快速胶体染料试纸条法检测弓形虫病IgG抗体的研究

目的研制一种检测弓形虫病IgG抗体的快速胶体染料试纸条法(DDIA),与国外进口试剂盒进行比较,以评价其临床应用价值。方法以本实验室筛选的胶体染料D1标记羊抗人IgG,以此标记物与弓形虫病人血清中的抗弓形虫IgG抗体结合,用点有弓形虫可溶性抗原的硝酸纤维膜通过层析捕获染料标记羊抗人IgG与弓形虫IgG抗体的复合物,采用正交试验确定DDIA的最佳检测条件;同时对弓形虫病流行病学筛查时的88份血清进行检测并与进口试剂盒的检测结果进行比较。结果DDIA的最佳检测条件为弓形虫可溶性抗原的点膜浓度为1mgml,人IgG点膜浓度为2mgml,血清用量为10μl和羊抗人IgG胶体染料检测液作1∶4稀释时的检测带清晰且背景较浅;DDIA与进口试剂盒的总符合率为97.7%,其中阳性和阴性符合率分别为100%(5252)和94.4%(3436)。结论DDIA与进口试剂盒有较高的符合率,表明该法具有较好的弓形虫病诊断价值,可进一步推广应用。     

Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae.

An enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae was developed. The EIA was evaluated on the basis of results in the M. pneumoniae complement fixation (MPCF) test and the cold agglutinin test. Serum samples from 430 patients with respiratory infections of known or unknown etiology, from 91 healthy children and adults and from 20 patients with rheumatoid factor, were investigated. By the criteria chosen for positive diagnostic EIA values, we found that the combined measurement of specific IgM and IgG gave a specificity of 99.7% and a sensitivity of 97.8%. If only IgM antibodies were measured, the specificity was 100% and the sensitivity was 88%. For IgG alone the specificity was 99.7%, but the sensitivity was only 46% because of the high EIA cutoff value chosen for IgG. We found no false positives among serum samples from patients with non-M. pneumoniae respiratory infection of known etiology, and there were no false IgM positives due to rheumatoid factor. In some cases the IgM EIA results became positive earlier in the course of illness than the MPCF titer. While children and teenagers responded predominantly with IgM antibodies, patients older than 40 years often had an IgG response only (56% of cases), probably because of reinfection. We conclude that this EIA is a good alternative to the combined MPCF and cold agglutinin tests in the diagnosis of M. pneumoniae infection.     

Detection of immunoglobulin G (IgG) and IgM antibodies to Toxoplasma gondii: evaluation of four commercial immunoassay systems.

A comparative evaluation of the following commercial immunoassays for the determination of antibodies to Toxoplasma gondii was performed: Behring Diagnostics OPUS Toxo G and Toxo M, Abbott Diagnostics IMX Toxo-IgG 2.0 and Toxo-IgM, Sanofi Diagnostics Pasteur Platelia Toxo IgG and Toxo IgM, and bioMérieux Vitek VIDAS Toxo IgG and IgM. Of 676 specimens that were tested for Toxoplasma-specific immunoglobulin G (IgG) antibodies, 26% were reactive by all methods while 8% displayed some discrepancy. Of 718 specimens that were tested for Toxoplasma-specific IgM antibodies, 3% were reactive by all methods while 10% displayed some discrepancy. Analysis of discrepant specimens revealed performance shortcomings with all IgM-specific assays. The impact of such shortcomings is magnified in a population with a low prevalence of toxoplasmosis.     

LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera.

We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.     

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population.     

Enzyme immunoassay for detection of immunoglobulin G (IgG), IgM, and IgA antibodies against type 6B pneumococcal capsular polysaccharide and cell wall C polysaccharide in chinchilla serum.

Conjugation of the capsular polysaccharides of Streptococcus pneumoniae to protein carriers has introduced a new generation of pneumococcal vaccines which may be efficacious in preventing pneumococcal otitis media during infancy. The chinchilla model has been used extensively for studying the pathogenesis of pneumococcal otitis media and for testing the efficacy of early pneumococcal capsular polysaccharide (PCP) vaccines, but immunologic studies in the chinchilla have been limited by the lack of antibodies against specific immunoglobulin isotypes. By using affinity-purified rabbit immunoglobulin G (IgG) anti-chinchilla IgG, IgM, and IgA, we developed a sensitive enzyme immunoassay that is highly specific for IgG, IgM, and IgA antibodies against type 6B PCP (anti-6B) and against C polysaccharide in chinchilla serum. Antibody titers increased in serum from five chinchillas immunized with a type 6B outer membrane protein complex vaccine. Increases of anti-6B IgG and IgM antibody titers were more striking than increases of anti-6B IgA or anti-C polysaccharide IgG, IgM, or IgA titers were.     

