Antibody Capture Radioimmunoassay Research Articles

HIV Surveillance By Testing Saliva

Saliva specimens were tested for HIV antibody (anti-HIV) by an immunoglobulin G (IgG) antibody capture radioimmunoassay (GACRIA) and three sensitive commercial assays. In tests on 460 seronegative subjects and 196 seropositive subjects GACRIA was 99.8% specific and 100% sensitive. The Wellcome HIV monoclonal and Abbott recombinant DNA enzyme-linked immunosorbent assays (ELISAs) were also highly specific (99.8%, 100%) but they were less sensitive (90.9%, 82.0%). The Fujirebio particle agglutination assay was sensitive (97.8%) but its specificity was poor (84.1%). In testing saliva specimens from populations with an anti-HIV prevalence greater than 0.5%, sampling by GACRIA alone could provide a good estimate of the true prevalence. For true prevalences less than 0.5% good estimates could only be obtained if positive GACRIA reactions were confirmed by another independent salivary assay. Salivary testing for anti HIV is a convenient and potentially an accurate epidemiological tool. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Evaluation of a commercial assay for the detection of mumps specific IgM antibodies in oral fluid and serum specimens

Background An IgM antibody capture radioimmunoassay (MACRIA) has been used in the UK Reference Laboratory for the detection of mumps specific IgM antibodies in oral fluid and serum samples. The method has proved both sensitive and specific and has been used for the surveillance of mumps since 1994. The use of radioactive labels has restricted the use of MACRIA to specialised laboratories and alternative, non-isotopic, sensitive assays capable of detecting the low levels of specific antibodies in oral fluid have not been available. Recently, a novel mumps specific IgM capture enzyme immunoassay (MACEIA) utilising recombinant mumps nucleoprotein (rMuVN) and monoclonal antibodies to the nucleoprotein, produced by Microimmune Limited, has been developed for use with both serum and oral fluid samples. Objectives In this study, we have evaluated the performance of the Microimmune MACEIA for both serum and oral fluid against specimens tested by MACRIA. Study design The panel consisted of matched serum and oral fluid specimens from 137 cases of suspected mumps received in the Virus Reference Department for routine investigation from March 2003 to October 2004. Results The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on serum samples compared to MACRIA were 98.8%, 100.0%, 100.0% and 98.5%, respectively. The sensitivity, specificity, positive and negative predictive value of the Microimmune MACEIA on oral fluid samples compared to Microimmune MACEIA and MACRIA consensus results on the paired serum samples were 90.3%, 97.6%, 98.3% and 87.1%, respectively. Conclusions The Microimmune MACEIA was found to be a rapid, sensitive and specific alternative to MACRIA for the detection of mumps specific IgM in sera and oral fluids.

Salivary diagnosis of measles for surveillance: a clinic-based study in Niterói, State of Rio de Janeiro, Brazil

This study was designed to investigate whether saliva could be a feasible alternative to serum for the diagnosis of recent measles infection in a clinic setting. Forty-two paired blood and saliva samples collected 1 to 16 days after onset of illness from 29 patients with clinical measles were tested for specific immunoglobulin (Ig) M by antibody-capture radioimmunoassay. Measles IgM was detected in all serum samples and in 39 (92·9%) saliva specimens. Between 1 and 3 weeks after illness onset, virus-specific IgM was detected in 100% of saliva samples. Measles IgM was also detected in 17 saliva specimens, not paired with blood samples, collected from study patients 5 days to 3 weeks after onset. Our results indicate that salivary IgM detection is a suitable non-invasive method for investigation of notified cases under conditions of routine clinic use.

Epidemiology of Epstein-Barr virus infection in pre-adolescent children: application of a new salivary method in Edinburgh, Scotland.

STUDY OBJECTIVE: To describe the epidemiology of Epstein-Barr virus (EBV) among primary school children by testing saliva with a new EBV capsid antigen "G" antibody capture radioimmunoassay (GACRIA). DESIGN: A...

Detection of parvovirus B19-specific IgM by antibody capture radioimmunoassay

A solid phase IgM-capture radioimmunoassay (MACRIA) for the detection of parvovirus B19-IgM is described (Cohen et al., 1983, J. Hyg. Camb. 91, 113–130). IgM from a dilution of patients serum is ‘captured’ onto a solid phase coated by anti-human IgM. To determine whether any of the IgM is specific for parvovirus B19, B19 antigen is added followed by a detector system. In the MACRIA described here the detector system comprises a mouse monoclonal antibody to parvovirus B19 and a 125I-labelled anti-mouse antibody. A calibration curve derived from a standard B19 IgM serum is used to quantify B19 IgM using a single dilution of test sera. The purpose of the protocol is the diagnosis of recent acute infection with parvovirus B19.

