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.     

Detection of rubella virus-specific immunoglobulin G (IgG), IgM, and IgA antibodies by immunoblot assays.

Immunoblot (IB) assays were developed for detection of rubella virus (RV)-specific immunoglobulin G (IgG), IgM, and IgA antibodies in human serum following natural infection or immunization. IB assays performed under nonreducing conditions were compared with those performed under reducing conditions and with immunoprecipitation assays. Significant loss of antigenicity (greater than 90%) of RV E1 and E2 proteins was observed when IB assays were performed in the presence of 2-mercaptoethanol as compared with assays under nonreducing conditions. In contrast, the antigenicity of RV capsid protein was not influenced by reducing agents. Sensitivity of IB for RV-specific IgG antibodies was determined to be 0.01 IU/ml under nonreducing conditions. In the determination of RV-specific IgM and IgA antibodies by IB, pretreatment of serum with protein G to remove competing high-affinity RV-specific IgG or rheumatoid factor significantly improved assay sensitivity. IB assays were observed to be superior to immunoprecipitation assays in their ability to better define the specificities of RV-specific antibodies and to detect antibodies of all immunoglobulin classes. However, the conformational sensitivity of RV protein antigenicity should be an important consideration in the interpretation of RV-specific antibodies by IB assays.     

Evaluation of commercial rubella immunoglobulin G avidity assays

We compared the performances of five commercial rubella virus immunoglobulin G (IgG) avidity assays. The Adaltis (kappa = 0.28) and Diesse (kappa = 0.33) assays showed poor correlation, the Behring assay (kappa = 0.68) showed good correlation, and the Euroimmun (kappa = 0.95) and Radim (kappa = 0.94) assays showed excellent correlation with a well-established in-house rubella virus IgG avidity assay. The Euroimmun and Radim assays were statistically significantly better than the other commercial assays (P < 0.01).     

Subclass distribution of IgG and IgA responses to rubella virus in man

Monoclonal anti-subclass antibodies were used in a micro-ELISA method to determine rubella-specific IgG subclass antibodies in serum from 22 subjects who had acute rubella or had been vaccinated, from 10 infants with congenital rubella, and in serum and synovial fluid samples from 21 patients with chronic arthritis. In nearly all samples IgG1 was the only type of IgG antibody detected. In acute infections it was present within 10 days of the onset of the rash. IgG4 antibody was detected in sera from two immune individuals. Rubella-specific IgA1 subclass antibody was detected by the same technique in sera from 6 of 12 subjects with acute rubella as early as 3 days but not later than 28 days after the appearance of the rash.     

Solid-phase radioimmunoassay of rubella virus immunoglobulin G and immunoglobulin M antibodies.

A solid-phase radioimmunoassay method has been developed for the detection of rubella virus-specific immunoglobulin G (IgG) and IgM antibodies in human serum specimens. Purified rubella virus was adsorbed onto polystyrene balls, and antibodies that attached to the virus-treated balls were detected by subsequent binding of 125I-labeled anti-human gamma or anti-human mu immunoglobulins. A total of 77 serum specimens were tested. Binding ratios between positive and negative sera were as high as 22 in the IgG assay but rarely exceeded 3 in the IgM assay. The sensitivity of the IgG assay was found to be 16 to 256 times higher than that of the rubella virus hemagglutination inhibition test. The IgG radioimmunoassay can be readily adopted for routine diagnostic use. The IgM radioimmunoassay, however, due to its lower sensitivity, must be modified before being routinely applied.     

