Type-specific Determinants Research Articles

Immunochemical analysis of antigenic determinants of Chlamydia trachomatis by monoclonal antibodies.

Monoclonal antibodies to outer membrane of Chlamydia trachomatis lymphogranuloma venereum (LGV) strain L1 (440-L) were used for antigen characterization. Two separate type-specific antigenic determinants (epitopes) and one species-specific epitope were represented in the major outer-membrane protein (MOMP, molecular weight 40 000) of homologous (440-L) and heterologous (IOL-1962, 810-B) L1 strains. One of the type-specific antibodies, the reactivity of which was not affected by oxidation or reduction of the antigenic sites, partially prevented the binding of species-specific antibody as determined by solid-phase radioimmunoassay. The binding of type- and species-specific antibody could be destroyed with proteolytic enzyme treatment. The reactivity of genus-specific monoclonal antibody originating from another fusion (L2, 434-Bu) was unaffected by proteolytic treatment but was sensitive to periodate, indicating the carbohydrate nature of the genus-specific epitope. Heating of antigens had no effect on the affinity of monoclonal antibodies studied.

Strain-specific determinants on the major phosphoprotein of murine mammary tumor viruses

The major phosphoprotein (p23) of murine mammary tumor virus (MuMTV) was purified for C3H virus, using a combination of alkyl agarose chromatography and gel filtration. Monospecific antiserum was raised against glutaraldehyde crosslinked polymers of purified p23, and the antiserum was then used with iodinated p23 to develop a competition radioimmunoassay. Phosphoprotein p23 was shown to contain both group- and type-specific antigenic determinants in assays using detergent-disrupted virions of MuMTV strains C3H, RIII, GR, and DBA/2 as competing antigens. Tryptic and chymotryptic peptide mapping analysis of p23 polypeptides isolated from MuMTV strains C3H, RIII, GR and DBA/2 showed that the p23s of each virus strain could be clearly distinguished from each other.

Quantitation of the relatedness of reovirus serotypes 1, 2, and 3 at the gene level

As judged by the ability of their genomes to hybridize, reovirus serotypes 1 and 3 are related to the extent of about 70% to each other and about 10% to serotype 2. Two techniques were developed to measure the extent to which the individual cognate genes of these serotypes are related. Both involve comparison of heterologous hybrid genes that contain plus and minus strands of genes of different serotypes with homologous hybrid genes (molecules formed by reannealing the separated plus and minus strands of the same gene). In the first technique, the amounts in such hybrids of material sensitive to ribonuclease under standard conditions were compared; in the second, their relative electrophoretic mobilities. The results obtained with the two techniques agreed well. They showed that for the three reovirus isolates examined (the Lang strain of serotype 1, the D5 Jones strain of serotype 2, and the Dearing strain of serotype 3), all 10 genes of serotypes 1 and 3 are much more closely related to each other than to the genes of serotype 2. Although this result relates to only three isolates of mammalian reovirus, it suggests that the gene sets of reovirus serotypes 1, 2, and 3 evolved independently of each other. Apparently double infection of hosts with strains of two reovirus serotypes, which would very likely yield recombinants, occurs infrequently and/or such recombinants have a lower survival advantage than strains containing “pure” gene sets. The results also show that the gene that has diverged most markedly during evolution is the S1 gene, the gene that encodes the minor outer shell capsid protein σ1 which is the reovirus cell attachment protein and hemagglutinin and possesses the most type-specific antigenic determinants. The serotype 1 and 3 S1 genes are about 10% homologous, serotype 2 and serotype 1 or 3 S1 genes about 3%. The genes that have diverged least are the three L genes (85–90% homology for the serotype 1 and 3 L genes). In all cases, the serotype 2 and serotype 1 or 3 genes exhibit no more than 20%, and often less than 10% homology. In spite of this high degree of divergence, the antigenic determinants on proteins encoded by genes of serotype 2 on the one hand and serotypes 1 and 3 on the other hand are, with the exception of those on proteins σ1, highly conserved, and the 60 to 80 nucleotides at the 5′- and 3′-termini of at least three sets of cognate genes (L3, M3, and S2) of all three serotypes, serotype 1 and 3 as well as serotype 2, are highly homologous and in some instances almost identical. Thus, while some regions of reovirus genes have diverged greatly during evolution, others have been highly conserved.

