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.     

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.     

Solid-phase enzyme-linked immunosorbent assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides.

Four recombinant antigens representing two distinct antigenic domains from two different strains of hepatitis E virus (HEV), were used individually to develop four ELISAs designed to detect antibodies to HEV. Both IgG and IgM class antibodies to HEV were detected in 7 of 8 pedigreed serum/plasma from known outbreaks of HEV in Mexico, Burma, Somalia and Pakistan. In addition, specific HEV-antibodies were detected in cynomolgus macaques following inoculation with various HEV strains. Anti-HEV was also detected in 8 of 386 (2.1%) randomly selected American blood donors. Supplemental tests utilizing both synthetic peptides and specific blocking assays provided additional serologic data confirming the presence of anti-HEV. Similar prevalence studies on a limited number of available sera from other geographical regions (Alaska, Japan, Germany, New Zealand, Thailand and Mexico) confirmed the presence of anti-HEV in at least 1.1 to 7.6% of the specimens.     

Identifying infections with respiratory syncytial virus by using specific immunoglobulin G (IgG) and IgA enzyme-linked immunosorbent assays with oral-fluid samples

Currently, respiratory syncytial virus (RSV) infection is identified in epidemiological studies by virus antigen or nucleic acid detection in combination with serology. Oral-fluid specimens may provide a noninvasive alternative to blood, and oral fluid is more suitable for sampling outside of the clinic setting. We evaluated an indirect enzyme-linked immunosorbent assay for the detection of RSV-specific immunoglobulin G (IgG) and IgA by using oral-fluid samples collected from individuals with RSV infections confirmed by an immunofluorescent antibody test. For five children sampled repeatedly from birth, antibody profiles in oral fluid quite consistently tracked those in paired sera, and RSV infections were detected by rising titers of antibodies of at least one Ig class. Specific IgG responses were generally more reliable than IgA responses, except in early infancy, where the reverse was sometimes true. For a further five young children from whom oral fluid was collected weekly following RSV infection, boosted antibody responses, frequently of a transient nature, lasting a few weeks, were observed; specific IgG responses were of longer duration and more pronounced than specific IgA responses. Our data show significant promise for the use of oral fluid alone in RSV infection surveillance. The observed rapid dynamics of the antibody responses are informative in defining study sampling intervals.     

The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella IgM and IgG antibodies

The interference of IgM rheumatoid factor (RF) in the indirect enzyme-linked immunosorbent assay (ELISA) for rubella IgM and IgG antibodies was evaluated quantitatively. In an ELISA for rubella IgM antibodies IgM RF produced false positive results in tests of sera with a rubella IgG concentration exceeding approximately 30 I.U./ml and an IgM RF concentration higher than 3.5 I.U./ml as determined by ELISA. Sera from 3 of 70 patients with recent rubella and sera from a similar proportion of blood donors contained IgM RF in a concentration exceeding this level.The false positive results were reproduced when an IgM RF preparation was added to sera containing rubella IgG. The rubella IgG values in ELISA, however, decreased after the addition of IgM RF . RF was absorbed by incubation of sera with a suspension of latex particles coated with aggregated human IgG. This method prevented the false positive results of the rubella IgM assay and increased the rubella IgM values of rubella convalescent sera. The activity in the rubella IgG assay also increased after absorption of RF. The interference of RF has been explained by a secondary binding of RF to rubella IgG-antigen complexes. The RF might subsequently be detected by the anti-IgM conjugate or compete with the anti-IgG conjugate.The common occurrence of IgM RF in concentrations sufficient to produce false positive results of the ELISA for specific IgM antibodies necessitates routine testing of IgM antibody positive sera for RF by a sensitive method and/or absorption of RF from such sera.     

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen. IgG or IgM antibodies were quantitated by the use of alkaline-phosphatase-conjugated anti-human IgG or IgM antibodies. Of the 24 randomly collected serum samples, 7 were positive in the IgG-ELISA test. One positive by the IgM-ELISA was found to contain rheumatoid factor. In 12 of 13 paired sera of clinical cases, IgM as well as IgG antibodies were detected in the second serum sample taken about 3 wk after the onset of clinical signs. The IgM positive sera were screened for rheumatoid factor (RF) on IgG-coated plates. None of them contained RF. Antibody titers obtained by ELISA showed a good correlation with titers obtained by hemagglutination inhibition, complement fixation, and neutralization tests. The ELISA tests for detection of IgM and IgG antibodies to WNV therefore can replace the other serological methods for epidemiological surveillance and diagnostic purposes.     