Evaluation of an indirect immunofluorescence assay for detection of immunoglobulin M (IgM) and IgG antibodies against yellow fever virus

The first commercial indirect immunofluorescence assay (IFA) using Euroimmun Biochip technology was evaluated for the serodiagnosis of immunoglobulin G (IgG) and IgM antibodies against yellow fever virus (YFV) and was compared with the plaque reduction neutralization test (PRNT), which is currently the gold standard test for YFV. An overall correlation between the tests of 98.7% was established based on the analysis of 150 sera from individuals after vaccination with the 17D yellow fever vaccine. The sensitivity and specificity, calculated using the 150 sera from vaccinees and 150 sera from healthy blood donors, were 95% and 95%, respectively, for the IgG IFA and 94% and 97% for the IgM IFA. Antibody titers found in the PRNT correlated poorly with the IgM and IgG titers detected by IFA. The analysis of preexisting heterologous flaviviral immunity revealed the presence of antibodies reactive with YFV, tick-borne encephalitis virus, West Nile virus, Japanese encephalitis virus, and dengue virus serotypes 1 to 4 in 20 out of the 150 vaccinees. The indirect IFA showed that nine of these individuals with previous flaviviral exposure who received 17D vaccine failed to produce detectable IgM antibodies. Despite this preexisting immunity, all vaccinees developed protective immunity as detected by PRNT and anti-YFV IgG antibodies as detected by IFA. The high specificity and sensitivity of the IFA make it a useful tool for rapid diagnosis of yellow fever during outbreaks, for epidemiological studies, and for serosurveillance after vaccination.     

Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis

PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis.     

IgG and IgM antibodies to rubella quantitated by enzyme immunoassay

A solid-phase enzyme immunoassay for rubella antibodies (of IgG, H & L type, IgM-type) is described that requires assay at usually one dilution of serum. Results are reportable in milligrams IgG (or IgM)--equivalents per liter serum and in approximate hemagglutination inhibition (HAI) titers. The method uses purified rubella virus immobilized by a special process in excess, a 16 to 18 h first incubation at 20 degrees C, and a glucose-oxidase labeled second antibody to obtain 97% agreement with HAI. IgM-class antibodies are assayed after a preliminary separation from IgG by ion exchange to obtain results equivalent to those obtained after sucrose gradient sedimentation.     

Enzyme immunoassay for detection of human immunodeficiency virus-specific immunoglobulin A antibodies.

Early diagnosis of human immunodeficiency virus (HIV) infection may be difficult in adults with acute or recent HIV infection and in infants with perinatally acquired HIV. Detection of HIV-specific immunoglobulin A (IgA) antibodies in infant serum by Western blot (immunoblot) has been suggested as a reliable method to identify HIV-infected infants, especially those over the age of 6 months, and as an adjunct to diagnosis of acute HIV infection in adults. We developed a simple enzyme immunoassay for detection of HIV-specific IgA, using standard commercially available reagents. Enzyme immunoassay was comparable to Western blot for detection of HIV-specific IgA in sera from adults (n = 216), older children (n = 49), and infants born to HIV-infected mothers (n = 65). Specificity was 100% and sensitivity ranged from 80 to 92%. IgA-enzyme immunoassay is a simple, highly sensitive method for detection of HIV-specific IgA antibodies and is easily adapted to the standard clinical laboratory.     

Detection of rubella virus immunoglobulin G (IgG) and IgM antibodies in whole blood on Whatman paper: comparison with detection in sera.

We compared detection of rubella virus hemagglutination inhibition (HI) antibody and rubella virus-specific immunoglobulin M (IgM) in dried whole blood spotted onto Whatman filter paper and serum samples, both of which were obtained from the same subject by venipuncture. Of 1,000 paired serum samples obtained to study HI antibodies, 807 dried blood samples had HI titers identical to those of the corresponding serum samples, and 193 dried blood samples showed 1 dilution difference. Storage of dried blood at room temperature for 28 days did not affect the HI antibodies. In a study of specific IgM by a solid-phase immunosorbent HI test done with blood from healthy subjects and patients with rubella, the result of the presence, positive or negative, of specific IgM from both blood sample sources corresponded when the dried blood samples were stored at room temperature from 5 h to 38 days. This study demonstrated that the use of Whatman filter paper as a transport medium for blood samples for the determination of rubella virus immunity and the diagnosis of rubella virus infection is possible.     

