Hemadsorption immunosorbent technique for determination of rubella immunoglobulin M antibody.

A highly specific and sensitive hemadsorption immunosorbent technique for measuring rubella immunoglobulin M (IgM) antibody is described. IgM from human sera was absorbed into anti-human IgM-coated wells in plates and rubella-specific IgM was detected by adding rubella virus hemagglutinin and a small quantity of sheep erythrocytes. Centrifugation of the plates facilitated reading of the test. Specific IgM-positive sera showed hemadsorption, whereas negative sera showed hemagglutination. Rheumatoid factor and rubella-specific IgG antibody did not interfere with the results. The test was clearly more sensitive than the solid-phase immunosorbent technique for detection of rubella IgM antibody by hemagglutination inhibition and at least as sensitive as the hemagglutination inhibition test on IgM fractions from a sucrose density gradient and the indirect immunofluorescence test for IgM antibody with absorbed serum. All of 40 sera from 17 rubella patients taken 4 to 49 days after the onset of rash were positive in the new test, with antibody titers ranging from 2,560 to 81,920 between 4 and 28 days. The test is reliable, practical, and suitable for general diagnostic use.     

Removal of nonspecific hemagglutination inhibitors,immunoglobulin G,and immunoglobulin A with streptococcal cells and its application to the rubella hemagglutination inhibition test

Summary A streptococcus, AW 43 strain, was found to bind nonspecific serum inhibitors of rubella virus hemagglutination (HA). This was demonstrated by titration of nonspecific HA inhibitors and by immunoelectrophoresis.Absorption of sera with the mixture of AW 43 cells, which bind IgA in addition to nonspecific HA inhibitors, and AR1 cells, another strain of streptococci which bind IgG, removed nonspecific HA inhibitors, IgG, and IgA simultaneously, leaving behind IgM and a trace of IgA.Pretreatment of sera with those streptococcal cells prior to the rubella hemagglutination inhibition (HI) test enabled to circumvent kaolin treatment of sera, which partially removes IgM antibodies, and to determine exclusively the early-appearing antibodies. The rise and fall of the HI antibodies thus determined correlated well with that of the IgM antibodies determined by enzyme-linked immunosorbent assay (ELISA).Thus, this modified rubella HI test may be useful for serodiagnosis of recent rubella virus infection.With 2 Figures     

Development of automated immunoassays for immune status screening and serodiagnosis of rubella virus infection

The fully automated IMx immunoassay analyzer was used to develop a system for the detection of IgG and IgM antibodies to rubella virus for immune status screening and diagnosis of primary infections. Reagents and assay protocol software were developed using rubella virus sensitized microparticles as the solid phase to capture specific antibodies from serum samples. Anti-human IgG or IgM antibody coupled to alkaline phosphatase enzyme followed by methylumbelliferyl phosphate substrate was used to detect the presence or absence of antibodies specific to the antigens on the solid phase. To evaluate the efficacy of the IMx rubella IgG assay, immune status screening was performed with a clinical patient population of 501 sera. When compared to an IgG specific enzyme immunoassay and passive hemagglutination assay the agreement was greater than 99%. The IMx rubella IgM assay was utilized to determine the presence of rubella specific IgM antibodies in 462 sera. These results were compared to IgM specific enzyme immunoassay results and also demonstrated greater than 99% agreement. Seroconversion following rubella vaccination of susceptible individuals was demonstrated by IgG and IgM antibody responses as early as two weeks postvaccination. In addition to automation, the IMx system offers rapid assay times and calibration curve storage without sacrificing clinical efficacy.     

Hemadsorption immunosorbent technique for the detection of dengue immunoglobulin M antibody.

We developed a highly specific, sensitive, and economical hemadsorption immunosorbent technique for the detection of dengue-specific immunoglobulin M (IgM) antibody. The technique is based on the reaction of human sera with anti-human IgM immobilized onto a solid phase followed by the detection of dengue-specific IgM by the addition of a known quantity of dengue virus hemagglutinin and goose erythrocytes. Dengue-specific IgM-positive sera showed hemadsorption. IgM antibody specific for dengue virus was detected in 22 of 39 (56%) convalescent-phase sera from primary dengue infections and 8 of 10 (80%) convalescent-phase sera from secondary dengue infections. Additionally, 32 of 76 single sera from patients were positive for dengue IgM; these sera were previously uninterpretable by the hemagglutination inhibition test, as only a single serum specimen was available. No false-positive results were obtained with sera that were negative by the hemagglutination inhibition test for dengue virus. Crude dengue virus hemagglutinin preparations could be used without purification. Dengue-specific IgG did not interfere with the results, nor was there any cross-reactivity between dengue hemagglutinins and IgM specific for other viruses. Some cross-reactivity of the dengue-specific IgM was observed with Japanese encephalitis virus hemagglutinins, but this did not present any problems in the interpretation of results. This test is specific, inexpensive, highly reproducible, and simple to perform.     

