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.     

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.     

Sensitive neutralization test for virus antibody

Summary A sensitive mumps virus plaque neutralization test has been developed based on the potentiation of virus-antibody complexes by heterologous anti-immunoglobulins (AIG). The enhanced neutralization test was approximately 100 times more sensitive than the conventional neutralization test or the hemagglutination-inhibition test. Using AIG against human immunoglobulin G (IgG) or human IgM permitted determination of the relative titers of the two classes of mumps antibody. The test does not require special equipment or expertise and can be readily introduced in virological laboratories.With 1 Figure     

Simple hemagglutination inhibition test for the diagnosis of toxoplasmosis.

A simple hemagglutination inhibition (HI) test for the serological diagnosis of toxoplasmosis has been developed and evaluated. A total of 84 human and 120 mouse serum samples were tested by the newly developed HI test and compared with an immunoglobulin G-indirect fluorescent antibody test. Statistical analysis of serum titers obtained by using the HI test and the immunoglobulin G-indirect fluorescent antibody test showed a correlation coefficient of 0.89. The diagnostic efficacy of HI when compared with the immunoglobulin G-indirect fluorescent antibody diagnostic test results was 96.43% for human sera and 100% for mouse sera. The unique hemagglutination antigen, derived from Toxoplasma gondii (Rh strain) exotoxin, spontaneously binds with mouse or rat erythrocytes, causing the hemagglutination reaction. In this study, 2, 4, or 8 hemagglutinating units of T. gondii exotoxin was used with Swiss/Webster mouse erythrocytes as an indicator for the HI assay. The results indicate that 8 hemagglutinating units is optimal because this concentration has the least unexplained variability. T. gondii exotoxin was stable for at least 18 months at -70 degrees C. The Toxoplasma HI test we report in this paper is shown to be a fast, easy, highly specific, and sensitive test for the diagnosis of toxoplasmosis.     

Antibody response after application of a new live attenuated mumps vaccine (Pariorix®) measured by enzyme-linked immunosorbent assay (ELISA)

Mumps virus specific antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA), hemolysis-in-gel (HIG) test, and hemagglutination-inhibition (HI) test in 17 adult volunteers before and after vaccination with a new live mumps vaccine, Pariorix®. The results of the present study indicate that the strain Urabe-Am 9 of the mumps virus (Pariorix) induces antibodies in 100% of seronegative adults when measured by ELISA and in 70.6% and 64.7% in HIG and HI, respectively. ELISA is therefore a very sensitive and reliable method for the demonstration of sero-conversion after vaccination. The vaccine was well tolerated and clinically acceptable in the group of adult volunteers.     

Hemadsorption immunosorbent technique for determination of mumps immunoglobulin M antibody

We used hemadsorption immunosorbent technique (HIT) to detect mumps immunoglobulin M (IgM) antibody. IgM from human sera was adsorbed into anti-human IgM-coated wells in plates, and mumps-specific IgM was detected by adding mumps virus hemagglutinin and guinea pig erythrocytes consecutively. Specific IgM-positive sera showed hemadsorption, whereas negative sera showed hemagglutination. All 81 patients with current mumps infections tested showed mumps-specific IgM antibody, with titers ranging from 160 to 327,680. In most cases IgM antibody was already present on day 1 or 2 after the onset of illness. IgM antibody persisted for 6 to 10 weeks. Of 57 patients with acute respiratory illnesses caused by parainfluenza virus, 5 showed cross-reactions in the hemadsorption immunosorbent technique assay for mumps IgM. The hemadsorption immunosorbent technique assay is specific for the IgM class of antibody and avoids false-positive results due to rheumatoid factor. This test is an efficient and sensitive method for rapid and early diagnosis of mumps infections.     

The effect of levamisole on cellular immunity in multiple sclerosis.

The lymphocyte stimulation test has been standardized in a normal human population using four virus cell-associated antigens (VCAA): human embryonic lung cells infected with the LEC and Norrby strains of measles virus, mumps virus, and vaccinia virus. Following 1 week of treatment with the immunopotentiating drug levamisole, a group of multiple sclerosis (MS) patients was found to have increased lymphocyte stimulation responses toward VCAA and increased delayed hypersensitivity responses towards a battery of skin test antigens. No change in the percentage of short- or long-incubation E rosettes occurred. Measles haemagglutinin inhibition (HI) antibody titres measured before and after the entire course of levamisole therapy (12 weeks) did not change. The neurological status of five out of seven MS patients deteriorated while they were taking levamisole.     

