A rapid, sensitive and reliable diagnostic test for scrub typhus in China

Purpose: To evaluate the performances for detection of IgM and IgG antibodies to Orientia. tsutsugamushi (Ot) using a gold conjugate-based rapid diagnostic test (RDT). Materials and Methods: The RDT employing mixture recombinant 56-kDa proteins of O. tsutsugamushi and the mIFA assay was performed on 33 patients from Fujian and Yunnan province respectively and 94 positive sera (36 from Hainan province and 58 from Jiangsu province) from convalescent stages of the patients with scrub typhus respectively and 82 negative sera from healthy farmers from Anhui province and Beijing City respectively in 2009. A comparison of the RDT and mIFA assay was performed by using the χ² test and the P level of ≤0.05 was considered to be significant. Results: Among these 94 positive sera from convalescent stages of the illness and 82 sera from control farmers, the specificity of RDT was 100% for both IgM and IgG tests. In 33 cases with scrub typhus, 5 cases were positively detected earlier by RDT than by mIFA for the IgM test, and 2 cases were positive for the IgG test. The sensitivities of RDT were 93.9% and 90.9% for IgM and IgG, respectively. Considering IgM and IgG together, the sensitivity was 100%. The geometric mean titre (GMT) of IFA and the RDT assay in diluted sera from confirmed cases were 1:37 versus 1:113 respectively (P<0.001) for IgM test and 1:99 versus 1:279 respectively (P<0.016) for IgG. Conclusions: The RDT was more sensitive than the traditional IFA for the early diagnosis of scrub typhus and was particularly suitable for use in rural areas.     

Primary interaction between antibody and components of Alternaria: II. Antibodies in sera from normal,allergic, and immunoglobulin-deficient children

Antibodies in sera from normal, allergic, and immunoglobulin-deficient children were studied for binding to radiolabeled components of Alternaria tenuis. Significant binding levels were found in 103 of 105 sera from normal children. The levels were age-dependent, rising from a low point in the 7- to 12-month age group to adult levels by the age of 8 years. Levels of binding to two antigens, a culture filtrate derivative (¹²⁵I-CLF) and a mycelial derivative (¹²⁵I-IIS), were similar. Sera from asthmatic children with strong immediate skin test reactions to Alternaria extracts bound significantly higher levels of ¹²⁵I-CLF than did sera from allergic children with negative skin tests or from control children. Binding levels in sera from children with hypogammaglobulinemia were significantly less than binding levels in sera from normal children in any age group. Sera from children with selective IgA deficiency bound ¹²⁵I-CLF at normal levels. The almost universal occurrence of anti-Alternaria antibodies in children was partly explained by the finding of partial cross-reactivity and/or shared antigens among several fungal species, including A. tenuis, Stemphylium sp., Curvularia sp., and Aspergillus fumigatus. The biological significance of these antibodies is not clear, but the procedures described lend themselves to further investigations.     

Evaluation of commercial enzyme linked immunosorbent assay for detection of B19 parvovirus IgM and IgG.

An indirect enzyme linked immunosorbent assay (ELISA) (Parvoscan-B19; Sweden) was compared with an in-house MACRIA for the detection of B19 specific IgM. A Parvoscan-B19 IgG test was also evaluated for its ability to detect a recent B19 infection in paired sera. Two hundred and twenty sera submitted to the laboratory for B19 serology and four MACRIA positive control sera were assayed for B19 IgM. Confirmation of the response of sera giving discordant results in the two assays was sought by the use of a "nested" polymerase chain reaction (PCR) for the detection of B19 DNA. The Parvoscan-B19 IgM test was 79% sensitive and 96% specific. Parvoscan-B19 was poor at detecting parvovirus infection in sera collected two to three months after the onset of symptoms. When sera collected more than seven weeks after the onset of symptoms were excluded from the analysis, Parvoscan-B19 IgM was 84% sensitive and 96% specific. Rubella specific IgM positive sera, rheumatoid factor positive sera, and heterophil antibody positive sera were also assayed for B19 IgM. No false positive results were encountered with these problematic sera. By using the cut off criteria for the Parvoscan-IgM test previously advocated by the manufacturers, 90% sensitivity and 87% specificity could be achieved. False positive results, however, occurred with six of the 17 rubella IgM positive sera, four of the 10 rheumatoid factor positive sera, and two of the 11 heterophil antibody positive sera tested. It is concluded that the Parvoscan-B19 was specific but insensitive when compared with in-house assays.     

