Moloney leukemia virus gene expression and gene amplification in preleukemic and leukemic BALB/Mo mice

Mice carrying the exogenous Moloney leukemia virus (M-MuLV) as an endogenous virus have been derived previously. This mouse strain (BALB/Mo) transmits the virus as a single Mendelian gene (Mov-1 locus) from one generation to the next. Molecular hybridization experiments were performed to identify the organs in which the M-MuLV gene is activated during postnatal life of BALB/Mo mice and to determine the age-dependent onset of M-MuLV gene expression. Spleen and thymus cells of BALB/Mo mice synthesize M-MuLV-specific RNA soon after birth and virus gene expression reaches high levels at 3–4 weeks of age. A substantial further increase in virus gene expression is not observed in leukemic tissues of older animals. Other organs, such as liver, brain, and kidneys, do not express M-MuLV-specific RNA throughout the animals' life. These observations define lymphatic tissues (spleen and thymus) as the target organs of M-MuLV expression in BALB/Mo mice. Virus gene expression was correlated with somatic amplification of M-MuLV-specific DNA sequences during the preleukemic and leukemic phase of the animals' life. DNA amplification occurs in two steps in target tissues of BALB/Mo mice. A first step to approximately two copies per haploid genome equivalent is observed in preleukemic mice and a second step to three to four copies is observed in leukemic tissues. Nontarget tissues carry one copy of M-MuLV-specific DNA sequences regardless of the age of the animal. These results suggest that M-MuLV-specific gene expression is not sufficient for leukemic transformation and is related to virus-specific DNA amplification in preleukemic animals. A second amplification of M-MuLV DNA sequences appears to be related to leukemic transformation.     

Transcription of endogenous C-type viruses in resting and proliferating tissues of BALB/Mo mice.

The effect of tissue proliferation on the genome expression of endogenous C-type viruses was studied. Liver cell proliferation was induced by partial hepatectomy of BALB/Mo mice. RNA sequences homologous to two classes of C-type viruses, to the Moloney leukemia virus (M-MuLV) endogenous in BALB/Mo mice, and to the xenotropic endogenous BALB 2 virus, were quantitated by molecular hybridization. RNA from resting liver tissue contained low concentrations of sequences homologous to either class of virus. No substantial increase in virus-specific RNA concentration was observed in livers regenerating between 18 hr and 5 days. Furthermore, the effect of liver cell proliferation on de novo infection with Moloney leukemia virus was studied. Viremic BALB/c mice infected with virus as newborns carry small amounts of M-MuLV-specific DNA copies in their liver. This value did not change subsequent to partial hepatectomy. These results indicate that neither tissue-specific expression of M-MuLV in BALB/Mo mice nor the organ-specific de novo infection with M-MuLV is dependent on the proliferative state of the cells. This is in agreement with the hypothesis that tissue-specific activation of the virus is dependent on regulatory mechanism involved in cellular differentiation.     

Characterization of the antibody response in vaccinated mice protected against Coxsackievirus B3-induced myocarditis

Adolescent male CD-1 mice can be rendered resistant to coxsackievirus B3 (CVB3m)-induced myocarditis following inoculation as neonates with a single dose of a temperature-sensitive mutant virus (ts1), derived from the prototype parent virus (CVB3m). Previously, anti-CVB3 neutralizing antibodies were not detected in sera of adolescent ts1 vaccinees by a standard plaque-reduction assay (Gauntt et al 1983. Infect. Immun. 39:851). However, a more sensitive cytopathic effects-reduction assay permitted detection of low titers of anti-CVB3m neutralizing antibodies of the IgG class prior to challenge with CVB3m. Following CVB3m challenge, serum anti-CVB3m neutralizing antibody titers of ts1 vaccines declined on days 1-2 post-inoculation (p.i.) then increased over the next 6 days. The neutralizing antibodies were of both the IgG and IgM classes. Normal mice challenged with CVB3 did not produce detectable serum anti-CVB3m neutralizing antibody until day 4 p.i. and by 8 days p.i. the neutralizing antibody was only of the IgM class. Thus, adolescent murine ts1 vaccines mount a secondary antibody response to CVB3m in both neutralizing IgG and IgM, but resistance to CVB3m-induced myocarditis is due to the presence of low levels of anti-CVB3m IgG neutralizing antibody in serum at the time of challenge with CVB3m.     