The incidence of epidemic mumps and the rate of antibody detection in different age groups of Moscow population]

Sera from Moscow residents of different ages were studied by the HI test for antibody to mumps virus. In newborn babies antibody was found in 94%. The number of subject possessing antibody decreased with age and was minimal among children of 3-4 years (40%) but then increased and reached 94% among adults (20 years or more).     

Antibody responses to mumps virus proteins in natural mumps infection and after vaccination with live and inactivated mumps virus vaccines

Paired sera from 20 patients with acute mumps infection, 16 from persons vaccinated with live attenuated mumps virus vaccine, and 12 from persons vaccinated with formalin-inactivated virus vaccine were studied for mumps antibodies by single radial hemolysis (SRH), hemagglutination inhibition (HI), and by enzyme immunoassays (EIA) specific for whole virus, envelope glycoprotein, and nucleocapsid antibodies. Mumps patients had diagnostic rises in serum mumps antibodies in 90–100% of the cases depending on the method of assay. Vaccination resulted in seroconversion in 75–88% (live vaccine) and in 92% (inactivated vaccine) of the cases as detected by SRH or EIAs, whereas HI detected seroconversion only in 38% and 58% of the cases, respectively. Immunoprecipitation analyses revealed that all sera from mumps patients and nearly all postvaccination sera had antibodies against the main structural proteins of mumps virus. By immunoblotting, antibodies against denatured hemagglutinin-neuraminidase (HN) and fusion protein (F) were detected in 15–25 % of mumps patients and persons vaccinated with live vaccine, whereas most postvaccination sera from those vaccinated with inactivated vaccine had HN (92%) and F (83%) protein antibodies, suggesting that antibodies against the denatured form of proteins are formed.     

Enzyme-linked immunosorbent assay for mumps and parainfluenza type 1 immunoglobulin G and immunoglobulin M antibodies

A solid-phase enzyme-linked immunosorbent assay (ELISA) for detection of mumps and parainfluenza type 1 antibodies (immunoglobulin G [IgG] and IgM classes) is described and compared with the conventional complement fixation (CF) test. A highly positive correlation was found between mumps IgG ELISA and the mumps CF test, whereas parainfluenza type 1 IgG ELISA had only a moderate positive correlation with the respective CF test. Mumps IgM antibodies could be demonstrated in all patients with serologically verified and clinically typical (parotitis, meningitis, or orchitis) mumps virus infection, but not in patients with rises in parainfluenza CF titers. Mumps IgM was already present in the acute-phase sera if they were not taken during the first 2 days after onset of disease. Mumps IgM was also found in some paired sera that were taken too late to demonstrate any significant increase in the antibody titers by CF. Therefore, mumps IgM ELISA provides an improvement over the conventional laboratory diagnosis of mumps infection, since the measurement of specific IgM antibodies in a single serum by ELISA is diagnostic, rather than the identification of a fourfold or greater rise in CF antibody titer. An unexpected finding was that parainfluenza type 1 IgM antibodies could not be demonstrated by ELISA in paired sera with rises in parainfluenza CF titers, suggesting a different antibody response from that occurring in mumps infection.     

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.     

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.     

Comparison of nonspecific reactivity in indirect and reverse immunoassays for measles and mumps immunoglobulin M antibodies.

Serum specimens collected from patients convalescing from acute measles or mumps infections, other viral infections, or rheumatoid arthritis and from blood donors were tested in indirect and reverse assays for measles and mumps immunoglobulin M (IgM) antibodies. All the samples from patients convalescing from acute mumps and measles infections gave positive IgM results in both tests. However, 6% of sera from patients recovering from other viral infections, 68.4% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive results by the indirect measles IgM enzyme immunoassay (EIA). By the indirect mumps IgM EIA, 9% of sera from other viral infections, 70.1% of sera from patients with rheumatoid arthritis, and 5.6% of sera from normal blood donors gave false-positive reactions. The reverse test system for measles IgM gave false-positive results in 1.5% of sera from the group with other viral infections, and the reverse mumps EIA gave false-positive results in 0.9% of the patients. Other sera groups did not react in either measles or mumps reverse IgM assays. The results indicated that although nonspecific reactions are frequent in indirect IgM tests for viral antibodies, such reactions are rarely encountered when reverse IgM EIA tests are employed.     