Detection of IgG antibody to Epstein-Barr virus viral capsid antigen in saliva by antibody capture radioimmunoassay

A ‘G’ antibody capture radioimmunoassay (GACRIA) to detect IgG to Epstein-Barr virus (EBV) viral capsid antigen (VCA) in saliva is described. The monoclonal antibody to EBV VCA used in the GACRIA bound non-specifically when testing saliva samples having a total IgG content of less than 2.0 mg/l, so giving false positive results. This problem was overcome by including 0.5% EBV-negative human serum in the monoclonal antibody diluent. The performance of the assay was then evaluated by comparing the GACRIA test on serum and saliva samples to indirect immunofluorescence assay (IFA) results on sera using a panel of paired serum/saliva samples. Compared to the corresponding serum IFA the saliva GACRIA had a sensitivity and specificity of 93.5 and 100%, respectively. Although less sensitive than IFA on serum samples, the saliva GACRIA is sufficiently sensitive to be used for epidemiological screening and will enable testing for anti-EBV VCA to be carried out easily and on a wide scale.

Evaluation of serological assays for identification of parvovirus B19 immunoglobulin M

Three different enzyme immunoassays (EIAs) (Parvoscan-B19, IBL parvovirus B19, and IDEIA parvovirus B19) and one immunofluorescence assay (Biotrin Parvo B19 IFA) were evaluated for detection of parvovirus B19 immunoglobulin M (IgM) antibodies in 203 clinical serum samples. An IgM antibody capture radioimmunoassay was used as a reference test. Serum specimens obtained from patients with clinical symptoms suggestive of parvovirus B19 infections were used to evaluate the sensitivities of the assays, which were shown to be comparable for the Biotrin IFA and IDEIA (97%) and lower for the other two EIAs (90%). In order to test the specificity of the assays, clinical serum samples with IgM antibodies against other viruses were examined, as well as sera with rheumatoid factor activity and sera from healthy pregnant women. The specificities of B19 IgM antibody detection were 96% for the Biotrin IFA, 96% for IDEIA, 90% for Parvoscan, and 88% for the IBL assay. These results show that all four assays can be recommended for diagnostic purposes, although false-positive results may be seen with other acute viral infections, healthy pregnant women, and rheumatoid factor-positive samples.

Salivary antibody detection in epidemiological surveys: a pilot study after a mass vaccination campaign against rubella in São Paulo, Brazil

The sensitivity and specificity of salivary rubella antibody detection was investigated using samples collected from 301 children after a mass vaccination campaign in the state of São Paulo, Brazil. Saliva samples were collected by 2 different methods: directly dribbling into a container or using a commercial collecting device. Corresponding finger-prick blood samples were collected on filter paper. Rubella specific immunoglobulin G (IgG) was measured in saliva by antibody capture radioimmunoassay and in blood samples by indirect enzyme-linked immunosorbent assay. The detection of salivary rubella specific IgG showed good correlation with the detection of rubella antibody in the blood samples. For both collecting techniques the predictive value for a positive saliva test was >99% compared with the results from the blood tests. However, the predictive value for a negative saliva test was only 58.3% for a dribbled sample, compared to 100% for saliva collected using the commercial device. Moreover, collecting saliva by dribbling from children less than 4 years old was difficult. The detection of rubella specific IgG in saliva collected using a commercial device proved to be sensitive and specific in this epidemiological study, encouraging its more widespread application as a means of surveillance after mass vaccination.

