Clinical evaluation of a rubella passive hemagglutination test system.

A recently marketed passive hemagglutination (PHA) test (Rubacell, Abbott Laboratories) was compared to the hemagglutination inhibition (HI) test for the detection of antibody to rubella virus. The performance of the PHA system in determining immunity to rubella was evaluated by comparing the PHA results and HI results on 1, 086 randomly selected sera submitted for routine premarital and prenatal testing. Of the 1, 079 specimens assayable by both procedures, 1, 053 of the results (97.6%) were in agreement on initial testing. When the 26 initially discrepant specimens were retested for clarification, there was final agreement in 1, 067 of the specimens (98.9%). Twelve specimens were classified as persistently discrepant (five were not retested by PHA) and seven were unassayable by HI.Seven of the specimens with discrepant results were PHA positive and HI negative, and five were PHA negative and HI positive. Discussion of the two tests with respect to technical difficulty, cost, and controls is also included.     

Single-serum diagnosis of rubella by combined use of the hemagglutination inhibition and passive hemagglutination tests.

We explored the feasibility of using the passive hemagglutination (PHA) test in combination with the hemagglutination inhibition (HI) test for the single-serum diagnosis of rubella. We found by sedimentation analysis of the serum that early 7S antibody produced within 1 month after the onset of the rash had high HI but much lower PHA titers, whereas the PHA titers of antibody produced later were slightly higher than the HI titers. (19S antibody in the early serum had some PHA activity.) This disparity between the HI and PHA activities of the early 7S antibody could be used for the routine diagnosis of rubella. A comparison of the HI titers of the test serum with the PHA titers of mercaptoethanol-treated serum constitutes a simple method for determining whether the serum sample was taken shortly or remotely after the infection.     

Evaluation of a rapid passive hemagglutination assay for anti-rubella antibody: comparison to hemagglutination inhibition and a vaccine challenge study

A rapid passive hemagglutination assay (Rubaquick) was developed that detects antibody to rubella virus in serum specimens. The test result is read visually after an incubation period of 15-30 minutes. When compared with a hemagglutination inhibition assay, the Rubaquick assay results obtained from 1,470 sera were greater than 99% specific, sensitive, and accurate. Studies of 179 paired serum specimens obtained before and 27 days after rubella vaccination showed that if antibody was detectable by the Rubaquick assay in the prevaccination specimens, the vaccine induced a secondary response consisting of increasing IgG antibody reactivity in the absence of a positive IgM response. In contrast to the positive prevaccination specimens, a negative prevaccination result was associated with IgM antibody in 98 of the 133 postvaccination specimens. Seroconversion was noted in all cases in which the prevaccination specimen was negative by the Rubaquick assay.     

Rubella antibodies detected by several commercial immunoassays in hemagglutination inhibition-negative sera

Although a very good correlation was found between the level of rubella antibodies measured by a standard hemagglutination inhibition (HI) test and by an enzyme-linked immunosorbent assay (ELISA) procedure (Cordia R), an appreciable proportion (31%) of ELISA-positive specimens were encountered among HI-negative sera. The reverse was rarely seen. Many of the HI-negative, ELISA-positive sera were also found to be positive for rubella antibodies by one or more other assay methods, including an immunofluorescence assay (IFA) procedure (FIAX), passive hemagglutination (PHA) (Rubacell and PHAST), latex agglutination (Rubascan), and a second ELISA procedure (Rubelisa). The specificity of all of the ELISA-positive HI-negative specimens was substantiated by absorption experiments. In these tests, the ELISA reactivities were blocked by rubella antigens, but not by a variety of tissue culture control antigens or by influenza virus grown on the same cell line. The findings indicate that many of the newer methods available for rubella antibody detection are more sensitive than HI for detecting low levels of rubella antibodies. Until more clinical information is available concerning the protective nature of these low levels of antibody, caution should be exercised in assessing the significance of these results.     