Serological diagnosis of parainfluenza virus infections: verification of the sensitivity and specificity of the haemagglutination-inhibition (HI), complement-fixation (CF), immunofluorescence (IFA) tests and enzyme immunoassay (ELISA).

Using four serological tests paired sera were examined of 117 patients with acute respiratory diseases, in whom parainfluenza viruses (PIV) infection was demonstrated by virus isolation, and of 41 patients with typical clinical mumps symptoms. Comparative analysis showed the high sensitivity of IFA and ELISA. A significant rise of antibodies in convalescent sera with homologous antigen of PIV was found in nearly 100 percent of cases. Only the sera of youngest children with high titres of persisting maternal antibodies remained without seroconversion. Cross heterologous antibody responses could be found by means of ELISA in 45% and by IFA in 10%, of patients who in the past experienced infection with one or more PIV or mumps virus--apart from homologous antibody reaction. HI and CF test proved to be less sensitive for detection of postinfections antibodies, especially in primoinfections with PIV types 1 and 2.     

The enzyme-linked immunosorbent assay (ELISA) for determination of IgG and IgM antibodies after infection with mumps virus

Under routine laboratory conditions ELISA was tested for suitability for serological demonstration of specific antibodies of the immunoglobulin classes G and M against mumps virus. Sera from patients with known clinical and virological data were used. The results of ELISA were compared with those of CFT. 45 paired sera were tested in ELISA IgG, 87 first sera in ELISA IgM. Both tests were highly sensitive, antibodies were detected earlier and with higher titers than with the CFT. The ELISA IgM is particularly suitable for early diagnosis of mumps infection with the first serum. In addition 23 paired sera from patients with acute parainfluenza virus infection were examined for cross-reacting antibodies. Low anti-mumps IgG antibody titers were found in some sera. These findings reduced the mumps specificity of the IgG test. In five serum samples from one patient — obtained before, during, and after an infection with mumps — the course of IgG and IgM antibodies could be demonstrated. Advantages and limitations of ELISA IgG and IgM are summarized.Behringwerke AG, Marburg, West Germany     

Characterisation of mumps virus genotype C among patients with mumps in India

Measles, mumps and rubella (MMR) vaccine failure had been reported globally and here, we report that it occurs in India now. MMR vaccinated people have developed acute mumps accompanied by anti-mumps immunoglobulin M. Genotypic characterisation revealed that the circulating mumps strain was genotype C, which is distinct from the vaccine strain of genotype N (L-Zagreb). This is the first report in India to suggest that genotype C is responsible for the present mumps infection. Thus, the present MMR vaccine must be revamped and optimised for its efficacy to prevent any future mumps epidemics.     

Cell-mediated immune response of chickens to Newcastle disease vaccines

The leukocyte migration inhibition (LMI) test was used to demonstrate cell-mediated immunity (CMI) and its relationship to immunoglobulin production, as assessed by the haemagglutination inhibition (HI) test, in chickens vaccinated with various Newcastle disease (ND) vaccines. Highest CMI levels were demonstrated in 3 or 7-week-old birds which had been vaccinated with live vaccine followed by an oil adjuvant vaccine 6 weeks later. There was no close correlation between LMI values and HI titres, LMI appearing earlier after primary vaccination and failing to give the strong secondary response seen in HI titres after challenge with live virus. There was a secondary CMI response after revaccination with oil adjuvant vaccine but this was not as strong as the humoral response.     

Assay of humoral immunity to mumps virus.

One hundred randomly chosen sera from blood donors from North London were assayed for antibodies to mumps virus by plaque reduction and microtitre neutralisation assay, haemagglutination inhibition and in-house ELISA. The assay reproducibility was determined, and there was reasonable agreement between antibody levels measured by the two neutralising methods. Neutralising antibody levels measured by either method were low but the strain used in the assay had a large effect on the antibody titres observed. Titres measured by neutralisation assay, HI assay and ELISA did not correlate well. Assessment of immunity to mumps virus remains problematical.