Widespread immunoglobulin E-mediated hypersensitivity in the Sudan to the “green nimitti” midge,Cladotanytarsus lewisi (diptera: Chironomidae): I. Diagnosis by radioallergosorbent test

A radioallergosorbent test (RAST) has been developed for the diagnosis of hypersensitivity to “green nimitti” chironomid midges of the species Cladotanytarsus lewisi. There was a high percentage binding of ¹²⁵I-anti-IgE to the allergen particle complex by serum from subjects who were clinically hypersensitive, and the RAST was inhibited following incubations of allergic sera with an extract of the allergen. In 104 hypersensitive subjects (i.e., those with a positive skin test or clinical history of bronchial asthma, with or without rhinitis) and 21 controls, the RAST appeared to be specific and of diagnostic value: (1) The percentage binding was appreciably higher in 38 symptomatic individuals (group I) with strongly positive skin tests as compared with 36 patients with moderate skin reactivity (group II). (2) Seven symptomatic subjects with negative skin tests (group III) had a positive (>6% binding) green nimitti RAST. (3) Positive RASTs were demonstrable in 16 and of 17 patients with positive skin tests in whom the history was equivocal (group IV). (4) Six asymptomatic individuals with positive skin tests (group V) had low RAST values. (5) Six asymptomatic Sudanese controls with negative skin tests gave similar values to those of the group V subjects. (6) All of the sera from 15 nonatopic United Kingdom controls gave less than 6% binding of ¹²⁵I-anti-IgE. There was no statistical correlation between the concentrations of total IgE and the green nimitti RAST values. These results suggest that the RAST may be useful diagnostic test in green nimitti hypersensitivity and may also be of value in studies on the epidemiology and in the monitoring of treatment of this important and widespread allergy problem in the Sudan.     

Experimental induction of rheumatoid factor-like substances in animal trypanosomiasis

Rheumatoid factor-like substances were induced in rabbits by infection with Trypanosoma equiperdum. There was a certain parallelism with the phenomena described earlier with T. gambiense infections in man. The anti-IgG globulins were IgM with a preference for heterologous (human) IgG in the latex fixation test. A correlation was found between the latex fixation titres and the IgM levels in the sera. A naturally occurring pre-infectious agglutinator was not of IgM nature. The anti-IgG globulins developed in all the infected animals, mostly within 2 weeks and often before the IgG levels in the sera started to increase.

The failure to induce rheumatoid factor-like substances in mice infected with a certain strain of T. gambiense indicates the importance of the host–parasite relationship for the formation of rheumatoid factors.

The single radial diffusion method according to Mancini, Carbonara & Heremans (1965) did not give valid results for the determination of IgM in the rabbit sera, but could be used for IgG. IgM was determined by an indirect method.

These experiments might form the basis of a model for investigating the nature of rheumatoid factor formation.

     

Improved immunoturbidimetric method for rheumatoid factor testing.

The performance of two immunoturbidimetric modifications for rheumatoid factor (RF) testing, which differ with respect to the means of complement inactivation (heat treatment and inactivation with polyvinyl sulphonate), were compared in serum samples from 87 patients with rheumatoid arthritis (RA) and from 403 healthy subjects. IgM-rheumatoid factor titres were also measured with an enzyme linked immunosorbent assay (ELISA). Both immunoturbidimetric tests gave positive reactions (rheumatoid factor > or = 20 IU/ml) in 74 out of the 87 (85%) RA sera. In cases with high RF concentrations the results after chemical inactivation tended to be slightly higher compared with heat inactivation. In healthy subjects rheumatoid factor was detected in 19/403 (4.7%) sera using heat inactivation and in 22/403 (5.5%) sera with chemical inactivation of complement. Interrun coefficient of variation in the chemical inactivation assay was 4.4%; with the heat inactivation method it was 8.1%. In the ELISA, a marginally better correlation was noted in the results obtained using chemical inactivation. Inactivation of complement by means of polyvinyl sulphonate offers the advantage of easier test performance and better reproducibility, and the results may reflect more accurately true rheumatoid factor concentrations.     

Screening test for rheumatic diseases: a combined enzyme immunoassay of rheumatoid factors and antibodies to DNA and extractable nuclear antigens.

