Radioimmunoprecipitation and immunoblot studies of antibodies to rubella virus in patients with chronic liver disease

Summary Patients with autoimmune chronic active hepatitis (AICAH) and some other chronic liver disorders often have very high titres of rubella HI antibodies. In the present study sera from 46 patients with chronic liver disease and controls were examined for rubella antibodies using radioimmunoprecipitation assay (RIPA) and Western blot. RIPA appeared to be more suitable than Western blot for the study of the individual antibody specificities provided that proteins (possibly actin) interfering with the resolution of the E2 glycoprotein band are identified. It was shown that patients with high rubella HI titres reacted strongly against the E1 glycoprotein and in general also against the core protein (C). Reactivity to the E2 glycoprotein was detected with all sera from patients with chronic liver disease but varied more in strength. Three patients with post-acute rubella showed very faint E2 reactivity, but strong E1 and C reactivities. Patients with primary biliary cirrhosis had normal HI titres and showed no increase in reactivity in RIPA. The present findings show that patients with chronic liver disease and high rubella HI antibody titres exhibit an enhanced specific antibody response to rubella virus structural proteins.     

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.     

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.     

Serological investigations on Indian kala-azar.

Detailed serological investigations were carried out in forty-nine active kala-azar (KA) cases in North Bihar, India. Various classes of immunoglobulin (IgG, IgA, and IgM) and third component of complement (C3) levels were determined in these sera and results were compared with those obtained in normal controls. Antibody titres were determined by the indirect haemagglutination (IHA) method using soluble Leishmania antigen. Immunoglobulin G and M class-specific antibody titres were also determined separately by the enzyme-linked immunosorbent assay (ELISA) method. Polyclonal hypergammaglobulinaemia with marked increase in serum IgG (and to a lesser extent in IgM) level was a rather common feature in the majority of these sera. Much of this immunoglobulin increase, however, appeared to be non-specific in nature and no absolute correlation could be noted between serum IgG or IgM levels and corresponding IgG or IgM antibody titres. Significant decrease in serum C3 level was observed in KA and this decrease was found to be independent of immunoglobulin levels or specific antibody titres. A fairly good correlation between aldehyde test and serum IgG level was evident from this study. Aldehyde-positive KA sera usually gave higher antibody titres than aldehyde-negative ones. Anti-leishmanial antibodies belonged mostly to IgG class although some IgM antibodies were also demonstrable. The latter class of antibodies probably appeared early in KA infection although their serological specificity was poorer to IgG antibodies. Out of forty-nine KA sera examined in this study thirty-six (73.5%) gave positive IHA titres while forty-six (93.9%) were positive by IgG-ELISA which appeared to be a highly specific and sensitive serodiagnostic method particularly for the early detection of KA cases.     

Detailed immunologic analysis of the structural polypeptides of rubella virus using monoclonal antibodies

A panel of murine monoclonal antibodies prepared against rubella virus is described. Fourteen of these monoclonal antibodies react with the E1 glycoprotein of rubella virus and define a total of six spacially separate epitopes in competitive inhibition assays. Antibodies binding to epitopes E1(a), E1(b), E1(c), or E1(e) inhibit the hemagglutinin function of the virus, while antibodies binding to epitopes E1(d) or E1(f) do not. Monoclonal antibodies binding to epitopes E1(c) or E1(d) prevent virus infectivity and identify antigen in distinct intracytoplasmic vacuoles of rubella virus-infected Vero cells by indirect immunofluorescence. Monoclonal antibody to epitope E1(f) localizes antigen primarily to the plasma membrane of infected cells, while antibodies binding to epitopes E1(a), E1(b), or E1(e) localize antigen throughout the infected cell's cytoplasm. A single monoclonal antibody is described which only reacts with the mature form of the virion E2 glycoprotein after rubella virus is treated with a disulfide-bond reducing agent. This antibody immunoprecipitates a 43,000 MW precursor to the E2 glycoprotein from lysates of infected cells and localizes its antigen throughout the cytoplasm of infected cells. The five remaining monoclonal antibodies react with the rubella virus C polypeptide. They define four topographically separate epitopes on the C polypeptide, C(a), C(b), C(c), and C(d), each of which is diffusely distributed throughout the cytoplasm of rubella virus-infected cells.     