Antibody responses in patients with rubella infection determined by passive hemagglutination, hemagglutination inhibition, complement fixation, and solid-phase radioimmunoassay tests

Antibody responses in serial serum specimens collected from 31 patients with an acute rubella infection were determined by passive hemagglutination (PHA), hemagglutination inhibition (HI), complement fixation (CF), radioimmunoassay (RIA) immunoglobulin G (IgG), and RIA immunoglobulin M (IgM) tests to evaluate the effectiveness of these tests in diagnosing a recent infection. The HI, RIA IgG, and RIA IgM antibodies appeared almost simultaneously and reached the maximum level about 1 week after the onset of rash. Compared to these, the CF antibodies developed only slightly later, whereas the development of the PHA antibodies was much more delayed. The RIA IgM response was shown to be transient, lasting approximately 1.5 to 2.5 months postinfection. The results of this study indicate that demonstration of specific IgM antibodies is the best method for diagnosing a recent infection, one within 2 months after the onset of the illness. If an IgM test is not available, a combination of the HI and PHA tests is recommended.     

Hemolysis-in-gel test in immunity surveys and diagnosis of rubella

The hemolysis-in-gel (HIG) technique was adapted for rubella antibody determinations. Use of sucrose gradient purified virus and its coupling with CrCl₃ to chicken erythrocytes resulted in gel plates that could be stored for several weeks and were suitable for reproducible antibody determinations. In a serological survey of young healthy adults the HIG values (range > 2–13 mm) were in close correlation to those obtained by the HI test (> 10 to 320). The HIG test seems well suited for screening the need of vaccination. Seronegative sera (HIG > 2, HI> 10) gave without heat inactivation hemolysis zones ranging from 4 to 6.5 mm. Although the present rubella HIG test did not measure IgM antibodies, the test, by virtue of its accuracy and sensitivity — extending to antibody levels corresponding to HI titers 2–10 — provides a simpler and more rapid means for diagnosis of rubella infections than the conventional HI and CF tests.     

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.     

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     

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.     

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.     

Passive hemagglutination test for measles immunity and serodiagnosis.

A passive hemagglutination (PHA) test for measles was evaluated in comparison with hemagglutination inhibition (HI) and neutralization (NT) tests. The PHA test determines exclusively the level of antibody directed to the hemagglutinin protein of measles virus. The ratio of PHA to HI titer was 1 to 32 (geometric mean, 6.5) for the first 5 weeks of infection but declined to near unity thereafter. It gradually increased again to 4 to 32 (geometric mean, 11.7) over several years. The initial high PHA titer relative to the HI titer was most likely due to the presence of the immunoglobulin M antibody known to be efficient in agglutination, because 2-mercaptoethanol (2ME) treatment of sera reduced the PHA titer to a level similar to that of the HI titer. The PHA titer in sera obtained after the convalescent phase was insensitive to 2ME, and the relative increase in the PHA over the HI titer was presumably a result of increased antibody avidity. In some individuals, the HI titer fell to below detectable levels several years after either natural infection or vaccination, but the PHA as well as the NT titer remained positive. The PHA titer was therefore a more reliable and more sensitive indicator of immune status against measles than the HI titer. The decrease in PHA titer by 2ME treatment provided evidence of a current or very recent infection. PHA was found to be useful both for assessing immunity status and for serodiagnosis.     

Direct enzyme immunoassay for detection of specific IgG antibody to rubella virus by use of labelled antigen

A new solid phase enzyme immunoassay (EIA) for detection of rubella-specific immunoglobulin G (IgG) antibody was developed. The test uses polystyrene microtiter strips coated with rabbit anti-human IgG immunoglobulins as the solid phase and an enzyme-labelled semipurified rubella antigen as indicator. The direct EIA was compared with hemagglutination inhibition (HI), single radial hemolysis (SRH), radioimmunoassay (RIA) and time-resolved fluoroimmunoassay (TR-FIA) using 52 serum specimens from patients with remote rubella infection. The overall agreement of direct EIA with HI was 96.1%, with SRH and RIA 98.1% and with TR-FIA 100%. The linear regression coefficient varied from 0.77 to 0.91, the best being obtained with direct EIA and SRH. The direct EIA was also suitable for diagnosis of acute infections, as a significant increase in antibody levels was detected in all paired specimens tested from patients with acute rubella infection. The sensitivity and were comparable to those of the assays employed. An advantage of the present assay is that the same method and same labelled antigen can be used to test for different classes of antibody using simply a solid phase with capture antibodies of different chain specificity.     