Prevalence of measles antibody among children under 15 years of age in Santa Cruz, Bolivia: implications for vaccination strategies

We conducted a community-based survey in Santa Cruz city, Bolivia, to determine the age-specific prevalence of measles antibodies, determine factors associated with absence of detectable measles antibodies, and to compare results of salivary and serum measles immunoglobulin G (IgG) antibody assays. Serum samples from 1654 children were assayed for measles IgG antibody using the haemagglutination inhibition (HI) assay, and salivary samples were also obtained from 187 children and tested for measles IgG antibody using an antibody capture radioimmunoassay. Reported measles vaccine coverage in children aged 12–35 months was 77% (95% confidence interval [CI], 72–81%). Eighty-seven percent (95% CI 85–89%) had detectable HI antibody, but a high proportion had antibody levels below 200 miu(30–40% of 2–14 years old children). Measles seronegativity was associated with not being vaccinated against measles, a negative history of measles disease, living in the inner city, being a lifetime resident of Santa Cruz, and young age. Of 212 children without detectable measles antibody, 58% had a positive history of vaccination or measles disease, so that historical information was not sufficiently reliable to identify susceptibles. The salivary measles antibody assay was not sufficiently sensitive to be used for population screening; only 54% of 171 salivary samples from children who had detectable serum HI antibody were positive. A mass measles vaccination campaign of all children under 15 years of age is planned in Bolivia in 1994. Although only 7% of school-age children in Santa Cruz were seronegative, the effectiveness of a mass campaign in this age group depends in part on the response to revaccination of children with low, but detectable, antibody levels.

Salivary diagnosis of measles: a study of notified cases in the United Kingdom, 1991-3.

To validate a method for salivary diagnosis of measles and to assess the diagnostic accuracy of notified cases of measles. Blood and saliva samples were collected within 90 days of onset of symptoms from patients clinically diagnosed as having measles and tested for specific IgM by antibody capture radioimmunoassay. 17 districts in England and one in southern Ireland during August 1991 to February 1993. 236 children and adults with measles notified by a general practitioner. Specific IgM was detected in serum in only 85 (36%) of the 236 cases. In cases associated with outbreaks and tested within six weeks of onset, 53/57 (93%) of samples were IgM positive, thereby confirming the sensitivity of serum IgM detection as a marker of recent infection. The serological confirmation rate was lower in cases with a documented history of vaccination (13/87; 15%) than in those without (70/149; 47%) and varied with age, being lowest in patients under a year, of whom only 4/36 (11%) were confirmed. Measles specific IgM was detected in 71/77 (92%) of adequate saliva samples collected from patients with serum positive for IgM. In cases where measles was not confirmed, 6/101 had rubella specific IgM and 5/132 had human parvovirus B19 specific IgM detected in serum. The existing national surveillance system for measles, which relies on clinically diagnosed cases, lacks the precision required for effective disease control. Saliva is a valid alternative to serum for IgM detection, and salivary diagnosis could play a major role in achieving measles elimination. Rubella and parvovirus B19 seem to be responsible for a minority of incorrectly diagnosed cases of measles in the United Kingdom and other infectious causes of measles-like illness need to be sought.

Detection of measles, mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay.

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MACRIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1-5 weeks after onset of illness as the optimum time for collection of samples.

Simple haemadherence test for the detection of class-specific immunoglobulins to hepatitis A virus.

The ability of hepatitis A virus (HAV) to agglutinate human erythrocytes was used to develop IgM and IgG antibody capture haemadherence tests (MACHAT and GACHAT). Haemadherence was dependent on the pH of the red cell suspension and was best in the pH range 5.4 to 5.8. The tests were applied to serum, urine, and saliva specimens from individuals susceptible to, or with recent or past infection with HAV. Haemadherence test reactivities were compared with results obtained with IgM and IgG antibody capture radioimmunoassay (MACRIA and GACRIA) and competitive radioimmunoassay (COMPRIA). For 339 serum specimens examined, the sensitivity and specificity of MACHAT were 98.2% and 99.6%, respectively, and of GACHAT 99.1% and 100.0%. For 303 urine specimens, the sensitivity and specificity of MACHAT were 99.1% and 100.0%, and of GACHAT 100% for both. On initial testing, accuracy on saliva specimens was considerably less. For 2,819 saliva specimens, the sensitivity and specificity of MACHAT were 85.7% and 97.2% and of GACHAT 90.4% and 94.7%. The haemadherence test is a simple, inexpensive method which is satisfactory for use on serum and urine specimens. MACHAT and GACHAT can be used for epidemiological investigations, e.g., hepatitis A outbreaks and, in conjunction with a confirmatory test, for clinical diagnostic testing.