Contribution to laboratory diagnosis of mumps and parainfluenza

Specific IgM and IgG antibodies to mumps virus (MV) were detected in sera of mumps-patients by ELISA in agreement with the results obtained by indirect immunofluorescence (IF). Of given sera 37.5% contained IgM reacting in indirect ELISA also with the antigens of parainfluenza virus (PiV) T3. In all patients with respiratory illness over 2 years of age, the significant increase of antibodies to PiV in haemagglutination inhibition (HI) test was in good correlation with serum IgM and IgG antibody levels to PiV T3 determined by ELISA; but, in addition, 30.7% of these sera cross-reacted with MV antigens. The cross-reactions were eliminated by using MV-nucleocapsid antigen in indirect ELISA, or in direct ELISA using the peroxidase-labelled whole virion antigen. In some children under two years of age a discrepancy was observed between the significant increase of serum antibodies in HI and the inability to detect specific IgM antibodies by means of ELISA in their sera. The low-avidity antibodies appearing after primary PiV infection were probably washed off during the ELISA procedure.     

Anti M-protein antibody response to type A or B natural influenza detected by solid phase enzyme linked immunosorbent assay and by complement fixation

Anti M-protein antibody response has been looked for in sera from individuals with serological evidence of A or B influenza infection using pure M-protein (M) in complement fixation tests (CF), in IgG and in IgA specific enzyme linked immunosorbent assays (ELISA). Mp ELISA (IgG specific) antibodies are not restricted to people with history of recent respiratory infection. Individuals under 15 years are less prone than those older to display M ELISA activity. Most M ELISA positive individuals are also nucleoprotein (NP) positive. There are more M than NP ELISA positives in the influenza A series whereas the reverse is observed in the influenza B series. Most of the M ELISA positives are also S CF (standard soluble antigen CF) positive indicating that M ELISA IgGs are related to recent infections. Some sera exhibit M ELISA activity with no other evidence of influenza experience than V CF (viral antigen CF) or HI (haemagglutination inhibition), suggesting that some recent influenza infections are better traced with M ELISA than with S CF. Amongst chronic bronchitis patients with V CF or HI antibodies to A2 influenza virus but no type A S CF activity, the proportion of M ELISA positives averages 40 per cent. This fact as well as two other features of that group i.e. cases with long lasting S CF activity and occasional virus isolation several months after the initial acute infection, suggest that influenza virus might cause prolonged infection in some patients with chronic bronchitis.     

Levels of immunoglobulins and antibodies to haemaglutinin and neuraminidase of influenza virus in nasal secretions after natural infection.

Nasal washings (NW) from 16 influenza patients in the course of an epidemic in November and December, 1974 were examined for the presence of influenza virus, immunoglobulins (Ig) and titres of haemagglutination inhibiting (HI) and neuraminidase inhibiting (NI) antibodies. Influenza virus identical with A/Port Chalmers/1/73 (H3N2), increased levels of IgA and occasionally IgG, and specific antibodies were detected in the NW. The dynamics of HI and NI antibody formation did not differ substantially, but there were individual differences in titres and persistence of antibodies. Convalescent sera always contained increased levels of HI and NI antibodies. In some cases, the titres of antibody to viral ribonucleoprotein did not increase.     

Radioimmunoassay of measles virus antibodies in SSPE

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.     

