Measurement of avidity of specific IgG for verification of recent primary rubella

Sixty-four subjects' serum samples, positive or equivocal by rubella IgM assays and containing rubella IgG, were examined for the avidity of rubella IgG. Four of the sera originated from rubella reinfections; others had false-positive IgM results due to interference by parvovirus infection or by other mechanisms; and the remaining were sera from the acute phase or convalescence of primary rubella. A novel IgG avidity test, avidity-ELISA, and a semiquantitative haemolysis typing assay were used. According to the avidity-ELISA, 29 subjects had recent primary rubella (low IgG avidity), and another 29 had previous rubella immunity (high IgG avidity), whereas 6 serum samples gave borderline avidity values. Comparison of these results with pre-existing clinical records and laboratory data showed that all samples with low IgG avidity were obtained during or shortly after acute primary rubella. All sera with high IgG avidity originated from the previously immune subjects; the rubella reinfections were confined within this group. Five of the six sera with borderline avidity values were obtained within 2 months from primary rubella. In conclusion, the measurement of IgG avidity is a powerful tool for the distinction of acute or recent primary rubella from pre-existing rubella immunity, including rubella reinfections.     

Humoral immune response after primary rubella virus infection and after vaccination

We measured rubella virus immunoglobulin G (IgG) and IgM levels, as well as IgG avidity indexes, in serum samples taken before or after 6 months either after infection or after vaccination. The results obtained indicate that humoral immune responses are different after primary infection and after vaccination. This may have important consequences on the serological diagnosis of rubella virus infection.     

Maturation of IgG avidity to individual rubella virus structural proteins.

BACKGROUND: the structural proteins of rubella virus, the capsid protein C and the envelope glycoproteins E1 and E2 were produced in lepidopteran insect cells using baculovirus expression vectors. The C-terminal ends of the corresponding proteins were fused to a polyhistidine tag for easy and gentle purification by metal ion affinity chromatography. OBJECTIVES: to investigate the maturation of natural and vaccinal IgG avidity against individual authentic and recombinant rubella virus (RV) structural proteins. STUDY DESIGN: the analysis was carried out using a modified immunoblotting technique where the purified baculovirus-expressed proteins were compared with authentic rubella virus proteins. Altogether, 47 well-characterised serum samples from both naturally infected patients and vaccines were studied. RESULTS: after natural RV infection, IgG antibodies specific for the E1 protein were predominant not only in terms of levels, but also in terms of rate and magnitude of avidity maturation. The avidity development of the IgG antibodies was much slower in vaccines than in patients after a natural RV infection. CONCLUSIONS: together, our results indicate that IgG avidity determination in conjunction with immunoblot analysis is useful in the diagnosis of a RV infection. The recombinant proteins showed similar reactivity patterns in the immunoblot analyses as compared with the authentic viral structural proteins, suggesting suitability for serodiagnostics.     

The immune response to influenza virus: III. Changes in the avidity and specificity of early IgM and IgG antibodies

Serum samples taken from rabbits 5 days after vaccination with SW influenza virus by the intravenous route contained high levels of IgM antibodies. IgG antibodies were either not detected or were present at very low levels. By the 10th day after vaccination both IgM and IgG antibodies were present in the serum. The early IgM antibodies were of high avidity while the early IgG antibodies were of very low avidity. The presence of low avidity IgG antibodies in whole serum caused a decrease in the average avidity of the antibodies in whole serum from the 5th to the 10th day post vaccination. The avidity of the IgM antibodies remained fairly constant for the first 20 days of the immune response but a slight increase was detected after secondary vaccination. The avidity of the early IgG antibodies increased during the test period of 20 days. The early IgM and IgG antibodies were heterogeneous with respect to avidity.

The highly avid IgM antibodies showed high cross-reactivity with related influenza viruses, i.e. they were of low specificity. The early IgG antibodies that were of low avidity cross-reacted with only one other influenza virus out of the four tested, i.e. they were more specific; as the avidity of the IgG antibodies increased so did their cross-reactivity.