The influence of dental status on the detection of IgG class anti-viral antibodies in human saliva

An antibody-capture radioimmunoassay was used to measure levels of IgG class antibodies to rubella and hepatitis A viruses in serum and saliva of 30 edentulous, 30 partially dentate and 31 dentate individuals. The prevalence of seropositivity for rubella was 98.9 per cent and for hepatitis A 73.6 per cent. The serum reactivities were generally greater than those for saliva. There were 8 false-negative results for saliva out of the 182 tests performed, of which 4 were in the edentulous group, 3 in the partially dentate and 1 in the dentate group. For both rubella and hepatitis A virus antibodies the (geometric) mean ratios between the saliva and serum reactivities were similar across the three dental groups. The values for sensitivity, specificity and positive predictive value suggest that assay of saliva for antiviral IgG antibody is a satisfactory technique regardless of dental status.

Prevalence of Antibodies against Parvovirus B19 in Norwegians with Congenital Coagulation Factor Defects Treated with Plasma Products from Small Donor Pools

The seroprevalence of antibodies against parvovirus B19 in 308 Norwegians with coagulation factor defects of different types and severities was assessed by an IgG antibody capture radioimmunoassay (GACRIA). The overall seroprevalence was 62%. The seroprevalence among subjects with different types of coagulation factor defects was related to the type and severity of the coagulation factor defect: severe hemophilia A 64%, moderate and mild hemophilia A 58%, severe hemophilia B 88%, moderate and mild hemophilia B 73%, and von Willebrand's disease 52%. The prevalence of parvovirus B19 antibodies among household contacts and blood donors was 49% and 42% respectively. This study confirms that replacement therapy with coagulation factors is accompanied by an increased risk for acquiring parvovirus B19 infection. However, the prevalence of parvovirus B19 antibodies among Norwegian hemophiliacs is well below the prevalence reported from other countries and probably reflects the small numbers of donors in plasma pools used for the preparation of coagulation factor concentrates.

The production of human parvovirus capsid proteins in Escherichia coli and their potential as diagnostic antigens

We have expressed a number of polypeptides derived from the capsid proteins of the human parvovirus B19 in Escherichia coli. These include native VP1 (84K) and VP2 (58K) proteins and also fusions to beta-galactosidase containing differing amounts of the amino terminus of the VP1/2 polypeptide. Although each of these was expressed at high levels and the majority were produced as full-length proteins, only one was soluble. This soluble polypeptide, p132, is a beta-galactosidase fusion protein that includes 145 amino acids from B19 which are entirely derived from the region unique to VP1. Despite containing such a small portion of VP1, which itself constitutes only 4% of total capsid protein, p132 reacted with all our known anti-B19 IgM-positive human serum samples. We conclude that this region contains epitopes which must be prominently exposed on the intact virus. We have demonstrated the use of this recombinant antigen in a simple diagnostic assay for B19-specific antibodies which can be used for initial screening of human serum samples. In a survey of 103 serum specimens, our ELISA positively identified all samples (19/19) which were positive by IgM antibody capture radioimmunoassay. The recombinant p132 antigen is efficiently produced and readily purified from E. coli, and its use as a diagnostic antigen should increase the availability of routine clinical testing for human parvovirus infection.

Evaluation of needle exchange in central London

From November 1987 to October 1988, numbers of clients, visits made and syringes dispensed and returned were monitored at the needle exchange of the Middlesex Hospital, London, UK. A sample of clients were interviewed 1 month after entry to the scheme and again 3 months later to evaluate changes in injecting and sexual risk behaviours for HIV infection. Clients were asked to donate saliva for anti-HIV immunoglobulin G (IgG) antibody capture radioimmunoassay (GACRIA). The rate of lending and borrowing used injecting equipment fell, both compared with rates prior to entry to the scheme and during the period of study. Frequency of injecting did not increase and there was reduced incidence of abscesses. There was a highly significant correlation between multiple sexual partners and condom use and a reduction in the proportion of clients with multiple partners. On entry to the study, seven out of 121 (6%) clients were anti-HIV positive; after 3 months, a further two clients tested were found to be anti-HIV positive. Anti-HIV positivity prevalence for the year of study was nine out of 121 (7%). The scheme attracts clients, reduces injecting-related risk for HIV infection and has high equipment return rates. Saliva testing is acceptable to clients. Continued monitoring of anti-HIV in saliva is indicated.

HIV surveillance by testing saliva.

