Rapid and sensitive heterologous enzyme immunoassays for densonucleosis virus (Parvoviridae)

Summary There exists a serious lack of rapid and sensitive methods to identify densonucleosis viruses and to discriminate among them. Two different enzyme-linked immunosorbent assays (ELISA) were adapted for this purpose, which were both significantly faster and more sensitive than currently used ELISA procedures. This increase in sensitivity was due to an improvement in the conjugation procedure of peroxidase to antibody, the establishment of the optimum conditions for the various incubations, an optimisation of the substrate (H₂O₂) concentration, and the use of a new H-donor. The speed of the assay could be considerably shortened by the use of polyethylene glycol-6000 (i.e. the total time of the assay needed for maximum sensitivity of the indirect assay was only 2 hours). The assays using peroxidase conjugates were found considerably more sensitive than those using alkaline phosphatase, which is very probably due to a more efficient and better controlled conjugation procedure for peroxidase. The virus could be detected in the pg to ng range in a large excess of nonspecific antigens and titers for the antisera usually exceeded 10⁶. Small differences in the viruses could be detected. Several factors, which may influence the sensitivity and specificity of these densonucleosis virus assays, were further investigated.With 10 Figures     

Mumps virus-specific antibody titers from pre-vaccine era sera: comparison of the plaque reduction neutralization assay and enzyme immunoassays

Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. However, assays used to detect neutralizing antibodies, such as the plaque reduction neutralization (PRN) assay, are labor- and time-intensive and consequently are often supplanted by the more rapid and inexpensive enzyme immunoassay (EIA) technique. For virus infections for which international antibody standards exist and are bridged to clinical studies of protection (e.g., measles and rubella), the EIA has been successfully used to determine immune surrogate endpoints, yet no such international reference exists for mumps serology. Since both virus-neutralizing and nonneutralizing antibodies are measured in the EIA, in the absence of a mumps serological standard, the EIA may be prone to yielding false-positive results when utilized for assessing surrogate markers of protective immunity. Moreover, since mumps virus-specific antibody titers are generally low in comparison to antibody levels induced by other viruses and EIA procedures often employ relatively high serum dilution factors, the EIA may be prone to yielding false-negative results. To examine these issues, a PRN assay and two commercially available EIA kits were used to evaluate wild-type mumps virus serological responses in human serum samples from the pre-mumps vaccine era. Our results indicate that the PRN assay is a more sensitive and specific method of measuring serological responses to wild-type mumps virus.     

Experiments on heterologous and homologous interference in LCM-infected cultures of murine lymph node cells

Summary Heterologous interference with the viruses of EEE and VS was not demonstrablein vitro in LCM-infected lymph node cells from tolerant or non-tolerant mice in spite of the fact that slight interference with these viruses occurs in the LCM-infected mouse.However, there was marked homologous interference in such cultures with strain WCC of LCM virus, which can be distinguished from the infecting strain W by its higher pathogenicity for newborn mice.It has not been possible to detect interferon in cultures in which homologous interference occurs and to identify such a substance as the cause of the fluctuations of the viral growth curve characteristic for lymph node cells from non-tolerant mice.     

Rescue of vesicular stomatitis virus from homologous and heterologous interferon-induced resistance in human cell cultures by poxviruses.

In human cell cultures the ability of poxviruses to rescue vesicular stomatitis virus from human interferon-induced resistance was significantly more efficient than the ability to rescue it from simian interferon-induced resistance. The sensitivity of the poxvirus to interferon was not related to its ability to rescue vesicular stomatitis virus.     

Serological detection of HBeAg and anti-HBe using automated microparticle enzyme immunoassays.

Fully automated microparticle enzyme immunoassays (EIA) were developed for the detection of HBeAg (IMx HBe) and antibodies against HBeAg (IMx anti-HBe), respectively. Specimens from blood donors, diagnostic and hospital patients and individuals with a variety of infectious and immune diseases were tested both in house and at four clinical sites. The overall agreement between IMx HBe and Abbott HBe RIA/EIA was 99.7% (2985 of 2994) and between IMx anti-HBe and anti-HBe RIA/EIA was 95.8% (2330 of 2432). Almost all anti-HBe discordant specimens (94.1%, 96 of 102) were reactive by IMx anti-HBe but negative by anti-HBe RIA/EIA. off anti-HBe discordant specimens were also reactive for anti-HBc. The IMx anti-HBe assay was 2- to 4-fold more sensitive than the current RIA as determined by serial dilution of anti-HBe reactive specimens. The ability of these IMx assays to detect HBeAg and anti-HBe in 199 HBsAg reactive specimens was also evaluated. 43.7% (87 of 199) and 66.3% (132 of 199) specimens were reactive for HBeAg and anti-HBe by IMx, respectively. Only one specimen was negative for both IMx assays compared to 14 (7.0%) non-reactive for both HBe and anti-HBe RIA. There were 24 specimens (12.1%) positive for both HBeAg and anti-HBe by IMx compared to 1 (0.5%) positive by the corresponding RIAs. This increased detectability of anti-HBe in HBsAg carriers using IMx anti-HBe may result from increased sensitivity for 'free' anti-HBe and/or increased ability to detect anti-HBe in immune complex. IMx anti-HBe also detected more reactives among volunteer blood donor specimens reactive for anti-HBc but negative for HBsAg (55.5%, 86 of 155), compared to RIA (38.7%, 60 of 155). IMx anti-HBe may be useful in confirming prior exposure to HBV in blood screened positive by Corzyme.     

