Evaluation of a radioimmunoassay for IgM-class antibodies against cytomegalovirus

A solid-phase radioimmunoassay (RIA-IgM) test for cytomegalovirus (CMV)-specific IgM-class antibodies is described and its potential diagnostic usefulness in adult patients is evaluated. IgM-class antibodies were readily detected by RIA in early convalescent sera from patients with serologically confirmed primary CMV infection, as well as in sera from patients with CMV mononucleosis. Serum specimens obtained several months after primary infection gave either negative results of low antibody titres, indicating that the IgM response is transient in these patients. CMV-specific IgM was also detected in 7 of 10 patients with symptoms and supportive serological evidence suggestive of recent active CMV infection. Conversely, 12 patients who were thought not to have experienced recent primary CMV infection, but whose sera gave high complement-fixing (CF) antibody titres, and 14 of 15 renal transplant recipients with reactivated CMV gave negative results in the RIA-IgM test. The results indicate that detectable IgM antibody production is a constant feature in patients with primary infection but not in patients who experience mild secondary attacks. The RIA-IgM test is highly virus-specific and can reliably be performed with unfractionated serum, provided that false reactivity due to rheumatoid factor (RF) is excluded. Thus RIA is a simple and specific technique which could be usefully applied as a diagnostic tool in a clinical situation and in research.     

An improved radioimmunoassay method for the detection of IgG antibodies against cytomegalovirus

The non-specific binding seen with human sera in a radioimmunoassay for the detection of IgG antibodies specific for CMV can be reduced greatly by using a murine monoclonal antibody as a radiolabelled detecting antibody. Such non-specific binding formerly obtained with a polyclonal detecting antibody was due to the binding of the polyclonal reagent to factors on the solid phase other than IgG molecules.     

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.     

Detection and differentiation of cytomegalovirus antibodies by radioimmunoassay

A sensitive radioimmunological method was adapted for the assay of anticytomegalovirus IgG and IgM antibodies. Binding of immunoglobulins G with cytomegalovirus (CMV) antigens was greatly reduced when an antigen prepared by sonication of the infected cells in glycine buffer was used in place of the antigen obtained by freezing and thawing of the cells. The application of labelled serum against the Fc fragment of IgG made it possible to identify selectively the antibodies bound with the virus antigen by antigenic determinants.     

Development of human monoclonal antibodies against human cytomegalovirus.

Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.     

Evaluation of three commercial enzyme immunoassays for toxoplasma and cytomegalovirus antibodies.

Three commercially available enzyme immunoassays (EIAs) were evaluated for specificity and sensitivity compared to immunofluorescent assays (IFAs) for detection of IgG and IgM antibodies to Toxoplasma gondii (Toxo) and Cytomegalovirus (CMV). A panel of 78 sera were tested, including specimens known to contain Toxo IgG or IgM, CMV IgM or IgM, antinuclear antibodies (ANA), antimitochondrial antibodies (AMA), and rheumatoid factor (RF). Linear correlation analysis comparing EIA and IFA results showed that the statistical relationship between the two assay methods was relatively weak (correlation coefficients ranging from 0.33 to 0.86). Qualitative correlation showed that the EIA methods agreed with IFA results in most cases. One manufacturer's products (Diamedix Corp.) gave consistently better sensitivity, specificity, and intra-assay reproducibility values than the other two systems (Clinical Sciences, Inc., and Whittaker Bioproducts). ANA, AMA, and RF did not appear to interfere with any EIAs.     

Direct radioimmunoassay for the detection of IgM antibodies against Mycoplasma pneumoniae

A direct solid-phase radioimmunoassay is described for detecting IgM-class antibodies against Mycoplasma pneumoniae. The assay achieves a prelminary separation of IgM from other serum proteins by immunoadsorption to anti-IgM-coated wells in microtitre plates. The IgM is then tested for antigen specificity by measuring its ability to bind radiolabelled M. pneunoiae. Positive results are confirmed by retesting sera after treatment with 2-metcaptoethanol. The assay is specific for IgM-class antibodies and specific for M. pneumoniae. It is not affected by competitive inhibition from IgG and avoids the use of density gradient centrifugation or gel filtration to separate IgM from other immunoglobulins. It is sensitive, reproducible, rapid, simple and requires very little serum.     

A solid-phase radioimmunoassay for anti-SNP antibodies.