A mu-capture immunoassay for detection of human herpes virus-6 (HHV-6) IgM antibodies in human serum.

BACKGROUND: Human herpes virus-6 (HHV-6) was first isolated in 1986. It has been shown to cause exanthema subitum and has been associated with various other diseases. HHV-6 infection is widespread, and more than 90% of the population have antibodies against HHV-6 at the age of 2 years. Once acquired, the virus remains latent in the body. This makes it difficult to draw any conclusions about a causal relationship between the demonstration of HHV-6 and a specific disease. OBJECTIVES: This work was to develop a mu-capture HHV-6 IgM enzyme linked immuno sorbent assay (ELISA) for use in routine diagnosis and for wide scale patient population analysis. STUDY DESIGN: A mu-capture HHV-6 IgM ELISA was established. A total of 682 sera consisting of 585 sera from Danish blood donors and 97 sera from patients with autoimmune antibodies were analysed in the HHV-6 IGM ELISA. One hundred and ninety-two sera had earlier been analysed for total HHV-6 antibody content in a competitive ELISA, 94 sera were analysed for cytomegalovirus (CMV) IgM and 57 sera for Epstein Barr virus (EBV) antibodies, using different ELISA assays. The results for 12 primary infections with HHV-6 are also reported. RESULTS: A HHV-6 IgM optical density (OD)-ratio was calculated according to a constant positive control. An empirical cut off of 0.5 HHV-6 IgM OD-ratio was chosen (with regard to the 10 HHV-6 seroconverters), which resulted in a specificity of 97.5% of the HHV-6 IgM ELISA. Two of the three donor sera with HHV-6 IgM OD-ratios more than 1.05 had total HHV-6 antibody titers significantly above the group with IgM OD-ratios below 0.7 consisting with HHV-6 reactivation. There was no cross reactions to EBV or CMV IgM positive sera. CONCLUSION: The HHV-6 IgM ELISA seems valid to diagnose primary HHV-6 infection in particular in combination with the HHV-6 total antibody assay.     

Microparticle enzyme immunoassay for determination of immunoglobulin G antibodies to human cytomegalovirus.

A microparticle enzyme immunoassay (LEIA) that uses passive latex agglutination was used to detect cytomegalovirus immunoglobulin G antibodies in 495 serum samples. LEIA was in excellent concordance with latex agglutination (97%) and an enzyme-linked immunosorbent assay (98.3%). The sensitivity and specificity of LEIA compared with those of the enzyme-linked immunosorbent assay were 100 and 92.5%, respectively.     

Detection of rubella virus-specific immunoglobulin G (IgG), IgM, and IgA antibodies by immunoblot assays.

Immunoblot (IB) assays were developed for detection of rubella virus (RV)-specific immunoglobulin G (IgG), IgM, and IgA antibodies in human serum following natural infection or immunization. IB assays performed under nonreducing conditions were compared with those performed under reducing conditions and with immunoprecipitation assays. Significant loss of antigenicity (greater than 90%) of RV E1 and E2 proteins was observed when IB assays were performed in the presence of 2-mercaptoethanol as compared with assays under nonreducing conditions. In contrast, the antigenicity of RV capsid protein was not influenced by reducing agents. Sensitivity of IB for RV-specific IgG antibodies was determined to be 0.01 IU/ml under nonreducing conditions. In the determination of RV-specific IgM and IgA antibodies by IB, pretreatment of serum with protein G to remove competing high-affinity RV-specific IgG or rheumatoid factor significantly improved assay sensitivity. IB assays were observed to be superior to immunoprecipitation assays in their ability to better define the specificities of RV-specific antibodies and to detect antibodies of all immunoglobulin classes. However, the conformational sensitivity of RV protein antigenicity should be an important consideration in the interpretation of RV-specific antibodies by IB assays.     

Evaluation of a commercial enzyme immunoassay for detection ofMycoplasma pneumoniae specific immunoglobulin G antibodies

A commercial enzyme immunoassay (Platelia Mycoplasma) infections was evaluated and found not to be suitable for the purpose. More than 80 % of healthy persons and patients with non-Mycoplasma pneumoniae respiratory infection, all with a negativeMycoplasma pneumoniae complement fixation test, had a positive EIA. Paired sera did not show the positive correlation between a rise in complement fixation titre and the EIA ratio reported by the manufacturer.     