Monoclonal and polyclonal antibody studies of VSV(hrMMTV) pseudotypes

By phenotypic mixing between the host range (hr) variant of mouse mammary tumor virus (MMTV) and the is-045 mutant of vesicular stomatitis virus (VSV), we were able to produce high titers of “complete” pseudotypes bearing the VSV genome and hrMMTV envelope. Four “strains” of pseudotypes designated VSV(C 3H), VSV(GR), VSV(RIII), and VSV(C 3Hf) were generated. To find out whether group-specific and type-specific neutralizing antigenic determinants (epitopes) were expressed on some of these pseudotypes, we utilized several monoclonal antibodies to MMTV-gp52 and sera from mammary tumor-bearing C 3H/HeN mice in neutralization assays. Four monoclonal antibodies to MMTV-gp52, which were group specific in solid-phase MMTV binding assays, were found to neutralize the infectivity of VSV(C 3H), VSV(GR), VSV(RIII), and VSV(C 3Hf), indicating the expression of group-specific “neutralizing epitopes” on these pseudotypes. One monoclonal antibody to MMTV-gp52 that was C3Hf type specific in MMTV binding assays was found to neutralize only the infectivity of VSV(C 3Hf) and not VSV(C 3H), VSV(GR), or VSV(RIII). In addition, polyclonal antibodies were found in sera of mammary tumor-bearing C 3H/HeN mice which neutralized specifically the infectivity of VSV(C 3H). Such antibodies were not found in the sera of normal C 3H/HeN mice or normal BALB/c mice. These results indicate that group-specific “neutralizing epitopes” were expressed on VSV(C 3H), VSV(GR), VSV(RIII), and VSV(C 3Hf). Furthermore, they suggest that type-specific “neutralizing epitopes” were expressed on VSV(C 3H) and VSV(C 3Hf) pseudotypes.

Identification of distinct antigenic determinants on dengue-2 virus using monoclonal antibodies.

Monoclonal antibodies directed against antigenic determinants of the New Guinea C strain of dengue-2 virus were obtained from lymphocyte hybridomas produced by fusing immune mouse lymphocytes with mouse myeloma cells. Hybridoma cell culture supernatants were screened by using a radioimmunoassay employing detergent-solubilized dengue-2 infected cell antigens. Monoclonal antibodies in ascitic fluids induced by 22 selected hybridomas were characterized by the hemagglutination-inhibition, plaque reduction neutralization, immunofluorescence, and complement-fixation tests. Both type-specific and broadly cross-reactive antibodies were observed, and immunoglobulin subclasses IgG1 and IgG2a were represented in both groups. At least three distinct antigenic determinants on the virion were defined using these antibodies. A single hybridoma produced antibody which recognized a dengue-2 virus type-specific determinant and exhibited high titered neutralization but had a low titer by hemagglutination inhibition. Four preparations reacted with a type-specific determinant and exhibited hemagglutination inhibition but did not neutralize. Seventeen hybridomas produced antibodies which were broadly cross reactive in all tests. Only two preparations reacted by complement fixation with dengue-2 antigens; both were cross reactive. Immunofluorescence specificity or cross reactivity correlated with neutralization and/or hemagglutination-inhibition. The dengue-2 virus type-specific antibody useful for identification of dengue-2 infected cells by immunofluorescence has been deposited in the Hybridoma Cell Bank of the American Type Culture Collection.

Effect of monoclonal antibodies on limited proteolysis of native glycoprotein gD of herpes simplex virus type 1.

We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one type-common group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.

Detection of type-specific antigenic determinants of murine mammary tumor viruses with cell-mediated immune assays.

Exposure of lymphoid cells from normal and mammary tumor-bearing Balb/cCrgl mice to MMTV antigens result in positive responses in blastogenic transformation and migration inhibition reactions. The levels of these responses appear to depend on the murine strain source of the viral preparations. Enhanced reactivities were observed in cultures exposed to purified MMTV from culture fluids of mammary tumor cells of spontaneous origin in the C3H strain when compared to purified MMTV derived from milk of mice from the RIII strain. To ensure the virus relatedness of the differences observed, MMTVs obtained from a common source, i.e., the Crandall feline embryo kidney cell line, were employed in blastogenesis assays. Parallel testing of feline cell-derived C3H, RIII, and GR mammary tumor viruses resulted in different levels of [3H]thymidine incorporation by spleen cells from Balb/cCrgl mice over a range of viral concentrations. These results indicate that it is possible to detect various types of MMTV variants with cell-mediated immune assays. The diverse functional reactivities elicited by these viruses may be related to the differences observed in their bioactivities as determined by incidence and development of mammary tumors and the potential of causing metastases.

