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.     

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.     

Evaluation of rubella immune status by three commercial enzyme-linked immunosorbent assays.

Three commercial indirect enzyme-linked immunosorbent assays (ELISAs) (Enzygnost-Rubella, RUBELISA, and ORTHO Rubella) were evaluated for the determination of immune status by testing 1,090 serum specimens, 410 of which were from nonimmune patients. In comparison with the standard reference technique, the hemagglutination inhibition (HAI) test, the sensitivities of ORTHO Rubella (100%) and Enzygnost-Rubella (99.26%) were excellent, whereas the sensitivity of RUBELISA (95.60%) was marginally lower because of the inability of this assay to detect antibody in 22% of the serum specimens with HAI titers of 10 and 11% of sera with HAI titers of 20. The specificity of all three systems was greater than 97%. There was a linear correlation between mean ELISA values and increasing HAI titers (r greater than or equal to 0.94). Both ORTHO Rubella and Enzygnost-Rubella were shown to be suitable replacements for the HAI test, provided that an equivocal zone is incorporated in the ORTHO system and only unheated sera are used in the Enzygnost system.     

Comparison of hemagglutination inhibition test and ELISA in quantification of antibodies to egg drop syndrome virus

A single-serum dilution ELISA for egg drop syndrome (EDS) virus-specific antibodies was developed. In testing 425 chicken sera it was found to have a 93.6% sensitivity and 98.7% specificity relative to a hemagglutination inhibition (HI) test. The correlation coefficient for ELISA and HI titers was 0.793. The ELISA was efficacious in quantification of both vaccinal and infection antibodies and could routinely be used for screening large numbers of field sera.     

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.     

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.     

Enzyme-linked immunosorbent assay for measurement of antibody against cytomegalovirus and rubella virus in a single serum dilution.

Enzyme-linked immunosorbent assays (ELISA) were developed for quantifying cytomegalovirus (CMV) and rubella antibodies using a single serum dilution (1/800) in conjunction with a standard curve. A near linear relation was found between the logarithms of absorbance values of sera at a dilution of 1/800 and the titres as determined by an end point dilution ELISA. The reproducibility of the single dilution ELISA was good; the within-test coefficients of variation averaged 7.5% for CMV antibody and 12.4% for rubella antibody. A close correlation was found between ELISA and complement-fixing (CF) antibody titres to CMV and between ELISA and haemagglutination-inhibition (HI) antibody titres to rubella virus. The titres in ELISA were 200 to 1000 times higher than in CF for CMV and 50 to 100 times higher than in HI for rubella virus.     

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.     

Latex agglutination test for rubella antibodies: report based on data from the College of American Pathologists surveys, 1983 to 1985

In the College of American Pathologists (CAP) rubella survey program, 45% of laboratories rely on the latex agglutination (LA) card assay for detecting rubella immunoglobulin G (IgG) antibodies. By using CAP survey data over a 3-year period, we compared LA results with hemagglutination inhibition (HI) and enzyme immunoassay (EIA) results. EIA indices were used to classify results into three categories: nonimmune, EIA index of 0.300 or less; borderline, EIA index of 0.300 to 0.619; and immune, EIA index of 1.700 or greater. There was 91% or more agreement between LA, HI, and EIA for categories i and iii. In category ii, the response from LA users varied, depending on the level of antibody present in the survey samples; at an EIA index of 0.346, 81% reported nonimmune status, whereas at an EIA index of 0.619, 48% reported nonimmune status. Less than 10% indicated borderline status. In testing of samples in the same category, approximately 40%, using the HI method, reported titers of less than 1:8 (nonimmune status). Among EIA users, 97 to 99% regarded the specimens as nonimmune. On analysis of specimens in the borderline category, the LA test showed a pattern of sensitivity and specificity comparable to that reported with the HI technique, whereas the EIA method showed a greater degree of precision. The LA card assay provides a rapid screening test in which LA is read macroscopically, and the procedure differs considerably from the fully quantitative HI and EIA methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Comparison of hemagglutination inhibition test and enzyme-linked immunosorbent assay for determining antibody to rubella virus.