Biological, immunological, and biochemical evidence that HIX virus is a recombinant between moloney leukemia virus and a murine xenotropic C type virus

Various parameters of HIX virus were examined to determine its origin and its relationship to other murine C type oncornaviruses. Envelope properties of HIX virus grown in cells of several species were subjected to analyses of host range, interference, and neutralization. Cloned amphotropic HIX virus was adapted to grow in human RD cells. After 6 months in culture, the resulting virus (HIX-RD) could enter mouse cells but essentially lost the capacity of propagating in mouse cells. Interference patterns of HIX and HIX-RD were identical to each other and unrelated to murine ecotropic MuLV interference. MSV(HIX) or MSV(HIX-RD) could not penetrate HIX-, HIX-RD-, or MuX-preinfected cells. However, infection with HIX exhibited a unique one-way interference in that MSV(MuX) could penetrate and transform HIX-preinfected cells. Neutralization of HIX and HIX-RD with relatively type-specific anti-gp70 sera showed that they resembled Moloney (M)-MuLV most closely. Significant neutralization was observed also with anti-Rauscher gp70 or BALB-2 MuX gp70 sera. Both HIX derivatives were acutely susceptible to inactivation with normal mouse sera, a characteristic of xenotropic viruses. Competition radioimmunoassays were performed to determine the antigenic relationship of HIX to other MuLV types. The highly type-specific phosphorylated p12 and the relatively type-specific gag region p15 of HIX were found to be identical to M-MuLV and less related to other murine C-type oncornaviruses. The examination of HIX gp70 with type-specific anti-M-MuLV or anti-C57L MuX gp70 sera showed that it was clearly different from either virus. Tryptic peptide maps of the gag region-p15 and p30 of HIX were identical to corresponding maps of M-MuLV proteins. The gp70 of HIX was unique and different from known eco-, xeno-, and amphotropic murine C type oncornaviruses. Based on known migration patterns of characteristic trypsin- and chymotrypsin-derived peptides of various eco-, and xenotropic MuLV's, it was concluded that gp70 of HIX was related to both MuX and M-MuLV. Tryptic fingerprint maps also revealed several significant differences between parental HIX and its HIX-RD variant. Comparative hybridizations of assorted high-molecular-weight (HMW) virus RNAs with complementary DNA from HIX virus showed that, with unfractionated probes, no significant differences could be seen between HIX and M-MuLV. Based on the above, HIX virus appears to contain predominantly M-MuLV-specific information except for its envelope gene which has been presumably derived from a recombinational event involving corresponding M-MuLV and MuX nucleotide sequences.     

Bile immunoglobulin of the duck (Anas platyrhynchos). II. Antibody response in influenza A virus infections

The capacity of the IgM-like bile immunoglobulin (IgX) of the duck (Anas platyrhynchos) to express antibody activity to H3N2 influenza A viruses, and the dependence of this activity on the co-existence of serum IgM antibodies were investigated. Ducklings infected orally and intranasally at 15-29 days of age with viruses isolated from different host species were examined for haemagglutination-inhibiting (HI) antibodies in biles and sera 16-29 days after infection (p.i.). All biles had antibodies associated with IgX; all sera had antibodies associated only with the 7.8S IgG. Following oral infection of birds 42-days-old with influenza A/duck/HK/7/75 virus, serum HI antibodies were an initial IgM response occurring from 5-12 days p.i., followed by the appearance of 7.8S IgG antibodies. Virus-neutralizing (VN) antibodies in serum were also biphasic; isotype classification was not attempted. Bile IgX developed HI and VN activity. HI antibodies reached peak titres 12 days p.i. and fell to low levels by 24 days p.i. VN antibodies also reached peak titres 12 days p.i., but thereafter persisted at quite high levels throughout the experiment. Development of high titres of antibody in bile coincided with the termination of virus excretion in faeces. These experiments confirm that bile IgX of the duck can function as antibody in response to influenza A viruses, and that its activity appears to be independent of serum IgM. Its possible relevance in determining survival of virus in the intestine is discussed.     

