A comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed.All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone.Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM.     

2007-2010年广州市海珠区疑似麻疹风疹病例血清学检测结果分析

目的分析广州市海珠区2007-2010年麻疹及风疹的流行特征。方法采用IgM抗体捕捉ELISA对广州市海珠区2007-2010年麻疹、风疹疑似病例血清标本进行IgM抗体检测。数据用SPSS13.0进行统计分析。结果麻疹IgM抗体总阳性率为44.30%,风疹IgM抗体总阳性率为13.09%,各年度麻疹、风疹的阳性率差异有统计学意义（χ2=171.708,P〈0.05;χ2=34.191,P〈0.05）。男女麻疹、风疹病例性别分布差异均无统计学意义。麻疹阳性病例年龄主要分布在8月龄～7岁及15～29岁,风疹阳性病例年龄主要分布在15～29岁。4年间麻疹、风疹阳性病例分别集中在3～7月、3～6月。结论海珠区2010年麻疹阳性率较前几年明显降低,说明防控措施有力,但2010年风疹的阳性率升高应引起足够的重视。麻疹的发病人群主要为学龄前儿童和青年,风疹的发病人群以青年为主。春季为麻疹、风疹的高发季节,应适时进行防控,避免暴发流行。     

Assessment of immune status against measles,mumps, and rubella in young Kuwaitis: MMR vaccine efficacy

Seroprevalence studies on measles, mumps, and rubella immunoglobulin G (IgG) antibodies after the implementation of the measles-mumps-rubella (MMR) vaccine are lacking in Kuwait. This study is an age-stratified serological study to assess the herd immunity to measles, mumps, and rubella among the young Kuwaiti population to evaluate the effectiveness of the MMR vaccine. IgG antibody titers to mumps, measles, and rubella were determined with commercial immune-assay in serum samples of 1000 Kuwaitis aged 5 to 20 years. The highest level of seropositivity was to measles (94.6%), which was significantly higher in females than in males. The highest seronegativity was for mumps (29%). The percentage of the young Kuwaiti population who were serologically positive for all the components of the MMR vaccine was 47%, and 2% of the individuals were without any protective antibodies to measles, mumps, and rubella. Females aged 5 to 10 years were best protected to rubella; however, seronegativity in 8.2% of 11- to 20-year-old females makes them vulnerable to rubella virus infection and congenital complications during pregnancy. The study provided insight into the effect of the MMR vaccine on seroprevalence of antibodies against measles, mumps, and rubella in Kuwait, which will contribute to the global knowledge base of vaccine coverage and help to inform elimination strategies. The findings strengthen the need for a third dose of MMR vaccine and catch-up campaigns for the young Kuwaiti population to increase vaccination coverage and prevent waning immunity, especially among those who received only one dose of the vaccine during childhood.     

Radioimmunoassay for the detection of virus-specific IgA antibodies in saliva

The use of a sensitive and versatile radioimmunoassay (RIA) for detection of mumps-specific IgA and measles-specific IgA in unconcentrated saliva samples is described. The samples were obtained either by expectoration or by swabbing of the oral cavity, with or without stimulation of secretion, and were inactivated and clarified before testing. Mumps-specific IgA antibodies were detected as early as one day after onset of illness and peaked at 1-2 weeks after onset. Measles-specific salivary IgA antibodies were detected in 15-month old children 2-3 weeks after immunization. These results suggest that the RIA technique may be useful for early diagnosis of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects.     

Solid-phase radioimmunoassay determination of virus-specific IgM antibody levels in a follow-up of patients with naturally acquired measles infections

The question of the exact disappearance time or possible persistence of measles-specific IgM antibodies after naturally acquired measles virus infections was studied with a sensitive solid-phase radioimmunoassay (RIA) method. A total of 30 patients were analyzed with follow-up times varying from 4.5 to 8 months; all were measles IgM positive in the first serum specimen obtained after the onset of rash. In 29 of 30 patients, the measles IgM declined to undetectable levels by approximately 90 days. The remaining patient developed postmeasles encephalitis, however, and was found to have a prolonged measles IgM antibody response. For comparison, the measles-specific IgG response was also studied and was found to develop only slightly later than the IgM response, with levels then remaining high and stable up to 8 months later. Although apparent measles IgM antibodies were found in 1 of 64 nonmatched adult controls, they were due to the presence of high levels of IgM-class rheumatoid factor. The data presented indicate that measles IgM antibodies begin to decline soon after the onset of rash and reach negative levels 1 to 3 months later; in complicated infections, however, measles IgM antibody synthesis may not terminate normally.     

