Detection of immunoglobulin M in cerebrospinal fluid from syphilis patients by enzyme-linked immunosorbent assay.

Cerebrospinal fluid (CSF) samples were evaluated in an immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) for syphilis with sonic extracts of Treponema pallidum coated on polystyrene plates. The ELISA procedure was reproducible, and T. pallidum antigens were stable., A total of 15 CSF samples from patients with neurosyphilis, 18 CSF samples from patients with syphilis, 12 CSF samples from patients treated for syphilis, and 494 CSF samples from patients with neurologic or other systemic diseases were tested. The IgM ELISA gave reactive results in all of six symptomatic and congenital neurosyphilitic patients and none of nine asymptomatic neurosyphilitic patients. Of 524 CSF samples from nonneurosyphilitic individuals, 513 were nonreactive, resulting in 98% test specificity. The IgM ELISA in CSF should prove to be useful for confirmation of symptomatic neurosyphilis.     

Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay.

We developed an enzyme-linked immunosorbent assay (ELISA) that was capable of detecting immunoglobulin M (IgM) antibody to vesicular stomatitis virus (VSV) in the sera of experimentally and naturally infected cattle and horses. The detection of IgM in the sera of these animals permitted an estimate of the recency of infection by VSV serotype New Jersey. A VSV serotype New Jersey epizootic strain isolated from a horse and passed once in an Aedes albopictus cell line was used to infect a horse and a calf. Sera from these animals were used to standardize the ELISA. This assay was used to test sera from cattle and horses involved in the 1982 VSV epizootic. Comparative antibody titrations were performed by three systems: the serum-dilution plaque-reduction neutralization, complement fixation, and indirect immunofluorescent tests. The antibody titers by neutralization and the ELISA were comparable for the period that IgM was present; when IgM ELISA titers diminished, the neutralization titers remained high. The complement fixation and indirect immunofluorescent antibody titers followed closely the IgM pattern determined by the ELISA. The capture IgM ELISA is applicable for the rapid detection of IgM antibody to VSV in cattle and horses and is a useful assay of recent infection.     

Evaluation of a West Nile virus immunoglobulin A capture enzyme-linked immunosorbent assay

An in-house-developed enzyme-linked immunosorbent assay detected West Nile virus (WNV) immunoglobulin A (IgA) in 65 of 68 sera from WNV-infected patients; 40 of 63 WNV IgM-positive, IgG-negative serum or plasma specimens; 65 of 67 WNV IgM-positive, IgG-positive specimens; 0 of 70 WNV IgM-negative, IgG-negative specimens; and 0 of 64 archived blood donation sera. WNV IgA is thus highly prevalent among WNV-infected patients and typically appears after WNV IgM but before WNV IgG.     

Epitope-blocking enzyme-linked immunosorbent assay to differentiate west nile virus from Japanese encephalitis virus infections in equine sera

West Nile virus (WNV) is now widely distributed worldwide, except in most areas of Asia where Japanese encephalitis virus (JEV) is distributed. Considering the movement and migration of reservoir birds, there is concern that WNV may be introduced in Asian countries. Although manuals and guidelines for serological tests have been created in Japan in preparedness for the introduction of WNV, differential diagnosis between WNV and JEV may be complicated by antigenic cross-reactivities between these flaviviruses. Here, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay that can differentiate WNV from JEV infections in horse sera. Under conditions well suited for our assay system, samples collected from 95 horses in Japan (regarded as negative for WNV antibodies), including those collected from horses naturally infected with JEV, showed a mean inhibition value of 8.2% and a standard deviation (SD) of 6.5%. However, inhibition values obtained with serum used as a positive control (obtained after 28 days from a horse experimentally infected with WNV) in nine separate experiments showed a mean of 54.4% and an SD of 7.1%. We tentatively determined 27.6% (mean + 3 x SD obtained with 95 negative samples) as the cutoff value to differentiate positive from negative samples. Under this criterion, two horses experimentally infected with WNV were diagnosed as positive at 12 and 14 days, respectively, after infection.     