Hemagglutination inhibition,single radial hemolysis,and ELISA tests for the detection of IgG and IgM to rubella virus

Hemagglutination inhibition (HI), single radial hemolysis (SRH) and enzyme-linked immuno sorbent assay (ELISA), performed with commercial antigen and reagents are described and were compared in the three distinct situations that require rubella antibody detection. Determination of immunity status was carried out on 156 sera. A degree of correlation > 0.9 was found when comparing the three methods. Analysis of a further 74 sera, from 31 primary infections and three congenital syndromes, was performed to compare the occurrence of the various classes of antibodies in the three tests: HI test and IgM-ELISA become positive the day after the rash, whereas SRH test is not positive before the sixth day. From our limited study bearing on a total of 230 sera, each test has a precise assignment. For the determination of immunity status, SRH is simpler, faster, and inexpensive; absence or evidence of past infection can be unequivocally obtained especially in cases of low (1:10, 1:20) residual immunity. In the seriodiagnosis of a rubella rash, SRH alone, due to the delayed rise in antibody titers, will demonstrate a complete seroconversion with a first serum collected up to the fifth day of the eruption. In case of absence of an early serum, of primary infection in a pregnant woman, of a newborn with suspicion of congenital syndrome, the measurement of rubella specific IgM is best obtained with ELISA, a procedure less time-consuming than HI following centrifugal, chromatographic, or electrophoretic separation. And “light” (8 S) RF with SRH test is discussed. Interference of IgM Rheumatoid Factor (RF) with IgM ELISA and IgG RF with SRH test is discussed.     

Detection of parainfluenza IgM antibody by hemadsorption immunosorbent technique

A hemadsorption immunosorbent technique (HIT) was developed for the detection of immunoglobulin M (IgM) to parainfluenza virus types 1, 2, and 3. Twenty-six (90%) of twenty-nine patients under 6 yr of age from whom parainfluenza virus was isolated showed parainfluenza IgM antibody in one or both of their paired sera, with titres ranging from 320 to 81,920. In about one third of the cases IgM antibody was demonstrated in the initial sera taken 1 to 3 days after the onset of illness. Heterotypic IgM antibody responses were observed in about 20% of the patients. The HIT test was more sensitive than the hemagglutination inhibition (HI) and complement fixation tests in detecting a seroresponse in the 29 virus-positive children. The results of studies in older patients with HI titre rises to parainfluenza virus suggested that reinfection probably induced IgM antibody in a proportion of cases. The HIT test proved to be specific for the IgM class of antibody and avoided false-positive results due to rheumatoid factor. It permits an early presumptive diagnosis in a proportion of patients with parainfluenza infection.     

Mumps-specific immunoglobulin M and G antibodies in natural mumps infection as measured by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay for the demonstration of mumps immunoglobulin G (IgG ELISA) and immunoglobulin M antibodies (IgM ELISA) in serum was compared with complement fixation (CF), hemagglutination inhibition (HI), and hemolysis-in-gel (HIG) tests. The antibody levels measured by IgG ELISA had a high positive correlation with the CF and HIG tests, whereas only a moderate correlation was found between IgG ELISA and HI. Similar patterns of antibody response were observed with IgG ELISA, CF, and HIG: the antibody titres increased rapidly after the onset of symptoms and reached the maximal values in about three weeks. The HI antibodies developed more slowly during the first week of disease, after which the titres increased rapidly up to the fourth week. IgM antibodies measured by ELISA developed soon after onset of symptoms; most patients had IgM antibodies from the second day, and the highest titres were reached within the first week. The antibody response in mumps parotitis did not differ from that in mumps meningitis/encephalitis, while relatively higher antibody titres were found in patients with orchitis/epididymitis. The diagnostic efficiencies of the methods were compared with serum specimens from 33 patients who had a serologically verified mumps infection by at least one of the five methods used (rising antibody titres in paired sera or detectable IgM): IgM ELISA detected all 33 cases, IgG ELISA 29, HIG 28, HI 23, and CF 13. In 27 cases, IgM antibodies were already present in the acute phase serum specimens. It was concluded that mumps IgM ELISA is a more rapid and sensitive means for the serological diagnosis of mumps infection than the conventional tests.     

Cost and performance analysis of haemagglutination inhibition, passive haemagglutination, radial haemolysis, and enzyme immunoassay for measuring rubella antibody

Cost and performance of non-commercial haemagglutination inhibition (HI) and radial haemolysis (RH) tests, and the commercially available passive haemagglutination (PHA) Rubacell and enzyme immunoassay (EIA) and Rubazyme assays were compared in their ability to detect rubella antibodies in 316 sera. Correlation coefficients were: HI to RH 0.96; HI to EIA 0.86. All 4 tests were in agreement on pre- and post-rubella immunization sera from 10 subjects. Eleven sera collected between 1 and 15 days after natural infection possessed clear HI titres whereas only 4 of them showed positive responses by PHA, RH or EIA. Immunity screening 285 sera identified 7 discordant results (positive in 2 of 4 tests). A detailed cost analysis for testing 100 sera showed a cost per test from +2.10 for HI to +3.71 for EIA. The labour component of the total cost was different for each assay and affected the unit cost of testing a single specimen. Results are discussed in view of antibody responses to specific rubella polypeptides and recommendations for diagnosis or immunity screening are made according to the findings.     