Characterisation of mumps virus genotype C among patients with mumps in India

Measles, mumps and rubella (MMR) vaccine failure had been reported globally and here, we report that it occurs in India now. MMR vaccinated people have developed acute mumps accompanied by anti-mumps immunoglobulin M. Genotypic characterisation revealed that the circulating mumps strain was genotype C, which is distinct from the vaccine strain of genotype N (L-Zagreb). This is the first report in India to suggest that genotype C is responsible for the present mumps infection. Thus, the present MMR vaccine must be revamped and optimised for its efficacy to prevent any future mumps epidemics.     

Investigation of measles and rubella outbreaks in Tamil Nadu, India-2003

The aims of the present study were to confirm measles outbreaks by detection of measles-specific IgM antibodies, isolation of measles virus, and genetic characterization to document the circulating genotypes in Tamil Nadu. Eight outbreaks were reported from six districts of Tamil Nadu, India during the period Jan-Dec 2003. Blood samples were collected for serology, urine, and throat swabs for virus isolation. Genotypic characterization of measles isolates was based on the sequence of the N gene. All the clinically suspected outbreaks (n = 8) were confirmed by serology; six out of the eight as measles and two as combination of measles and rubella highlighting the need to carry out rubella serology on measles-negative samples. Genetic characterization of three isolates obtained revealed one as genotype D4 and two as D8. Measles genotypes D4 and D8 were found to circulate in three districts of Tamil Nadu. It is necessary to be aware of the circulating genotypes within the geographical area. The information would be valuable to evaluate control measures and identify viral transmission and importation.     

Detection of early hemagglutination inhibitory antibodies to rubella virus by pretreatment of sera with streptococcal cells

A technique that is based on absorption of sera with streptococcal cells and hemagglutination inhibition (HI) was evaluated for its feasibility for serologic diagnosis of recent rubella. The mixture of AR1 and AW43 cells removes IgG and IgA from the kaolin-treated sera, leaving IgM and a trace of IgA, probably oligomeric IgA. Consequently, after the absorption with streptococci, the HI antibodies are detectable exclusively in the early sera of patients with rubella. The streptococci (AR1 and AW43) have several advantages as the absorbent over the staphylococcus (Cowan I) that has been used routinely.     

The detection of antibodies to cytomegalovirus in the sera of renal transplant patients by an igm antibody capture assay

An IgM antibody capture assay for detection of cytomegalovirus (CMV) IgM antibody (MACRIA) was developed. It was shown to be of similar sensitivity to the indirect immunofluorescence test but has the advantage that rheumatoid factor does not react in it and pretest fractionation of serum is not required. It does, however, give false results with some Paul Bunnell-positive sera. The assay was used to measure the IgM response in 28 renal transplant patients followed prospectively. Seven patients (100%) with primary infections and six of 13 (46%) patients with secondary infections developed IgM by MACRIA. Nine of 13 (69%) patients with CMV IgM-positive sera had symptoms other than pyrexia associated with CMV infections, while only one of seven (14%) IgM-negative infections were symptomatic. Four of seven irreversible rejection episodes were associated with CMV IgM. The possible significance of CMV IgM production is discussed.     

Detection of IgM antibodies against rubella virus: comparison of two indirect ELISAs and an anti-IgM capture immunoassay

A commercial antibody capture enzyme immunoassay (Rubenz M) was compared to two commercial indirect enzyme immunoassays (Enzygnost IgM, Rubazyme-M) for the detection of rubella-specific IgM. Five hundred and fifty-two sera collected between the day of onset and 272 days after the onset of the exanthem of primary rubella were tested. Rubenz M was more sensitive early and late after the onset of the exanthem than the two indirect ELISAs. Rubenz M also appeared more sensitive when 240 sera were examined from patients with possible rubella in pregnancy, reinfection in pregnancy, suspected intrauterine infection, and recent vaccination. However, 5.5% of 968 pregnant women with no history of rubellalike symptoms or recent vaccination, the majority with elevated HAI titers, gave a low-positive or borderline result with Rubenz M. None of these women delivered a congenitally infected child. Therefore, borderline and low-positive results must be interpreted with caution, as for any assay for rubella-specific IgM.     

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.     

广州市海珠区2007-2010年麻疹、风疹检测结果分析

目的分析广州市海珠区2007-2010年麻疹、风疹流行特征,为麻疹、风疹的预防和控制提供可靠依据。方法对辖区内2007-2010年的麻疹、风疹病例的实验室检测结果进行统计分析。结果辖区内2007-2010年间麻疹年平均发病率为15.61/10万;风疹年平均发病率为2.57/10万。麻疹病例多集中在婴幼儿及中青年人群;风疹病例以10～25岁青少年为主,且麻疹、风疹的病例发生具有明显的季节性。结论要达到消除麻疹、控制风疹发病率的目标,应继续做好计划免疫工作,提高免疫覆盖率,严密监测,预防暴发。     