The hemagglutination inhibition test (HAI) and the enzyme-linked immunosorbent assay (ELISA) for detecting antibody to rubella virus were compared by testing 25 sets of paired sera taken before and after infection and 10 sets of sera taken during acute and convalescent stages of the disease and by screening 700 serum samples from the Collaborative Perinatal Project, NIH/NINCDS. The tests were found to be comparable in their ability to detect positive and negative sera, rises in titers, and seroconversions. When a purified antigen and carefully prepared reagents were used, ELISA was found to be as accurate and reliable as HAI. ELISA required no pretreatment of serum, could easily be automated, and was less time-consuming than HAI.     

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.     

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.     

Use of hemolysis in gel in the serodiagnosis of rubella. Comparison with hemagglutination inhibition]

The hemolysis in gel test (HIG) was compared with the hemagglutination inhibition test (HI). This new test is simple and time-sparing since it does not require dilution or serum pre-treatment, and can be measured directly in mm. It is not more expensive than the HI method. It has proved sensitive and is not affected by non specific serum hemagglutination inhibitors and is conclusive whenever HI titers are looked upon with suspicion (titers of less than 1/20). It is therefore well adapted to mass screening of immunity against rubella but in the field of recent infections HI method still has its role to play. Using the HIG test as a reference, we measured the loss of Ig to rubella following the three most commonly employed methods for removal of inhibitors: the mean values of 16 sera were 25% of antibodies lost after kaolin pre-treatment, 62% lost after heparine-Mn-Cl2 procedure 75% lost after dextran-CaCl2 treatment.     

Quantitative and qualitative assay of rubella IgA antibody in breast milk

Breast milk contains immunological factors, such as IgA antibody, which help to prevent infectious diseases. A total of 197 paired samples of colostrum and breast milk was collected from postpartum mothers in Gunma City, Japan, and examined for anti‐rubella IgA antibody by enzyme‐linked immunosorbent assay (ELISA) and Western blotting (WB). The anti‐rubella virus IgA ranged from 0.5 to 78.5 U/ml with a mean of 6.05 U/ml and a median of 3.6 U/ml in colostrum, and from 0.5 to 32.7 U/ml with a mean of 2.74 U/ml and a median of 2 U/ml in milk. The differences between the means of titers of total IgA and anti‐rubella virus IgA in colostrum and in milk were significant statistically. The levels of anti‐rubella virus IgA in both colostrum and breast milk correlated positively with the anti‐rubella virus hemagglutination inhibition (HI) titers in the sera of mother, indicating that the levels of these different classes of antibodies correlated. Based on WB, anti‐rubella virus IgA in both colostrum and breast milk reacted with the rubella viral protein E1 and C, but not with the E2 protein. J. Med. Virol. 82:1475–1479, 2010. © 2010 Wiley‐Liss, Inc.     

A study of ELISA systems incorporating pooled viral and Mycoplasma antigen preparations for antibody screening of chicken sera

Enzyme linked immunosorbent assays (ELISAs) incorporating up to three different antigens for screening of avian sera for antibodies to several viruses or mycoplasma are described. A triple antigen test comprising Newcastle disease virus (NDV), infectious laryngotracheitis (ILT) virus and avian influenza (AI) antigens for screening sera normally negative for antibodies to these viruses, was shown to be as sensitive as the corresponding single antigen ELISA in detecting seroconversion in experimentally inoculated birds and was also as sensitive as the haemagglutination-inhibition (HI) test for NDV and AI, and the serum neutralisation test for ILT virus. Sensitivity was also demonstrated by comparison of end-points in serially diluted NDV, ILT or AI positive sera. A Mycoplasma synoviae ELISA was shown to be as sensitive as HI test for detection of MS and M. gallisepticum (MG) antibodies in experimentally inoculated birds and in field sera, and this antigen combined with NDV detected antibodies to MG, MS and NDV with sensitivity equivalent to the HI test in each case. The advantages of using pooled ELISA preparation for screening large numbers of sera which are normally negative for the pathogens concerned are discussed.     

Detection of rubella antibodies by hemagglutination inhibition, indirect fluorescent-antibody test, and enzyme-linked immunosorbent assay.