Rubella serology by solid-phase radioimmunoassay: its potential for screening programmes

Sera from 269 adult females who had experienced naturally acquired or vaccine-induced infection by rubella virus, including immune persons challenged intranasally with rubella vaccine (RA27/3) as well as sera from 100 patients attending antenatal clinics, were tested for rubella antibodies by the conventional haemagglutination inhibition tests (HAI), as well as a newly developed solid-phase radioimmunoassay (RIA) for rubella immunoglobulin G (IgG) antibodies. Following both naturally acquired and vaccine-induced infection, titres by RIA were approximately ten-fold higher than by HAI. The RIA test was particularly useful in assessing the true immune status of those with apparently low levels of HAI antibody and has the added advantage that pre-treatment of sera to remove inhibitors of haemagglutination and red cell agglutinins is unnecessary. The RIA test has potential for the large-scale screening programmes which need to be carried out if the Department of Health and Social Security recommendation, that women attending antenatal and family planning clinics be screened for rubella antibodies, is to be effectively met.     

IgM antibodies in sera from patients with chronic active hepatitis reacting with the measles virus matrix protein and nucleoprotein

The antigenic specificity of measles virus IgM antibodies in sera from patients with chronic active hepatitis not caused by hepatitis B virus has been examined. An immunosorbent column containing antihuman IgM covalently bound to Sepharose was used to pick up IgM from the sera. Radiolabelled measles virus antigens were then allowed to react with the IgM antibodies. The immune complexes were eluted and analysed by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis. Four sera from patients with hepatitis B surface antigen [HBsAg]-negative chronic active hepatitis with high measles virus haemagglutination inhibition [HI] and complement fixation [CF] antibody titres and positive enzyme-linked immunosorbent assay [ELISA] for measles-virus-specific IgM were examined. The results were compared with those obtained using sera from patients with an acute measles virus infection and from healthy controls. In both patient groups, IgM antibodies with specificity against the matrix protein represented the major portion of the measles virus IgM. IgM antibodies against the measles virus nucleoprotein and probably against host-cell-derived actin were also present. The patient sera contained only traces of IgM antibodies with specificity against the measles virus haemagglutinin or fusion protein. No specific IgM antibodies were found in sera from healthy controls.     

Nonspecific inhibitors of coronavirus OC43 haemagglutination in human sera

Antibodies against the human coronavirus OC43 in human sera were measured by haemagglutination inhibition (HI), complement fixation (CF), and radial diffusion-haemolysis in gel (HIG) techniques. The apparent HI titres in a fraction of sera with no antibodies detectable by the two other methods were found to be reduced considerably after treating the sera with phospholipase C (PLC). The PLC treatment also reduced the apparent HI titres in some sera containing variable amounts of CF and/or HIG antibodies, but did not affect antibody determinations by the two latter methods.These results suggest that false positive results can be obtained in the OC43 HI test unless the sera are treated with phospholipase C before the assay.     

Humoral immunity in congenital rubella

1. Rubella complement-fixing (CF) and neutralizing antibodies are reported for eighty-one patients with congenital rubella, six adults with acute rubella, and a control series of fifty-four children. In some of the congenital rubella patients virus isolation and serum immunoglobulin levels are recorded, together with maternal antibody and immunoglobulin levels.

2. In children infected with rubella in utero (with neutralizing antibody), the incidence and titres of CF antibody are significantly higher in those with defects than those without defects.

3. Whereas neutralizing antibody persists in congenital rubella and is probably a combination of IgG produced by both mother and child, and IgM produced by the child alone, CF antibody presents a more complex picture. It is nearly always detectable at some time in patients with congenital rubella, but may show a rising, falling or constant titre. The titre correlates significantly with the IgG concentration, and this and other data suggest that it may be predominantly IgG produced by both mother and child.

4. There is no obvious relationship between virus elimination and either CF or neutralizing antibodies or levels of IgG or IgM.

     

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.     

Diagnosis of recent primary rubella virus infections: significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference μ-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5-100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis.     