Evaluation of a rapid assay as an alternative to conventional enzyme immunoassays for detection of hepatitis C virus-specific antibodies

A rapid membrane flow-through immunoassay to detect antibodies to hepatitis C virus was compared with a commercial enzyme immunoassay (EIA) and microparticle enzyme immunoassay (MEIA) using 2,590 serum samples. Sensitivity and specificity of the "rapid assay" in comparison to the EIA/MEIA were 99.3 and 99.0%; the correlation coefficient being 0.91. This assay is suitable where infrastructure and laboratory expertise are limited.     

Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies

A two-site sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Monoclonal anti-beta-hCG antibody was used for coating microtitration plates and monoclonal anti-alpha-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or beta-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r = 0.968) with the values obtained by radioimmunoassay.     

Adsorption of interferon to homologous and heterologous cells

Summary Mouse and human interferons adsorbed well both to human and mouse cells. There was no difference in the recovery of homologous and heterologous interferons from the cells. Pretreatment of the cells with heterologous interferon did not prevent adsorption of subsequently applied homologous interferon and did not interfere with the antiviral activity of homologous interferon.     

Measurement of monoclonal antibody concentrations in hybridoma cultures: Comparison of competitive inhibition and antigen capture enzyme immunoassays

Competitive inhibition and antigen capture enzyme immunoassays were compared for the measurement of mouse monoclonal IgG1 antibody produced by a hybridoma culture. Both methods yielded standard curves that were linear over several orders of magnitude, and both were comparable in sensitivity (10 ng/ml). However, the slope of the antigen capture curve was flatter than the slope for competitive inhibition. This difference in slope, coupled with a larger average standard deviation for each point on the standard curve for antigen capture, resulted in a significantly larger range of variability in IgG1 levels. It is concluded that the competitive inhibition enzyme immunoassay method is better suited to the precise quantification of mouse monoclonal antibodies in hybridoma culture supernatants.     

Cell cultures for diagnosis of arbovirus infections in livestock and wildlife

Summary Arboviruses can be isolated in serially propagated cells derived from various vertebrates and invertebrates. Cell cultures can be used for direct detection of antigen by fluorescent antibody and enzyme-linked immunosorbent assays, for nucleic acid hybridization, and for visualization of viruses with electron microscopy. Reagents for enzyme-linked immunosorbent assays for IgM and IgG antibodies, hemagglutination-inhibition, complement fixation, and serum dilution-plaque reduction neutralization tests can be prepared in cell cultures infected with these viruses. Thus, cell cultures can be used as laboratory hosts for essentially all isolation, identification, and serodiagnostic procedures for arboviruses. This paper outlines current methods for diagnosis of arbovirus infections in livestock and wildlife, describes certain of these techniques, and provides references for others.     

Local and systemic immune responses in humans againstHelicobacter pyloriantigens from homologous and heterologous strains

The capacity ofHelicobacter pylorito induce strain specific immune responses was studied in adult Swedish volunteers. Sera and gastric aspirates from 11H. pylori-infected subjects were tested for specific antibody levels against, respectively, lipopolysaccharides (LPS) and total membrane preparations (MPs) prepared from the study subjects» own strains, as well as with corresponding antigens from two referenceH. pyloristrains or heterologous strains collected from other subjects within the study. It was found that sera from five of the 11 subjects had significantly higher IgA antibody titres against LPS from the homologous strain than against LPS from either of the reference strains and in five cases sera reacted with higher IgG titres against the homologous LPS than with LPS preparations from the reference or heterologous patient strains. Analyses of specific titres against MPs revealed that six sera had higher IgA titres and four sera had higher IgG titres against MPs prepared from the subjects» own strains than against MPs from either of the two reference strains. Determination of specific antibodies in gastric aspirates revealed significantly higher IgA titres against LPS from the homologousH. pyloriisolate than against LPS from the two reference strains in five cases, and six aspirates reacted in higher IgA titre with the homologousH. pyloriMPs. Results from immunoblotting analyses of sera support induction of strain specific immune responses againstH. pyloriLPS. By means of specific monoclonal antibodies againstH. pyloriLPS, antigenic heterogeneity between the different LPS preparations tested was confirmed.     