Immunoglobulin responses to echovirus type 11 by enzyme linked immunosorbent assay: Single-serum diagnosis of acute infection by specific IgM antibody

An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgM and IgG antibodies to echovirus type 11 in a single dilution of serum was developed using partially purified echovirus type 11 bound to microplates. Whole serum was used for IgG antibody but prior to assaying for IgM antibody interfering IgG was removed by ion exchange chromatography. The ELISA for echovirus type 11 IgG antibody was a more sensitive, rapid, technically easier and less costly alternative to the neutralisation test. With the IgG ELISA 12 of 132 sera (10.6%) known to contain enterovirus antibodies other than echovirus type 11 were positive but it could not be determined to what extent this was due to the greater sensitivity of the ELISA or cross-reactions. The IgM ELISA was even more sensitive than the IgG ELISA with acute sera, and showed a reactivity in 4 of 36 sera (11.1%) with no detectable echovirus type 11 neutralising antibodies. Echovirus type 11 IgM antibody was detected in all sera collected after the first week of infection and up to 30 days after infection. However, it was only detected in 58% of sera collected during the first week after onset thus limiting its use for rapid diagnosis. The echovirus type 11 IgM ELISA appears to have considerable laboratory diagnostic potential when a rising antibody level cannot be demonstrated in paired sera or when virus is not cultured.     

Isotypic and clonal variations in the interactions between model monoclonal immune complexes and the human erythrocyte CR1 receptor.

Erythrocytes (E) play a central role in handling circulating immune complexes (IC) in primates. E capture IC via complement receptors, type 1 (CR1) which can bind to C3b and C4b ligand sites generated on IC during activation of the complement cascade. The present study was designed to explore how the immunochemical properties of IC affected their interactions with human E. Model IC were constructed by combining murine monoclonal anti-dinitrophenyl (DNP) antibodies with DNP-bovine serum albumin. A panel of 10 independently-derived monoclonal IgG1, IgG2a, IgG2b, IgG3, IgM and IgA antibodies were used to construct IC and their interactions with human E were examined in vitro. The data reveal that IC constructed with the different monoclonal antibodies differed with respect to their rate of binding to E, the peak magnitude of IC binding to E, and the rate and extent of IC release from E. IC containing IgG1 antibodies (IgG1 IC), IgG2a IC, IgG2b IC, and IgA IC all bound rapidly to E, whereas IgG3 IC and IgM IC were bound relatively slowly to E. The peak magnitude of IC binding to E correlated directly with their binding rate. There was an inverse correlation between the antigen/antibody ratio of the IC and the magnitude of IC binding to E. The rate of release of the various types of IC from E also differed. IgG2a IC and IgG2b IC displayed the most rapid maximum release rates while IgG3 IC had the slowest peak release rate. IgM IC and IgA IC were also released relatively slowly from E. IgG1 IC had an intermediate release rate. There was no direct correlation between the maximum release rate and either the maximum binding rate or the peak magnitude of IC binding to E. While there were some clonotypic differences in binding and release rates between IC made with different IgG2a, IgG3 and IgM antibodies, antibody isotype appears to be of fundamental importance with respect to both the binding of IC to E and the release of IC from E. These data indicate that the immunochemical properties of IC can profoundly affect their interactions with human E and that the panel of IC constructed with monoclonal antibodies can serve as a useful model to explore these interactions.     

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay

The development of a solid phase radioimmunoassay which is able to detect virus-specific IgG and IgM antibodies in serum specimens from patients with enterovirus infections is described. Viral antigen partially purified from infected tissue culture fluid was adsorbed passively to individual polystyrene microtiter wells. Dilutions of sera were incubated on these antigens and bound anti-viral antibodies were monitored by the addition of 125-iodine labeled anti-human-IgG or anti-human-IgM antibody. Specificity of the assay to detect virus-specific IgM antibody was ensured by highly specific anti-IgM antibody which did not cross react with IgG and 2-mercaptoethanol sensitivity of IgM antibody titers. Changes of IgM antibody titers clearly indicated a current infection by that virus strain which was isolated as etiological agent. Advantages and restrictions of the introduced radioimmunoassay are discussed.This study was supported by the Bundesministerium für Forschung und Technologie (ZA/NT/SS 0313/6)     

Conformation of free and of integrated Moloney leukemia virus proviral DNA in preleukemic and leukemic BALB/Mo mice