     

Diagnosis of recent primary rubella virus infections: significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference μ-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5-100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis.     

Simultaneous IgM reactivity by EIA against more than one virus in measles, parvovirus B19 and rubella infection.

BACKGROUND: A clinical diagnosis of rash-causing infections is not always possible and reliance has to be placed on serological evidence of infection, especially on the presence of specific immunoglobulin (Ig)M. However, despite the use of modern serological methods and validated commercial kits, reports appear in the literature of simultaneous IgM reactivity against more than one virus in cases of Epstein Barr virus, rubella, cytomegalovirus, human parvovirus B19 (HPV B19) and measles infections, all with implications for the pregnant woman. OBJECTIVES: We decided to evaluate the extent of the problem in rubella, measles and HPV B19 infections in a routine diagnostic laboratory. STUDY DESIGN: We tested sera from cases with initial clinical and serological evidence of infection with measles, HPV B19 or rubella for evidence of simultaneous IgM reactivity against more than one virus. We confirmed primary infections with specific-IgG antibody avidity tests, and subjected sera with IgM reactivity against more than one virus to avidity tests to identify which, if any, of the three viruses was the cause of the primary infection. Groups of monoreactive IgM sera were randomly selected from the presented sera to demonstrate that the avidity of the IgG specific for the other two viruses would be of high avidity compared with the low avidity of the IgG specific for the virus against which specific IgM had been detected. RESULTS: Our results confirm that simultaneous IgM reactivity against more than one virus does occur in these three infections, and that this is unlikely to be caused by the presence of rheumatoid factor. CONCLUSIONS: In the absence of seroconversion, reliance on specific IgM results alone for diagnosis of these infections should be avoided and tests such as specific IgG antibody avidity should also be employed. The simultaneous occurrence of IgM reactivity against more than one virus is also important for epidemiological and surveillance reasons as the widespread use of the mumps, measles and rubella vaccine makes its impact on the population. Falsely diagnosed cases of apparent measles or rubella could throw into question the efficacy of the vaccine.     

Development of a rapid and convenient method for determination of rubella virus-specific immunoglobulin G avidity

We describe here a rapid and semiautomated method for the determination of rubella virus immunoglobulin G (IgG) avidity with the VIDAS instrument. A total of 153 serum samples from persons with naturally acquired rubella virus infections (n = 98), from vaccinated persons (n = 44), and from patients with autoantibodies (n = 11) were included in this study. The rubella virus-specific IgG avidity assay we developed for the VIDAS instrument was evaluated by comparison with an in-house method. Results obtained with the VIDAS instrument allow considering this method valuable to help confirm or exclude acute primary infection or recent vaccination.     

Persistence of specific IgM and low avidity specific IgG1 following primary rubella.

Persistence of specific IgM in sera following primary rubella infection was compared with the maturation of the specific IgG1 response. 206 sera, from 171 patients with primary rubella, taken 1 day to 2.5 years after onset of illness, were tested. Rubella-specific IgM was detected by M-antibody capture radioimmunoassay in 100% of sera taken 15-28 days after onset, but in only 9% taken 3-4 months after onset. However, using the diethylamine (DEA) shift value (DSV) method, low avidity specific IgG1 was detected in 91% sera taken at 3-4 months and at 5-7 months 21% of sera remained positive. Using an avidity index method, with urea in the wash buffer, none of the sera were positive for low avidity specific IgG1 beyond 3 months after onset. With DEA in the wash buffer, the number of sera positive rose to 38% at 3-4 months. Thus, the DSV method for detecting low avidity specific IgG1 is a useful additional test for confirming or refuting a diagnosis of primary rubella and is of particular value for assessing pregnant patients.     

