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.     

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.     

Direct radioimmunoassay for the detection of IgM antibodies against Mycoplasma pneumoniae

A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a prelminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneunoiae. Positive results are confirmed by retesting sera after treatment with 2-metcaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum.     

Seroprevalence of immunoglobulin M (IgM) and IgG antibodies to polysaccharides of Streptococcus pneumoniae in different age groups of Ecuadorian and German children.

The age-specific prevalence of serum immunoglobulin M (IgM) antibody to capsular polysaccharides of Streptococcus pneumoniae, as detected by enzyme-linked immunosorbent assay, was studied in 1,301 Ecuadorian children enrolled in a national nutrition and health survey. This prevalence was 6% in infants < 6 months old and increased to 28% in children 6 to 11 months old, 49% in those 12 to 17 months old, and 58% in those 18 to 23 months old. About 80% of the 5-year-old children had this antibody. When tested separately against six different capsular polysaccharides, serum IgM antibody reacted with decreasing frequency with serotype 3, 8, 19, 6, 23, and 1 capsular polysaccharides. We did not observe a broadening of the antibody response with increasing age in the sense that more and more serotypes were recognized. A similar age-related prevalence was found for IgM antibody to the species-specific C-polysaccharide of S. pneumoniae and for IgG antibody to capsular polysaccharides of S. pneumoniae. A smaller German serum collection showed a comparable age-related prevalence of pneumococcus-specific serum IgG and IgM antibodies. The highest incidence of respiratory diseases was observed in 1- and 2-year-old Ecuadorian children. It thus seems that acquisition of serum antibody to S. pneumoniae reflects more the developmental maturation of an immune response than an actual exposure to different pneumococcal serotypes.     

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.     

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.     

Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay.     

Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus.

A sensitive, solid-phase enzyme immunoassay for the detection of immunoglobulin M antibodies to cytomegalovirus is described. The results of the enzyme immunoassay correlated well with those obtained by an indirect immunofluorescence method. Horseradish peroxidase proved to be a more sensitive label than alkaline phosphatase. Nonspecific reactions, occurring with commercially available cytomegalovirus antigens, could be avoided by using a nuclear antigen prepared from sonically disrupted nuclei of cytomegalovirus-infected cells.     

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.     

Gold blot for detection of immunoglobulin M (IgM)- and IgG-specific antibodies for rapid serodiagnosis of melioidosis.

Gold blot tests for rapid serodiagnosis of melioidosis were developed and evaluated with sera from 40 melioidosis patients and 159 normal controls. The sensitivity and specificity were 87.5 and 88%, respectively, for the immunoglobulin M (IgM) test and 100 and 91%, respectively, for the protein A test for IgG. Combination of the IgM gold blot and protein A gold blot yielded 97.5% sensitivity and 94.3% specificity. The tests were rapid and simple.     

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.     

Use of monoclonal antibodies to quantify subclasses of human IgG. II. Enzyme immunoassay to define antigen specific (anti-filarial) IgG subclass antibodies

Because of their single epitope specificity, monoclonal antibodies (Mcabs) may perform with different levels of efficiency in immunoassays depending on the accessibility of the particular epitope recognized. In order to develop assays capable of detecting specific antibodies of each of the four human IgG subclasses, we have evaluated by ELISA the performance characteristics of a panel of Mcabs raised to the subclass proteins. At least one Mcab to each of the four subclasses was identified that was specific in its ability to capture its own relevant IgG subclass without any associated light chain, allotype or isoallotype activity and that was able to function effectively as a probe in an optimized, quantitative ELISA. When IgG subclass antibodies were measured in sera from patients with filariasis using specific filarial antigen, the sensitivities of each subclass antibody assay varied; for IgG1 and IgG4 antibodies the sensitivity of detection was 50 ng/ml and for IgG2 and IgG3, 10 ng/ml. The potency of the Mcab, determined by its titration for use as a probe, did not correlate with the sensitivity of the assay. These Mcabs were also capable of defining IgG subclass antibody responses qualitatively in immunoblot analyses with little or no non-specific binding. The availability of such highly characterized Mcabs for use in quantitative and qualitative definition of specific IgG subclass antibody responses should greatly improve our detection and subsequent understanding of the role of these IgG subclasses in various disease states.     

Development of an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay for detection of equine and swine IgM antibodies to vesicular stomatitis virus

An immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MC-ELISA) was developed for the detection of primary infection of vesicular stomatitis virus (VSV) in equine and swine sera. The test was based on the use of biotinylated sheep antibodies against equine or swine IgM molecules bound to a streptavidin-coated ELISA plate. The captured IgM antibodies were detected by application of antigens prepared from the New Jersey and the Indiana VSV serotypes (VSV-NJ and VSV-IN, respectively) and mouse polyclonal antibodies against VSV-NJ and VSV-IN. The MC-ELISA was compared to a competitive ELISA (C-ELISA) and the standard microtiter serum neutralization (MTSN) assay by testing serum samples from horses and pigs experimentally infected with VSV-NJ or VSV-IN. The MC-ELISA detected specific homologous IgM antibodies from equine and swine sera as early as 5 and 4 days postinfection (DPI), respectively, and as late as 35 DPI. The MTSN test also detected antibodies as early as 5 DPI and as late as 160 DPI. In a similar fashion, the C-ELISA detected antibodies from 6 to 7 DPI and as late as 160 DPI. These results demonstrated that the MC-ELISA is a useful test for serodiagnosis of primary VSV infection in horses and pigs.     