Restriction enzyme analysis has been used to characterize the structure of Moloney leukemia virus (M-MuLV) DNA in preleukemic and leukemic BALB/Mo mice. Leukemogenesis in these mice is accompanied by a somatic amplification of M-MuLV-specific DNA sequences which are derived from the germ line-transmitted Moloney leukemia virus genome. Quantitation by DNA-DNA annealing in solution has shown that this increase of M-MuLV-specific DNA sequences occurs in two steps, a first step from 1 to approximately 2 copies in spleen and thymus of preleukemic mice and a second increase to 3–4 copies following leukemic transformation. Using a specific cDNA probe for restriction enzyme analysis, no somatically acquired M-MuLV copies are detectable in tissues of preleukemic BALB/Mo mice. This indicates that virus integrated at many different chromosomal sites in individual cells. In contrast, restriction enzyme analysis of DNA from leukemic tissues shows 5–8 integrated copies in addition to the endogenous M-MuLV genome. Identical integration patterns are observed in leukemic tumors arising at different anatomical sites in individual animals. Tumors from different animals, however, have distinct integration patterns and no specific integration site common to all tumors can be identified. These results suggest that in BALB/Mo mice transformed cells originate among a population of preleukemic target cells which is heterogenous with respect to integration sites of newly acquired proviral copies. Selection of one transformed cell clone leads to monoclonal disease. Similar results are obtained with BALB/c mice infected with virus as newborns. Gel analysis indicated that unintegrated forms of M-MuLV viral DNA appear in target tissues of preleukemic BALB/Mo mice. Quantitation by quantitative hybridization in solution showed that up to 0.5 copy per cell can be present in thymic DNA from 1? to 5-month-old mice but not in spleens of the same animals. Small amounts of unintegrated proviral DNA are occasionally detected in thymus, spleen, and bone marrow of animals younger than 4 weeks of age. These results provide the first evidence for occurrence of free proviral DNA in animals and suggest that superinfection of target cells with endogenous virus is involved in the process of leukemic transformation.     

Diagnosis of Colorado tick fever virus infection by enzyme immunoassays for immunoglobulin M and G antibodies.

An immunoglobulin M (IgM) capture enzyme immunoassay technique was adapted for the detection of antibody to Colorado tick fever virus in sera from 84 individuals for whom diagnosis had been confirmed by virus isolation or neutralization test. Titers were compared with those for IgG and neutralizing antibodies in these Colorado tick fever cases. IgM antibody titers were higher than neutralizing antibody titers, but neither appeared until 1 to 2 weeks after the onset of illness. Neutralizing antibodies were detected earlier than IgM antibodies, and both were detected with greater frequency than IgG antibodies. Late-convalescent-phase sera contained both neutralizing and IgG antibodies, but IgM was all but undetectable by 2 months after onset. Although the neutralization test may remain the serological test of choice, the enzyme immunoassay for IgM antibody offers a simple and more rapid method of serodiagnosis; the enzyme immunoassay is, however, less sensitive than the neutralization test. Furthermore, because there was a sharp decline in IgM antibody after 45 days, the presence of IgM antibody in a single serum sample provides a basis for the presumptive serodiagnosis of recent Colorado tick fever virus infection.     

The importance of antibody isotype in HIV-1 virus capture assay and in TZM-bl neutralization

The binding of murine IgM mAbs to five different clades of HIV-1 was examined using a modified ELISA-based virus capture assay. Two murine multispecific IgM mAbs that exhibit both lipid and gp41 epitope specificities, and one murine IgM mAb that exhibits lipid-binding specificity, were utilized. The binding of the IgG and the IgM isotypes of human mAb 2F5 to clades A through AE were also evaluated. The binding of 2F5 to HIV-1 was dependent upon the antibody isotype. Monoclonal IgM antibodies bound significantly lower amounts of HIV-1 than the corresponding IgG isotype. Although murine IgM mAbs bound HIV-1 to varying degrees in the virus capture assay, they failed to neutralize HIV-1 in a TZM-bl pseudovirus assay. In contrast, 2F5-IgM mAb bound certain HIV-1 isolates, and also neutralized them, although not as efficiently as the 2F5-IgG isotype. Studies on the relationship between virus binding and neutralization in a TZM-bl pseudovirus assay indicated that in most cases, mAbs that exhibited neutralization also bound the virus.     

Measurement of antibody-producing capacity in man. V. Immunoglobulin classes of antibodies to flagellin

