in both the retrospective and prospective studies, and specificities were 98.39% in the retrospective study and 100% in the prospective study, respectively. The sensitivities of the other three Toxo IgG assays were 100%, and the specificities ranged from 96.77% to 100%. For the Vidia Toxo IgM assay, the sensitivity and specificity were 100% in both the retrospective and prospective studies. The overall sensitivities and specificities of the other three Toxo IgM assays ranged from 80% to 100% and from 99.44% to 100%, respectively. In our study, the Vidia system revealed excellent sensitivity (100% for both IgG and IgM assays) and good specificity (99.25% for IgG and 100% for IgM assays).     

Local production of mumps IgG and IgM antibodies in the cerebrospinal fluid of meningitis patients

Immunoglobulin G (IgG) and M (IgM) antibodies against mumps virus were measured by an enzyme-linked immunosorbent assay (ELISA) in the serum and cerebrospinal fluid (CSF) specimens of patients with mumps meningitis. The CSF IgG antibodies correlated well with the respective antibody titers in serum. On the contrary, in only about half of the patients a moderate correlation was found between the CSF and serum IgM antibody titers, while the other patients did not have detectable mumps IgM antibodies in CSF irrespective of intermediate to high titers in serum. Two different immunologic mechanisms may be involved in these two groups which, however, did not show any clinical differences. The lack of IgM antibodies in the CSF of many patients diminished the value of CSF in the laboratory diagnosis of mumps meningitis compared to use of serum specimens. Intrathecal synthesis of mumps IgG antibodies was demonstrated in 83% of the patients, and of IgM antibodies in at least 67% of those patients with detectable IgM antibodies in CSF. The ratio between mumps IgG and IgM antibodies was higher in CSF than in serum, suggesting that the synthesis of IgG antibodies in central nervous system was more efficient than that of IgM antibodies.     

Particle counting immunoassay (PACIA)--VI. The determination of rabbit IgG antibodies against myelin basic protein using IgM rheumatoid factor

PACIA, which is based on latex agglutination and instrumental counting of residual non-agglutinated particles, is a practical method for the determination of IgG antibodies against myelin basic protein (MBP). These antibodies agglutinated latex to which MBP had been covalently bound by carbodiimide. The agglutination was greatly enhanced by rheumatoid factor, the final reaction being directly proportional to the amount of IgG antibody attached to the latex bound MBP. To avoid interference by endogenous IgM rheumatoid factor each sample was reduced with dithiothreitol. The antibody content of a rabbit antiserum used as a calibrator for both PACIA and a solid phase radioimmunoassay (RIA) was estimated by the Farr precipitation technique using ¹²⁵I-labelled MBP. The sensitivities of PACIA and RIA were similar i.e. 10 ng/ml IgG anti-MBP antibodies. However, the presence of serum proteins tended to decrease the agglutination reaction. The correlation coefficient between methods was 0.93. The worst coefficient of variation achieved by PACIA over daily analyses during 5 days was 23%. Despite this imprecision, which for antibody titration is still acceptable, PACIA seems an attractive method for large epidemiological studies because of its sensitivity, its short incubation time (30 min instead of 24 hr for RIA), facility of application and stability of reagents.