Subclass distribution of salivary secretory immunoglobulin A antibodies to oral streptococci

The ability of specific secretory immunoglobulin A (S-IgA) antibodies to inhibit bacterial colonization of mucosal surfaces may be neutralized by the activity of bacterial IgA1 proteases. Because of the resistance of the IgA2 subclass to these enzymes, the biological effect of IgA1 proteases in vivo may depend on the subclass distribution of the bacterium-specific antibodies. We have estimated the subclass distribution of S-IgA antibodies in saliva samples from 13 individuals against IgA1 protease-producing (Streptococcus sanguis and Streptococcus oralis) and nonproducing (Streptococcus gordonii and Streptococcus mitis bv. 2) oral streptococci. IgA1 was found to be the predominant subclass of antibodies against these four bacteria in most of the saliva samples, corroborating previous data suggesting a role of IgA1 proteases in plaque formation. However, variation in the subclass distribution of S-IgA antibodies against the same strain was observed. In one individual, IgA2 was the predominant subclass of antibodies against all four streptococci and of total salivary S-IgA, pointing to the possible significance of genetic variations. The study also addresses methodological problems related to the quantitation of salivary antibodies by solid-phase immunoassays.     

Detection of rubella virus-specific immunoglobulin M antibodies with a baculovirus-expressed E1 protein.

The structural proteins of rubella virus (RV) were expressed in insect cells by using the baculovirus expression vector system. The recombinant E1 envelope glycoprotein was purified by immunoaffinity chromatography and used to detect RV-specific immunoglobulin M antibodies in a time-resolved fluoroimmunoassay. Correlation analysis between the reactivities of antibodies against this recombinant E1 and the reactivities against authentic RV antigen shows that purified E1 can detect RV antibodies of the immunoglobulin M type.     

Measles virus-specific immunoglobulin G isotype immune response in early and late infections

A total of 154 human serum samples (32 acute-phase and 22 convalescent-phase serum samples obtained within a week and between days 8 and 26 after the onset of rash, respectively, and 100 samples drawn from healthy immune adults) were processed by an immunofluorescence assay for the detection of immunoglobulin M (IgM), total immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 measles virus-specific antibodies. In the acute phase, IgG1 was seen first, followed by IgG2, IgG3, and IgG4 responses, the mean seropositivity of which gradually increased during convalescence, reaching 100% (standard deviation [SD], 84 to 100%), 57% (SD, 34 to 80%), 86% (SD, 66 to 100%), and 86% (SD, 66 to 100%), respectively. IgG2 rose and fell in connection with IgG3 subclass antibodies, showing a rate of detection of IgG2 and/or IgG3 subclass antibodies of 95.5% (range, 100 to 86.5%) in the convalescent phase of infection. The mean percentage of measles IgG2 and IgG3 seropositivity dropped significantly during the memory phase, to 2% (range, 2 to 6%) and 3% (range, 3 to 7%), respectively (P < 0.05); meanwhile IgG1 and IgG4 subclass responses remained relatively unmodified in samples obtained years after infection (mean 100% [SD, 96 to 100%] and 86% [SD, 79 to 93%], respectively). Results obtained defined two highly different immune isotypic response patterns. One pattern is restrictive to IgG2 and/or IgG3 in the convalescent phase and is kinetically similar to the IgM antibody response, so its detection could be referred to as a recent viral activity. On the other hand, IgG1 and IgG4 were detected in both the convalescent and memory phases of the immune response, but their isolated occurrence without IgG2 and IgG3 could be related to the long-lasting immunity.     

Persistence of immunoglobulin G and immunoglobulin M antibodies after postnatal rubella infection determined by solid-phase radioimmunoassay.

The appearance and persistence of immunoglobulin M (IgM) and IgG antibodies in postnatal rubella infections were studied by employing a solid-phase radioimmunoassay test. Altogether, 222 serial serum specimens from 51 patients with acute rubella infection were tested. Both IgG and IgM antibodies developed rapidly and appeared in all patients within 4 days after the onset of rash. In some patients, the IgM antibodies clearly preceded the IgG antibodies; however, the reverse situation was also noticed in a few cases. The IgG antibodies showed only minor changes after 8 to 10 days from the onset of rash. The IgM titers also reached a maximum level at approximately 8 to 10 days after the onset of rash, after which time a rapid decrease was normally seen. The mean half-life of IgM antibodies after 15 days from the onset of rash was 4.5 days, giving for IgM antibodies persistence times from 43 to approximately 80 days. Two patients with a prolonged IgM antibody response were detected. One of these patients had bilateral arthritis of the knee as a complication, whereas in the other patient no complication caused by rubella virus was detected. The IgM antibody response and its value in diagnosis are discussed.     