The immunoglobulin classes of antibody to flagellin were determined in twenty-eight persons injected with flagellin from Salmonella adelaide. Sera were examined from non-immunized persons and from persons given primary and secondary immunization. Results obtained by titration before and after treatment of whole serum with 2-mercaptoethanol (ME) were compared with those obtained after fractionation of serum by gel filtration on Sephadex G-200 and by radio-immunoelectrophoresis (RIEP). The ME procedure, whilst not fully reliable for determining class of specific antibody in individual sera, is suitable for routine analyses of many sera. After gel filtration, ME-sensitive antibodies were mostly contained in the IgM protein peak whereas ME-resistant antibodies were mainly associated with the IgG protein peak. Using RIEP, specific antigen binding to the IgM, IgG and IgA precipitin lines was demonstrated. Binding of flagellin to the IgM precipitin line correlated with the presence in whole serum of ME-sensitive antibody and binding to the IgG line with ME-resistant antibody. Antigen binding to the IgA line did not correlate with results from ME-sensitivity studies using whole sera.Natural antibody in serum before immunization was entirely IgM. The antibody after primary immunization was mainly IgM but varying amounts of IgG were also produced; a change to the synthesis of entirely IgG antibody during the primary response was rare. The antibody after secondary immunization was mostly IgG; there was no evidence of increased IgM synthesis. IgA antibodies to flagellin were detected during both primary and secondary responses in about half of the subjects tested, but were not quantified.     

Class-specific antibody responses to Mycoplasma pulmonis in sera and lungs of infected and vaccinated mice.

Class-specific antibodies were measured by a solid-phase microradioimmunoassay in the sera and lung washings of mice after intranasal or intravenous inoculation with live Mycoplasma pulmonis and after systemic, intranasal, or combined vaccination with Formalin-inactivated mycoplasmas. After intranasal or intravenous inoculation with live organisms, serum antibodies were first detected in immunoglobulin M (IgM) followed by IgG2, IgG1, and IgA classes, but significant levels of IgA developed only in those mice inoculated intranasally. The appearance of antibodies in lung washings was later than in serum, but again these were predominantly IgG2 and IgG1. After inoculation with killed organisms, serum antibodies were predominantly IgG1, although IgG2, IgM, and, in intranasally vaccinated mice, IgA were also present. Only IgG1 was detected in lung washings from mice vaccinated systemically, but IgA and IgG2 were present in addition in animals vaccinated intranasally. Immunofluorescence studies indicated that some antibody in lung washings from the latter group of animals was produced locally. A comparison of the levels of various class-specific antibodies and resistance to intranasal challenge suggested that local antibody of any immunoglobulin class is capable of mediating resistance in the lungs to M. pulmonis infection.     

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     

Detection of Japanese encephalitis virus immunoglobulin M antibodies in serum by antibody capture radioimmunoassay.

An assay for detecting human immunoglobulin M (IgM) antibodies to Japanese encephalitis (JE) virus was developed by using the antibody capture solid-phase radioimmunoassay approach (JE IgM ACRIAAA). Heavy-chain-specific goat antihuman IgM was first bound to the wells of a polyvinyl microtiter plate, and successive steps involved sequential binding of test sample IgM, acetone-extracted mouse brain JE antigen, and (125)I-labeled flavivirus hyperimmune human IgG. Among 20 patients hospitalized in Bangkok with clinical diagnoses of acute encephalitis, and with acute flavivirus infections proven by hemagglutination inhibition (HAI) serology, 16 had detectable (positive/negative [P/N] ratio, greater than 3.0) JE IgM ACRIA antibodies in the acute-phase serum specimen, and 19 had such antibodies in the convalescent-phase serum specimen. Convalescent patient sera regularly had higher P/N values than the corresponding acute-phase sera (mean +/- 1 standard deviation = 13.0 +/- 9.3 with acute-phase sera and 25.8 +/- 19.6 with convalescent-phase sera). JE virus-infected patients with HAI serological responses indicative of a primary flavivirus infection had higher JE IgM ACRI P/N responses than did those patients whose serological response indicated past exposure to other flaviviruses. None of 70 serum specimens from healthy Thai adults and children with serum JE HAI antibodies had detectable JE IgM ACRIA activity (P/N ratios all less than or equal to 3.0). Biological false-positives with low P/N ratios (range, 3 to 15) were found in sera from patients with acute or recent infections with flaviviruses other than JE virus but could be differentiated by the fact that these sera gave higher P/N ratios with homologous antigens than with JE virus. False-positive reactions with low P/N ratios (range, 3 to 6) due to serum rheumatoid factor activity were differentiated by testing with control antigen. The JE IgM ACRIA technique permits a rapid, accurate diagnosis of acute JE virus infections in both patients with and those without previous exposure to other flaviviruses.     