Immunochemistry of the moloney MuLV gp71 molecule—I. Distribution of antigenic determinants on the products of digestion by Staphylococcus aureus protease

The antigenic characterization of Moloney murine leukemia virus gp71 was carried out by cleavage with Staphylococcus aureus protease. The whole molecule and two fragments of 43,000 and 32,000 daltons were immunoprecipitated with monospecific anti-type, group and interspecies antiserum. The three native molecules were recognized by anti-type antibodies, but only the gp71 and the 32,000-dalton molecule seemed to retain group-specific antigenic determinants. No interspecies determinants were detected on the two products of digestion. A similar pattern of precipitation was found with SDS-denatured antigens. The results suggest that sequential structures contribute to the type-specific antigenic determinants, because they were not destroyed by SDS unfolding or by the change in the protein conformation that follows enzymatic cleavage. Interspecies determinants seem to be mainly conformational, since they disappeared from the whole molecule and the two peptides after the same treatment. Group-specific determinants appear to be intermediate: they disappeared after the conformational change on the native 43K but not on the 32K peptide and the monospecific anti-group antibody showed an intermediate ability to precipitate the SDS-unfolded antigens.

Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4.

Monoclonal antibodies were prepared against herpes simplex virus type 1 (strain 14012) by two immunization procedures. Procedure A utilized infectious virus propagated in mouse cells, and procedure B utilized mouse cells infected with herpes simplex virus in the presence of cycloheximide and harvested 1 h after removal of the inhibitor. A total of 52 monoclonal antibodies were obtained against 10 herpes simplex virus proteins, including four glycosylated proteins (a 110,000-molecular-weight protein, gB, gC, and gD) and six nonglycosylated proteins (a 68,000-molecular-weight protein, ICP 9, ICP 8, ICP 6, ICP 5, and the immediate-early ICP 4). The antibodies were assayed against herpes simplex virus types 1 and 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioimmunoprecipitates, immunofluorescence, and neutralization. Using the reagents prepared, we concluded that the 110,000-molecular-weight protein, gD, ICP 9, ICP 9, ICP 6, and the 68,000-molecular-weight protein express both type-specific and cross-reactive antigenic determinants. In contrast, nine antibodies against gB all cross-reacted with herpes simplex virus type 2, whereas eight antibodies to gC all reacted type specifically.

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL.

Preparation and characterization of antisera to electrophoretically purified SA11 virus polypeptides.

Antisera to SA11 virus proteins were prepared by immunizing rabbits with individual polypeptides separated by polyacrylamide gel electrophoresis under reducing or nonreducing conditions; the resulting antisera were characterized by four immunological methods. Results of complement fixation tests with double-shelled rotavirus particles and sera raised against reduced or unreduced proteins of the outer shell of the virus suggested the presence of common antigenic determinants in the outer capsid layers of SA11 and the Northern Ireland strain of calf rotavirus. In this test, antisera to outer shell polypeptides gp34 (O2) and gp25 (O4) cross-reacted with calf rotavirions, whereas those to p62 (O1) and p26 (O3) reacted only with the homologous virus. Antisera to the reduced outer shell proteins of the virus did not neutralize viral infectivity, nor did they possess hemagglutination inhibition activity. Evidence suggesting the presence of type-specific antigenic determinant(s) in the major inner protein p42 (I4) of SA11 virus, capable of inducing neutralizing antibody, is presented and discussed. Antisera produced against unreduced gp34 and p26 polypeptides of the virus contained type-specific neutralizing antibodies. Polypeptide gp34 was also capable of inducing hemagglutination inhibiting antibody. All of the antisera to unreduced polypeptides had agglutinating activity against double-shelled particles of homologous and heterologous rotaviruses.

Herpes simplex virus type 1 and 2 intracellular p40: type-specific and cross-reactive antigenic determinants on peptides generated by partial proteolysis.