Rapid separation of immunoglobulin M from immunoglobulin G antibodies for reliable diagnosis of recent rubella infections.

Chromatography on controlled pore glass was adapted for the separation of immunoglobulin M (IgM) and immunoglobulin G (IgG) rubella antibodies from 0.3-ml samples of human serum. An extremely sharp separation of IgM from IgG antibodies could be obtained within 40 min. Nonspecific inhibitors were removed before chromatography by precipitation with high-molecular-weight dextran sulfate, and the titer of rubella antibodies in the different classes of immunoglobulins were assayed with a modified hemagglutination inhibition technique. The combination of these methods is recommended for routine tests. It permits an accurate diagnosis of recent rubella infection within a few hours.     

Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.     

Detection of rubella virus-specific polymeric immunoglobulin A by enzyme-linked immunosorbent assay in combination with streptococcal pretreatment of serum.

An enzyme-linked immunosorbent assay combined with streptococcal treatment of serum was assessed for its ability to detect serum polymeric immunoglobulin A. This technique detects rubella virus-specific polymeric immunoglobulin A antibody, which appears for only a short time after infection, and it is useful for serodiagnosis of recent rubella virus infection.     

Dried blood spots versus sera for detection of rubella virus-specific immunoglobulin M (IgM) and IgG in samples collected during a rubella outbreak in Peru

Most persons with rubella virus-specific immunoglobulin M (IgM)- or IgG-positive sera tested positive (98% [n = 178] and 99% [n = 221], respectively) using paired filter paper dried blood spot (DBS) samples, provided that DBS indeterminate results were called positive. For persons with IgM- or IgG-negative sera, 97% and 98%, respectively, were negative using DBS.     

Serological detection of varicella-zoster virus-specific immunoglobulin G by an enzyme-linked immunosorbent assay using glycoprotein antigen

Since the introduction of varicella vaccination in several countries, there has been an urgent need for commercially available test procedures that allow highly sensitive and specific quantitative determination of the varicella-zoster virus (VZV)-specific immune status, including immunity postimmunization. This study compared the performance of two enzyme-linked immunosorbent assays (ELISAs) for the sensitive and specific determination of VZV-specific immunoglobulin G (IgG) in seronegative and latently infected persons, as well as in vaccinees. One ELISA is based on the detection of antibody to VZV-specific envelope glycoproteins (gp), and the other comprises the whole antigen extract prepared from VZV-infected cells. A modified standard fluorescent-antibody-to-membrane-antigen (FAMA) assay was used as a reference. An excellent sensitivity (100%) in relation to FAMA was demonstrated for the gpELISA (Virion\Serion), while the non-gpELISA (Dade Behring) had a lower sensitivity (83%) when sera from latently infected persons were tested. After postvaccinal immunity was measured, a sensitivity of 87% was achieved with gpELISA, whereas the ELISA incorporating antigen extract of VZV-infected cells had a sensitivity of 78%. Excellent specificity (100%) was calculated for both the gpELISA and the non-gpELISA. In conclusion, SERION ELISA classic VZV IgG is useful for the sensitive and specific quantitative determination of VZV immune status after natural infection. The test can also be recommended for measuring antibody response after varicella vaccination, particularly after the cutoff value was optimized.     

Clinical evaluation of the sensitivity and specificity of a commercially available enzyme immunoassay for detection of rubella virus-specific immunoglobulin M.