A highly sensitive and discriminatory solid-phase radioimmunoassay has been developed to detect anti-SNP antibodies in sera. Polystyrene tubes are coated with SNP and incubated with the test sera. The fixed antibodies are detected by a double layer technique using rabbit anti-human γ-globulin antiserum followed by incubation with a ¹²⁵I-labelled sheep anti-rabbit γ-globulin antiserum. Results are expressed in ng of the ¹²⁵I reagent fixed by 30 ωl of serum. The mean binding of 47 normal human sera was 22 ng ± 12 ng: 22/50 SLE sera gave over 34 ng binding. The specificity of the assay was studied in 3 different types of experiment: inhibition of the binding of positive sera either by pre-incubation with NDNA, SNP or RNA, DNAse I digestion of the SNP coated tubes, and incubation of SNP coated tubes with sera of known reactivity. It was shown that the solid-phase assay detects mainly antibodies directed against NDNA; however antibodies directed against the DNA-protein complex or the protein alone can easily be detected. Our results obtained with the solid-phase assay correlated well with those of the Farr assay. However, this new assay presents major improvements: it is simple, highly reproducible, and avoids the need for labelled antigen. A single labelled antiglobulin reagent allows identification of the class or subclass of reactive antibodies in a given species. Quantitation is more precise, particularly for sera containing high amounts of antibodies.     

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.     

Comparison of solid phase test systems for demonstrating antibodies against hepatitis A virus (anti-Hav) of the IgM-class

Three methods were compared for determining anti-HAV of the IgM class. In the first method flat-bottomed microtiter plates coated consecutively with anti-HAV of the IgG class and HAAg were incubated with patient serum and, after washing, peroxidase conjugated anti-mu was added. After subsequent incubation with substrate the enzymatic reaction was stopped and the optical density was measured. In the second method the solid phase was coated first with antibodies to IgM and after incubation with patient serum and subsequent incubations with HAAg and 125I anti-HAV of the IgG class radioactivity was counted. These two methods were compared with reorienting sucrose gradient ultracentrifugation, an established method for demonstrating specific IgM antibodies. The persistence of IgM anti-HAV in 103 sera drawn at different times after onset of jaundice was evaluated. Sera drawn up to 30 days after onset of hepatitis A were IgM anti-HAV positive with both of the first two methods. Forty-one to 90 days after onset of illness IgM anti-HAV could be demonstrated with the first method in 47% of the patients, in 94% with the second method, and in 82% with gradient centrifugation. The second method was most sensitive and could be adjusted so that at a serum dilution of 1:10(4) anti-HAV IgM was detected only up to six months after infection. In contrast to the first method, nonspecific reactions caused by rheumatoid factor were not detected with the second method. During a one-year period about 15,000 sera of patients with clinical diagnoses of acute hepatitis were tested; the positive results correlated well with the clinical data, and there was no indication of nonspecific positive results.     

Enzyme-linked immunosorbent assay for measurement of antibodies against pneumococcal polysaccharide antigens: comparison with radioimmunoassay.

An enzyme-linked immunosorbent assay (ELISA) for measuring antibodies against each of 14 polysaccharides in contemporary pneumococcal vaccine is described, and the findings of tests of paired sera from vaccinated human subjects are compared with those obtained by radioimmunoassay. The findings were in very poor agreement, and this appears to be due to the lesser ability of the ELISA procedure to measure antibody of low avidity. The ELISA procedure described here is not considered to be a satisfactory substitute for radioimmunoassay for measuring antibody responses to pneumococcal vaccine.     

A new radioimmunoassay using a commercially available solid support for the detection of IgE antibodies against muscle relaxants.

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anesthesia. In this study, we developed an in vitro test with a commercially available solid phase for the detection of specific IgE directed to the tertiary or quaternary ammonium groups of neuromuscular-blocking drugs. The solid-phase complex was P-aminophenylphosphoryl-choline (PAPPC) immobilized on agarose, and an RIA was performed with an antihuman IgE labeled with 125I. The results, expressed as the percentage of 125I-labeled anti-IgE linked to the solid phase, were at 0.41 +/- 0.19 for 34 control sera from nonallergic healthy adults, with an upper limit estimated at 1%. The values obtained with the sera of 31 allergic patients ranged from 0.6% to 41% with a sensitivity of 97%. The specificity and the positive predictive value of the PAPPC RIA were 97% and 94%, respectively. These results were compared with results of other RIAs with morphine, trimethylamine, triethylamine immobilized on epoxy-activated Sepharose, and choline hydrochloride immobilized on Sepharose (quaternary ammonium Sepharose RIA) and with Phadebas RAST succinylcholine and Phadebas RAST alcuronium. The PAPPC RIA appears to be the most efficient test to screen sera for the presence of IgE antibodies directed to neuromuscular-blocking drugs. One major advantage is that this solid phase is commercially available and ready to use. This advantage will improve the accuracy in the comparison of the results with results from different laboratories.     

A solid-phase radioimmunoassay for quantitative measurement of class-specific antibodies against tick-borne encephalitis virus

Antigen-coated polystyrene spheres are used for a solid-phase RIA for IgM and IgG antibodies against tick-borne encephalitis virus (TBE). The use of highly purified anti-mu and anti-gamma antibodies permitted the construction of standard curves from which quantitative values for TBE-specific IgG and IgM could be obtained. An antibody-blocking test identifies non-specific results.     

Preparation of human monoclonal antibodies against a cytomegalovirus glycoprotein complex of 130 and 55 kDa.

