Further evaluation of a rubella passive hemagglutination test

Clinical experience with the Rubacell passive hemagglutination (PHA) test over a one-year period has shown the test to be a rapid, reliable, and economical method for determining antibody to rubella. The data from two separately administered rubella proficiency surveys showed 100% correlation between the PHA and the hemagglutination inhibition (HI) qualitative results with 24 reference specimens. Also, the PHA titers appeared to be generally higher than the HI in these specimens and in the sera of immune individuals. The efficacy of detecting HI antibody in the absence of PHA antibody as an indication of recent infection was compared to the HI paired sera method and to a rubella-specific immunoglobulin M (IgM) test based on protein A absorption. From the results obtained with the sera of 76 rubella patients, the efficacy of the three diagnostic methods was of the following order: protein A IgM test > positive HI/negative PHA > HI paired sera method.     

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.     

A method for coupling cytomegalovirus antigens to aldehyde-fixed erythrocytes for use in passive hemagglutination

A method for coupling cytomegalovirus (CMV) antigens to aldehyde-fixed erythrocytes for use in a passive hemagglutination assay for antibody to CMV is described. This method uses acid buffer instead of tannic acid for coupling CMV-complement fixation (CF) antigen to the stabilized cells. The coated cells are stable for at least 6 mth at -70 degrees C and for 5 days at 4 degrees C. This provides an inexpensive reagent having a sensitivity for anti-CMV detection which compares favorably with that of the CF test and enzyme immunoassay.     

Evaluation of a rubella hemagglutination inhibition test system.

Two systems for measurement of rubella hemagglutination inhibition antibodies were compared. One had chick erythrocytes as indicator and the other, which is available in kit form ("Rubindex," Ortho-Diagnostics), uses human group O erythrocytes. Correlation to within one dilution of each other was noted in 92% of the tests. The Rubindex system has the advantage of longer shelf life of reagents and also does not require one of the absorption steps necessary for the other method, thus saving technologist's time.     

Specificity of the passive hemagglutination test for antibody to rubella virus and the passive hemagglutination response after vaccination.

Nonspecific reactions by the passive hemagglutination test for rubella viral antibody occurred with 1.5% of sera tested. Rises in passive hemagglutination titers (greater than or equal to 4X) occurred with some serum samples taken 14 days after vaccination.     

Covalent coupling of proteins to erythrocytes by isocyanide. A new,sensitive and mild technique for identification and estimation of antibodies by passive hemagglutination

A mild method for covalent coupling of protein-antigens to the surface of red blood cells (RBC) is described. The method is based on the formation of covalent bonds between aldehyde groups of oxidized RBC and amino or carboxyl groups of proteins in the presence of isocyanide.Very low concentrations of antibodies (8–30 ng/ml) could be detected in antisera with the specifically sensitized RBC as reagents in passive hemagglutination with no or very low non-specific agglutination.     

Direct HIG test for rubella antibodies

A modification of the haemolysis-in-gel test for rubella is described. This involves using, instead of sera, whole blood samples taken from the earlobe and placed directly onto the test plates without any pretreatment. A comparison was made between this method and the HI test. Samples from 461 individuals were tested by both methods. The results of these two tests were in good agreement. No false negatives or false positives were observed by the direct HIG test. The direct HIG test is particularly well suited for screening large groups for rubella antibodies in connection with a vaccination program.     

An evaluation of ELISA kits for rubella IgG- and IgM-antibodies

Two ELISA kits for rubella IgG- and IgM-antibodies, respectively, were compared with a haemagglutination inhibition (HAI) test (after ultracentrifugation of the sample if IgM was to be detected). When screening 535 samples from pregnant women, 99.6% agreement was found between the IgG-ELISA and HAI; one true IgM-positive was encountered in this group with the IgM-ELISA. An agreement of 95.5% was found between the IgM-ELISA and HAI after ultracentrifugation. In at least some of the discrepant samples the IgM-ELISA may be assumed to have given the correct answer.     

Reactivity of nonsensitized control erythrocytes in rubella passive hemagglutination assays.

Two commercially available, nonsensitized control erythrocytes for use in rubella antibody passive hemagglutination assays detected nonspecific reactions in 10 of 600 (1.7%) and 9 of 500 (1.8%) sera tested. Reactive sera were positive with one or the other cell type, but not both. The probability of obtaining a false-positive result owing to a nonspecific passive hemagglutination reaction was estimated to be 0.17% for either cell type.     

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.     

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.     