Three hundred and one sera from patients with rheumatic and other diseases were investigated using a simple enzyme immunoassay for screening of rheumatoid factors and antinuclear antibodies. The assay had a sensitivity of 77% for systemic lupus erythematosus, 90% for the primary sicca syndrome, and 89% for rheumatoid arthritis. Only 13% of sera from patients with chronic non-rheumatic diseases were positive. The test was further evaluated in a group of patients with suspected rheumatic disease who were followed up for six to 12 months. The test was positive in 16 of 17 sera from patients with connective tissue diseases but in only seven of 36 sera (19%) from patients with non-inflammatory joint diseases. None of the four patients with reactive arthritis was positive by this test. The sensitivity of the assay was comparable with that of the agglutination and immunofluorescence tests for rheumatoid factors and antinuclear factors. For the screening of rheumatoid factor and antinuclear antibodies this kind of test panel offers a simple alternative to the conventional tests for small clinical laboratories and for those in which the autoantibody tests could be automated, as the assay can be performed in one working day and only one dilution of serum is needed to obtain a quantitative result.     

Evaluation of Primary Binding Assays for Presumptive Serodiagnosis of Swine Brucellosis in Argentina

An indirect enzyme-linked immunosorbent assay (IELISA), a competitive ELISA (CELISA), and a fluorescence polarization assay (FPA) for the presumptive serological diagnosis of swine brucellosis were evaluated using two populations of swine sera: sera from brucellosis-free Canadian herds and sera from Argentina selected based on positive reactions in the buffered antigen plate agglutination test (BPAT) and the 2-mercaptoethanol (2-ME) test. In addition, sera from adult swine from which Brucella suis was isolated at least once for each farm of origin were evaluated. The IELISA, CELISA, and FPA specificity values were 99.9, 99.5, and 98.3%, respectively, and the IELISA, CELISA, and FPA sensitivity values relative to the BPAT and the 2-ME test were 98.9, 96.6, and 93.8%, respectively. Actual sensitivity was assessed by using 37 sera from individual pigs from which B. suis was cultured, and the values obtained were as follows: BPAT, 86.5%; 2-ME test, 81.1%; IELISA, 86.5%; CELISA, 78.5%; and FPA, 80.0%.     

Surface immunofluorescence assay for diagnosis of Lyme disease.

A surface immunofluorescence assay (SIFA) was analyzed and compared with a conventional indirect immunofluorescence assay (IFA) and whole-cell enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin G (IgG) antibodies to Borrelia burgdorferi in sera from patients with Lyme disease. Fifty-five patients with syphilis and 33 patients with rheumatoid arthritis were used as disease controls. The sensitivity of the SIFA was low during the acute phase of Lyme disease (sera from seven of nine patients presenting with erythema chronicum migrans were negative during the first 2 months of illness); later, seroconversion was observed in all patients at various times during convalescence. Sera from five patients with complicated Lyme disease were strongly positive. SIFA was found to be highly specific, since sera from all patients with secondary or latent syphilis and patients with rheumatoid arthritis did not react in the test. Strong cross-reactivity occurred when these sera were tested in conventional IFA and ELISA; sera from 38 (69%) patients with syphilis were positive by IFA and sera from 51 (93%) patients were positive by ELISA, whereas 7 (21%) and 12 (36%) of the serum samples from patients with rheumatoid arthritis were positive by IFA and ELISA, respectively. Immunoblot analysis of SIFA-positive sera showed that the 31- and 34-kDa outer surface proteins (proteins A and B, respectively) of B. burgdorferi were the major reactive antigens involved in the test. The results support a role for SIFA in the investigation of complicated Lyme disease as well as in the differentiation of Lyme disease from other diseases associated with B. burgdorferi cross-reactive antibodies.     

The Trypanosoma lewisi immunofluorescence test: A new simple technique for simultaneous determination of total antinuclear antibodies and the detection of antibodies to double-stranded DNA

An indirect immunofluorescence technique was developed for the detection of antibodies to dsDNA and the simultaneous assessment of antinuclear antibodies ‘in toto’ (ANA). This assay was based upon the use as substrate of smears of peripheral blood derived from rats infected with Trypanosoma lewisi. T. lewisi possesses a giant kinetoplast posteriorly to the nucleus. Enzyme digestion and absorption experiments provided strong evidence that T. lewisi kinetoplast contains dsDNA uncontaminated by other nuclear antigens. The T. lewisi immunofluorescent test was evaluated on a total of 130 sera (30 from patients with SLE) and compared with radioimmunoassays for antibodies to dsDNA ([¹²⁵I]dsDNA-RIA) and antibodies to ssDNA ([¹²⁵I]ssDNA-RIA). Excellent correlation was found between kinetoplast immunofluorescence and [¹²⁵I]dsDNA-RIA, whereas no non-SLE sera showing significant ssDNA binding activity gave kinetoplast staining. With a single exception, only SLE sera reacted with T. lewisi kinetoplast. Sera containing auto-antibodies other than ANA did not induce fluorescene of any part of the parasite, including the flagellum and its base. These results indicated that the T. lewisi immunofluorescence test is specific and reliablem and combines the advantages of Crithidia luciliae with those of Trypanosoma gambiense. It may be used routinely for evaluating of total ANA and simultaneous detection of antibodies against dsDNA.     