Solid-phase radioimmunoassay of rubella virus immunoglobulin M antibodies: comparison with sucrose density gradient centrifugation test.

The solid-phase radioimmunoassay (RIA) method developed in our laboratory for demonstrating rubella virus-specific immunoglobulin G (IgG) antibodies (Kalimo et al., 1976) was further developed for demonstrating IgM antibodies. A total of 188 serum specimens were tested. The statistical probability of obtaining a false-positive IgM result, based on determinations of 100 rubella-negative sera, was below 0.001. Nonspecific inhibitors and IgM antibodies against other viruses tested did not interfere in the assay. In 2 out of 20 (10%) serum specimens with rheumatoid factor, a false-positive IgM result was obtained. The new RIA method was compared with sucrose density gradient centrifugation, followed by hemagglutination inhibition testing of the separated immunoglobulins with respect to demonstrating IgM antibodies. In patients with acute rubella infection, IgM antibodies were demonstrated by RIA in 9 out of 20 acute-phase sera and in all 20 early-convalescent-phase sera, compared with 7 out of 20 acute-phase sera and 19 out of 20 early-convalescent-phase sera by sucrose density gradient centrifugation. The results obtained indicate that the RIA method is reliable and sensitive and suitable for routine diagnostic use.     

Variables of the rubella hemagglutination test employing freeze-dried erythrocytes

Summary The variables which affect the interaction between freeze-dried one-day-old chick erythrocytes and rubella hemagglutinin prepared from rubella-infected porcine kidney cells were defined and evaluated. The sensitivity of the hemagglutination (HA) reaction is much greater at pH 6.0 to 6.2 than at pH 7.0 to 7.5. HEPES (N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid) diluent with added Ca²⁺ or Mg²⁺ ion gave four- to eightfold higher HA titers than one without divalent cations.The development of agglutinated and non-agglutinated erythrocyte patterns depended much upon the concentrations of gelatin and albumin in the HEPES diluent. Gelatin especially was essential to obtain stable and clearly distinguishable patterns.Optimal conditions for the agglutination of freeze-dried erythrocytes by rubella hemagglutinin were provided when a HEPES-buffered saline at pH 6.2, containing 10^–3 m CaCl₂, 0.2 per cent bovine serum albumin, and 0.0025 per cent gelatin was employed throughout as a diluent for serum, hemagglutinin, and freeze-dried erythrocyte suspension. This diluent gave maximally sensitive and reproducible results in rubella HA and hemagglutination-inhibition (HI) tests employing freeze-dried erythrocytes.With 1 Figure     

Improved rubella hemagglutination inhibtion test: inactivation of non-immunoglobulin hemagglutination inhibitors by phospholipase C.

A method using phospholipase C (PL-C) for removing nonspecific inhibitors (NSI) of rubella virus hemagglutinin is described. PL-C was found to hydrolyze NSI without altering the hemagglutination inhibition (HI) activity of the specific antibody and could be used to remove NSI in the rubella HI test by using formalinized erythrocytes, which resisted the enzymatic action; fresh erythrocytes were lysed by PL-C. The HI test using PL-C treated sera gave true measurements of actual rubella antibody content, and HI titers of PL-C treated sera were identical or equivalent (+/-1 dilution) to those of sera treated with dextran sulfate and CaCl2 (DS-C). Thus, the PL-C method gave results as reproducible and reliable as the DS-C method and was more convenient.     

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.     

Detection of cell surface rubella virus antigens in Vero cells with Staphylococcus aureus rich in protein A

The presence of cell surface rubella antigen was used to verify and monitor viral replication in Vero cell monolayers. Viral antigen was observed in infected cells by the adherence of Staphylococcus aureus sensitized with immune anti-rubella sera. The staphylococci specifically bound to infected cells were Gram-stained and observed using light microscopy. The minimum titer of IgG antiviral hemagglutinin required for sensitizing the bacteria was 3.9 IU/ml. The specificity of the assay was demonstrated by treating the infected cells with bacteria sensitized with normal sera, by treating the mock-infected cells with staphylococci sensitized with either immune or normal sera, and by sensitizing the bacteria with immune sera from which anti-rubella antibodies had been removed. Viral antigens were detected from day 2-9 post-infection. The sensitivity of the assay in verifying and monitoring viral propagation was comparable to the titration of viral particles of hemagglutination. The assay is specific, rapid, simple and can be performed in laboratories with minimal equipment.     