Antigenic differences among multiply charged Moloney murine leukemia virus p30 polypeptides found inside infected cells

At least three Moloney murine leukemia virus (M-MuLV) p30 polypeptides (p30's), viz., a major species at pI 6.3 and two minor ones at pI 6.1 and pI 6.6, have previously been identified in purified virions by 2-dimensional gel electrophoresis and chromatofocusing (Katoh, I., Yoshinaka, Y. and Luftig, R.B. (1984) J. Gen. Virol. 65, 733-741). We have observed a similar, but distinctive pI pattern for [35S]methionine-labeled MuLV p30's in lysates from chronically infected (MuLV) cells. The variation in pI pattern of the intracellular MuLV p30's was dependent on the type of p30 reactive antibody used for immunoprecipitation. Specifically: a p30 spot with pI 6.3 was always precipitated as the major spot with three different antibodies, minor spots with pI 6.0 and 6.6 were variably seen dependent on the antibody used, and an intracellular p30 spot at pI 6.1 was only precipitated with a rat p30 monoclonal antibody but not with monospecific mouse or intact MuLV cross-reacting p30 sera. These results indicate that first, there are differences between the pI pattern of virion and intracellular MuLV p30's, and second, the antigenic determinants of intracellular p30's vary dependent on the antibody used for immunoprecipitation.     

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.     

Antibody response to rabies virus in Syrian hamsters.

Syrian hamsters were injected with inactivated, attenuated, and virulent rabies virus (RV), and the antibody response was quantified by a neutralization test and the immunoglobulin class of the virus antibody was characterized by indirect fluorescent microscopy. Serum antibodies to RV were found to be predominantly of the immunoglobulin G2 (IgG2) class, although IgG1 anti-RV also were detected in high-titered sera obtained after secondary challenge. Brain extracts of hamsters inoculated intracerebrally with RV contained only IgG2 anti-RV. IgA and IgM anti-RV were not detected. The preferential IgG2 response to RV is in marked contrast to the isolated IgG1 response detected after inoculation of hamsters with soluble purified protein antigens.     

Relationship of in vitro phagocytosis of serotype 14 Streptococcus pneumoniae to specific class and IgG subclass antibody levels in healthy adults

The role of specific IgG2 antibody in the protection against serious infection with Streptococcus pneumoniae is unclear. We therefore decided to investigate the relationship between serum antibody levels and opsonization and phagocytosis of this microorganism. We have measured serum IgM, IgA and IgG subclass antibody specific for pneumococcal capsular polysaccharide and in vitro phagocytosis of serotype 14 pneumococcus by polymorphs, in healthy adults before and after immunization with Pneumovax II. IgM and IgG2 were the predominant anti-pneumococcal antibodies seen, IgA and IgGl being present at low titre. No significant relationship of phagocytosis with specific IgM and IgA antibodies was found. However, both specific IgG 1 and IgG2 antibodies in post-immunization sera correlated significantly with phagocytosis of the pneumococcus in the presence of complement (r= 0.57, P= 0.029 and r= 0.59, P= 0.022 respectively). After heat-inactivation, the remaining opsonic activity of sera correlated only with levels of specific IgG2 antibody (r= 0.61, P = 0.0006). Whereas phagocytosis supported by specific IgG 1 and IgG2 antibody to serotype 14 pneumococcus after immunization is mediated by complement activation, IgG2-specific antibody in high titre may also be able to function by complement-independent interaction with Fcγ receptors on polymorphs.     

Double-conjugate enzyme-linked immunosorbent assay for immunoglobulins G and M against Treponema pallidum

An enzyme-linked immunosorbent assay (ELISA) for the simultaneous measurement of immunoglobulin G (IgG) and IgM was developed to detect antibodies to Treponema pallidum. Wells of polystyrene microtiter plates were coated with T. pallidum antigen, diluted patient serum was added, and IgG and IgM which bound to the T. pallidum antigen were measured by the simultaneous addition of alkaline phosphatase-labeled anti-human IgG and horseradish peroxidase-labeled anti-human IgM. Bound IgG was detected first, followed by bound IgM. After development of the procedure, 145 categorized sera were evaluated: 60 from individuals without syphilis; 62 from patients with syphilis, including 22 with primary, 20 with secondary, and 20 with latent phases of syphilis; and 23 from patients with rheumatoid arthritis. Of the 60 sera from individuals without syphilis, 100% were nonreactive for IgG antibody and 16% were reactive for IgM. Of the 23 sera from patients with rheumatoid arthritis, 3 were reactive for IgG and 3 were nonreactive for IgM. Of the 62 sera from patients with syphilis, 61 (98%) were reactive for IgG antibody with increased titers as the stage of syphilis increased, whereas IgM reactivity decreased. This enzyme-linked immunosorbent assay appears to be a simple method for the simultaneous measurement of antibodies under equal assay conditions.