Measurement of BK papovavirus IgG and IgM by radioimmunoassay (RIA)

Current techniques for the measurement of BK papovavirus (BKV) specific IgM include sucrose density gradient centrifugation followed by hemagglutination inhibition (HAI) or indirect immunofluorescent (IF) staining of BKV infected cells using a fluorescein conjugated anti-human IgM antibody. These techniques are cumbersome and labor intensive and do not lend themselves to testing large numbers of sera. A solid phase radioimmunoassay (RIA) was developed to facilitate the measurement of BKV IgG and IgM in large numbers of sera. Solid phase antigen was prepared by adsorbing CsCl purified BKV antigen to polyvinyl chloride microtiter plates. Following reaction with serum, bound immunoglobulin was detected with iodinated goat anti-human IgG or IgM. RIA for the measurement of BKV IgG was sensitive with titers approaching 10⁻⁶. Determination of IgG titers by RIA and HAI showed good agreement (P < 0.01, correlation coefficient = 0.74). Measurement of BKV IgM was not affected by the presence of BKV IgG as evidenced by sucrose density gradient fractionation of IgM positive sera, removal of IgG by treatment with S. aureus protein A, and addition of BKV IgG to BKV IgM. Rheumatoid factor (RF) gave false positive IgM titers in the presence of BKV IgG when RF titers were ≥ 1:640 by latex agglutination testing and BKV IgG levels exceed 1:256 by HAI. False positives due to RF could be eliminated by treatment of sera with sheep anti—human IgG antisera. RIA for BKV IgM was specific as sera containing JCV-, cytomegalovirus (CMV)-, rubella-, or hepatitis B core antibody (anti HBc)—IgM were negative by RIA. RIA detected BKV IgM in several sera from renal dialysis or allograft palienls with titers ranging from 1:400 to 1:128,000 and demonstrated that BKV IgM persisted in sera of renal allograft patients for as long as 343 days post transplantation.     

Antisera to late cytomegalovirus nuclear antigens produced in cynomolgus monkeys

Cynomolgus monkeys were immunized with human cytomegalovirus Ad.169-infected fibroblast nuclei taken 10–15 days after infection. Antisera were obtained which had high titers of cytomegalovirus (CMV) antibody in complement fixation (CF), indirect immunofluorescence (IF) and enzyme-linked immunosorbent assay (ELISA). The antisera were used in ELISA-inhibition assays for determination of small amounts of CMV antigens. The antisera were specific for CMV and did not significantly cross-react with herpes simplex virus (HSV), varicella or control antigens from uninfected human lung cells or green monkey kidney cells.     

Rapid detection of cytomegalovirus by fluorescent monoclonal antibody staining and in situ DNA hybridization in a dram vial cell culture system.

By using dram vial cell culture methods, three commercially available tests for cytomegalovirus (CMV) detection were compared: direct fluorescent monoclonal antibody staining for CMV-specific early and late antigens (direct FA), indirect fluorescent monoclonal antibody staining for a CMV-specific early antigen (indirect FA), and in situ DNA hybridization with a biotinylated CMV-specific DNA probe kit (DNA probe). Of those tests, only the indirect FA provided consistent, reliable virus detection within the initial 24 h postinfection for serial 10-fold dilutions of CMV AD169 (laboratory strain) and for three selected urine samples. However, when used prospectively, the indirect FA failed to detect virus within the initial 10 days postinfection in 15 of 78 consecutive specimens that were eventually positive by cell culture. Although the indirect FA was more sensitive than the direct FA or DNA probe, its utility appeared limited to specimens with high CMV concentrations. On the basis of these data, we recommend that indirect FA be reserved as an adjunct to standard cell culture for selected samples in diagnostic hospital laboratories.     

Physicochemical and serological characteristics of respiratory virus fluorescein-isothiocyanate conjugates for fluorescent-antibody diagnosis.

