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PubMed1990

Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages.

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Animals↗
PubMed1991

Resistance to LDL oxidative modifications of an N-terminal apolipoprotein B epitope.

The immunoreactivity of human apolipoprotein B (apo B) towards 5 monoclonal antibodies was studied by enzyme immunoassay in native and in vitro oxidized low density lipoproteins (LDL). LDL oxidative modifications were obtained by incubation with either copper ions or an association of lipoxygenase and phospholipase A2. The monoclonal antibodies used in the inhibition analysis were directed to epitopes located in the amino-terminal region (1D1), in the middle part (2D8, L7, 4G3) and in the carboxy-terminal region (L3) of the apo B molecule. The results demonstrated that the immuno-reactivity of 1D1 epitope was little affected by LDL oxidation with copper ions or lipoxygenase plus phospholipase A2, whereas the immunoreactivity of the other epitopes were markedly decreased by these LDL modifications. Immunoreactivity changes were more important in L3 and L7 epitopes than in 2D8 and 4G3 epitopes. Since it is known that L3 and L7 epitopes are located in apo B domains rich in lipid-associated peptides whereas 1D1 is in a domain poor in such peptides, these results suggest a relationship between the lipid environment of an apo B epitope and its susceptibility to alteration by LDL oxidation.

Antibodies, Monoclonal↗
PubMed1991

Stability of monoclonal antibody-defined epitopes.

Epitope instability can limit the applications of monoclonal antibody (mAb) technology in laboratory and clinical research. We exposed a group of representative antigens on human hepatocellular carcinoma (HCC) cells to physiochemical insults to study epitope stability as measured by mAb immunoreactivity. Each epitope was found to have a unique pattern of instability which serves to biophysically characterize the antigen and defines the conditions to which the antigen can be exposed during laboratory and clinical investigations. Individual antigens were found to be unstable within a surprisingly well defined window of solvent polarities while being stable on either side of that window. Several antigens were observed to be unstable when exposed to transient changes in pH. When a critical temperature between 42 degrees C and 65 degrees C was achieved, epitopes which were thermosensitive underwent a sudden loss in immunoreactivity. This critical temperature was found to be pH dependent. The effects of polarity, pH, and temperature on epitope stability are consistent with changes in protein structural conformation. In addition, we found that certain fixatives cause a time and concentration dependent loss of epitope immunoreactivity. This study provides a rapid and easy determination of monoclonal antibody-defined epitope stability; the results of which serve to guide further studies on the antigen and to characterize the antigen on the basis of its unique physiochemical stability.

Animals↗
PubMed1992

Multiple epitope interactions in the two-step sandwich immunoassay.

The 'hook' effect as related to the two-step sandwich immunoassay has been investigated experimentally and theoretically. The multiple epitope interactions between the analyte and the labeled antibody cause a 'hook' in the two-step sandwich immunoassay. Three different analytes and monoclonal antibodies were chosen to carefully demonstrate the effect of the analyte characteristics on this immunoassay. Two monoclonal antibodies against two different epitopes of biosynthetic human growth hormone (hGH) was the simplest model for this study. The sandwich immunoassay for hGH shows no 'hook' effect. The non-covalent dimeric form of hGH (D-hGH) possesses two repeating epitopes which is the simplest model for an analyte having a discrete number of repeating epitopes. The D-hGH assay demonstrated a 'hook' effect in the two-step sandwich immunoassay if the labeled antibody was allowed to interact with more than one epitope. In a third system multiple epitope interactions with the labeled antibody were observed using ferritin. The effect of the analyte concentration and the liquid-phase antibody have been examined to elucidate the nature of these various interactions. The cause of the 'hook' effect in the two-step sandwich immunoassay is attributed to the desorption of the bound analyte due to a conformational change after the labeled antibody interacts with several epitopes of the adsorbed analyte.

Antibodies, Monoclonal↗
PubMed1995

Topographical characterization of the DNA polymerase from Thermus aquaticus. Defining groups of inhibitor mAbs by epitope mapping and functional analysis using surface plasmon resonance.