The use of homologous virus in the haemagglutination-inhibition assay after vaccination with Newcastle disease virus strain La Sota or Clone30 leads to an over estimation of protective serum antibody titres.

We evaluated the influence of the use of the Newcastle disease virus (NDV)-strains Ulster and La Sota in the haemagglutination inhibition (HI) assay for the measurement of antibody titres after NDV vaccination. The use of the homologous La Sota antigen in the HI assay after Clone30 and La Sota vaccination of SPF-chickens, resulted in significantly higher titres than the use of the heterologous Ulster virus. The mean difference was 1.4 on a log 2 scale (2.6-fold). A significant difference was also found in virus neutralization (VN) assays. The virus strain in the HI or VN test did not influence the resulting titres after Ulster vaccination. When HI antibody titres after vaccination were related to VN titres measured with virulent Herts NDV or to survival after virulent challenge, it was found that the use of La Sota antigen in the HI assay after vaccination with Clone30 or La Sota resulted in an overestimation of protective serum antibody titres. Also in commercially derived White Leghorns vaccinated with Clone30, significantly higher HI titres were obtained when La Sota antigen was used in the HI test. Our data have direct implications for potency testing of inactivated vaccines as the European Pharmacopeia does not prescribe the antigen to be used in the HI test.     

Analysis of antibody assay methods and classes of viral antibodies in serodiagnosis of cytomegalovirus infection.

Forty-nine serum pairs with antibody to cytomegalovirus (CMV) were evaluated for rises in antibody titer (greater than or equal to fourfold) by indirect hemagglutination (IHA) and complement fixation (CF), using a freeze-thaw antigen (FT) and a glycine extract antigen (GE). In this sample CF-FT detected more rises in antibody titer than did CF-GE. IHA detected the least number. The apparent reason for stationary antibody titers with CF-GE and IHA was the presence of high antibody titers in the first serum specimen. Separation of immunoglobulin classes of 20 serum pairs by sucrose gradient centrifugation indicated that these antibodies with IHA were of the immunoglobulin M (IgM) class and those with CF-GE were of the IgG class. By separation of immunoglobulin classes, rises in IgG CMV antibody titers were seen with IHA, rises not observed in the whole serum because of high IgM antibody titers in the first serum specimen. Absence of rises in antibody titers with CF-FT was due in part to too early sampling of the second serum specimen (less than 21 days) and in part to an apparent inability of some individuals to respond with antibody reactive with FT antigen. CF-GE and CF-FT antibodies of the IgM class were detected in some sera, usually in specimens collected more than 10 days after the onset of symptoms. Although reactive with CMV antigen, the specificity of these IgM antibodies in relation to rheumatoid factor requires clarification.     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies.

The interference of IgM-class rheumatoid factor (RF) in the solid-phase radioimmunoassay (RIA) of rubella virus IgM antibodies was studied. Acute rubella infections did not significantly activate RF. False-positive rubella antibody results were obtained, however, when patients with raised RF levels were tested. If a low rubella IgG antibody titre was present, a high level of RF was required to cause a false-positive IgM result; conversely, in sera with high IgG titres, only a low level of RF was required for interference. Although the false-positive IgM titres obtained were generally low, thet did show a positive correlation to both RF levels and rubella IgG titres. False-positive results were successfully avoided by removing the RF by absorption with heat-aggregated human gamma globulin. The absorption procedure did not affect true rubella IgM antibody titres.     

An enzyme linked immunoassay for estimation of antibodies to Newcastle disease virus strains

The sensitivity of enzyme linked immunoassay (ELISA) was compared to that of haemagglutination inhibition (HI) test in detection of humoral antibodies in birds vaccinated with Newcastle disease virus (NDV). The highest antibody titre at day 21 post-vaccination as detected by ELISA in the sera of vaccinated birds was 2560 by all strains in contrast to the HI titres of 640 to 1280. The antibody titre was higher in all pre- and post-vaccination sera as determined by ELISA than detected by HI test.