Intracellular p40 is a class of protein ranging in molecular weight from 39,000 to 45,000 that is immunoprecipitated from herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell extracts by mouse monoclonal antibodies or guinea pig antisera against HSV-1 and HSV-2 nucleocapsid p40. Analysis by a two-dimensional gel system showed that HSV-1 and HSV-2 intracellular p40 each consisted of three major components. However, these HSV-1 and HSV-2 proteins differed in charge and size. Analysis of Staphylococcus aureus V8 protease partial digests by two-dimensional gel electrophoresis indicated that none of the peptides of HSV-1 and HSV-2 intracellular p40 were identical. Immunoprecipitation of the partial digest products of intracellular p40-1 and p40-2 with homologous and heterologous guinea pig antisera resulted in the precipitation of various combinations of peptides indicating the presence of either type-specific or cross-reactive antigenic determinants.

Immunological characterization of a mammary tumour virus from Swiss mice: multiple epitopes associated with the viral gene products.

The major antigens of a mouse mammary tumour virus (MMTV) isolated from the milk of exogenously infected MB+ Swiss mice were compared with the viral components of other MMTV strains by using the methodology developed by Teramoto et al. (1977 a). The anti-gp47 (Swiss) serum differentiated between type- and group-specific antigenic determinants on the major glycoprotein; two distinct type-reactivities were demonstrated, one of them being shared by the MMTVs (Swiss) and (RIII), and the other by the MMTVs (C3H) and (GR). The MMTVs (Swiss) and (RIII) also reacted identically in a 'type-specific' assay using an anti-p28 (Swiss) serum, but a distinct reactivity was observed with the MMTV (C3H). In the isologous test where an anti-(total RIII) serum was used to bind intact, externally labelled RIII virions, the Swiss virus was seen to possess a type-specific determinant on its surface which distinguished itself from both MMTVs (RIII) and (C3H). The Swiss/fC57BL mice (which are devoid of the milk virus and for this reason are referred to as MB-), expressed only the internal virus antigens in their mammary glands. Under the 'type-specific' assay conditions, the p28 antigen present in the mammary gland extracts of the MB+ mice was indistinguishable from the p28 antigen purified from the Swiss virion, but was clearly distinct from the p28 reactivity present in the mammary gland extracts of the MB- mice. The p28 (MB-) antigen may thus represent the expression of an endogenous virus sequence different from the milk virus genome.

Use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease.

Monoclonal antibodies HCl and HD1, directed against herpes simplex virus type 1 (HSV-1) glycoproteins gC and gD, respectively, were evaluated for their ability to passively immunize mice against acute virus-induced neurological disease after footpad inoculation with HSV-1 or herpes simplex virus type 2 (HSV-2). Control virus-infected mice receiving a single intraperitoneal injection of normal serum died within 7 to 10 days after the spread of virus from footpad to spinal cord and brain. However, a single intraperitoneal injection of either HCl or HD1 antibody protected mice from neurological illness and death when administered to HSV-1 (strain HTZ)-infected mice at either 2 h before virus challenge or at 24 h after virus inoculation. To determine the in vivo specificity of the antibodies, passive transfer studies were performed with mice infected with the MP strain of HSV-1, a mutant of HSV-1 (mP) which is defective in the production of glycoprotein gC. Whereas HD1 antibody decreased the incidence of neurological illness in MP- and mP-infected mice, HCl antibody, which protected mP-infected animals, failed to protect mice infected with the MP strain. When HD1 antibody was administered to HSV-2 (strain G)-infected mice at either 2 h before virus challenge or at 6 h (but not 24 h) after virus inoculation, 100% of the infected animals receiving HD1 antibody survived. In contrast, 100% of HSV-2 (strain G)-infected animals passively immunized with HCl antibody developed neurological illness and died. These results provide in vivo evidence that the HSV-induced glycoprotein gC expresses type-specific antigenic determinants, whereas glycoprotein gD expresses type-common determinants.