Evaluation of Chlamydia immunoglobulin M (IgM), IgG, and IgA rELISAs Medac for diagnosis of Chlamydia pneumoniae infection.

Chlamydia pneumoniae is an important pathogen responsible for a variety of respiratory diseases in humans. Cell culture remains the most specific method for C. pneumoniae diagnosis, but it is labor-intensive and time-consuming. Thus, serology, particularly microimmunofluorescence (MIF) testing, is frequently utilized. However, the MIF test has a significant subjective component. We evaluated a new serological test: Chlamydia Immunoglobulin M (IgG, IgA, and IgM rELISAs Medac, based on a recombinant Chlamydia-specific lipopolysaccharide (LPS) fragment, for the diagnosis of C. pneumoniae infection. The results of this study demonstrated that the use of rELISAs Medac with single sera does not appear to be sensitive or specific for diagnosis of C. pneumoniae infection compared to culture. In children, sensitivities of the rELISAs compared to culture did not exceed 34.2%, and the specificities ranged from 68.4% (IgG) to 91.2% (IgA). In adults, the sensitivities of the rELISAs were slightly higher, up to 77.8% (IgA or IgG), but the specificities ranged from a very low 20.8% for IgA or IgG to 81.1% for IgM. When multiple sera were tested, the results of the rELISAs Medac correlated with culture results in five of eight (62.5%) patients. However, this offers only a retrospective diagnosis, which makes it difficult to manage these patients prospectively.     

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.     

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.     

Analysis of eight commercial enzyme immunoassay tests for detection of antibodies to Mycoplasma pneumoniae in human serum

Mycoplasma pneumoniae is an important etiologic agent of primary atypical pneumonia in children and adults. The diagnosis of M. pneumoniae infection is commonly confirmed through serologic testing. In this study, we used paired sera from 51 patients (all with confirmed M. pneumoniae infection and positive complement fixation [CF] titers) to compare the results of eight enzyme immunoassays (EIAs) available commercially in the United States. We compared two single-use EIAs and six plate-type EIAs. Results from acute-phase sera ranged from only 7 (14%) positive by ImmunoWELL (GenBio) immunoglobulin M (IgM) EIA to 23 (45%) positive by Zeus IgG EIA. When both the acute-phase and convalescent-phase serum samples were examined, positive results ranged from 20 (39%) by the ImmunoWELL (GenBio) IgM assay to 45 (88%) positive by the Remel IgG-IgM EIA. In this study, the single-use EIAs by Remel and Meridian were more reliable than were the plate-type EIAs. Among the plate-type EIAs, the Zeus and DiaSorin assays (which detect antibodies to protein antigens) were more sensitive than the ImmunoWELL assay (which detects antibodies to glycolipid antigens). In general, IgG EIAs on convalescent-phase sera were more concordant with one another than were IgM EIAs with one another. Scatter plot analysis of convalescent-phase sera showed that, as the CF titer dropped, the IgM assays identified fewer positive convalescent-phase sera. In contrast, the IgG assays provided fairly consistent positive results for convalescent-phase sera with CF titers of 64 and above. Results of individual tests and overall limitations of serodiagnostics for M. pneumoniae infections are discussed.     

Single antigen detects both immunoglobulin M (IgM) and IgG antibodies elicited by all four dengue virus serotypes

The resurgence of dengue (DEN) virus infections in the last few decades coupled with the lack of a preventive vaccine and specific antiviral drugs has jointly contributed to making this a significant global public health problem. Currently, symptomatic supportive treatment and fluid replacement therapy are the only means available to minimize DEN-induced mortality. As the clinical symptoms associated with DEN virus infections are indistinguishable from those of many other viral, bacterial, and parasitic infections, specific diagnostic tests assume critical importance in the unequivocal identification of DEN virus infections. We have designed a novel chimeric antigen based on envelope domain III (EDIII), a critical antigenic region of the major structural protein of DEN viruses. We fused EDIIIs corresponding to each of the four DEN virus serotypes using pentaglycyl linkers, overexpressed the resultant tetravalent chimeric protein in Escherichia coli, and affinity purified it in high yields, obtaining approximately 30 mg protein of >95% purity per liter of culture. We show that this tetravalent antigen could specifically recognize anti-DEN virus antibodies of both the immunoglobulin M (IgM) and IgG classes. Using a large panel of IgM antibody capture-enzyme-linked immunosorbent assay- and hemagglutination inhibition-confirmed DEN virus-infected and uninfected patient sera (n = 289), we demonstrate that this tetravalent antigen can function as a diagnostic tool of high sensitivity and specificity.