Fluorescein-isothiocyanate (FITC) conjugates were prepared by improved methods from standard reference antisera to influenza A and B, mumps, parainfluenza 1, 2, 3, and 4, herpesvirus, respiratory syncytial virus, and adenovirus hexon. The antisera, prepared in a variety of animals, were fractionated three times with selected optimal concentrations of ammonium sulfate and yielded gamma globulins of adequate purity for conjugation with FITC. Conjugates containing optimal fluorescein-to-protein ratios of between 5 and 10 were produced in 2 h by dialysis labeling. Serological titers of each antiserum and conjugate were determined by complement fixation, hemagglutination-inhibition, serum neutralization, and indirect hemagglutination tests where appropriate. When corrected for dilution, the serological titers of the FITC conjugates were identical to those of the starting antisera. The fluorescent-antibody staining titers correlated well with one of the serological parameters of the original serum. The conjugates stained homologous antigens specifically and were free of nonspecific staining at the working dilution. Undesired staining of host cells which was a problem with some of the conjugates produced from sera containing cellular antibodies was removed by absorption with packed cells. These physicochemical and serological findings were then used as a guide in preparing high quality reagents for fluorescent-antibody identification of respiratory viruses.     

Evaluation of anti-complement immunofluorescence test in cytomegalovirus infection.

The anti-complement immunofluorescence (ACIF) technique was evaluated for the diagnosis of human cytomegalovirus (CMV) infection in a group of sera derived from renal transplant recipients and donors by comparing it with the indirect immunofluorescence (FA) and complement fixation (CF) TESTS. The ACIF and FA tests yielded similar results. However, the ACIF test had a distinct advantage over the indirect FA test, since it eliminated the nonspecific cytoplasmic staining that may result in false positive readings in inexperienced hands. Both the indirect FA and ACIF tests were more sensitive than the CF test. In primary CMV infection, the FA and ACIF antibodies appeared earlier and had significantly higher titer than corresponding CF titers. This difference in titers was not seen in seropositive individuals who lacked overt infection. Our previously reported correlation between the seropositivity of the donor and CMV infection in seronegative recipients has been confirmed.     

Sensitivity of a radioimmunoassay method for detection of certain viral antibodies in sera and cerebrospinal fluids.

An indirect solid-phase radioimmunoassay (RIA) was applied to titration of serum and cerebrospinal fluid (CSF) antibodies against a variety of viruses including rubella, mumps, measles, herpes simplex, varicella-zoster, and vaccinia. The test used fixed, virus-infected cells as a source of antigen, and conditions for optimal production of viral antigen were determined for each virus-host cell system. In acute, uncomplicated viral infections, sera taken 2 to 5 days after onset generally had low homotypic RIA titers ranging from less than 1:100 to 1:500, whereas convalescent-phase titers ranged from 1:128,000 to 1:512,000. Rubella and measles antibody titers as high as 1:256,000 were demonstrated by RIA in CSF from patients with chronic panencephalitis, whereas homologous antibody titers of 1:4,000 were detected in CSF from acute mumps, herpes simplex, and varicella-zoster virus infections with central nervous system involvement. Some heterotypic antibody was demonstrable by RIA in CSF, but, with the exception of herpes simplex antibody in a mumps virus infection, titers were markedly lower than those to the infecting virus type. RIA generally demonstrated titers at least 1,000 times higher than those obtained by conventional assays such as complement fixation, hemagglutination inhibition, neutralization, and immunofluorescent staining.     

Monoclonal antibody to Japanese encephalitis virus cross-reacting with histones present in the cell nuclei.

An immunoglobulin G (IgG2b) class of monoclonal antibody (MoAb, NHA-1) raised against Japanese encephalitis virus (JEV) E glycoprotein, reacted with the viral antigen expressed in cytoplasm of the infected cells and also with the cell nuclei, by an indirect fluorescent antibody technique (FA). The NHA-1 reactivity to nuclei was found to be due to its recognizing a JEV cross-reactive epitope present on the nuclear histones. Adsorption with calf thymus histones (type II-AS) showed a drop in NHA-1 reactivity to both JEV and histones by an enzyme-linked immunosorbent assay (ELISA) and indirect FA; the drop was higher against the histones. The MoAb recognized specifically the viral antigens expressed on the infected porcine kidney cell surface by a modified indirect FA. ELISA carried out with glutaraldehyde-fixed antigens showed an almost 2-fold increase in the reactivity over unfixed JEV antigen but none for the histones. Thus, the results indicate that histones share a sequential homology with E glycoprotein of JEV, which might lead to an autoimmune disorder induced due to the molecular mimicry between these two antigens.     