L'avidité des IgG: implications en infectiologie

IgG avidity : role in infectious diseases. The IgG avidity is the strength of the binding between multivalent antigens and the corresponding IgG antibodies. In immunocompetent patients, the measurement of the IgG avidity index is used as a complementary test to help in datation of infections. The use of this test is based on the fact that after primary infection, the antibody response matures from low avidity to high avidity over a period of several weeks to several months. The maturation of the IgG avidity depends on the nature and the dose of the dispensed antigen as well as on somatic mutations of immunoglobulins genes. Different methods can be used to measure the avidity index : among them, the most currently developed tests are based on the use of proteins denaturing agents. The measurement of the avidity index is very useful for the datation, in pregnant women, of materno-fetal infections. It could also help in the differenciation of pre- and post-natal infections. Furthermore, it has been published that the IgG avidity could play a protecting role : the more the avidity index is high, the more the patient would be protected.     

Avidity maturation following immunization with two human cytomegalovirus (CMV) vaccines: a live attenuated vaccine (Towne) and a recombinant glycoprotein vaccine (gB/MF59)

Two human cytomegalovirus (CMV) vaccines have been previously evaluated for their immunogenicity: a recombinant gB/MF59 vaccine and an attenuated strain of CMV (Towne). In healthy adults, we measured the antibody avidity maturation indices that occurred after vaccination with each. For Towne, administered as a single dose, the rise in IgG antibody avidity to CMV glycoprotein gB occurred slowly and continued for 24 months post-immunization. For gB/MF59, administered as two priming doses and a booster dose given at 6 months, the booster rapidly induced IgG antibodies to gB whose avidity was maximal at 7 months after the initial priming dose. Both vaccines induced antibody levels and avidity maturation indices that equaled those induced by wild-type virus suggesting that both vaccines may be effective in controlling CMV infections.     

Comparison of various serological methods and diagnostic kits for the detection of acute, recent, and previous rubella infection, vaccination, and congenital infections

The antibody development after natural rubella infection and rubella vaccination has been followed in 802 sera from 493 patients and 71 sera from 22 vaccinees. Also examined were 67 sera from 28 infants with rubella embryopathy and sera from 50 children with presumed prenatal infection. In addition, 777 sera from 641 patients tested for routine rubella diagnosis were studied. Anamnestic information was available from all these patients. These sera were assayed for IgM antibody detection by sucrose density gradient (SDG), the commercial ELISAs (Enzygnost IgM and Rubazyme M), and the non-commercial anti-my-hemadsorption immunosorbent technique (HIT). For the determination of IgG antibodies the hemagglutination inhibition test (HAI), the commercial ELISAs (Enzygnost IgG, Rubazyme), and a single radial hemolysis test (SRH) were used. The SDG and HIT were less sensitive for IgM antibody detection than the two ELISAs, particularly when IgM concentrations were low. In total 26.5% of the IgM results with the newer tests were discordant with SDG, but only 0.5-1.3% of these results were not explicable when the clinical data was considered. Problems were encountered with all IgM assay systems used. For the detection of rubella antibodies after acute infection and vaccination the ELISA Enzygnost IgG was as sensitive as the HAI whereas the ELISA Rubazyme and SRH detected antibodies with some delay. Corresponding results with all tests were found more than 25 days after acute infection and more than 50 days after vaccination. All methods can be used for detection of antibodies in infants with rubella embryopathy. The results of this study suggest that certain combinations of tests can be used for the reliable detection of rubella infection.     

Humoral immune response to primary rubella virus infection

An assay capable of distinguishing between the immune response generated by recent exposure to rubella virus and the immune response existing as a result of past exposure or immunization is required for the diagnosis of primary rubella virus infection, especially in pregnant women. Avidity assays, which are based on the premise that chaotropic agents can be used to selectively dissociate the low-avidity antibodies generated early in the course of infection, have become routinely used in an effort to accomplish this. We have thoroughly investigated the immunological basis of an avidity assay using a viral lysate-based assay and an enzyme-linked immunosorbent assay (ELISA) based on a peptide analogue of the putative immunodominant region of the E1 glycoprotein (E1208-239). The relative affinities of the antibodies directed against E1208-239 were measured by surface plasmon resonance and were found to correlate well with the avidity index calculated from the ELISA results. We found that the immune response generated during primary rubella virus infection consists of an initial low-affinity peak of immunoglobulin M (IgM) reactivity followed by transient peaks of low-avidity IgG3 and IgA reactivity. The predominant response is an IgG1 response which increases in concentration and affinity progressively over the course of infection. Incubation with the chaotropic agent used in the avidity assay abolished the detection of the early low-affinity peaks of IgM, IgA, and IgG3 reactivity while leaving the high-affinity IgG1 response relatively unaffected. The present study supported the premise that avidity assays based on appropriate antigens can be useful to confirm primary rubella virus infection.     

