May 21, 1984 - MAX A. CHERNESKY,l* LORRAINE WYMAN,2 JAMES B. MAHONY,1 SANTINA CASTRICIANO,1 JOHN T. UNGER,2 JOHN W. SAFFORD,2 ...
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1984. p. 400-404 0095-1137/84/090400-05$02.00/0 Copyright © 1984. American Society for Microbiology
Vol. 20, No. 3
Clinical Evaluation of the Sensitivity and Specificity of a Commercially Available Enzyme Immunoassay for Detection of Rubella Virus-Specific Immunoglobulin M MAX A. CHERNESKY,l* LORRAINE WYMAN,2 JAMES B. MAHONY,1 SANTINA CASTRICIANO,1 JOHN T. UNGER,2 JOHN W. SAFFORD,2 AND PEYTON S. METZEL2
McMaster University Regional Virology Laboratory, St. Joseph's Hospital, Hamilton, Ontario, Canada L8N 4A6,1 and Abbott Laborcatories, Nor-th -Chicago, Illinois 600642 Received 9 March 1984/Accepted 21 May 1984
A solid-phase capture antigen enzyme immunoassay (Rubazyme-M) was evaluated for sensitivity and specificity on sera from 1,200 blood donors, 51 patients with rubella, 2 infants with congenital rubella, 104 patients with other infections, and 126 patients with immunological abnormalities. The sensitivity was 100% for sera tested between days 3 and 40 after the onset of symptoms of rubella virus infection. Rubella virusspecific immunoglobulin M was detected at birth in sera from congenitally infected infants and persisted for several months. Positive Rubazyme-M responses were observed in some patients in the absence of rubella diagnosis (one blood donor, three other infections, and two immunological abnormalities), providing a test specificity of 99.6%. None of 67 patients with rubella virus-specific immunoglobulin G antibody and high levels of rheumatoid factor were positive in the test.
Although immunization
programs
in North America and
IgM, performed during 1982 by testing a large number of sera from the general population, rubella virus-infected persons, congenitally infected children, and individuals with other infections or immunological abnormalities. MATERIALS AND METHODS EIA procedure. The EIA for IgM antibody to rubella virus, Rubazyme-M (Abbott Laboratories), was supplied as a kit by the manufacturer and is a capture antigen system based upon the procedures of Voller and Bidwell (35) and Gravell et al. (13). A plastic bead coated with rubella virus was incubated with a diluted test specimen. The bead was washed and then incubated with goat anti-human IgM conjugated with horseradish peroxidase. The beads were again washed and incubated with the enzyme substrate o-phenylenediamine * 2 Hcl to develop a color, which was read in a quantum spectrophotometer (Abbott). A control specimen was adjusted to a titer at the limit of sensitivity in the rubella IgM SDG procedure (Centers for Disease Control method) by using HAI. The ratio of absorbance of the test specimen to that of the control yielded an index, which was a quantitative result. Specimens with an index of greater than 1.090 were considered to be positive. Those with an index of less than 0.910 were considered to be negative, and an index between the two values was considered equivocal. Specimens. A total of 1,200 sera from normal blood donors with no indication of recent rubella virus infection were assayed. Also analyzed were 116 sera from 51 patients with recent rubella virus infection, determined by clinical symptoms and a fourfold increase between paired sera in a rubella HAI test, by the presence of rubella virus-specific IgM in a SDG fraction, or both. Serially collected sera from two congenitally infected infants (proven by symptoms and isolation of rubella virus in early voided urine) were also analyzed. Sera from patients with other infections were collected within 2 months of onset of symptoms and included 12 with cytomegalovirus (CMV), 17 with Epstein-Barr virus (EBV), 24 with hepatitis A virus (HAV), 3 with herpes simplex virus (HSV), 13 with mumps, 10 with measles, 6
western Europe have reduced the incidence of rubella and congenital rubella syndrome (CRS), 10 to 20% of these