Indirect fluorescent-antibody test for human cytomegalovirus infection in the absence of interfering immunoglobulin G receptors.

The presence of immunoglobulin G receptors in human fibroblasts infected with human cytomegalovirus (CMV) resulted in a nonspecific cytoplasmic reaction in the indirect fluorescent-antibody test. Both CMV antibody-positive and antibody-negative sera from human or other animal species produced the cytoplasmic reaction. The substitution of a simian CMV strain for the human virus successfully eliminated this cytoplasmic reaction and, thus, allowed for the observation of virus-induced fluorescent intranuclear inclusions. With the latter system, CMV antibody titers in human sera were equivalent to those obtained by using the human virus and, in addition, allowed for the detection of relatively low-titered serum samples in which antibody measurement was difficult when human CMV-infected cells were used in the indirect fluorescent-antibody test.     

Localization of chlamydial group Antigen in McCoy cell monolayers infected with Chlamydia trachomatis or Chlamydia psittaci.

Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cells during the chlamydial developmental cycle.     

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells.

The presence of a chlamydia-specified antigen associated with the plasma membrane of infected cell lines was demonstrated by indirect immunofluorescence staining with a monoclonal antibody, designated 47A2, specific for the chlamydial genus-specific lipopolysaccharide (LPS) antigen. Staining of HeLa, L-929, and McCoy cells infected with the L2 or F serovar of Chlamydia trachomatis was observed either without fixation or following aldehyde fixation and brief drying. The 47A2-reactive antigen appeared to be present on the plasma membrane, on bleb-like structures on the host cell surface, and on proximal processes of neighboring uninfected cells. Antibodies to chlamydial protein antigens such as the major outer membrane protein produced no surface staining under similar conditions. Membrane vesicles elaborated from infected cells were enriched for the 47A2-reactive antigen. Superinfection of chlamydia-infected cells with vesicular stomatitis virus, an enveloped virus which buds from the plasma membrane, allowed purification of progeny virions that were enriched with chlamydial LPS. These results are consistent with the presence of chlamydial LPS in the plasma membranes of infected host cells.     

Anti-complement immunofluorescence test for antibodies to human cytomegalovirus.

An anti-complement immunofluorescence (ACIF) test that detects human cytomegalovirus (CMV) antigen in the nuclei of infected cells was used for assay of CMV antibodies in human sera. Various factors influencing the sensitivity and specificity of the ACIF test system were investigated, and results were applied to the development of a procedure which could be completed in a relatively short length of time and gave reproducible results. Results obtained in the ACIF test were compared with those obtained in complement fixation, indirect hemagglutination, and neutralization tests, and the ACIF test was shown to be suitable for detection of significant antibody titer rises and stationary levels of CMV antibody. Heterotypic antibody responses were not seen with sera from other human herpesvirus infections. The nonspecific cytoplasmic staining that occurs in indirect immunofluorescence tests for CMV did not occur in the ACIF system, and sera that were anti-complementary in complement fixation tests could be examined satisfactorily by ACIF. Thus, the test is a valuable supplemental or back-up procedure for the serodiagnosis of CMV infection.     

Antigenic characteristics of Marek's disease tumour cells

Surface antigens of Marek's disease lymphoblastoid cells were studied by indirect immunofluorescence (FA) tests and by cytotoxic antibody tests. The results of FA tests revealed genetic differences between cell lines of different sources with respect to their histocompatibility antigens. JMV and MSB-1 cells shared one of their alloantigens detectable by B blood group antisera. Differences between the cell lines were more pronounced when the respective hyperimmune sera were examined by cytotoxicity tests rather than by FA tests. The results of cross-absorptions of cell line antisera with cells of the different lines suggested that no identical common tumour-specific cell surface antigen was detectable serologically in addition to histocompatibility antigens or other normal cell surface antigens. The results of vaccination experiments with chicken embryo fibroblasts and with cells from various lymphoid tissues indicated that resistance against JMV lymphoblastic leukaemia could be induced by a number of different antigens which were not specific to Marek's disease.     