Recent rubella virus infection indicated by a low avidity of specific IgG

Rubella-specific IgG in acute-phase sera produces a characteristically altered zone termed soft hemolysis in the radial hemolysis test. Here, the soft hemolysis was shown to be a product of the purified IgG₁ subclass isolated from acute-phase sera. In contrast, ordinary hemolysis was produced by IgG₁ isolated from sera of previous rubella immunity, indicating that the subclass composition of IgG was not involved in the mechanism of soft hemolysis. A novel type of solid-phase immunoassay was developed for the avidity of virus-specific IgG. Acute-phase IgG (with soft hemolysis) was dissociated from rubella antigen in an enzyme immunoassay (EIA) test by hydrogen-bond disrupting agents under conditions where IgG of previous immunity (showing ordinary hemolysis) remained mostly bound. These data suggest that the mechanism of soft hemolysis is the avidity of rubella-specific IgG. The new quantitative avidity EIA was tested with sera taken from 169 subjects. Recent infection could be shown from sera taken weeks or months after primary rubella.     

HHV‐6 IgG4 isotype response following measles infection

Human herpesvirus 6 (HHV‐6) is widespread in the human population by infecting most individuals in early childhood. After primary infection, HHV‐6 establishes a latent infection by remaining in circulating mononuclear cells of healthy individuals. The HHV‐6 antibody titer increases after primary infection with measles virus. The present study was undertaken to determine the specific antiviral IgG1, IgG2, IgG3, and IgG4 subclass response patterns to HHV‐6 in HHV‐6‐seropositive individuals with natural measles virus infection, measles vaccination, and rubella virus infection. The purpose of this study was to examine HHV‐6‐specific IgG isotype response in patients with acute virus coinfection. Serum samples were obtained from individuals who were seropositive for HHV‐6 after natural primary infection with measles virus during an outbreak, measles vaccination, or rubella virus infection, and from healthy individuals. Sera were examined by indirect immunofluorescence assays for detection of HHV‐6‐specific IgG1, IgG2, IgG3, and IgG4 antibodies. A high percentage (69%) of those infected with measles virus had an HHV‐6 IgG1 and IgG4 response (P < 0.001, χ² test), whereas persons vaccinated against measles, those infected with rubella, and healthy individuals showed an HHV‐6 IgG1 response. These results demonstrate that natural measles virus infection induces an HHV‐6 IgG isotype response, which suggests a shift in immune activity from a Th1 to a Th2 response. J. Med. Virol. 82:396–399, 2010. © 2010 Wiley‐Liss, Inc.     

Significance of avidity and immunoblot analysis for rubella IgM-positive serum samples in pregnant women

A total of 512 IgM-positive serum samples from 449 pregnant women referred by microbiologists and medical laboratories for additional testing and final interpretation were collected over a 3-year period. Employment of an IgM capture enzyme immunoassay (EIA) confirmed only 31% of the initial EIA-IgM-positive samples. In order to discriminate acute rubella virus infections, which are associated with increased risk of fetal infection and embryopathy, from persistent or non-specific IgM, IgG avidity index and the presence of IgG with specificity to rubella virus E2 glycoprotein was determined by Western immunoblot. In only six patients (1.3%), a primary infection with rubella virus was diagnosed on the basis of IgM positivity, low avidity IgG, absence of E2-specific IgG in immunoblot concordant with clinical findings as well as consistent changes in follow-up samples. The serological results were not compatible with rubella re-infection. The infection status in 14 patients (3.1%) remained inconclusive even when both avidity assay and immunoblot were used, while restriction to either test did not allow a conclusive interpretation in 11.6% of patients. The use of both assays is clearly better, and therefore, recommended in IgM-positive samples.     