Among 24 unique monoclonal antibodies (mAb) generated against Taq polymerase (TaqPol) 13 are potent inhibitors of polymerase activity. These antibodies have been sorted into groups defined by their topographical or functional properties using surface plasmon resonance-based methods to examine three different types of interactions. An epitope map of all the pairwise interactions among all 24 antiTaqPol antibodies revealed the surface of TaqPol as a complex space populated by isolated antigenic domains with no evident relationship to each other. 11 discrete epitopes or epitope clusters were defined and potent inhibitors bound to sites within seven of them. The second method examined the ability of antiTaqPol mAbs to bind to recombinant forms of the constituent functional domains of TaqPol, the N-terminal 5'-nuclease domain and the C-terminal polymerase domain. Most of the antibodies demonstrated a clear specificity for one domain or the other. The third method measured the ability of each mAb to block the interaction of TaqPol with a preformed, immobilized primer:template complex (PTC). Some antibodies had no effect on this interaction while others effectively blocked it. Together these latter two methods resolved many of the antibodies into five distinct groups. In addition, antibodies that bound to overlapping epitopes in the pairwise interaction analysis were members of the same group by their interaction with the polymerase fragment and PTC. Three groups of polymerase inhibitors were clearly resolved by these analyses: (1) those that recognize an epitope on the 5'-nuclease domain and have no effect on the interaction of TaqPol with PTC; (2) those that recognize an epitope on the polymerase domain and block the interaction of TaqPol and PTC; and (3) those that recognize an epitope on the polymerase domain, but have no effect on the interaction of TaqPol with PTC. The surface of TaqPol bears at least three antigenic regions that are topographically and functionally distinct and may correspond to sites for inhibition of different steps in the enzymatic activity of TaqPol.

Antibodies, Monoclonal↗
PubMed1985

The neutralization site on the E2 glycoprotein of Venezuelan equine encephalomyelitis (TC-83) virus is composed of multiple conformationally stable epitopes.

The neutralization (N) site on the gp56 (E2) surface glycoprotein of the TC-83 vaccine strain of Venezuelan equine encephalomyelitis (VEE) virus has been characterized using monoclonal antibodies. Five new epitopes (E2d-h) were identified three of which could be mapped into the critical N site by using a competitive binding assay (CBA). Antibodies reactive with these three epitopes had either N or N and hemagglutination-inhibition activity. All epitopes contained within this N site elicited monoclonal antibodies that could protect mice from peripheral virus challenge. Antibodies reactive with the N site on other subtypes of VEE virus (IC and II) bound to, but failed to neutralize, TC-83 virus. Epitopes defined by these antibodies could be located outside of the N site on TC-83 virus by CBA. Antigenic activity of all epitopes except E2d was resistant to treatment with 2% SDS, 3% beta-mercaptoethanol, or cleavage with Staphylococcus aureus V8 protease. Those antibodies which defined epitopes located within the N site of TC-83 with CBA bound the same V8 fragments in immunoblots. Those antibodies which defined epitopes not located within the N site bound a different set of fragments than neutralizing antibodies. These results indicate that there is a specific N site on the E2 of VEE virus which undergoes significant antigenic drift while maintaining structural and functional integrity.

Animals↗
PubMed1988

Point mutations in glycoprotein gene of vesicular stomatitis virus (New Jersey serotype) selected by resistance to neutralization by epitope-specific monoclonal antibodies.

Antigenic variants of the New Jersey serotype of vesicular stomatitis virus (VSV-NJ) were isolated and cloned by selecting virus plaques resistant to neutralization by high-titered monoclonal antibodies (MAbs) directed to glycoprotein (G) epitopes V, VI, VII, or VIII. The G proteins of each neutralization-resistant virus variant also exhibited markedly reduced antigenic reactivity with each corresponding epitope-specific MAb as determined by enzyme-linked immuno-absorbent assay and by Western blot analysis. Loss of antigenic reactivity of certain mutant G proteins to a MAb other than the one used to select the mutant virus suggested close antigenic proximity, particularly for epitopes VI and VII. The virion RNAs coding for the entire G gene of the wild-type virus and 10 MAb-induced mutants were sequenced by primer DNA extension using the dideoxy method. Each mutant G gene exhibited only a single nucleotide change, leading in each case to a single amino acid substitution, as follows: Glu210----Lys for all three mutants selected by MAb14 (epitope VII); Pro268----Thr for one mutant selected by MAb12 (epitope VI); Ser277----Lys for all three mutants selected by MAb15 (epitope VIII); and Glu364----Lys for all three mutants selected by MAb11 (epitope V). These neutralizing MAb-selected mutations are clustered in the middle third of the 517-amino acid VSV-NJ G protein, presumably resulting in conformational changes that alter recognition of one or more antigenic determinants by a specific monoclonal antibody.