Nine human monoclonal antibodies (MAbs) with neutralization activity against cytomegalovirus (CMV) were obtained by screening human MAbs using a CMV glycoprotein complex of 130 and 55 kDa (gp130/55). The gp130/55 antigen was purified by immunoaffinity chromatography and the purified antigen used to detect anti-gp130/55 MAbs in an enzyme-linked immunosorbent assay. Relatively few of the human anti-CMV MAbs were directed against gp130/55 but all showed high neutralization activities to a variety of clinical isolates with titers (ED50 values) ranging from 0.15 to 7.9 micrograms/ml. Six of the nine anti-gp130/55 MAbs required complement for virus neutralization. Such human MAbs may prove to be useful for passive immunotherapy against CMV infection.     

Evaluation of solid-phase immunofluorescence for quantitation of antibodies to herpes simplex virus and cytomegalovirus.

A recently developed semiautomated technique based on solid-phase immunofluorescence (FIAX) was compared with complement fixation for the determination of antibody levels to herpes simplex virus and cytomegalovorus in human serum samples. The results demonstrated that the FIAX method was in aggreement with the complement fixation technique for 97% of the serum samples tested. Reproducible titers were obtained from multiple FIAX determinations of representative sera within the same experimental run and between separate experimental runs. However, variability was rather high for patient sera with low (less than or equal to 1:5) antibody levels to cytomegalovirus. Hence, the results obtained by the FIAX technique were reproducible, and the FIAX system was as sensitive as complement fixation for the determination of antibody levels to herpes simplex virus and cytomegalovirus.     

Solid-phase radioimmunoassay for the detection of antibodies to collagen.

A solid-phase radioimmunoassay has been developed using polystyrene tubes for the detection of class-specific antibodies to collagen. The optimal conditions for the adsorption of collagen onto the tubes, followed by incubation with the antisera and finally the radiolabelled antibody to the class-specific antibody are described. Studies carried out on antisera raised in rats and rabbits using this assay confirm its sensitivity and applicability.     

Comparison of antibodies for rapid detection of cytomegalovirus.

Antibodies were compared for use in the spin-amplified shell vial method for rapid cytomegalovirus detection. Commercial antibodies by Du Pont Co., Whittaker M.A. Bioproducts, Bartels Immunodiagnostic, Virostat, and Serono Diagnostics were compared at incubation times of 16 to 48 h on 22 patient specimens. Only the Du Pont antibody showed 100% sensitivity and specificity and no nonspecific reactions.     

Surveillance of mice for antibodies to murine cytomegalovirus.

The sera of 256 mice from nine commercial sources were screened for antibodies to murine cytomegalovirus (MCMV) because a surveillance of this virus has not been reported in the literature for over a decade. Although no evidence of antibodies to MCMV were detected by complement fixation or nuclear anticomplement immunofluorescence, 54.7% of these sera did have antibodies that were detected by enzyme-linked immunosorbent assay. These data emphasize the need for proper containment of laboratory mice to prevent the potential outbreak of acute MCMV infection. Including MCMV antibody surveillance by enzyme-linked immunosorbent assay in routine health monitoring of mice and imparting these findings in an analysis of the role of MCMV on interpretation of experimental results is advised.     

Partially purified antibodies used in a solid-phase radioimmunoassay for detecting candidal antigenemia.

The development of a solid-phase radioimmunoassay procedure for the detection of Candida albicans antigens in serum of mice is described. Antibodies against C. albicans that were used in the radioimmunoassay procedure were partially purified from immune serum by a C. albicans antigen-coupled affinity column. Elution of anti-C. albicans antibodies from the column was by glucose and mannose; 4 mg of protein was recovered per ml, which contained 50% of the candidal agglutinin activity of immune serum. Also, 81% of the protein (partially purified antibody) recovered was adsorbed by whole C. albicans cells. Anti-C. albicans antibodies were either coupled to Sepharose 4B for use as the solid phase to bind candidal antigen in serum of infected animals, or radioiodinated (125I) for use as a tracer molecule to bind to the candidal antigen solid-phase complex. Although control experiments indicated that at least 100 ng of candidal antigen should be present in a serum specimen for a positive radioimmunoassay test, candidal antigenemia was detected in 70.4% of infected mice even in cases where blood cultures for C. albicans were negative. With further refinement and adaptability to human serum, the radioimmunoassay test may become a helpful tool for use in the diagnosis of systemic candidiasis.     

Monoclonal antibodies directed against human myoglobin: characterization and application in a bideterminant radioimmunoassay

Monoclonal hybridoma cell lines secreting antibodies directed against human myoglobin were selected. Two of these cell lines were grown in mouse ascitic fluid resulting in the production of large quantities of antibody. Antimyoglobin antibodies isolated from the ascitic fluids were employed in the development of the sensitive solid-phase, bideterminant radioimmunoassay for human myoglobin that uniquely recognizes two different epitopes on the same molecule.