Rapid diagnosis of echovirus 33 infection by neutralizing specific IgM antibody

In 1982, we isolated 20 strains of echovirus type 33 (EV33) from 18 patients. We studied the humoral response to EV33 of 2,437 subjects from whom at least one serum was available during the same year. In 388 subjects with the neutralizing antibody level at 64 or more, we assayed the EV33 IgM antibodies by seroneutralization test after fractionation of sera by ion exchange chromatography. One hundred ninety-five subjects (8.0%) had a high titre (greater than or equal to 32) of EV33 IgM antibodies, which was considered as evidence of recent infection. The EV33-positive IgM fractions were assayed against five other enteroviruses. Sixty percent of the IgM fractions did not cross-react with any of the five serotypes, 8.7% cross-reacted with at least one serotype but with predominant EV33 IgM response, and 31.3% had an equivalent or greater amount of non-EV33 IgM antibodies; the type specificity of the assay was directly related to the age of the subjects. These findings suggest that determination of neutralizing specific IgM antibody is a sensitive and rapid test for the diagnosis of enterovirus infections, especially in young people.     

Rapid diagnosis of echovirus type 33 meningitis by specific IgM detection using an enzyme linked immunosorbent assay (ELISA)

During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.     

Detection of IgM antibodies against cytomegalovirus: comparison of two radioimmunoassays, enzyme-linked immunosorbent assay and immunofluorescent antibody test

The sensitivity and specificity of direct antibody radioimmunoassay (RIA), M-antibody capture RIA (MACRIA), enzyme-linked immunosorbent assay (ELISA), and the immunofluorescent antibody (IFA) test for the detection of CMV-specific IgM was compared using 40 sera selected from different groups of patients. RIA, MACRIA, and ELISA gave concordant results with thirty-two sera but discordant results with eight sera, of which three were cord sera from congenitally infected babies, three were from immunocompromised patients with recurrent CMV infections, and two were from patients with lymphadenopathy and Paul-Bunnell-positive mononucleosis, respectively. RIA, MACRIA, and ELISA were of similar sensitivity with sera from adult patients, but ELISA was apparently less sensitive than RIA and MACRIA for the detection of CMV IgM in cord serum. By comparison IFA was significantly less sensitive than the other three tests. Rheumatoid factor is reactive in RIA, ELISA, and IFA but can efficiently be removed by absorption with latex-IgG beads or cross-linked human IgG.     

用ELISA方法检测埃可病毒感染的特异性IgM抗体

目的 研究埃可病毒感染的无菌性脑膜炎的诊断方法。方法 采用埃可病毒混合抗原包被酶标板，用抗人γ链处理人脑脊液标本，通过酶标二抗及底物显色，建立了抗埃可病毒IgM的间接ELISA检测方法；并用特异性试验、对照和重复性试验证实方法的可靠性和实用性。结果 在临床诊断为无菌性脑膜炎的78例患者脑脊液中有14例阳性（17.9%)，而细菌性脑膜炎的36例患者脑脊液中仅有1例阳性（2.8%)，28例脑外伤患者脑脊液均为阴性，ELISA阳性的5份脑脊液中和试验4例阳性，而ELISA阴性的5份脑脊液中和试验均为阴性；该方法与脊髓灰质炎病毒、柯萨奇A组病毒7型和柯萨奇B组病毒1-6型无交叉反应；ELISA阳性的6份标本经特异性IgM破坏和阻断试验均全部转为阴性。结论 本方法快速，简便、可靠、适合于临床早期特异性诊断。     

Detection of rotavirus-specific IgG antibodies by immunoperoxidase assay and enzyme-linked immunosorbent assay

An indirect immunoperoxidase assay (IPA) has been developed for determination of IgG antibodies to rotavirus. The technique employed as antigen, SA-11 infected MA 104 cells, which were air-dried on glass slides and acetone-fixed. In parallel, rota-specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA). Specific IgG antibodies to rotavirus were determined in sera of healthy children and in sera of patients suffering from gastroenteritis. A good correlation (r = 0.92) and (r = 0.98) for healthy children and patients, respectively, was found between IPA and ELISA techniques. The IPA technique is rapid and simple and positive results, because of the intensive staining, are easily read by low-power light microscope. The potential application of IPA and ELISA methods in serodiagnosis of rotavirus infections is discussed.     

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.     

Serum IgA, IgG, and IgM responses to different enteroviruses as measured by a coxsackie B5-based indirect ELISA.