Type-specific determinants on proteins of an endogenous C3H mouse mammary tumor virus (MMTV) distinguish this virus from highly oncogenic exogenous MMTVs

The genetically transmitted endogenous MMTV isolated from C3H mice after removal of the milk-transmitted virus by foster nursing is designated C3Hf MMTV to distinguish it from the highly oncogenic milk-transmitted exogenous virus designated C3H MMTV. We have isolated a MMTV-expressing C3Hf mammary tumor cell line which has no exogenous proviral sequences detectable by analysis of DNA fragments generated by Pst I restriction endonuclease. This cell line produced sufficient C3Hf MMTV to allow purification of the major proteins and an antigenic comparison of this virus with highly oncogenic exogenous MMTVs from C3H, GR, and RIII strains of mice. The envelope glycoproteins, gp52 and gp36, purified from the C3Hf MMTV, were found to have both group- and type-specific reactivities. Only C3H MMTV gave incomplete competition in the gp36 assay and, therefore, could be distinguished from C3Hf, RIII, and GR MMTVs which gave complete competition. Unique antigenic determinants exist on the gp52 of C3Hf MMTV since it is the only virus to give complete competition in the gp52 radioimmunoassay. GR MMTV competed only 60%, whereas C3H and RIII MMTVs gave 80% competition. This was the first demonstration that RIII and C3Hf MMTVs were immunologically distinct. Only group-specific reactivity was found with the gag-coded MMTV p27; however, group and class antigenic determinants were found on the gag-coded MMTV p10.

Herpes simplex virus antigens and antibodies: a survey of studies based on quantitative immunoelectrophoresis.

Herpes simplex virus type 1 and type 2 antigens extracted from infected cells with use of nonionic detergent produce a number of distinct immunoprecipitates in crossed immunoelectrophoresis (CIE) with hyperimmune rabbit antibodies to herpes simplex virus. The analytic power of CIE combined with the use of single immunoprecipitates made it possible, for a number of individual antigens, to determine (1) the immunogenicity of the antigens in experimentally infected animals and naturally infected humans, (2) the distribution of type-common and type-specific antigenic determinants on the antigens, (3) the biochemical structures of the antigens, (4) the localization of the antigens on the infectious virus particle and in infected cells, and (5) the ability of the antigens to act as targets for neutralizing antibodies and for antibody-complement and antibody-dependent cellular cytotoxicity. Two herpes simplex virus type-common, membrane-bound glycoproteins, Ag-11 (gA + gB) and Ag-8 (gD), which are localized on the surface of the viral envelope and in the membranes of infected cells, are the principal immunogens in infected humans. The monospecific antibodies against these two antigens neutralize the virus and cause cytolysis of infected cells in the tests mentioned above. Antibodies to glycoproteins carrying herpes simplex virus type-specific antigenic determinants also are present in infected humans. These antigens can be utilized in herpes simplex virus type-specific human serology, and the corresponding monospecific antibodies can be used for the typing of herpes simplex virus isolates.

Radioimmunoassay for infectious primate retrovirus reverse transcriptase: characterization, comparison with conventional immunologic assays and applicability to cellular extracts.

SSV reverse transcriptase (RT) was purified to homogeneity and used in a radioimmunoassay. Following iodination, the homogeneity of the protein and its identity with RT were confirmed by several criteria: (1) its molecular weight on an SDS-polyacrylamide gel; (2) its precipitation by anti-SSV RT but not by antisera to other SSV proteins; (3) its cross-reactivity in RIA with antisera to other retroviral polymerases; (4) its competition in RIA by active homogeneous SSV RT but not by other purified SSV proteins; and (5) its competition in RIA by only those fractions from a poly(U)-Sepharose column possessing SSV RT activity. Competition of the labelled probe with disrupted retroviruses of the infectious primate group showed that, while a homologous RIA detected only type-specific enzyme determinants, it did not distinguish the various woolly-gibbon retroviral DNA polymerases. A more broadly reactive heterologous assay utilizing an antiserum to R-MuLV RT detected group- but not interspecies-specific enzyme determinants. A comparison of immunologic assays for RT showed that: (1) highly purified RT is not essential for reliable results in enzyme neutralization or enzyme binding assays; (2) the greater sensitivity of enzyme binding compared to enzyme neutralization assays is a function of the antibody, not of the antigen. Competition RIAs using extracts of virus-infected cells showed that infectious primate retrovirus RT could be measured in a crude system and that cellular DNA polymerases alpha, beta and gamma did not compete with the labelled probe.