Performance of two commercial enzyme-linked immunosorbent assay kits using recombinant glycoprotein G2 antigen for detection of herpes simplex virus type 2 specific antibodies

For 93 stored serum samples tested by HerpeSelect2 and the Euroimmun enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies, the concordance of positive and negative results was 100%. Moreover, all the results that were equivocal by HerpeSelect2 (negative by Euroimmun) were confirmed as being negative by a Western blot assay.     

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.     

Evaluations of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.     

Ability of enzyme-linked immunosorbent assays to detect early immunoglobulin G antibodies toToxoplasma gondii

Two ELISA procedures, one using sonicated antigen coated with carbonate buffer and the other formalin fixed trophozoites with dry coating, differ in their ability to detect early antibodies in toxoplasmosis. In order to identify factors responsible for this difference, seven ELISA systems differing from each other in antigen used and/or coating procedure were compared. Both fixation of the trophozoites with formalin and air-drying of the antigen in the microtiterplate were important factors determining the ability of the assay to detect IgG antibodies in the early stage of infection. Differences in the results of the two ELISA procedures can be used to distinguish between the acute and chronic stages of infection.     

Properties and use of mumps viral antigen for detection of specific IgG and IgM antibodies in enzyme-linked immunosorbent assay

Specific mumps virus antigens of three purification degrees have been prepared and their quality was evaluated in the enzyme-linked immunosorbent assay (ELISA) with specific human sera. Two enzyme immunoassays were elaborated, namely a simple sandwich method for IgG and the IgM-capture technique for IgM detection. Four different lots of mumps antigen were tested in these two ELISA systems. All antigens of corresponding purification degrees showed practically the same properties.     

Evaluation of rubella virus immunoglobulin G (IgG) and IgM assays with the new Vidia instrument

For rubella virus immunoglobulin G (IgG) and IgM detection, Vidia assays were compared to Vidas, AxSYM, and Liaison assays with 419 serum samples. Only Vidia produced a sensitivity of 100% for IgG and IgM. Vidia specificities were 98.4% for IgG and 99.8% for IgM, versus Vidas specificities of 100 and 99.3%. Vidia IgG and IgM assays performed equally well.     

Serological detection of varicella-zoster virus-specific immunoglobulin G by an enzyme-linked immunosorbent assay using glycoprotein antigen

Since the introduction of varicella vaccination in several countries, there has been an urgent need for commercially available test procedures that allow highly sensitive and specific quantitative determination of the varicella-zoster virus (VZV)-specific immune status, including immunity postimmunization. This study compared the performance of two enzyme-linked immunosorbent assays (ELISAs) for the sensitive and specific determination of VZV-specific immunoglobulin G (IgG) in seronegative and latently infected persons, as well as in vaccinees. One ELISA is based on the detection of antibody to VZV-specific envelope glycoproteins (gp), and the other comprises the whole antigen extract prepared from VZV-infected cells. A modified standard fluorescent-antibody-to-membrane-antigen (FAMA) assay was used as a reference. An excellent sensitivity (100%) in relation to FAMA was demonstrated for the gpELISA (Virion\Serion), while the non-gpELISA (Dade Behring) had a lower sensitivity (83%) when sera from latently infected persons were tested. After postvaccinal immunity was measured, a sensitivity of 87% was achieved with gpELISA, whereas the ELISA incorporating antigen extract of VZV-infected cells had a sensitivity of 78%. Excellent specificity (100%) was calculated for both the gpELISA and the non-gpELISA. In conclusion, SERION ELISA classic VZV IgG is useful for the sensitive and specific quantitative determination of VZV immune status after natural infection. The test can also be recommended for measuring antibody response after varicella vaccination, particularly after the cutoff value was optimized.     

Detection of BK virus IgM antibodies by two enzyme-linked immunosorbent assays (ELISA) and a hemagglutination inhibition method

We have used an antigen solid-phase enzyme-linked immunosorbent assay (SP-ELISA) and an IgM antibody capture ELISA (MACELISA) for detecting IgM antibodies to human polyomavirus BK (BKV). These tests were compared with the standard hemagglutination inhibition test (HAI) of IgM serum fractions following sucrose density gradient fractionation. The SP- and MACELISA were not influenced by concomitant BKV-IgG, but high levels of both BKV-IgG and rheumatoid factor could cause false positive results by SPELISA, but not by MACELISA. The MACELISA gave much higher positive to negative ratios than the SPELISA. The sensitivity and specificity of the two tests were high compared to the IgM-HAI method. The sera could be tested in a single dilution (1:160), and thus the ELISA-tests are useful for testing large numbers of sera.     