Amino Acid Sequence↗
PubMed1990

Epitope-specific antibody response to the surface antigen of duck hepatitis B virus in infected ducks.

In order to investigate the immune response to duck hepatitis B virus (DHBV) infection, newly hatched DHBV DNA negative ducklings were injected with infectious serum of sufficiently low DHBV-DNA titer to allow clearance of viremia. Of 20 injected ducklings, 13 (65%) became viremic. Of these, 6 (46%) cleared virus from the serum 3 to 22 weeks postinjection. The convalescent sera of these 6 animals were tested for an epitope-specific antibody response in a highly specific competitive inhibition assay using a panel of monoclonal antibodies against duck hepatitis B surface antigen (DHBsAg) that had been well-characterized. All 6 animals recovering from DHBV infection developed antibodies to epitopes on the preS and S proteins of DHBV. Antibody responses were highly variable with marked differences between animals in the extent and specificity of the antibody response. The humoral response to DHBsAg was prolonged in some animals but transient in others. No antibody to preS or S was detected in either preimmune sera or sera of control animals from an uninfected flock. Infected animals that did not clear viremia also remained antibody negative. The humoral responses to neutralizing preS epitopes III and V were weak but antibodies to two immunodominant epitopes on the preS region (II and B) were present in all 6 animals. The humoral response to the two epitopes in the S region was transient and of lower titer when compared to the two immunodominant preS epitopes. The two immunodominant preS epitopes may play an important role in clearance of DHBV infection in ducks.

Animals↗
PubMed1990

Vaccination and protection from a lethal viral infection: identification, incorporation, and use of a cytotoxic T lymphocyte glycoprotein epitope.

The outcome of infection by lymphocytic choriomeningitis virus (LCMV) is determined largely by the cytotoxic T lymphocyte (CTL) response of the host. In H-2b mice, the anti-glycoprotein (GP) response is directed to at least two epitopes, one located at GP aa 272-286 and a second in GP-1. Here we show that the second epitope can be minimally identified by amino acid residues GP 34-40 (AVYNFAT). The epitope is restricted by the Db class I glycoprotein. Characterization of these CTL epitopes allowed us to address the role(s) played by each epitope when expressed singly in the control of a lethal challenge with LCMV. Here we show that a single immunization with a recombinant vaccinia virus (VV) vaccine expressing LCMV GP aa 1-59 confers protection to H-2b mice from lethal LCMV infection. In contrast, a VV expressing LCMV GP aa 272-293, although recognized by CTL, does not protect. We show that the success or failure of protective immunization is determined by the ability of the immunizing sequences to prime for CTL in vivo. Although the GP 278-286 epitope when contained as a "minigene" fails to induce CTL, when incorporated in the normal GP "backbone" it successfully elicits CTL. These observations suggest that the "minimal" recognition sequence alone may not be sufficient to induce a protective CTL response in vivo. Thus a single CTL epitope can protect against a lethal virus infection, but to achieve an effective vaccine, the immunizing sequences must be carefully selected.

Amino Acid Sequence↗
PubMed1992

Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus.

Current serological assays using whole rubella virus (RV) as a target antigen for detecting RV-specific antibodies fail to define specific RV proteins and antigenic determinants such as hemagglutinin (HA) and virus-neutralizing (VN) epitopes of rubella virus. A panel of E1 deletion mutants and a subset of E1-specific monoclonal antibodies (MAb) were used for the initial analysis of HA and VN epitopes of E1 glycoprotein. A peptide region (E1(193) to E1(269)) was found to contain HA and VN epitopes. Using both overlapping synthetic peptides and truncated fusion proteins within this region, the HA epitope defined by MAb 3D9F mapped to amino acid residues E1(214) to E1(240), while two VN epitopes defined by MAb 21B9H and MAb 16A10E mapped to amino acid residues E1(214) to E1(233) and E1(219) to E1(233), respectively. The epitopes defined in this study are recognized by antibody whether or not the epitopes are glycosylated.

Amino Acid Sequence↗
PubMed1996

Localization of a calcium-dependent epitope to the amino terminal region of the Gla domain of human factor IX.