Monoclonal Antibodies Identify Individual Determinants on Mouse Mammary Tumor Virus Glycoprotein gp52 with Group, Class, or Type Specificity

Hybrid cell lines producing monoclonal antibodies against the C3H strain of mouse mammary tumor virus (C3H MMTV) were prepared by the fusion of mouse myeloma cells with the lymphocytes of BALB/c mice that were immunized with C3H MMTV. Approximately 10% of the hybrid cells initially plated after cell fusion produced immunoglobulins that reacted in antibody-binding assays with C3H MMTV; 40 of these cells were cloned, and 6 eventually yielded stable cell lines. High concentrations of monoclonal antibodies (5 to 20 mg/ml) were obtained from serum and ascites fluid of syngeneic mice inoculated with the hybrid cells. All of the monoclonal antibodies were directed against the envelope glycoprotein gp52. Three of the hybrid cell lines produced immunoglobulins of the immunoglobulin M subclass and three produced immunoglobulin G2a. The monoclonal antibodies showed limited charge heterogeneity in light and heavy chains when analyzed by high-resolution, two-dimensional gel electrophoresis. Three serologically distinct specificities were observed when these ascites fluids were tested against different strains of MMTV. The antigenic determinants detected were the following: (i) a type-specific determinant unique to the C3H strain of MMTV; (ii) class-specific determinants shared between C3H and GR MMTVs; and (iii) a group-specific determinant found on C3H, GR, RIII, and the endogenous C3H (C3Hf) MMTVs. Because monoclonal antibodies recognize single antigenic determinants, these results demonstrate for the first time that the three patterns of antigenic reactivity for MMTV are related to individual determinants on the gp52 molecule and also clearly show that one strain of MMTV can be distinguished from other strains.

Heterogeneity of type-specific and cross-reactive antigenic determinants within a single M protein of group A streptococci.

The heterogeneity of a pepsin extract of type-24 M protein (pep M24) was demonstrated by absorption of type-specific and cross-reactive human antisera with M protein fragments and heterologous serotypes of M proteins, pepsin extract of type-5 M protein (pep M5) and pepsin extract of type-6 M protein (pep M6). 2 of 12 individuals immunized with pep M24 developed significant rises in antibody titers against pep M5 and pep M6, as measured by the enzyme-linked immunosorbent assay. The sam individuals also developed opsonic antibodies against type-6, but not type-5, streptococci, which suggested the development of cross-protective immunity. Inhibition studies of one of these sera with the heterologous pep M proteins showed that the cross-reactive antibodies against pep M6 could not be blocked by high concentrations of pep M24, the immunizing antigen; these antibodies could be blocked, however, by cyanogen bromide-derived peptide fragments of pep M24, which suggested that the cross-reactive antibody was raised against an inaccessible site(s) in the pep M24 molecule. Inhibition studies of type-specific immune sera with pep M24 and peptides derived therefrom indicated that the M protein molecule contained multiple distinct as well as identical type-specific antigenic determinants that are unequally distributed among the seven derived peptide fragments.

Effect of chemical modification and fragmentation on antigenic determinants of internal protein p30 and surface glycoprotein gp70 of type C retroviruses.

The effects of protein modification on the antigenic determinants of p30 and gp70 of type C retroviruses were investigated by using solid-phase competition radioimmunoassays. Proteins were modified by reduction with 2-mercaptoethanol and subsequent carboxymethylation of SH groups with iodoacetamide or by amidination of alpha and epsilon amino groups with methylacetimidate. The type-specific determinants of gp70 were found to be conformational in nature, as they were destroyed by these chemical modifications. Group- and interspecies-specific determinants of gp70 antigens, however, appear to be sequential and do not involve residues susceptible to these chemical reagents. Conformation-dependent type-specific determinants of p30 were affected only by methylacetimidate. Group- and interspecies-specific determinants of p30 are similar to those of gp70 in that they also appear to be sequential antigenic sites. Therefore, the broadly reactive group- and interspecies-specific determinants of gp70 and p30 can be followed into small peptides. Accordingly, a cyanogen bromide cleavage fragment derived from the carboxyl-terminal one-third of Rauscher leukemia virus p30 was found to carry group-specific determinants but no detectable interspecies-specific determinants. In contrast, a peptide obtained by limited trypsin cleavage of p30, which was derived from the NH(2)-terminal region of the protein, contained at least one of the interspecies determinants shared with feline leukemia virus p27.