Saliva specimens were tested for HIV antibody (anti-HIV) by an immunoglobulin G (IgG) antibody capture radioimmunoassay (GACRIA) and three sensitive commercial assays. In tests on 460 seronegative subjects and 196 seropositive subjects GACRIA was 99.8% specific and 100% sensitive. The Wellcome HIV monoclonal and Abbott recombinant DNA enzyme-linked immunosorbent assays (ELISAs) were also highly specific (99.8%, 100%) but they were less sensitive (90.9%, 82.0%). The Fujirebio particle agglutination assay was sensitive (97.8%) but its specificity was poor (84.1%). In testing saliva specimens from populations with an anti-HIV prevalence greater than 0.5%, sampling by GACRIA alone could provide a good estimate of the true prevalence. For true prevalences less than 0.5% good estimates could only be obtained if positive GACRIA reactions were confirmed by another independent salivary assay. Salivary testing for anti HIV is a convenient and potentially an accurate epidemiological tool.

Detection of specific IgM in varicella and herpes zoster by antibody-capture radioimmunoassay.

A simple and sensitive M antibody-capture radioimmunoassay (MACRIA) is described which utilizes crude commercial VZV antigen and a single monoclonal anti-VZV antibody. This was compared to the immunofluorescence (IF) test for IgM antibody and was used to study IgM responses in sera from 261 patients with varicella and 220 patients with herpes zoster. With MACRIA, IgM antibodies were detected in all patients with varicella. The IgM antibodies appeared shortly after onset of rash, reached peak levels between 1 and 4 weeks after onset and then declined to low or undetectable levels in most, though not all, patients after 3 months. IgM antibodies were also detected in 98.2% of patients with herpes zoster, but the levels of IgM were significantly lower than after varicella and there was wider individual variation both in magnitude and duration of the IgM responses, in some cases only lasting 2-3 weeks. Comparison between MACRIA and IF showed good agreement in the detection of IgM antibodies following varicella. Discordant results were obtained with 13% of sera, of which 81% were taken either early or late after onset of rash and contained very low IgM levels. In contrast, 62 (28%) of the 220 sera from patients with zoster gave discordant results in the two tests, all except five being MACRIA-positive but IF-negative. The largest proportion of discordant results were obtained with sera taken more than 3 months after onset of rash, but 18 (29%) contained high IgM levels and were taken during the period of peak IgM responses. The diagnostic applications of the VZV MACRIA are discussed.

The prevalence of antibody to human parvovirus B 19 in England and Wales

The prevalence of antibody to human parvovirus B19 (anti-B19 IgG) in England and Wales was measured by an antibody-capture radioimmunoassay. Over 2000 sera were examined; 1422 from the general population, 374 from unselected children admitted to hospital and 300 from women attending an antenatal clinic. Waning levels of maternally-derived antibody were found in infants under 1 year old. In children 1-5 years old, 5-15% had anti-B19 IgG and this rose to 50-60% in older children, young adults and women of child-bearing age. In older people, the prevalence of anti-B19 IgG increased with age, rising to more than 85% in those over 70 years old.

IgM antibody to hepatitis B core antigen in children with chronic type B hepatitis.

IgM antibody to hepatitis B core antigen (anti-HBc IgM) was investigated by an antibody-capture radioimmunoassay (serum dilution 1:4000) in serum samples from 31 untreated children with chronic hepatitis B who were followed prospectively for 1-7 years. At the start, all patients were positive for hepatitis B e antigen (HBeAg), and anti-HBc IgM was detected in 23 cases, including 15 out of 16 with chronic active hepatitis and 7 out of 14 with chronic persistent hepatitis. A significant positive correlation was found between anti-HBc IgM levels and severity of liver damage (P less than 0.05), while an inverse relationship was found between anti-HBc IgM levels and distribution of hepatitis B core (HBcAg) antigen in the liver as detected by immunofluorescence. In fact 75% of anti-HBc IgM positive patients showed a focal HBcAg pattern (less than 40% positive nuclei), whereas 87% of antibody negative cases exhibited a diffuse HBcAg expression (more than 60% stained nuclei). During follow-up, seroconversion from HBeAg to anti-HBe with subsequent remission of liver disease occurred in 82% of patients presenting with detectable levels of anti-HBc, including three out of seven cases with chronic persistent hepatitis, but in none of the cases that were initially negative (P less than 0.01). These results indicate that during the natural course of chronic hepatitis B in children, anti-HBc IgM levels in serum reflect the degree of host immune response to infected hepatocytes. The close correlation between anti-HBc IgM seropositivity and seroconversion from HBeAg to anti-HBe suggests that anti HBc IgM may have a prognostic value during the follow-up of children with chronic HBeAg positive hepatitis B.