A solid-phase capture antigen enzyme immunoassay (Rubazyme-M) was evaluated for sensitivity and specificity on sera from 1,200 blood donors, 51 patients with rubella, 2 infants with congenital rubella, 104 patients with other infections, and 126 patients with immunological abnormalities. The sensitivity was 100% for sera tested between days 3 and 40 after the onset of symptoms of rubella virus infection. Rubella virus-specific immunoglobulin M was detected at birth in sera from congenitally infected infants and persisted for several months. Positive Rubazyme-M responses were observed in some patients in the absence of rubella diagnosis (one blood donor, three other infections, and two immunological abnormalities), providing a test specificity of 99.6%. None of 67 patients with rubella virus-specific immunoglobulin G antibody and high levels of rheumatoid factor were positive in the test.     

The distribution of anti-thyroglobulin antibodies in the immunoglobulin G subclasses

Using the coprecipitation technique with rabbit antisera specific for the human IgG subclasses, the antigen–binding capacity for thyroglobulin has been investigated in patients with Hashimoto''s disease. Anti-thyroglobulin activity was found in all four subclasses. The proportion of the total activity in each subclass closely paralleled the relative amounts of each subclass in normal serum.     

Comparison of a whole-virus enzyme immunoassay (EIA) with a peptide-based EIA for detecting rubella virus immunoglobulin G antibodies following rubella vaccination.

A total of 250 human serum samples were tested for rubella virus immunoglobulin G antibodies by two enzyme immunoassays (EIAs), one using whole rubella virus antigen and the other based on the use of synthetic peptide antigen. The samples were taken from 125 volunteers before and after their immunization with the RA 27/3 rubella vaccine. This study indicates that a synthetic peptide-based EIA can favorably replace current viral lysate-based EIAs to detect rubella virus antibodies following immunization. Because the synthetic peptide used in this newly developed EIA represents a putative neutralization epitope of the rubella virus, it could also be instrumental in determining rubella immune status and in assessing vaccine program efficiency.     

Evaluation of rubella virus immunoglobulin G (IgG) and IgM assays with the new Vidia instrument

For rubella virus immunoglobulin G (IgG) and IgM detection, Vidia assays were compared to Vidas, AxSYM, and Liaison assays with 419 serum samples. Only Vidia produced a sensitivity of 100% for IgG and IgM. Vidia specificities were 98.4% for IgG and 99.8% for IgM, versus Vidas specificities of 100 and 99.3%. Vidia IgG and IgM assays performed equally well.     

Enzyme-linked immunosorbent assay for rubella immunoglobulin G: new method for attachment of antigens to microtiter plates

Many of the enzyme-linked immunosorbent assay (ELISA) techniques previously described for detection of rubella-specific antibodies employ complex technology not available in routine diagnostic laboratories. The method described allows the use of commercially available rubella hemagglutination inhibition (HI) antigen. Passive adsorption of these antigens to plastic is variable, but with the use of albumin as a bridge, it is possible to attach the antigen reliably to the plastic wells. Over 1,500 sera were tested by both HI and ELISA techniques to detect the presence of rubella antibodies. These sera were selected with a bias towards those with low levels of rubella-specific antibody, since it has been demonstrated that it is in this range that discrepancies are more likely to occur between HI and ELISA techniques. In 99% of the sera tested, the results of both techniques were in agreement. On the basis of these results, the technique offers a useful alternative to the routine rubella HI test and other ELISA techniques which need sophisticated antigen preparations.     

Detection of low-avidity immunoglobulin G in oral fluid samples: new approach for rubella diagnosis and surveillance

Low-avidity rubella immunoglobulin G (IgG) was detected in oral fluid samples from 30 of 32 rubella IgM-positive patients (sensitivity, 94%) and from 4 of 34 IgM-negative patients (specificity, 88%). Measuring IgG avidity in oral fluid samples could improve the reliability of rubella surveillance when the incidence of the disease and the positive predictive value of IgM tests are low.