Development of an enzyme-linked immunosorbent assay for immunoglobulin M antibodies against measles virus

Measles is a highly contagious respiratory virus infection, with typical clinical symptoms including maculopapular rash, fever, cough, coryza, and conjunctivitis. Despite implementation of widespread vaccination programs throughout the world, the rates of global morbidity and mortality are still considerable. This study was performed to design a reliable indirect enzyme-linked immunosorbent assay (ELISA) to measure measles-specific immunoglobulin M (IgM). First, human IgM was purified, and then an anti-IgM antibody was produced in rabbits and purified in a multistep process. The rabbit IgG against human IgM was conjugated with peroxidase. Measles virus-infected Vero cells produced viral antigen. One hundred serum samples from infants of 9 to 18 months of age, mostly vaccinated, were evaluated for determining the presence of specific IgM antibodies against measles virus. The samples were also evaluated for neutralizing antibodies against measles virus by a microneutralization test (MNT). By comparing the results of the ELISA with those of MNT, it was demonstrated that ELISA had a sensitivity and specificity of 100 and 92%, respectively. On the other hand, when the results obtained by our ELISA system were compared with those of an imported measles virus IgM ELISA kit (EIAgen; Adaltis Italia SPa, Bologna, Italy), a high level of agreement was shown (k = 0.926).     

Evaluation of an immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Orientia tsutsugamushi infection

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.     

Epitope-blocking enzyme-linked immunosorbent assays for detection of west nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.     

Interlaboratory evaluation of indirect enzyme-linked immunosorbent assay, antibody capture enzyme-linked immunosorbent assay, and immunoblotting for detection of immunoglobulin M antibodies to Toxoplasma gondii.

One hundred and fifteen serum samples from healthy laboratory personnel and 50 consecutive samples from 19 patients with anamnestic clinical signs of toxoplasmosis were assayed by four laboratories for the presence of immunoglobulin M antibodies to Toxoplasma gondii by an indirect enzyme-linked immunosorbent assay (ELISA), an antibody capture assay with peroxidase-labeled toxoplasma antigen, and an immunoblotting assay. In addition, a commercially available antibody capture ELISA was used. Highly significant correlation coefficients were obtained between the four laboratories and the commercial test. The indirect ELISA and antibody capture ELISA showed equal sensitivity in detection of immunoglobulin M antibodies to toxoplasma in early-stage serum samples. However, in this study, the antibody capture assay discriminated better between serum samples obtained at early or late stages of toxoplasma infection.     

Evaluation of immunoglobulin M and G capture enzyme-linked immunosorbent assay Panbio kits for diagnostic dengue infections.

BACKGROUND: Serological assays are widely used to confirm dengue virus infections and to differentiate between a primary and a secondary infection. OBJECTIVE: Two commercial dengue diagnostic kits, Panbio Dengue IgM Capture and Dengue IgG Capture ELISA (Brisbane, Australia) were evaluated. STUDY DESIGN: Three hundred and seventy-three serum samples were tested. Panel sera included samples from dengue confirmed cases (representing both primary and secondary infections), from non-dengue infectious diseases, and from healthy individuals. The MAC-ELISA/Dengue IPK was used for the detection of anti-dengue virus IgM antibody in the sera and the ELISA inhibition method (EIM/Dengue IPK) was used to differentiate between primary and secondary infections. Both these reference assays, which were previously developed in the Arbovirus Laboratory at the "Pedro Kouri" Tropical Medicine Institute, were employed as the gold standard. RESULTS: High sensitivity (96.8%) and specificity (99.4%) were found with the commercial diagnostics when compared to the reference methods. Furthermore, high concordance 95.5% in classifying dengue infection types (primary or secondary infections) was observed. CONCLUSIONS: The Panbio Dengue IgM and IgG assays offer a good alternative for dengue diagnosis. They are easy to perform and results can be obtained in less than 3h.     