populations remain susceptible, and diagnostic laboratories continue to be called upon to diagnose rubella virus infection (21). Infections may be diagnosed by performing a combination of serological tests (32), including neutralization, hemagglutination inhibition (HAI), complement fixation, fluorescent antibody, passive hemagglutination, latex agglutination, radial hemolysis, mixed hemadsorption, radioimmunoassay, enzyme immunoassay (EIA), or time-resolved fluoroimmunoassay, and paired sera are generally required. The presence of rubella virus-specific immunoglobulin M (IgM) in a specimen has been used for a definitive diagnosis of recent infection. Because traditional methods for measurement of rubella virus-specific IgM (sucrose density gradient [SDG], gel filtration, mercaptoethanol treatment, staphylococcal protein A adsorption) are time consuming, costly, or not totally specific, efforts have been made to design solid-phase assays which separate and measure rubella virus-specific IgM in the same test, maintaining sensitivity and specificity. The earliest of these assays used infected cells as a solid phase and immunofluorescent reagents to detect IgM but were plagued with nonspecific reactions due to rheumatoid factor (RF) and Fc receptors on the surface of virus-infected cells (16). Assays that use rubella virus antigen on a solid phase (capture antigen) have been previously reported (17, 24, 33, 36), using antibodies labeled with radioisotopes or enzymes. Assays that use antibodies to IgM on the solid phase (capture antibody) have been previously described, employing rubella virus antigen which is followed by an indicator system, such as rubella antiserum and erythrocytes (solid-phase immunosorbent technique) (10, 18, 30) or enzyme or radioactive probes (4, 11, 15, 27, 31). This study describes an evaluation for sensitivity and specificity of a capture antigen EIA for rubella virus-specific *
Corresponding author. 400
EVALUATION OF EIA FOR RUBELLA VIRUS-SPECIFIC IgM
VOL. 20, 1984
with mycoplasma infections, 17 with chronic non-A, non-B hepatitis, and 2 with varicella-zoster virus. Single sera from patients with immunological abnormalities included 29 with antinuclear antibodies (ANA), 7 with elevated immunoglobulins, 10 with elevated IgM, 13 with multiple myeloma (MM), and 67 with elevated RF. All bloods were drawn without anticoagulant, and sera were separated and then stored at -20°C until testing. Sera were fractionated on SDG as previously described (8). Other serological methods. Rubella HAI antibodies were determined by the kaolin adsorption method, employing pigeon erythrocytes (8). Complement-fixing antibodies for CMV, HSV, and varicella-zoster virus were measured by a standard technique, using commercially available antigens (29), as were the HAI tests for measles and mumps (3). Patients with HAV infections possessed HAV-specific IgM by radioimmunoassay, and those with non-A, non-B chronic hepatitis were negative for hepatitis B virus markers and HAV-specific IgM and lacked serological evidence of EBV or CMV infections. EBV-infected patients showed serological evidence by viral capsid and early-antibody assays (2), and mycoplasma infections were determined by the presence of high levels of cold agglutinins. ANA levels were measured by a fluorescence technique (7). MM and total serum IgM were measured by nephelorhetry, using a Technicon automated immunoprecipitation technique (5). RF was determined by a latex agglutination kit (Behring Diagnostics). Isolation of rubella virus. Urines and throat swabs were inoculated onto grivet monkey kidney cells, challenged 10 days later with echovirus 11, and then read 3 days postchallenge (28). RESULTS Normal blood donors. Figure 1 summarizes the distribution of Rubazyme-M indexes for the blood donors. Of the 1,200 tested, 1,192 had negative indexes, 2 had indexes in the equivocal range, and 6 had positive responses. Five of six with positive indexes and one of two in the equivocal range showed rubella virus-specific IgM in the SDG test. Patients with rubella virus infection. Sera from 51 patients who had clinical symptoms compatible with rubella and demonstrated a fourfold or greater antibody titer increase in paired sera by HAI or the presence of rubella virus-specific IgM in the SDG test were assayed by Rubazyme-M. EIA test sensitivity was assessed in terms of the number of positive specimens as a function of the number of days from onset of rubella symptoms (Table 1). Twenty-four sera (100%) drawn
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FIG. 1. Distribution of Rubazyme-M indexes for 1,200 blood donors. Rubazyme-M indexes of >1.090 were considered positive, and indexes of