Comparison between a commercial ELISA, Rubazyme, and hemolysis-in-gel test for determination of rubella antibodies

A commercial enzyme-linked immunosorbent assay, Rubazyme, was compared with the hemolysis-in-gel (HIG) test for antibodies to rubella in 826 sera. The results were in agreement for 99.4% of the 725 sera tested for immunity. However, the Rubazyme assay was no more efficient that either the hemagglutination-inhibition or HIG test in discriminating between sera with low levels of antibody and negative sera. Thus, it was concluded that the HIG test is the method of choice for immunity testing because of the low cost and simplicity. Rubazyme may be of value to confirm equivocal HIG results.     

Detection of early hemagglutination inhibitory antibodies to rubella virus by pretreatment of sera with streptococcal cells

A technique that is based on absorption of sera with streptococcal cells and hemagglutination inhibition (HI) was evaluated for its feasibility for serologic diagnosis of recent rubella. The mixture of AR1 and AW43 cells removes IgG and IgA from the kaolin-treated sera, leaving IgM and a trace of IgA, probably oligomeric IgA. Consequently, after the absorption with streptococci, the HI antibodies are detectable exclusively in the early sera of patients with rubella. The streptococci (AR1 and AW43) have several advantages as the absorbent over the staphylococcus (Cowan I) that has been used routinely.     

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     

Persistent rubella-specific IgM reactivity in the absence of recent primary rubella and rubella reinfection.

Between two and seven sera from cases of persistent detection of rubella-specific IgM for periods in excess of 2.5 months, but in the absence of recent primary rubella or rubella reinfection, were examined for rheumatoid factor, heterophile antibody, and IgM reactivity against toxoplasma and a number of viruses. The relative avidity of the rubella-specific IgG1 has been assessed in all the sera by two methods. None of the sera contained rheumatoid factor or heterophile antibody, nor did any contain detectable concentrations of IgM specific for any of the panel of antigens apart from five sera which contained low concentrations of IgM specific for some coxsackieviruses B. No sera were positive for low avidity specific IgG1 although three did give equivocal results with one avidity test and one gave equivocal results with the second avidity test.     

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.     

Nonspecific reactions in the hemagglutination inhibition test for detection of rubella antibodies.

Approximately 7% of the sera tested to determine the presence of rubella-specific antibodies by the hemagglutination inhibition test demonstrated abnormal patterns of reactivity, rendering the test unreadable. Another 3% of sera were shown to have false-positive titers as high as 1:128. When these abnormally reacting and false-positive sera were heated at 56 degrees C for 30 min after chemical treatment they always converted to negative, indicating the absence of specific rubella hemagglutination-inhibiting antibody. These results were confirmed by fractionation of the sera after sucrose gradient centrifugation. It was established that manifestation of these nonspecific results was dependent on the concentration of Ca2+ or Mn2+. The heat-labile inhibitor(s) responsible for abnormal and false-positive reactions was found not to be complement. This inhibitor(s) was detected in the light fractions of sera and when added to negative sera was capable of reproducing the abnormal patterns of reactivity. These results emphasize the necessity of heating sera for the rubella hemagglutination inhibition test after the chemical removal of nonspecific inhibitors.     

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.     

Application of molecular and serological assays to case based investigations of rubella and congenital rubella syndrome

Rubella and congenital rubella syndrome continue to be important health problems worldwide. The detection of rubella RNA directly in clinical specimens is a critical factor in early laboratory diagnosis of recent or congenital infection, in addition to detection of rubella-specific IgM. In order to comply with recent WHO recommendations for establishing uniform genetic analysis protocols for rubella virus we have developed a new block based PCR assay (PCR-E317), which extends the sequence generated by the block based PCR-E592 currently in use, to cover the minimum acceptable 739 nucleotides (nt) window at the E1 gene. In addition, a real-time PCR assay has been developed to allow rapid detection of the virus in the laboratory. The assays were applied to a number of clinical specimens collected from patients including recent rubella incidences in the UK, Ethiopia and Turkey, two prenatal and two congenital rubella syndrome cases. Rubella RNA was detected in specimens from two patients that were collected too early for IgM detection, in two amniotic fluids for prenatal diagnosis and in the follow up specimens from the two infant with congenital rubella syndrome tested for viral secretion. At least four genotypes were identified among these patients. The results showed that molecular assays are important tools in the early diagnosis of rubella and congenital rubella syndrome, in the provision of molecular epidemiological information for tracking transmission pathways and in adding to the knowledge of rubella strain distribution worldwide.