Development of immunoglobulin M capture enzyme-linked immunosorbent assay to differentiate human flavivirus infections occurring in Australia

We report the development of a flavivirus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (MAC-ELISA) which improves the determination of an infecting flavivirus serotype over that by current serological methods. A panel of 165 IgM-positive sera from flavivirus patients with specific diagnostic results was tested by the flavivirus MAC-ELISA using a panel of 10 antigens. For 134 of these sera (81.2%), the highest reactivity was demonstrated against the infecting virus, which was consistent with the original diagnostic result. Specific antibody reactions inconsistent with the original diagnosis were found for six sera (3.6%). In our experience, the flavivirus-serotyping ELISA provides a rapid and accurate alternative to other serological tests, such as hemagglutination inhibition, for the specific diagnosis of flavivirus infections.     

Detection of immunoglobulin M antibodies to hepatitis A virus by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay for the detection of immunoglobulin M (IgM) antibodies to hepatitis A virus is described. The test uses the principle of binding of IgM antibodies to anti-IgM-coated microtiter plates to determine whether the IgM antibodies attached have specificities for hepatitis A virus. In three patients with hepatitis type A followed up to 12 months, IgM antibodies to hepatitis A virus could be demonstrated from the onset of illness and during the following 2 to 3 months. When acute-phase sera from 48 patients with acute hepatitis were tested, IgM antibodies to hepatitis A virus could only be demonstrated in 18 patients previously classified as type A, whereas 30 patients with type B and non-A non-B hepatitis were negative. IgM antibodies to hepatitis A virus could not be demonstrated in 108 normal sera nor in 55 sera containing rheumatoid factor. These results indicate that the enzyme-linked immunosorbent assay for IgM antibodies to hepatitis A virus is useful in the serodiagnosis of acute hepatitis type A on a single serum sample taken during the acute phase of illness.     

Specific immunoglobulin M enzyme-linked immunosorbent assay for confirming the diagnosis of measles.

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the detection of measles virus-specific immunoglobulin M (IgM) (MIgM). The ELISA was standardized by deriving a seronegative range of values from sera which should not contain MIgM (24 cord sera, 59 sera from immune health care workers, and 47 sera from infants before the administration of measles vaccine). These values were separable from those obtained from individuals convalescing from measles. Twenty sera containing rheumatoid factor were MIgM seronegative. Of 30 acute-phase sera from suspected measles cases, 26 contained MIgM; those that were seronegative were obtained on day 0, 0, 2, or 9. All 25 convalescent-phase samples contained MIgM. Of the 25 paired samples, 22 were IgG positive at the first sampling; 3 of the 22 did not show a rise in IgG titer. The MIgM ELISA can be used for confirming suspected measles cases, often requiring only a single serum specimen.     

Solid-phase enzyme-linked immunosorbent assay for detection of hepatitis A-specific immunoglobulin M.

A solid-phase enzyme linked immunosorbent assay was developed for the detection of immunoglobulin M antibody to hepatitis A virus. The system was capable of detecting hepatitis A-specific immunoglobulin M in a single dilution of serum and appears to be a reliable and rapid means of establishing a diagnosis of hepatitis A infection. Specific immunoglobulin M was only detected in patients with serologically confirmed hepatitis A and not in patients with other forms of hepatitis, chronic liver disease, or autoimmune disease. In patients with hepatitis A, specific immunoglobulin M was usually detectable for 6 weeks after the onset of dark urine, and the longest period for which it was present in any patient was 115 days. This enzyme-linked immunosorbent assay is rapid, simple to perform, and does not require complicated equipment. Provided adequate supplies of purified reagents can be obtained, this enzyme-linked immunosorbent assay procedure is likely to simplify hepatitis A serology, because the same antibody-coated plates can be utilized to detect hepatitis A virus, anti-hepatitis A virus, and hepatitis A-specific immunoglobulin M.     