Immunological properties of isolated IgG and IgM anti-gamma-globulins (rheumatoid factors)

Anti-gamma-globulins of the IgG and IgM classes have been isolated from sera of normal individuals and from patients with osteoarthritis, rheumatoid arthritis and juvenile rheumatoid arthritis. All of the isolated antibodies gave precipitin curves with heat-aggregated, reduced and alkylated gamma-globulin. IgM anti-gamma-globulins gave a positive latex fixation test at 4°C and 37°C while IgG anti-gamma-globulins generally gave a positive test only at 4°C. Anti-gamma-globulins from normals did not fix complement but IgG and IgM anti-gamma-globulins from rheumatoids fixed complement to a similar degree. This in vitro complement fixation could account at least in part for the diminished complement levels seen in many rheumatoid synovial effusions.     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.     

Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody.

The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the ¹²⁵I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, ¹²⁵I-labelled Clq BA and Raji-cell radioimmunoassay (RIA).

Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application.

     

Performance of the Epstein-Barr Virus and Herpes Simplex Virus Immunoglobulin M Assays on the Liaison Platform with Sera from Patients Displaying Acute Parvovirus B19 Infection

Acute parvovirus B19 infection has been reported to cause false-positive results frequently in the Epstein-Barr (EBV) and herpes simplex virus (HSV) immunoglobulin M (IgM) assays from DiaSorin performed on the Liaison platform. We tested 65 sera from patients with a presumptive or conclusive diagnosis of acute parvovirus B19 infection in both assays and obtained no false-positive results in the EBV IgM test and 10.4% nonspecific reactivities in the HSV IgM assay. Our data support the specificity of both assays in this clinical setting.Biological diagnosis of febrile diseases of viral origin commonly relies on serological tests, demonstration of seroconversion being the “gold standard.” Convalescent-phase sera are often unavailable, so that the presence of virus-specific immunoglobulins M (IgMs) in acute-phase sera is the only marker supporting the diagnosis, unless sufficient levels of virus-specific IgGs are detected, and thus avidity index (AI) values can be determined. The interpretation of an isolated positive IgM test is frequently complicated by the concurrent appearance in sera of IgMs against heterologous viral agents. The presence of IgMs against Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, measles virus, and rubella virus has been reported to occur in sera from patients with acute human parvovirus B19 infection (3, 5-7). IgM antibodies against unrelated viruses appearing in the setting of acute parvovirus B19 infection may be either cross-reactive antibodies or truly virus-specific antibodies secreted as a result of heterologous virus reactivation driven either directly or indirectly by parvovirus B19 infection. Alternatively, false-positive results in viral IgM assays may occur as a result of spurious binding of nonspecific serum antibodies to the solid phase. In this context, it has been recently reported (1) that an exceedingly high percentage of sera drawn from patients with either a conclusive or a presumptive diagnosis of acute parvovirus B19 infection gave a false-positive result in several IgM immunoassays (EBV, CMV, herpes simplex virus, and Borrelia burgdorferi sensu lato) from DiaSorin (Saluggia, Italy) performed on the Liaison platform. The false-positive reactivities were apparently due to nonspecific binding of IgMs to the antigen-coated beads used in the assay (2) and were found to be partially eliminated (although sera remained positive) by adding polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) to the dilution buffer. The diagnostic relevance of the above findings prompted us to evaluate the performance of the DiaSorin EBV and HSV IgM assays with sera from acutely parvovirus B19-infected patients in our setting. Sixty-five sera from 65 patients (45 females and 20 males, aged 4 to 70 years, median age of 15 years) with a presumptive clinical and/or biological diagnosis of acute parvovirus B19 infection sent to our laboratory from January 2005 to January 2009 were retrieved for analysis. These sera had been tested in the parvovirus B19 enzyme immunoassay (EIA) from Biotrin International (Dublin, Ireland) and found to be IgG and IgM positive (n = 53) or IgG negative and IgM positive (n = 12). In the Biotrin assay, antibodies against a baculovirus-expressed VP2 conformational protein are detected. The B19-specific IgM assay is a mu capture EIA, while the IgG assay is an antigen capture EIA. This immunoassay has 89.1% sensitivity and 99.4% specificity for IgM detection (4). Clinical data were available for 41 patients. These patients displayed fever and one or more of the following clinical or biological signs compatible with acute parvovirus B19 infection: exanthema, arthralgia, and mono- or pancytopenia. To confirm the acute nature of parvovirus B19 infection, IgG avidity tests were performed. In brief, parvovirus B19 IgG avidity was determined with the Biotrin assay. The first step of the assay was modified to include two washes (5 min each) with a washing buffer containing urea (4 M). The AI value (as a percentage) was calculated as follows: (absorbance of parvovirus B19 IgGs in the presence of urea/absorbance of parvovirus B19 IgG in the absence of urea) × 100. AI values of <25% are seen early after infection, and AI values of >80% are observed in past infections (8). In our experience, AI values of <40% should be considered indicative of a recent infection when using urea at 4 M in the washing buffer (unpublished observation). IgG AI values were determined for 20 of the 53 parvovirus B19 IgG-positive sera of which a sufficient sample volume was available for analysis. All 20 sera gave AI values of <40% (median, 32%; range, 6.6 to 39.42%), thus confirming the acute nature of the parvovirus B19 infection in these patients. In 8 of the 12 patients with an isolated IgM reactivity profile in the acute-phase serum specimen, acute parvovirus B19 infection was confirmed by demonstration of seroconversion in convalescent-phase sera. No follow-up samples were available for the remaining four patients. Thirty-four of these sera had been tested for viral capsid antigen IgM antibodies (Captia VCA IgM; Trinity-Biotech, Bray, Ireland) as requested and found to be negative. Fifty-five sera were tested for HSV IgMs (Herpes simplex 1 + 2 IgM test; Vircell, Granada, Spain). Forty-four sera had a negative result, seven displayed a positive result, and four had an indeterminate result. The sera were tested in the EBV IgM (n = 65) and HSV IgM (n = 55) assays from DiaSorin on the Liaison platform. Interpretation of results was done according to the instructions of the manufacturer. Data are shown in Table ​Table1.1. None of the sera tested positive in the EBV IgM assay. Five of the 44 HSV IgM-negative sera (in the Vircell assay) tested positive in the DiaSorin HSV IgM assay. These sera, however, had a negative result when PVP (0.1%) and PVA (0.005%) (both purchased form Sigma-Aldrich) were added to the dilution buffer. Of the seven sera testing positive in the HSV IgM assay from Vircell, five did so in the DiaSorin HSV IgM assay. A number of sera (n = 12) were also tested in the B. burgdorferi sensu lato IgM assay from DiaSorin and found to be negative.