An enterovirus-specific indirect ELISA, based on a single local isolate of coxsackie B5 as antigen, was used to study the IgA, IgG, and IgM responses in 19 patients with a recent or current enterovirus infection. Twelve different enterovirus serotypes were isolated from 15 patients. Paired serum samples were available from 10 and a single serum from 5 of these 15 patients. In addition, 4 patients diagnosed by a significant titer rise of complement fixing antibodies to enterovirus were included. A serological diagnosis, defined as an increase in titer of enterovirus IgG and/or presence of enterovirus IgM, were established in all 14 patients with paired sera. Enterovirus IgM was present in either a single serum or in both sera in 13 of them. Out of 5 patients with a single serum sample only, enterovirus IgA or enterovirus IgM was found in 4. Specific IgA was present in either a single serum or in both sera in 14 of the 19 patients. Seven of the 10 enterovirus isolate patients with paired sera had a significant titer rise of complement fixing antibodies; however, all 10 were diagnosed by ELISA. Among 64 healthy controls 2 had enterovirus IgA and none had enterovirus IgM. In conclusion, the use of a single antigen-based ELISA was found to be reliable for the diagnosis of recent and current enterovirus infections.     

人生长激素的生物素—新和素放大酶联免疫法的建立

为建立有生物素和亲和素放大系统的人血清生长激素 (hGH)酶联免疫分析法 (BA ELISA) ,以微量的GH抗原免疫BALB/C小鼠 ,利用杂交瘤技术制备出 2 9株分泌抗hGH单克隆抗体 (MCA)的杂交瘤株。选择最佳配对MCA ,利用亲和素与生物素高亲和力的特性 ,建立了血清hGH双位点的BA ELISA法。我们的MCA滴度为 (0 5～ 5 0 0 )× 10 4,亲和常数Ka =(0 13～ 1 40 )× 10 10 L/mol。血清hGH的ELISA的灵敏度为 0 0 4± 0 0 1μg/L ;在血清中分别加入 1,2 5 ,5 μg/L的hGH ,其回收率为 90 7%～ 10 7 6 % (平均为 10 1 2 0 %± 0 0 3 % ) ;hGH含量为 2 8、10 0、2 9 8μg/L的血清批内、批间变异系数分别为 4 9%、3 8%、7 9%和 6 2 %、3 5 %、9 5 % ;用本法测定了正常儿童、生长激素缺乏症 (GHD)患儿和活动性肢端肥大症患者空腹血清hGH水平 ,分别为 1 87± 3 0、0 30± 0 42和 8 71± 6 95 μg/L。上述结果表明 ,本ELISA方法灵敏、特异、稳定、准确 ,能确切反映hGH分泌功能 ,在诸多方面均优于RIA。     

An anti-human μ chain monoclonal antibody: Use for detection of IgM antibodies to toxoplasma gondii by reverse immunosorbent assay

A precipitating anti-human μ chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1κ) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, λ and κ chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the Cμ2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the Cμ2 domain, such a McAb coulb be used for IgM capture in the detection of specific IgM to Toxoplasma gandii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human μ chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.     

Effects of bovine serum albumin on antibody determination by the enzyme-linked immunosorbent assay

The effect of bovine serum albumin (BSA) post-coating and addition of BSA to serum diluent was studied in an IgG and IgA Candida antibody enzyme-linked immunosorbent assay (ELISA). BSA post-coating decreased the background readings and increased the specific Candida antibody activity in most sera. However, in a few sera post-coating alone increased background readings and this effect was probably due to the occurrence of BSA antibodies. It could be abolished by the addition of BSA to serum diluent. It is therefore suggested that BSA post-coating should only be used in combination with BSA addition to serum dilution buffer in ELISA.     

Recent enterovirus infection in type 1 diabetes: evidence with a novel IgM method