Diagnosis of acute toxoplasmosis by an enzyme immunoassay for specific immunoglobulin m antibodies

A recently developed enzyme-linked immunosorbent assay for detection of immunoglobulin M (IgM) class antibodies to Toxoplasma gondii was evaluated with respect to specificity and sensitivity. By using an antibody capture principle and F(ab')2 conjugates, interference of rheumatoid factors was absent. No cross-reactions with anti-toxoplasma IgG occurred, and no interference with antinuclear antibodies was found. A large-scale study with about 1,500 clinical specimens revealed a 100% specificity. By testing 79 sera from patients with acute-phase acquired toxoplasmosis, sensitivity was found to be 97%. In routine clinical practice, the IgM-enzyme-linked immunosorbent assay proved to be a more sensitive tool for diagnosis than the immunofluorescent-antibody test. The course of IgM-enzyme-linked immunosorbent assay antibodies in acute patients was studied; IgM reached peak levels within 1 month after onset of illness, and could be demonstrated up to an average of 8 months after onset.     

Escherichia coli do not express Fc-receptors for human immunoglobulin G (IgG)

Gram-positive bacterial pathogens express immunoglobulin (Ig) binding proteins that perturb Fc-dependent functions such as the interaction with complement or phagocytic Fc-receptors. The possession of such molecules by gram-negative bacteria, including Escherichia coli (E. coli), has also been documented. In many such studies, the detection of Ig binding has relied on the use of polyclonal antibodies as detecting reagents. These are not ideal since such preparations may be contaminated with E. coli specific IgG, allowing for the potential misinterpretation of specific F(ab')(2) binding as non-specific Fc mediated binding. Here we use mono-specific recombinant antibodies to develop a novel assay for Ig binding non reliant on traditional polyclonal antibodies, allowing us to demonstrate the unequivocal absence of Fc binding proteins from E. coli.     

Fast Dipstick Dye Immunoassay for Detection of Immunoglobulin G (IgG) and IgM Antibodies of Human Toxoplasmosis

A dipstick dye immunoassay (DDIA) was developed to detect immunoglobulin G (IgG) or IgM antibodies of toxoplasmosis infection in humans. The assays employ a blue colloidal dye particles (D-1) conjugated to sheep anti-human IgG and rabbit anti-human IgM as the visualizing agents and a soluble antigen of tachyzoites of Toxoplasma gondii strain RH (TSA) as the detective antigen. The mixture of dye-labeled anti-human antibody-special human antibody was captured by TSA onto a nitrocellulose membrane dipstick by means of immunochromatography. The assays are rapid (the whole test can be completed within 15 min), simple, and cheap, and they don't require any equipment. They are sensitive and specific for the detection of anti-Toxoplasma IgG or IgM antibodies and generally agree closely with the results from the enzyme-linked immunosorbent assay. The assays are especially suitable for field applications.     

A sandwich enzyme immunoassay for the detection of human IgG antibodies to dog allergens

A double-sandwich enzyme immunoassay (DSEIA) was developed for the detection of human IgG antibodies to dog dander and hair (DH) allergens. Since DH allergens are immunogenic in rabbits, the gamma-globulin fraction of rabbit antiserum to DH (RGG-a-DH; 10 micrograms/ml) was used for coating of polystyrene microtiter plates. Dog allergens (1 microgram/ml of DH extract) were bound to RGG-a-DH. Binding of human IgG Ab to nonimmune RGG (10 micrograms/ml)--used as a background control--was substracted from the DH specific one and nonspecific binding was further eliminated by using 0.5% of normal rabbit serum in the dilution buffer. Sera were diluted 1:10 in this buffer. Specificity of the assay was shown by absorption experiments: protein A, anti-IgE discs (Phadebas PRIST) and dog or birch (e5, t3; Phadebas RAST) allergen discs were used to remove total and allergen specific IgG and IgE, respectively. The results were expressed as net absorbances or as a percentage of a reference serum. A significant correlation (r = 0.65, p less than 0.001) between IgG (DSEIA) and IgE (RAST) antibodies to dog allergens was observed in 40 untreated dog allergic subjects. The DSEIA was found to be more sensitive than conventional ELISA in detecting IgG Ab in 15 asthmatic children during hyposensitization: a significant rise was observed in 14 compared to 12 with ELISA, while no significant increases were observed in the placebo-treated group.