Isolation of an avian sarcoma virus-specific protein kinase from virus particles

Avian sarcoma viruses (ASV) of the Schmidt-Ruppin strain (SR) contain a protein kinase activity which specifically phosphorylates the IgG of sera from tumor-bearing rabbits (TBR). The amount is comparable with that from transformed cells. The activity is thermolabile in two mutants with a temperature-sensitive lesion in the sarcoma (src) gene. Ion-exchange column chromatography on DEAE-cellulose and phosphocellulose allowed a 250-fold purification of enzymatically active protein kinase with a molecular weight of 60,000 (60K). It phosphorylated casein and the heavy chain of IgG of TBR sera but not of control sera. Phosphorylation of casein could be completely inhibited by TBR serum and resulted in phosphorylation of IgG. Purification of the protein kinase from a mutant virus, OS122, and its wild-type SR-D revealed a threefold higher thermolability for the mutant enzyme. Partial proteolytic digest of the [³⁵S]methionine-labeled 60K protein obtained from the phosphocellulose column by immune precipitation was indistinguishable from that of pp60^STC precipitated from SR-D-transformed cells.     

Solid phase enzyme immunoassays of pertussis toxin using peroxidase or biotin labelled antibodies

The quantitative determination of pertussis toxin (PT) is generally estimated by biological tests which are time-consuming, cumbersome and unsuitable for simultaneous testing of a large number of samples. The present work describes a rapid and sensitive ELISA procedure allowing PT assay based on a sandwich technique amplified via avidin-biotin interaction. As low as 0.1 ng of PT in 0.1 ml sample could be detected by the procedure described.     

Effect of dilution on the growth of bacteria from blood cultures

Artificial blood culture systems were set up to find the dilution with medium necessary to overcome the natural antibacterial effect of blood. The results indicate that blood should be diluted at least 1 in 50 in medium unless an additive such as Liquoid is used.     

Isolation of Mycoplasma hominis from extragenital cultures

In order to document the characteristics of extragenital Mycoplasma hominis infections, the clinical features of 36 cases in which M. hominis was isolated from extragenital sites of adult patients were reviewed. In most cases, the organism was detected in conventional bacterial cultures (from specimens obtained from surgical and immunosuppressed patients) that had been incubated for at least 72 h. The results indicate that in cases in which M. hominis involvement is suspected, prolonged incubation or specialized microbiological techniques for detecting Mycoplasma spp. should be employed.     

Evaluation of six immunoassays for detection of dengue virus-specific immunoglobulin M and G antibodies

The performance of six commercially available immunoassay systems for the detection of dengue virus-specific immunoglobulin M (IgM) and IgG antibodies in serum was evaluated. These included two IgM and IgG enzyme immunoassays (EIA) from MRL Laboratories and PanBio, a rapid immunochromatographic test (RIT) from PanBio, immunofluorescence assays (IFA) from Progen, a dot blot assay from Genelabs, and a dipstick EIA from Integrated Diagnostics (INDX). For this study a panel of 132 serum samples, including 90 serum samples from patients with suspected dengue virus infection and 42 serum samples from patients with other viral infections, was used. In addition, serial serum samples from two monkeys experimentally immunized and challenged with dengue virus type 2 were used. Results were considered conclusive when concordant results were obtained with four of the six antibody-specific assays. Based on this definition, the calculated overall agreement for the human serum samples for the respective IgM immunoassays was 97% (128 of 132), with 34% (45 of 132) positive serum samples, 63% (83 of 132) negative samples, and 3% of samples (4 of 132) showing discordant results. The calculated overall agreement for the IgG assays was 94% (124 of 132), with 49% (65 of 132) positive, 45% (59 of 132) negative, and 6% (8 of 132) discordant results, respectively. The sensitivities of the dengue virus-specific assays evaluated varied between 71 and 100% for IgM and between 52 and 100% for IgG, with specificities of 86 to 96% and 81 to 100%, respectively. The relative sensitivities of the respective IgM assays measured with the monkey serum samples were comparable with those obtained with 12 serial serum samples from humans. Overall performance, based on the sum of the agreement, sensitivity, specificity, and Kappa statistics of the IgM and IgG immunoassays, showed that the antibody detection systems from INDX and Genelabs and the MRL and PanBio EIA are useful and reliable assays for dengue virus serodiagnosis.