Enterovirus (EV) infection has been associated with Type 1 (T1D) diabetes and on a few occasions virus could be isolated at onset of the disease. Using two such isolates as antigens in a quantitative PCR enhanced immunoassay (T1D-EV-QPIA) we have measured IgM antibodies against such potentially diabetogenic viruses in serum from 33 newly diagnosed T1D children, 24 siblings, and 27 healthy children. Sera were also analysed with regard to autoantibodies against GAD65, the cytokine TNF-alpha and the chemokine IP-10. EV-RNA detection was performed on peripheral blood mononuclear cells (PBMC). IgM antibodies against this "new" EV antigen were more frequent in serum from T1D children than in serum from siblings and/or controls (P < 0.001). EV-RNA was detected more frequently in PBMC from T1D children than in healthy control children (P < 0.001) and also compared to the siblings (P < 0.003). The cytokine TNF-alpha was less frequently detected in serum from the T1D children compared with serum from siblings and/controls (P < 0.001). A positive correlation was found between the results obtained with the T1D-EV-QPIA and the EV-PCR (P < 0.001). These findings are in line with earlier findings of an increased frequency of enteroviral infections in newly diagnosed T1D patients. In addition, we found that T1D children at onset of the disease had lower frequencies of the chemokine TNF-alpha in their serum than age- and sex-matched controls had, suggesting an impaired immune response.     

Immunoglobulin and specific antibody responses to Rhodococcus (Corynebacterium) equi infection in foals as measured by enzyme-linked immunosorbent assay.

Humoral immune response to intestinal Rhodococcus (Corynebacterium) equi in horses was studied by enzyme-linked immunosorbent assay. Anti-R. equi immunoglobulin M (IgM), IgG, and IgA antibodies were demonstrated in the healthy horse population. Adult horse levels of anti-R. equi IgM and IgG antibodies were reached by 5 to 9 weeks of age in two healthy newborn foals. R. equi was recovered from the foals in the range of 10(3) to 10(4) per g of intestinal contents. A 1-week-old foal was infected with R. equi by mouth daily for 9 weeks. The foal did not show any clinical signs of illness. Anti-R. equi IgM antibody values in the foal increased about 5 to 8 weeks after initial inoculation, similar to the naturally occurring immune response to intestinal R. equi. There were differences among the antibody responses to R. equi in healthy horses, foals with suspected infection, and infected foals. These results suggest that exposure to R. equi is widespread in the horse population and that intestinal R. equi is the most important source of antigenic stimulation for a naturally occurring immune response in horses.     

Use of a monoclonal antibody in a double-sandwich ELISA for detection of IgM antibodies to Toxoplasma gondii major surface protein (P30)

A double-sandwich ELISA, developed for detection of IgM antibodies to the major surface protein of Toxoplasma gondii (P30), is proposed for the diagnosis of acute acquired toxoplasmosis. The method is based on the capture of serum IgM antibodies, which are revealed indirectly by the sequential addition of a Toxoplasma extract and a beta-galactosidase-conjugated anti-P30 monoclonal antibody. All 57 patients tested with serological characteristics of recently acquired toxoplasmosis showed high levels of IgM anti-P30 antibodies. In addition, 5 out of the 24 patients with chronic toxoplasmosis and all 7 patients with a clinical acute infection in which the classical IgM serology was negative, also presented significant anti-P30 IgM antibodies. Patients with either rheumatoid factor or antinuclear antibodies were all negative. In view of its simplicity, specificity and sensitivity, this method is recommended for the current diagnosis of T. gondii infection.     

Quantitative assessment of human leukocyte antigen-G protein in amniotic fluid by a double-determinant enzyme-linked immunosorbent assay using anti-human leukocyte antigen-G-specific antibody '87G'.

PROBLEM: The objective of this study was to establish an enzyme-linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)-G protein in amniotic fluid. METHOD OF STUDY: We established a double-determinant ELISA system using the anti-HLA-G specific mouse monoclonal antibody '87G' as a capture antibody and the horseradish-peroxidase labeled rabbit anti-human beta2-microglobulin antibody as a detection antibody. We then measured the concentration of HLA-G protein in amniotic fluid samples from nine normal second-trimester pregnant women and in serum samples from eight normal males. RESULTS AND CONCLUSION: HLA-G protein was detected in amniotic fluid at a concentration of 275 ng/ml (197-343 ng/ml) (median value and 95% confident range), whereas the concentration of HLA-G protein in male serum was below the minimum detection level.     

Rapid diagnosis of acute Epstein-Barr virus infection by an indirect enzyme-linked immunosorbent assay for specific immunoglobulin M (IgM) antibody without rheumatoid factor and specific IgG interference.