Using hemagglutination inhibition (HAI) as a reference method, 292 (40 nonimmune, 252 immune) human serum samples were tested by indirect fluorescent antibody (IFA) and enzyme-linked immunosorbent assay (ELISA) for immune status and quantitation of rubella antibodies. The overall agreement with HAI for immune status was 99.7% (291/292) with IFA and 98.6% (288/292) with ELISA. Two specimens (0.7%, 2/292), negative by HAI, were equivocal by ELISA. Initially a 6.5% (19/292) overall disagreement was obtained for immune status evaluation between HAI and IFA, which was reduced to 0.3% (1/292) upon repeat testing. All of these samples were near the immune/nonimmune cutoff point (95 samples), reflecting an initial disagreement of 20% (19/95) in this category (HAI titers less than 1:20). Likewise, an initial overall disagreement of 4.5% (13/292) was obtained between HAI and ELISA which was reduced to 0.7% (2/292) upon repeated testing. Eleven of the 13 samples were near the immune/nonimmune cutoff point, reflecting an initial disagreement of 11.6% (11/95) with sera having an HAI antibody titer of less than 1:20. Quantitation of rubella antibodies by IFA showed an overall correlation with HAI of 86.6% within less than twofold titer and 99.3% within less than fourfold titers. In testing the ability of ELISA to quantitate antibody, a correlation coefficient (r) of 0.996 was obtained by plotting the measured average optical density (405 nm) of ELISA against the corresponding log of HAI titer. Both IFA and ELISA showed good correlation with HAI for immune status evaluation and for quantitation of rubella antibodies. Technically the HAI was the most cumbersome to perform, whereas IFA was the least technically demanding. Originally, 308 samples were tested; 16 samples (5.2%) could not be evaluated by IFA because of a high level of nonspecific fluorescence. The strict requirement of controlling the temperature range (23 to 24 degrees C) during substrate hydrolysis proved to be a problem with the ELISA test in our laboratory.     

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     

New tests for characterization of mumps virus antibodies: hemolysis inhibition, single radial immunodiffusion with immobilized virions, and mixed hemadsorption.

Hemolysis inhibition (HLI), single radial immunodiffusion (SRID) with immobilized virions, and mixed hemadsorption tests were used for measuring antibodies against mumps virus. Rabbit hyperimmune sera against mumps and early and late human convalescent sera were analyzed. All three tests identified antibodies against both hemagglutinin and the second major envelope component, hemolysin (fusion factor). The sensitivity of the HLI test corresponded to that of the hemagglutination inhibition (HI) test, but in some sera HLI antibodies occurred in greater quantity than HI antibodies. The SRID test readily identified rises in antibody titers in connection with acute infection. Due to its simplicity and lack of sensitivity to nonspecific inhibitors, it is recommended for use in this context. The mixed hemadsorption test showed a high sensitivity for specific identification of mumps antibodies. It therefore may be suitable for use in screening for immunity to mumps.     

Immunofluorescence tests for HIV antibody and their value as confirmatory tests

The enzyme-linked immunosorbent assay (ELISA) is currently being used as a sensitive screening test for HIV antibody. The immunoblot assay (IBA) and indirect immunofluorescence (IF) techniques are two recommended confirmation tests for EIA-positive sera. An indirect IF test has been developed by various laboratories using acetone fixed mixtures of uninfected and HIV-infected cells, which facilitated the reading, since nonspecific reactions were easily differentiated from specific staining. Similar results have been obtained with H9-, CEM-, and HUT78-HIV-infected and uninfected cells. Anti-nuclear antibodies and auto-antibodies resulting in false-positive EIA results, could easily be differentiated by the IF test. Aspecific fluorescence can be removed by absorption of the specimens with non-infected cells. However, IF is not suitable for the screening of large series of specimens. IF is especially well suited for quantitative analysis of serum antibody levels. Whereas serum antibody titers rise initially after infection, they decrease as AIDS develops. Heat inactivation of sera did not affect reactivity in IF, in contrast to a high rate of false-positive results obtained with heat inactivated sera in some ELISAs. A well characterized serum from an AIDS patient can be used to perform IF in order to monitor HIV infection of susceptible cells. It has been claimed that titers of neutralizing antibodies significantly correlate with the levels of IF anti-HIV antibodies. An overall correlation of 99% between IF and IBA was reported by different laboratories, when HIV ELISA-reactive European and North American sera were tested. The concordance with IBA was 97% when HIV ELISA-reactive African sera were tested.(ABSTRACT TRUNCATED AT 250 WORDS)