Assessment of IgM enzyme immunoassay and IgG avidity assay for distinguishing between primary and secondary immune response to rubella vaccine

The primary test for the laboratory confirmation of rubella is IgM serology. It is important to distinguish IgM reactivity caused by primary infection from that caused by reinfection or persistence, especially in pregnant women; as termination of pregnancy is considered when primary rubella is diagnosed during the first trimester.

In this study, the performance of rubella IgM enzyme immunoassay (IgM-EIA) and rubella IgG avidity assay were compared using well-defined panels of sera from persons vaccinated against rubella and commercial rubella IgM and IgG enzyme immunoassay kits (Dade Behring, Marburg, Germany).

The sensitivity and specificity of rubella IgM-EIA were found to be 77.4 and 97.9%, respectively, while the results for rubella IgG avidity assay were 100 and 100%. IgG avidity assay showed higher positive and negative predictive values than the IgM-EIA (100 and 100% compare to 96.9 and 82.9%).

In conclusion, the rubella IgG avidity assay is more sensitive and specific than IgM-EIA for differential detection of primary rubella infection from rubella reinfection.     

Multicenter Evaluation of a Rapid and Convenient Method for Determination of Cytomegalovirus Immunoglobulin G Avidity

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.     

Multicenter evaluation of a rapid and convenient method for determination of cytomegalovirus immunoglobulin G avidity

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.     

Differential IgG avidity to rubella virus structural proteins.

Avidity maturation of IgG antibody responses directed against the structural proteins of rubella virus (E1, E2, and C) as well as whole rubella virus (RV) was assessed at sequential time intervals in 7 individuals following serologically confirmed wild rubella infection. Individual structural proteins were purified from tissue culture supernatants by differential centrifugation, followed by preparative SDS-PAGE under non-reducing conditions. Avidity of IgG anti-rubella responses was measured by using the 8 M urea elution technique and results expressed as an elution ratio [ER(%)]. A low mean ER(%) of 23% was determined for E1-specific IgG responses during the 10-20 day period following onset of clinical rubella, with subsequent maturation of avidity ER(%) values to 52%, 75%, and 84% at 3 months, 1 year, and 2 years, respectively, post-rubella. In contrast, IgG anti-E2 responses showed minimal avidity maturation with ER(%) values of 20%, 29%, 30%, and 31% over the same time intervals. Similarly, responses to the capsid protein (C) remained at low avidity ER(%) values of 21%, 29%, 36%, and 35% over the 2 year follow-up period. The avidity maturation values for IgG directed against whole RV preparations paralleled observations for E1-specific responses with ER(%) values of 23%, 52%, 85%, and 87%, respectively. These data support the need to assess individual protein-specific antibody avidities in order to more fully understand viral-specific immune responses.     

Immunoglobulin G avidity in the serodiagnosis of congenital rubella syndrome

The avidity of specific IgG was investigated in three infants with serologically verified congenital rubella infection. Two sera were taken from each infant: the first soon after birth and the second at the age of 23 to 31 months. Avidity of specific IgG was measured by a protein-denaturing enzyme immunoassay using urea as the elution factor, and avidity then determined by the end-point ratio (derived from antibody titration) and the avidity index methods. Rubella-specific IgM was present in the first sera of all patients, but not in the second sera. However, low avidity of specific IgG persisted in two children until age 23 to 31 months, as determined by the end-point ratio method. These data are in agreement with the findings of previous studies of avidity in congenital rubella, and show the usefulness of the protein-denaturing IgG-avidity assays employing the end-point ratio method for serological diagnosis of congenital rubella even after disappearance of specific IgM.