Development and evaluation of a capture enzyme-linked immunosorbent assay for determination of rubella immunoglobulin M using monoclonal antibodies.

A capture enzyme-linked immunosorbent assay (ELISA) for detection of virus-specific immunoglobulin M (IgM) antibody was developed which used a panel of labeled monoclonal antibodies to rubella virus hemagglutinin. The rapidity of the test system was increased by using, after 1-h incubation of the test serum, a second 1-h incubation of the serum with a mixture of viral antigen and labeled monoclonal antibody. The new assay was tested for specificity on 371 human sera from people without any recent contact with rubella virus; of these, 66 were sera selected from people with rheumatoid factor or IgM antibody to human cytomegalovirus, Epstein-Barr virus, or other viruses. In parallel, the new assay was performed on 191 sera from patients having recent contact with rubella virus. Results were compared with those obtained by an indirect ELISA method on IgM serum fractions, using purified rubella virus as a solid phase. Of the 371 sera tested for specificity, 5 (1.3%) gave false-positive results with indirect ELISA (1 rheumatoid factor, 2 heterophil antibody, and 2 human cytomegalovirus sera positive for IgM), and none were false-positive with the capture assay. Two sera from a patient with primary cytomegalovirus infection, which were positive for rubella IgM antibody with both methods and were initially interpreted as false-positive, were finally considered to be true-positive, since they were reactive only in the presence of IgM antibody and viral antigen. Of the 191 sera from 92 patients (84 patients with acute rubella, four newborns from mothers with rubella during pregnancy, and four vaccinees), 136 (71.2%) were found to be positive for IgM by direct ELISA, and 128 (67.0%) were positive by capture ELISA; 12 sera drawn during the first 2 days of disease, or at least 40 days after onset (or after vaccination), were detected only by indirect ELISA, and 4 sera were detected only by capture ELISA. Thus, specificity and sensitivity, respectively, were 100 and 91.4% for capture ELISA and 98.6 and 97.1% for indirect ELISA. However, when the number of patients was considered, 86 were detected as IgM positive by indirect ELISA, and 87 were detected positive by capture ELISA. The overall agreement between the two assays was 96.2%. Capture ELISA using monoclonal antibody appears preferable over indirect ELISA on IgM serum fractions because of its higher specificity and shorter time for test performance; furthermore, there is no need for serum fractionation or virus purification for the capture ELISA.     

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.     

Diagnosis of recent rubella virus infection by demonstration of specific immunoglobulin M antibodies: comparison of solid-phase reverse immunosorbent test with sucrose density gradient centrifugation.

A solid-phase reverse immunosorbent test (SPRIST) based on the addition of an excess of rubella virus hemagglutinin was evaluated for the demonstration of rubella-specific immunoglobulin M (IgM), and the results were compared with those of the density gradient centrifugation technique. In a retrospective study in which 157 sera were tested, the two techniques yielded identical results (55 IgM-positive and 102 IgM-negative samples). In a prospective study, 592 sere were examined; 8 IgM-positive results by SPRIST corresponded to a recent rubella infection or vaccination. Neither rheumatoid factor nor heterophil antibody seemed to interfere with the results of SPRIST. This test would be a useful and rapid routine technique for demonstration of the presence of virus-specific IgM in serum samples, particularly for viruses with a hemagglutinin. Except for anti-human IgM, no more reagents are needed than for widely used hemagglutination inhibition procedures.     

Diagnosis of postnatally acquired rubella by use of three enzyme-linked immunosorbent assays for specific immunoglobulins G and M and single radial hemolysis for specific immunoglobulin G.

Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (RUBELISA, Enzygnost-Rubella, and Rubazyme) and a commercial single radial hemolysis (SRH) test (Rubazone) were evaluated for the diagnosis of acute rubella by testing 41 acute- (less than 7 days postonset) and convalescent-phase (8 to 82 days postonset) serum pairs from cases of rubella previously confirmed by significant change in the hemagglutination inhibition test titer. Specificity was tested by using 10 acute- and convalescent-phase serum samples from patients with rash not confirmed as rubella (control group). In testing for rubella-specific immunoglobulin G (IgG) antibody, Enzygnost-Rubella and Rubazyme confirmed infection in 40 and RUBELISA in 39 of the 41 proven rubella patients. For one patient all three ELISAs failed to show a significant titer rise. No false-positive diagnoses occurred in the control group, although a suspected infection was shown by Rubazyme in one patient. No specific IgM could be detected in this case. Single radial hemolysis confirmed infection in 39 of the 41 proven rubella patients, and one false-positive diagnosis occurred in the control group patients. Of the 43 convalescent-phase serum samples, rubella-specific IgM was detected in 42 by Enzygnost-Rubella, in 41 by RUBELISA M, and in 39 by Rubazyme-M. For a rapid diagnosis with acute-phase sera, specific IgM detection by ELISA was most reliable in hemagglutination inhibition test-positive sera; of 18 such serum samples IgM was shown in 15 by Enzygnost-Rubella, in 13 by RUBELISA M, and in 11 by Rubazyme-M. False-positive specific IgM results were shown by Rubazyme-M in two serum samples from one patient in the control group. These serum samples were negative with the other two ELISA methods.     

New passive hemagglutination assay kit that uses hemagglutinin-sensitized erythrocytes for detection of rubella antibodies.

A new passive hemagglutination assay for the detection of antibodies to rubella virus hemagglutinin (PHAST-Rubella) was compared with the hemagglutination inhibition (HI) test and another passive hemagglutination test that uses a soluble rubella virus antigen (SA-PHA). When the immune responses of vaccinated individuals were monitored, similar rises in antibody titer were detected by HI or PHAST-Rubella, whereas the rise in titer detected by SA-PHA was delayed. Early-phase vaccine-induced immunoglobulin M antibody analyzed by sucrose gradient fractionation was detected to the same degree by HI and PHAST-Rubella, but early-phase immunoglobulin G antibody reacted more strongly in the HI test. When acute and convalescent serum pairs from rubella-infected individuals were evaluated, a fourfold rise in titer was detected by PHAST-Rubella and HI in 15 of 15 pairs, whereas SA-PHA, which is not intended for detecting antibody titer rises in acute infections, detected a rise in titer in only 3 of 15 pairs. In studies to determine rubella immune status, testing of 1,078 premarital and random serum specimens resulted in 98.6% agreement among the three methods in identifying rubella antibody-positive and -negative individuals. For the quantitative PHAST-Rubella procedure, a coefficient of correlation of 0.93 was obtained, in comparison with HI, when a panel of 40 characterized sera were tested. These results indicate that PHAST-Rubella reagents can detect rubella antibodies as well as HI reagents and thus may be used as a fast and accurate means of determining rubella immune status and for the quantitation of rubella antibodies.     

The detection of rubella-specific IgM antibodies by radioimmunoassay.

An indirect solid-phase radioimmunoassay (RIA) has been developed for the detection of immunoglobulin (Ig) class-specific rubella antibodies. A commercial rubella haemagglutinin is dried and fixed on to the wells of flexible microtitre plates and allowed to react with serial dilutions of whole or fractionated human sera. Class-specific rubella antibodies are then detected by determining the specific binding of 125I-labelled anti-human IgG or IgM. The RIA was first evaluated by comparison with the haemagglutination-inhibition (HI) test for the detection of rubella-specific IgM in gel-filtration fractions. RIA was found to be as specific as HI but 10-150 times more sensitive. Rubella-specific IgG antibodies did not interfere in specific IgM determinations by RIA and therefore the latter technique was applied to unfractionated sera. The results obtained indicate that RIA on unfractionated sera is a practical, sensitive and specific technique which could provide a reliable method for the diagnosis of rubella. The rubella-specific IgM titres obtained by RIA were not increased by the removal of IgG by pretreatment of sera with Staphylococcal Protein A.     

Detection of rubella virus immunoglobulin G (IgG) and IgM antibodies in whole blood on Whatman paper: comparison with detection in sera.

We compared detection of rubella virus hemagglutination inhibition (HI) antibody and rubella virus-specific immunoglobulin M (IgM) in dried whole blood spotted onto Whatman filter paper and serum samples, both of which were obtained from the same subject by venipuncture. Of 1,000 paired serum samples obtained to study HI antibodies, 807 dried blood samples had HI titers identical to those of the corresponding serum samples, and 193 dried blood samples showed 1 dilution difference. Storage of dried blood at room temperature for 28 days did not affect the HI antibodies. In a study of specific IgM by a solid-phase immunosorbent HI test done with blood from healthy subjects and patients with rubella, the result of the presence, positive or negative, of specific IgM from both blood sample sources corresponded when the dried blood samples were stored at room temperature from 5 h to 38 days. This study demonstrated that the use of Whatman filter paper as a transport medium for blood samples for the determination of rubella virus immunity and the diagnosis of rubella virus infection is possible.     