We have used site-directed mutagenesis to define the epitope of calcium-dependent monoclonal antibodies to human factor IX. We demonstrate that the calcium-specific epitope includes residues 1-11 of factor IX, with apparent contributions from other regions of the protein. Antibodies JK.IX-1, -3, and -4 had critical portions of their epitopes in the first eleven amino acids of the Gla domain. These three antibodies could bind to a chimera containing the factor VII Gla domain, in which surface residues 3, 4, 5, 9, 10, and 11 were modified to those of factor IX. In contrast, the epitope of JK.IX-2 was unaffected by mutations in residues 3-11 of factor IX, but was dependent on the amino terminal tyrosine residue. Furthermore, the calcium-dependent monoclonal antibodies, JK.IX-1, -3, and -4, whose epitope include residues 3 through 11. inhibit factor IX's binding to endothelial cells, for which the binding site on factor IX has been localized to this region. Our results, together with previous studies, confirm the existence of discrete calcium and metal-dependent epitopes within the Gla domain of factor IX and show that the calcium-specific epitope lies near the amino terminus region of this domain.

1-Carboxyglutamic Acid↗
PubMed1986

Human p85 glycoprotein bears three distinct epitopes defined by several monoclonal antibodies.

A glycoprotein of apparent mol. wt 85,000 isolated from human cells of B lineage by affinity to 50B4-IgG immunoadsorbent was shown previously to express two spatially distinct epitopes identified with monoclonal antibodies 50B4 and 50E6 [Letarte et al. (1985) Molec. Immun. 22, 113-124]. It is now demonstrated that the p85 glycoprotein is homologous to the F10-44-2 antigen, defined initially as a T-lymphocyte-granulocyte-brain antigen [Dalchau et al. (1980) Eur. J. Immun. 10, 745-749], the A1G3 human medullary thymocyte antigen [Haynes et al. (1983) J. Immun. 131, 1195-1200] and the A3D8 antigen defined on erythrocytes [Telen et al. (1983) J. clin. Invest. 71, 1878-1886]. The 50B4 antigen purified either from a B lymphoblastoid cell line or a leukemic cell line, at a concn ranging from 0.25 to 2.0 micrograms protein/ml, could block the binding of either F10-44-2 and F-10-62-1 (a related antibody) or A1G3 and A3D8 antibodies to human leukemic cells. All of these antibodies immunoprecipitated, from the purified antigenic preparation, a single glycosylated polypeptide chain of apparent mol. wt 85,000. Competitive binding studies indicated that these antibodies define at least three epitopes. The F10-44-2 and A3D8 antibodies blocked the reactivity of monoclonal 50E6-IgG with human leukemic cells and thus bind to an epitope identical or spatially related to the 50E6 epitope. Antibody F10-62-1 competed for the binding of 50B4-IgG to leukemic cells and thus recognizes the 50B4-like epitope. The A1G3 antibody blocked 30% of the binding of 50E6-IgG but did not inhibit the binding of 50B4-IgG: it is thus reacting with a third epitope on the p85 glycoprotein distinct from but close to the 50E6 epitope.

Antibodies, Monoclonal↗
PubMed1991

Epitope expression and partial structural characterization of F62 lipooligosaccharide (LOS) of Neisseria gonorrhoeae: IgM monoclonal antibodies (3F11 and 1-1-M) recognize non-reducing termini of the LOS components.

F62 LOS of Neisseria gonorrhoeae consists of two components. The higher molecular weight (MW) component is recognized by monoclonal antibody (MAb) 1-1-M and the smaller MW component by MAb 3F11. Epitope expression of the two LOS components and their partial structures were investigated by treating the F62 LOS with several glycosidases and then monitoring their antigenicity with the two mouse IgM MAbs. The 1-1-M-defined LOS component was cleaved with both beta-N-acetylhexosaminidase and endo-beta-galactosidase, and each cleavage resulted in the loss of expression of the 1-1-M-defined epitope. The N-acetylhexosamine (HexNAc) released by the hexosaminidase was found to be GalNAc, and the smaller oligosaccharide released by the endo enzyme was identified to be a dimer GalNAc beta----Gal. In contrast, the MAb 3F11-defined LOS component was not digested by the endo galactosidase, but it was cleaved with alpha and beta-galactosidase, and expression of the MAb 3F11-defined LOS epitope expression of the MAb 3F11-defined LOS was abolished by the treatment with each of two exo enzymes. MAb 3F11 bound to the 1-1-M-defined LOS component resulting from the removal of the beta-GalNAc residue, and the resulting LOS was further cleaved with beta-galactosidase, but not with alpha-galactosidase. From these results, we conclude the following: (1) MAbs 1-1-M and 3F11 both recognize the non-reducing termini of the LOS components; (2) the 1-1-M-defined LOS component has the GalNAc beta----Gal beta 1----4-Glc (or GlcNAc) structure, and the GalNAc beta----Gal residue is involved in the MAb 1-1-M-defined epitope; (3) the MAb 3F11-defined LOS component may not have a Gal beta 1----4GlcNAc beta 1----4Gal beta 1----4Glc structure within the molecule. However, it has beta-Gal residue at its non-reducing terminus, and this residue is involved in the MAb 3F11-defined epitope; (4) the two LOS components share a similar antigenic structure, and the 3F11-defined epitope structure is present in the MAb 1-1-M-defined LOS component. Expression of this epitope within the 1-1-M-defined LOS molecule is blocked by the beta-GalNAc residue; however, the beta-GalNAc residue at the non-reducing end may be not the only structural difference between the two components.