TABLE 1.

Performance of the DiaSorin EBV, HSV, and B. burgdorferi sensu lato IgM assays with sera from patients with acute parvovirus B19 infection

Rapid indirect enzyme linked immunosorbent assay (ELISA) for detecting antitoxoplasma IgG: comparison with dye test.

A rapid and simple enzyme linked immunosorbent assay (ELISA) for the detection of specific IgG against Toxoplasma gondii was compared with the dye test on 533 serum samples. In general, results were comparable but not with sera that contained high concentrations of toxoplasma specific IgM or that had been heated at 56 degrees C. There were no false positive results with sera containing rheumatoid factor or anti-nuclear factor. It is concluded that if a dye test is not to be performed then the serum should be tested for both toxoplasma specific IgG and IgM to avoid misleading results. Heat inactivated serum should also not be tested in this type of specific IgG assay.     

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.     

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.     

Comparison of a New Immunochromatographic Test to Enzyme-Linked Immunosorbent Assay for Rapid Detection of Immunoglobulin M Antibodies to Hepatitis E Virus in Human Sera

An immunochromatographic test for rapid detection of IgM antibodies in patients with acute hepatitis E infection was developed utilizing the well-characterized recombinant protein EP2.1 and monoclonal antibody 4B2. The new rapid test based on a novel reverse-flow technology was able to generate a positive result within 2 to 3 min. Our study showed that this test was able to detect anti-HEV IgM antibodies in 96.7% of the patient samples tested (n = 151) while maintaining an excellent specificity of 98.6% with samples from various patient or healthy control groups (total n = 208). Furthermore, this rapid test gave a good specificity of 90.9% when tested with rheumatoid factor (RF)-positive sera (RF value of ≤850 IU/ml; n = 11) although a higher concentration of RF in samples might cause cross-reactivity. The new test has a good agreement of 97.2% with a kappa value of 0.943 when compared with a reference enzyme-linked immunosorbent assay. The positive predictive value and the negative predictive value for the rapid test thus reached 98.0 and 97.6%, respectively. This is the first rapid, point-of-care test for hepatitis E and will be especially useful for the diagnosis of acute hepatitis E virus infection in field and emergency settings and in resource-poor countries.