Evaluation of the hemolysis-in-gel test for the screening of rubella immunity and the demonstration of recent infection.

The hemolysis-in-gel method for detection of antibodies to rubella virus gave results which correlated well with results of hemagglutination inhibition and neutralization tests. With a diffusion time of 24 or 48 h, a linear correlation was obtained between the logarithm of antibody concentration and the diameter of the hemolytic zone. Fourfold, and even twofold, differences in serum antibody concentrations were shown to give statistically significant differences in hemolytic zone diameters. It could therefore be concluded that the hemolysis-in-gel test is well suited for the serological diagnosis of primary rubella infection, as well as of reinfection. The sensitivity of the hemolysis-in-gel test was comparable to that of the hemagglutination inhibition test. Pigeon erythrocytes were superior to sheep erythrocytes for use in the test. Studies of the antibody response after natural rubella infection or vaccination showed that the appearance and persistence of antibodies measured by hemolysis in gel is similar to that of hemagglutination inhibition antibodies.     

Evaluation of a simplified sucrose gradient method for the detection of rubella-specific IgM in routine diagnostic practice

Rubella-specific IgM was measured in a single fraction of serum from a sucrose density gradient. Haemagglutination inhibition (HAI) tests were performed on paired aliquots of the fraction untreated and after treatment with 2- mercaptoethanol, dilutions of the aliquots being incubated over night with rubella antigen before the addition of red cells. Of 822 sera tested, specific IgM was found in 249, but not in 492. When first tested, the remaining 81 sera gave unsatisfactory results because of contamination of the IgM fraction with IgG (6.0%), probable aggregation of IgG (3.5%), or the persistence of chick red cell agglutinins (0.4%). Tests were performed on 134 patients with rubella confirmed by a rise of HAI antibodies. Rubella-specific IgM was found at a titre of more than eight in the sera taken from 62 of 64 patients between 10 and 29 days after the onset of the rash but in only one of the sera taken between 80 and 119 days, and in none taken later. However, specific IgM was still to be found at lower titre in the sera of 13 patients collected between 80 and 162 days after the onset of the illness. In routine diagnostic tests over three years on the serum from 479 patients with suspected acquired rubella, specific IgM was found at a titre of more than eight in 51 patients and in only 10 instances (2.1%) did a lower level pose a problem in interpretation.     

Clinical validation of an antibody-capture anti-rubella IgM-ELISA

An antibody-capture IgM-ELISA using monoclonal antibodies for conjugate was subjected to clinical validation with respect to sensitivity and specificity. In 103 serum specimens, known to contain anti-rubella IgM by a sucrose density gradient method, IgM was found by the ELISA in 99 sera. In a second study, 16 out of 17 acute rubella infections were detected by the IgM-ELISA. In 17 out of 17 vaccinees, a specific IgM response could be demonstrated.

Specificity of the antibody-capture ELISA was found to be high; no interference was seen in 60 rheumatoid-factor positive sera, in 100 highly positive IgG sera or 10 sera with anti-CMV IgM, Only one out of 100 sera with heterophile antibodies showed a positive response.

In acute rubella infections, IgM was shown to be detectable from 1 to 4 days after onset of illness up to about 12 wk, with peak values at about 1 wk after onset.     

Enzyme-linked immunosorbent assay for detection of human antibodies to Salmonella typhi Vi antigen

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to Salmonella typhi Vi antigen in human serum, and the results were compared with those from a previously described hemagglutination assay (HA). The ELISA detected Vi antibodies at a titer of greater than or equal to 20 in 40 (52%) of 77 sera from typhoid fever patients, whereas the HA gave titers of greater than or equal to 20 in 35 (47%). Determination of titers of serum specimens from 170 persons without typhoid fever revealed Vi antibody titers of greater than or equal to 20 in 4 (2.3%) by the ELISA and 3 (1.7%) by the HA. Unlike the sensitized erythrocytes used in the HA, the ELISA reagents have a shelf life of greater than or equal to 1 year. The ELISA may be preferred by some laboratories, especially those already performing other ELISA tests.