Production of polyclonal antisera against cytomegalovirus and varicella-zoster virus by use of affinity-purified viral antigens

Specific animal antisera to cytomegalovirus (CMV) and varicella-zoster virus (VZV), devoid of antibodies to host cell components, have been difficult to prepare because of the cell-associated nature of these viruses. A simple method for the purification of CMV and VZV antigens is described in this paper. The viral antigens were purified from commercially available crude antigens by affinity chromatography with human antibodies against CMV and VZV. Rabbits were immunized with the purified antigens. The produced antisera were specific for the respective viruses and did not contain antibodies to host cell components, as determined by immunofluorescence microscopy and enzyme-immunoassay. The antisera have been used for direct detection of viral antigens in clinical specimens and for identification of cytopathic effect in cell cultures by immunofluorescence microscopy. The CMV antiserum could also be used for the early detection of CMV in cell cultures inoculated with patient specimens, but it was less sensitive than a monoclonal antibody to the early antigen of CMV.     

Determination of IgA antibodies to human cytomegalovirus by enzyme-linked immunosorbent assay (ELISA)

A sensitive enzyme-linked immunosorbent assay (ELISA) is described for detection of IgA antibodies cytomegalovirus (CMV). The antigen consisted of a sonicated extract of CMV infected human embryo cells. The tested sera were absorbed with staphylococcus aureus (strain Cowan 1) before analysis. Rabbit antihuman IgA peroxidase conjugate was used to detect human IgA bound to viral antigen. In parallel, Igm and IgG antibodies to CMV were studied by ELISA and by the immunoperoxidase antibody to membrane antigen (IPAMA) technique, respectively. CMV IgA was detected in high titers by ELISA in eight of nine CMV patients. The maximal IgA titers were generally lower than the IgM titers detected by ELISA. No CMV IgA antibodies (titer less than 20) were detected by ELISA in 57 control sera (healthy adults, hospitalized patients with various other diseases), paired sera of five patients with acute herpes simplex, infection, two patients with Epstein-Barr infections, one patients with varicella, and one with zoster infections. The potential application of the ELISA CMV IgA technique in serodiagnosis of CMV infections is discussed.     

Characterization of the human cytomegalovirus-induced chromatin factor responsible for activation of host cell chromatin template.

One of the chromatin-associated factors induced in human embryonic lung cells at early stages of human cytomegalovirus infection stimulates template activity of cell chromatin. This factor coincides with a major component of “pre-early nuclear antigen” that is detectable by anticomplement immunofluorescence staining of infected cells within 1 hr after infection with regard to molecular size, elution properties in DNA-cellulose and DEAE-Sephadex chromatography, and reactivity to specific antibody.     

Seroconversion to virus-specific pre-early nuclear antigens in infants with primary cytomegalovirus infection

Antibodies to human cytomegalovirus (CMV)-specific antigens were determined in sera serially collected from 10 infants with primary CMV infection. Antibodies to pre-early nuclear antigens (PENA), which are detectable in human embryonic lung cells within 3 h of CMV infection by anticomplement immunofluorescence staining, developed in all the patients. However, in contrast to the early response of anti-early antigens (EA), anti-late antigens (LA), and immunoglobulin M antimembrane antigens (MA), seroconversion or the maximum antibody response to PENA was usually observed 1 or more months later. Immunoglobulin M antibody to MA became undetectable soon after recovery from illness, followed by a decrease in anti-EA, anti-PENA, and then anti-LA titers. Results indicated analogy of the clinical significance of anti-PENA in CMV infection to that of anti-Epstein-Barr nuclear antigen in infectious mononucleosis and support the idea that parallel determinations of anti-PENA and IgM anti-MA antibodies can be useful for identifying the acute or chronic phase of primary CMV infection.     