An indirect enzyme-linked immunosorbent assay (ELISA) for detection of Epstein-Barr virus-specific immunoglobulin M (IgM) antibody was developed with commercial reagents. Sera containing rheumatoid factor (RF) (as little as 0.5 IU/ml) coupled with specific IgG resulted in false-positives in the ELISA. This interference was eliminated by the use of anti-human IgG antibodies to remove RF and IgG. Thus, pathogen-specific IgG complexes to which IgM-RF could be bound during the subsequent test were inhibited, and competition between specific IgG and IgM was also prevented. Of the 1,672 serum specimens tested, 353 were found to be Epstein-Barr virus IgM antibody positive by indirect immunofluorescence (IF). Compared with the IF test, the ELISA showed 96.6% sensitivity, 99.7% specificity, and 99% accuracy. Further evidence indicated that most of the 12 ELISA false-negatives were IF false-positives. There was a linear correlation between mean ELISA values and increasing IF titers (r = 0.96). However, the IF test has the disadvantages that it lacks automated reading and requires considerable technical expertise, both of which restrict the range of laboratories performing the test. The indirect ELISA has the advantages that it is simple and rapid and can be automated. All the reagents used in this assay are commercially available, have been prestandardized, and are stable.     

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the β₂-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on β₂, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to β-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes.

Using this test, it is shown that, of the 7 anti-β₂ monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.     

IgM antibody detection of ppUL80a and ppUL32 by immunoblotting: An early parameter for recurrent cytomegalovirus infection in renal transplant recipients

The value of IgM detection for the early diagnosis of an active cytomegalovirus (CMV) infection in renal transplant recipients was evaluated prospectively. Sequential serum samples obtained from 22 allograft recipients with active CMV infection were tested for the presence of CMV-specific immunoglobulin M antibodies (IgM) by an enzyme-linked immunosorbent assay (ELISA) and a microparticle enzyme immunoassay (MEIA) and were compared with the Western-immunoblotting technique (IB). The time course of CMV IgM antibody detection was evaluated in relation to the shell vial assay (SVA), CMV disease, and immunosuppressive regimen. By IB, IgM antibodies against the capsid protein ppUL80a and the basic matrix phosphoprotein ppUL32 were detected in all 22 recipients with active CMV infection. Using the MEIA and the ELISA, the presence of CMV IgM antibodies was detected in 17 (77%) and ten (46%) of these 22 recipients, respectively. The SVA was the earliest parameter for detection of primary CMV infection in seven of nine (78%) recipients, in contrast to two of 13 (15%) patients with recurrent CMV infection (P < .05). The detection of IgM antibodies by IB was the earliest parameter for detection of recurrent CMV infection in seven out of 13 (54%) recipients in contrast to one out of nine (11%) patients with primary CMV infection (P < .05). During a primary CMV infection, the development of an abundant IgM antibody response was associated with recovery from CMV disease and the end of the viremic phase. © 1996 Wiley-Liss, Inc.     

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the β2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the β₂-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on β₂, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to β-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes.Using this test, it is shown that, of the 7 anti-β₂ monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.     

Comparison of antibody measurements against Aspergillus fumigatus by means of double-diffusion and enzyme-linked immunosorbent assay (ELISA)

IgG antibody titers against Aspergillus fumigatus from different sera were measured by means of a standardized ELISA and compared with precipitates measured by double diffusion (D.D.). There was a significant correlation between the number of precipitates and the ELISA IgG titers in the 758 sera examined. However, in several individual sera within this group, large deviations between these two immunologic parameters were found. Further analysis indicated that ELISA detects antibodies against nonprecipitating antigenic components in addition to the antibodies detected by D.D. Furthermore, not all precipitating antigenic components appear to play a role in the detection of antibodies against A. fumigatus by ELISA. Patients with aspergillosis largely show increased titers of IgG antibody by ELISA even when the results of D.D. are negative, except those of patients with Aspergillus-provoked asthma which fall within a normal range.     

Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay

A simple, general procedure is described for the determination of the dissociation constant (KD) of antigen-antibody equilibria in solution. First the monoclonal antibody is incubated in solution with the antigen until the equilibrium is reached; then the proportion of antibody which remains unsaturated at equilibrium is measured by a classical indirect ELISA. The experimental values of KD found by this ELISA procedure for 2 monoclonal antibodies are shown to be very close to those obtained by conventional methods (immunoprecipitation of the radiolabeled antigen, or fluorescence transfer). Moreover, it is shown that, provided the measurements are made under conditions where the total antigen concentration is in large excess over the total antibody concentration, the dissociation constant of antibody-antigen complexes can be determined even with crude preparations of monoclonal antibody. The sensitivity of the ELISA used permits the detection of very small concentrations of antibody and the determination of KD values as small as 10(-9) M. This method also offers the great advantage of dealing with unmodified molecules since no labeling of either the antigen or the antibody is required.