Antibody response to rubella virion (V) and soluble (S) antigens in rubella infection and following vaccination with live attenuated rubella virus

Summary The antibody response to rubella virion antigen and rubella S antigen was studied in natural rubella infection and after vaccination with live attenuated rubella virus by the complement fixation (CF), platelet aggregation (PA), and hemagglutination inhibition (HI) techniques.In natural rubella infection CP and HI antibodies to rubella virion appeared early and rapidly reached high titers. The CP and PA antibody responses to rubella S antigen were more delayed and great individual variation was seen. Generally CF-S titers were several times lower than CF-V titers. The CF antibody response to rubella S antigen is thus different from the immune response to nucleoprotein S antigens of paramyxoviruses, supporting the concept that rubella S antigen is a subunit of virus envelope. Use of virus-free rubella S antigen preparations in routine CF test is recommended for detecting later rises of antibody titer.After rubella vaccination a 95% seroconversion rate was recorded in both HI and CF-V tests, but the titers were lower than after natural infection. The CF-S and PA antibody responses were weaker and measurable antibodies developed in only 50% and 25% of the cases respectively.Rubella-specific IgM antibodies could be detected in rubella infection by both sucrose gradient fractionation (followed by HI titration) and fluorescent antibody (FA) techniques. The former was a little more sensitive. In the seronegative vaccinees IgM antibodies became demonstrable in 50% of the cases between the 20th and 55th day after vaccination.     

Comparison of methods for detecting specific IgM antibody in infants with congenital rubella.

Serum specimens from 14 infants with congenital rubella were examined for specific IgM antibody by six different methods. IgM-containing fractions were separated either by sucrose density-gradient centrifugation or by gel filtration through Sephadex G-200, and were then tested by the indirect immunofluorescence technique and by the haemagglutination-inhibition (HI) test (long-and short-incubation methods). Immunofluorescence staining of density-gradient fractions detected specific IgM in all 14 infants. The HI test (long method), applied to density-gradient fractions, was almost as sensitive, detecting antibody in 13 infants; the short method was less sensitive. The gel-filtration technique proved to be generally less satisfactory than sucrose density-gradient centrifugation. Evidence was obtained for the occurrence of as yet unclassified non-specific inhibitors in the serum of some infants. These inhibitors were deposited with the IgM on sucrose-density gradients and they could have been mistaken for rubella-specific IgM antibody, particularly in the HI test (long method).     

Single radial hemolysis test for rubella immunity and recent infection.

The single radial hemolysis (SRH) test was compared with the hemagglutination inhibition (HI) test for establishing rubella immune status and diagnosing recent infection. Correlation between mean SRH diameters and HI titers greater than or equal to 1:8 was high (R = 0.99). It is suggested that a level of greater than or equal to 5 IU represents low-level antibody and that greater than or equal to 15 IU is a conservative threshold for designation of immunity. Of 343 sera tested, only 1 false-positive was found by SRH with the 5 IU cutoff level. The SRH test could detect serum antibody levels as low as 2.5 IU, whereas 15 IU was generally the limit of resolution of the HI test. Data from sucrose density gradient fractionation of serum demonstrated that neither rubella-specific immunoglobulin M (IgM) nor early postinfection HI-reactive IgG was detected by SRH. However, diagnostic changes in antibody titer measured by SRH were in general greater than those measured by HI. The SRH test showed diagnostic titer changes in some sera containing specific IgM for which no such changes were detected by the HI test. A 2.5-mm difference in hemolytic zone diameters (a fourfold rise in international units) between acute- and convalescent-phase serum pairs was chosen as being of diagnostic significance. This difference was less than the minimum observed difference of 2.9 mm from 59 serum pairs showing diagnostic changes by HI and far exceeded (greater than 3.6 standard deviations) the within-test and individual variability seen for 95 pregnant women from whom samples were obtained during each trimester.