Serological studies on rabbit antibodies to the rabbit anterior pituitary

Rabbits immunized with suspensions or extracts of rabbit anterior pituitary in Freund adjuvant may develop specific antibodies to components of the rabbit pituitary. Immunofluorescent staining with such antisera occurred in isolated cells of the anterior pituitary. These correspond to cells stained with acid fuchsin, i.e. acidophils or alpha cells. Some of the pituitary antisera fix complement with pituitary extracts. A tanned-cell haemagglutination test using pituitary extracts as coating antigen yielded positive reactions with some of the pituitary antisera. The rabbit antisera appeared to be specific for the anterior pituitary within the limits of the rabbit organs tested. Hog, guinea-pig, dog and beef pituitaries share the antigen, but monkey and human pituitaries fail to react.

Immunofluorescent staining revealed that antisera reacted with the pituitary of the antibody producing rabbit, i.e. the sera contain pituitary autoantibodies. No direct or indirect evidence for pathological changes in the autoantibody producing animals could be found.

     

Antibody response to Ehrlichia risticii and antibody reactivity to the component antigens in horses with induced Potomac horse fever.

The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type antibody response was detected in 30% of the horses by ELISA. In the primary antibody response, a distinct titer was observed at 2 weeks postinoculation (PI), when the immunoglobulin M (IgM)/IgG ratio was 2 to 5, and the overall antibody titer peaked at 6 to 8 weeks PI. The secondary-type antibody response exhibited a characteristic titer at 1 week PI, the IgM and IgG titers were about equal at 2 weeks PI, and the overall antibody titer peaked at 6 weeks PI. A transient depression in the IgG response at 4 weeks PI was observed in both response types. The antibody was maintained at a high titer for over a year in all horses. Western immunoblot reactivity showed that the antisera collected from these infected horses at 4 to 5 weeks PI recognized some or all of the six major E. risticii component antigens (70, 55, 51, 44, 33, and 28 kilodaltons), all of which were apparent surface components. The 6- to 8-week PI antisera recognized up to 16 component antigens, including 9 major antigens (110, 86, 70, 55, 51, 49, 44, 33, and 28 kilodaltons). However, the PrI sera of these horses showed reactivity at various intensities with one to seven of the component antigens. There was no apparent correlation between this reactivity pattern and the subsequent antibody response types.     

Immunoelectron microscopic identification and localization of Streptococcus sanguis with peroxidase-labeled antibody: localization of surface antigens in pure cultures.

An indirect method of localizing antigens with horseradish peroxidase-labeled antibody was used to identify and localize surface antigens of Streptococcus sanguis at the ultrastructural level. An electron dense layer surrounding the cell wall could be distinguished without any additional electron microscope staining. This labeled layer represents an immune complex consisting of bacterial surface antigens, specific rabbit antisera, and peroxidase-labeled goat anti-rabbit globulins. Although with undiluted antisera slight cross-reactions occurred with S. salivarius and S. miteor (mitis), these could be readily distinguished from the more intense homologous reaction by their patchiness and the difference in distribution of the label. These cross-reactions were eliminated by appropriate dilutions of the antiserum. No cross-reactions occurred with S. mutans, S. faecalis, Actinomyces species, or Bacterionema, microorganisms wents indicated that horseradish peroxidase can become non-specifically adsorbed to the membrane of certain bacterial cells. Appropriate controls must, therefore, be included for localization of membrane associated antigens with horseradish peroxidase.     

Cytomegalovirus infection of the human placenta: an immunocytochemical study.

In congenital cytomegalovirus (CMV) infection histologic evaluation of the placenta is often unrevealing. In the present study immunocytochemistry to CMV immediate early and early nuclear antigens was used to characterize placental involvement in six cases of symptomatic intrauterine CMV infection. Histologic examination had demonstrated diagnostic viral inclusions in one placenta and non-specific villitis in another. However, immunocytochemistry revealed CMV infection in five of the six placentas, including three with no pathologic changes on routine histologic evaluation. Infected cells were located primarily in the villous stroma. In one case immunoperoxidase staining showed infection in the syncytiotrophoblast. Infected endothelial cells were demonstrated by double staining for CMV and factor VIII antigen. No double-stained cells were seen in tissue sections stained for CMV immediate early nuclear antigen or the human macrophage-associated CD68 antigen, which is expressed in Hofbauer cells. In conclusion, specific immunoperoxidase staining was more sensitive for demonstrating placental CMV infection than was histologic examination and it aided in the characterization of infected cells.