Detection of Ross River virus immunoglobulin M antibodies by enzyme-linked immunosorbent assay using antibody class capture and comparison with other methods

An enzyme-linked immunosorbent assay based on antibody class capture was developed for the detection of Ross River virus-specific immunoglobulin M antibodies (RRV IgM). The assay was specific, reproducible and precise. When compared with conventional tests for the detection of RRV IgM, such as hemagglutination inhibition following sucrose density gradient centrifugation and indirect enzyme-linked immunosorbent assay, the class capture assay was more sensitive. In 186 sera which were collected from 39 patients with RRV infection over a period of 1-4 yr from onset of initial symptoms, RRV IgM persisted for at least 1-2 yr. Sera were tested both at a single dilution from which the results were expressed as a binding index and in a dilution series in which they were expressed as an antibody titre. Binding index values gave better discrimination between sera collected during acute and later phases of the disease and may be of greater value than antibody titres in the diagnosis of RRV infection.     

Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of >/=2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.     

Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay

The emergence of West Nile (WN) virus in New York and the surrounding area in 1999 prompted an increase in surveillance measures throughout the United States, including the screening of sentinel chicken flocks for antibodies. An enzyme-linked immunosorbent assay (ELISA) for the detection of chicken immunoglobulin M (IgM) to WN virus was developed, standardized, and characterized as a rapid and sensitive means to detect WN viral antibodies in sentinel flocks. Serum specimens from experimentally infected chickens were analyzed by using this assay, and IgM was detected as early as 3 to 7 days postinfection. Persistence of IgM varied from at least 19 to more than 61 days postinfection, which indicates the need to bleed sentinel flocks at least every 2 weeks for optimal results if this method is to be used as a screening tool. The ELISA was compared to hemagglutination-inhibition and plaque reduction neutralization tests and was found to be the method of choice when early detection of WN antibody is required. House sparrows and rock doves are potential free-ranging sentinel species for WN virus, and the chicken WN IgM-capture ELISA was capable of detecting anti-WN IgM in house sparrow serum samples from laboratory-infected birds but not from rock dove serum samples. The chicken WN IgM-capture ELISA detected anti-WN antibodies in serum samples from naturally infected chickens. It also detected IgM in serum samples from two species of geese and from experimentally infected ring-necked pheasants, American crows, common grackles, and redwinged blackbirds. However, the test was determined to be less appropriate than an IgG (IgY)-based assay for use with free-ranging birds. The positive-to-negative ratios in the ELISA were similar regardless of the strain of WN viral antigen used, and only minimal cross-reactivity was observed between the WN and St. Louis encephalitis (SLE) IgM-capture ELISAs. A blind-coded serum panel was tested, and the chicken WN IgM-capture ELISA produced consistent results, with the exception of one borderline result. A preliminary test was done to assess the feasibility of a combined SLE and WN IgM-capture ELISA, and results were promising.     

Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA.     

Quantitation of immunoglobulin E antibody to cytomegalovirus by antibody capture enzyme-linked immunosorbent assay

An antibody capture enzyme-linked immunosorbent assay was developed for detection of immunoglobulin E antibody to cytomegalovirus (CMV-IgE). Affinity-purified anti-human IgE-coated microtiter plates were used to separate IgE from other classes of antibody in serum. Virus-specific IgE was detected by subsequent incubation with horseradish peroxidase-labeled CMV antigen and substrate. The assay was shown to be very sensitive, since in most positive sera CMV-IgE was still detected at a dilution of 1:5,000. Of 45 patients with primary CMV infection, 43 (96%) were found to produce CMV-IgE. In contrast, CMV-IgE was detected in only 4 (9%) of 44 patients with recurrent CMV infection and in 1 of 144 healthy controls. Furthermore, the level of CMV-IgE in patients with recurrent CMV infection appeared to be lower than that in patients with primary infection. Preliminary examination of successive sera suggested that CMV-IgE is produced somewhat slower than CMV-IgM and -IgA but persists for a shorter period. These results suggest that CMV-IgE may be used as an indicator of primary CMV infection.     

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to west nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WNV or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.1112G potentially discriminated between WNV and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2B2 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.67G, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.