West Nile virus immunoglobulin A (WNV IgA) detection in cerebrospinal fluid in relation to WNV IgG and IgM reactivity.

BACKGROUND: Diagnostic criteria for neurologic involvement in WNV infection include WNV IgM detection in CSF; however, WNV IgM can persist in CSF >6 months. CSF IgA characterizes other flavivirus infections, but WNV IgA in CSF has not been evaluated. WNV IgM in CSF correlates with IgM in serum but the presence of WNV IgA in CSF compared to serum is unknown. OBJECTIVES: Evaluate WNV IgA detection in CSF as a marker of WNV neuroinvasive infection, initially with samples pre-selected based on WNV IgG and IgM reactivity and subsequently with all available CSF samples submitted for WNV antibody testing over an entire WNV season. STUDY DESIGN: Selected CSF samples and CSF/serum pairs previously tested for WNV IgG and IgM were assayed for WNV IgA. Subsequently, all available CSF samples tested for WNV antibodies during the 2005 season were tested for WNV IgA, including those where paired sera were available and tested for IgA, IgG and IgM. RESULTS: For most samples, including paired CSF and serum, the IgA result qualitatively agreed with the IgM result, regardless of the IgG result. CONCLUSION: IgA detection is equivalent to IgM detection as a marker of WNV infection in CSF.     

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient — obtained before, during, and after an infection with mumps — the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized.Behringwerke AG, Marburg, West Germany     

Recombinant nucleoprotein-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

The full-length nucleoprotein of Crimean-Congo hemorrhagic fever virus (CCHFV; 482 amino acid residues) was expressed as a His-tagged recombinant protein (His-CCHFV rNP) in the baculovirus system. The His-CCHFV rNP was efficiently expressed in insect cells and purified by Ni(2+) column chromatography. Using this substrate, an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was developed. We evaluated the sensitivity and specificity of the IgG ELISA, using serum samples previously determined to be antibody positive or negative by immunofluorescence tests on CCHFV-infected Vero E6 cells. We found very good correlation between the two tests: 87% for the positive sera (13 of 15) and 99% for the negative sera (107 of 108). These results indicate that the new IgG ELISA using His-CCHFV rNP has high sensitivity and specificity for detecting CCHFV antibodies. The CCHF patients' sera with high titers reacted only with the NP fragment containing amino acid residues between 201 and 306 in Western blotting. It is known that amino acid homologies are high in this region among various isolates. Thus, it is expected that this ELISA can detect antibodies not only for Chinese strains of CCHFV but also for other strains circulating in the world. These results suggest that the IgG ELISA system developed with the recombinant CCHFV NP is a valuable tool for diagnosis and epidemiological investigations of CCHFV infections.     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.     

Performance of commercial enzyme-linked immunosorbent assays for detection of antibodies to Bordetella pertussis

Measuring antibodies to Bordetella pertussis antigens is mostly done by enzyme-linked immunosorbent assays (ELISAs). We compared the performance of ELISA kits that were commercially available in Germany. Eleven measured IgG antibodies, and nine measured IgA antibodies. An in-house ELISA with purified antigens served as a reference method. Samples included two WHO reference preparations, the former Food and Drug Administration (FDA)/Center for Biologics Evaluation and Research (CBER) reference preparations, serum samples from patients with clinically suspected pertussis, and serum samples from patients having received a combined tetanus, diphtheria, and pertussis (Tdap) vaccination. Kits using pertussis toxin (PT) as an antigen showed linearity compared to the WHO Reference preparation (r2 between 0.82 and 0.99), and these kits could quantify antibodies according to the reference preparation. ELISA kits using mixed antigens showed no linear correlation to the reference preparations. Patient results were compared to results of in-house ELISAs using a dual cutoff of either ≥100 IU/ml anti-PT IgG or ≥40 IU/ml anti-PT IgG together with ≥12 IU/ml anti-PT IgA. The sensitivities of kits measuring IgG antibodies ranged between 0.84 and 1.00. The specificities of kits using PT as an antigen were between 0.81 and 0.93. The specificities of kits using mixed antigens were between 0.51 and 0.59 and were thus not acceptable. The sensitivities of kits measuring IgA antibodies ranged between 0.53 and 0.73, and the specificities were between 0.67 and 0.94, indicating that IgA antibodies may be of limited diagnostic value. Our data suggest that ELISAs should use purified PT as an antigen and be standardized to the 1st International Reference preparation.