Synthesis of measles virus-specific IgM antibodies and IgM-class rheumatoid factor in relation to clinical onset of subacute sclerosing panencephalitis

Thirty-seven serum and 37 cerebrospinal fluid (CSF) specimens from 27 patients with subacute sclerosing panencephalitis (SSPE) were tested for measles virus (MV) IgM antibodies and IgM-class rheumatoid factor (RF) to determine if a temporal relationship exists between SSPE onset and these immunoglobulins. Sensitive solid-phase radioimmunoassays were used for immunoglobulin detection. Five of six MV IgM-positive CSF, both MV IgM-positive sera and six out of eight sera with elevated RF levels were collected within 1 yr of SSPE onset. Also collected during this time were six other sera having high binding in the MV IgM assay, which was not due to MV-specific IgM antibodies. Two of these sera had as high or higher binding of IgM to a Vero cell control antigen, suggesting involvement of an IgM-class autoantibody. The other four sera had false-positive MV IgM assay results due to RF interference. RF interference was dependent on both the titer and avidity of the MV IgG antibodies involved. Three conclusions can be drawn from these results. First, MV IgM antibodies and elevated RF levels are not markers for acute SSPE, despite the tendency for their synthesis at this stage of the disease. Second, immunoassay analysis of viral IgM antibodies must employ an appropriate control antigen to account for background IgM binding. Finally, even if RF levels are normal or near normal, a false-positive IgM immunoassay result can still occur if antigen-specific IgG antibodies in the same sample have the right combination of titer and avidity.     

Assessment of RNA amplification by multiplex RT‐PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella

Sanz JC, Mosquera M, Ramos B, Ramírez R, de Ory F, Echevarria JE. Assessment of RNA amplification by multiplex RT‐PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella. APMIS 2010; 118: 203–9. The aim of the study was to compare RNA amplification using multiplex RT‐PCR and IgM detection by means of indirect and capture ELISAs for the diagnosis of measles and rubella. A total of 229 cases of maculopapular rash with serum and throat swab samples were included. Specific serological IgM to measles and rubella was determined by Enzygnost^® (Siemens) and Platelia^TM (Bio‐Rad). Both viruses were researched using multiplex RT‐PCR performed on throat samples. Criteria for inclusion of measles or rubella cases were a positive RT‐PCR result for one virus and negative for the other; and/or a positive IgM result for one virus by both ELISAs and negative RT‐PCR for the other virus. A total of 74 cases were classified as measles and 54 as rubella. In measles, sensitivity and specificity were 93.2% and 100% for RT‐PCR, 97.3% and 98.1% for Enzygnost^®, and 90.5% and 95.5% for Platelia^TM. For rubella, these values were 42.6% and 100% for RT‐PCR, 100% and 97.1% for Enzygnost^®, and 94.4% and 98.3% for Platelia^TM. Enzygnost^® and Platelia^TM are useful techniques for detecting IgM against measles and rubella. RNA amplification by RT‐PCR was both sensitive and specific for the diagnosis of measles; however, for rubella, the sensitivity of this technique must be improved.     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

Genotyping of measles and mumps virus strains using amplification refractory mutation system analysis combined with enzyme immunoassay: a simple method for outbreak investigations

A simple method, based on a modification of the amplification refractory mutation system (ARMS), for genotyping outbreak strains of measles and mumps viruses and detecting these in a simple enzyme immunoassay (EIA) is described. Fifty-three measles strains circulating at the time of an outbreak in London in 2000 and 26 strains circulating at the time of a mumps outbreak in Accrington, UK, in 1999 were investigated. All strains were genotyped by direct sequencing. ARMS primers were then designed to amplify the outbreak strain. The ARMS-EIA for measles and mumps detected all 36 measles outbreak strains as genotype D6, and all 15 mumps outbreak strains as genotype F, respectively. The sensitivity and specificity of both the measles D6 and Mumps F genotype ARMS EIA was 100% compared with direct sequencing. The results show that ARMS-EIA can be used as a rapid alternative to genotyping by direct sequence analysis in outbreak situations.     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.     

Persistent rubella-specific IgM reactivity in the absence of recent primary rubella and rubella reinfection.

Between two and seven sera from cases of persistent detection of rubella-specific IgM for periods in excess of 2.5 months, but in the absence of recent primary rubella or rubella reinfection, were examined for rheumatoid factor, heterophile antibody, and IgM reactivity against toxoplasma and a number of viruses. The relative avidity of the rubella-specific IgG1 has been assessed in all the sera by two methods. None of the sera contained rheumatoid factor or heterophile antibody, nor did any contain detectable concentrations of IgM specific for any of the panel of antigens apart from five sera which contained low concentrations of IgM specific for some coxsackieviruses B. No sera were positive for low avidity specific IgG1 although three did give equivocal results with one avidity test and one gave equivocal results with the second avidity test.