Acetylglucosaminidase↗
PubMed1996

Statistical comparison of established T-cell epitope predictors against a large database of human and murine antigens.

Identification of T-cell epitopes within a protein antigen is an important tool in vaccine design. The T-cell epitope prediction schemes often are exploited by workers but have proved unreliable in comparison with experimental techniques. We compared published T-cell epitope predictors against two databases of human and murine T-cell epitopes. Each predictor was assessed against random cyclic permutations of epitopes in order to determine significance. Predictor performance was expressed in terms of two parameters, specificity and sensitivity. Specificity is an expression of the quality of predictions, whereas sensitivity is an expression of the quantity of epitopes predicted. Against the human data set, the strip-of-hydrophobic helix algorithm [Stille et al., Molec. Immun. 24, 1021-1027 (1987)] was the only significant predictor (p < 0.05), whereas against murine data only, the Roth2 pattern [Rothbard and Taylor, EMBO J. 7, 93-100 (1988)] was significant (p < 0.05). Not only were the majority of algorithms no better than random against both data sets, against the murine data two schemes were significant (p < 0.05) anti-predictors. This report indicates which predictors are relevant statistically and is the first to describe anti-predictors which can themselves be useful in the identification of T-cell epitopes.

Algorithms↗
PubMed1986

Immunocytological localization of cell adhesion molecules L1 and N-CAM and the shared carbohydrate epitope L2 during development of the mouse neocortex.

The expression of the two adhesion molecules L1 and N-CAM and their shared carbohydrate epitope recognized by monoclonal antibody L2, was studied during development of the embryonic mouse neocortex by immunohistology at light- and electron-microscopic levels between embryonic days 9 and 18. Throughout this time period N-CAM is expressed in all layers of the telencephalic anlage. L1 antigen shows a more restricted expression than N-CAM. It is not detectable at day 9. From day 10 onward it is expressed on young neurons in the marginal zone, but not in the ventricular layer. At embryonic day 13 L1 antigen appears also in the intermediate zone on afferent fibers from subcortical structures and on migrating neurons. Neuronal cell bodies in the cortical plate and subplate express L1 antigen only transiently on embryonic days 13-16. These observations suggest that L1 antigen does not play a prominent role in the initiation of neuronal migration in the ventricular zone, but could be functional during later stages of migration and in the aggregation of neuronal cell bodies at their final position in the cortical plate. The L2 epitope also shows a more restricted expression than N-CAM during the time period studied. Similar to L1 antigen, it first appears at embryonic day 10 in the marginal zone and remains undetectable in the ventricular layer also at later stages. In the marginal zone the L2 epitope is strongly expressed on neuroepithelial endfeet at the basal lamina. The basal lamina itself is L2 epitope-negative. From embryonic day 10 onward the L2 epitope is most strongly expressed in the marginal zone and subplate and more weakly in the cortical plate and intermediate zone. In the subplate it is not only associated with the surface membrane, but also with the extracellular matrix. These observations support previous biochemical data which show that the L2 epitope is not present on all N-CAM molecules of the embryonic or adult forms and suggest that the independent regulation or L2 epitope expression may have functional implications during development.

Animals↗
PubMed1989

Selection of antibody epitopes in an immunopathogenic neural autoantigen.

Results with two well-characterized self-antigens, cytochrome c and myelin basic protein, have led to differing opinions regarding the predominant specificities of autoantibodies, whether regions of sequence diversity or 'structurally inherent features' of a protein determine favored antigenic sites. To further examine this question, 16 antibody epitopes have been mapped on a highly immunopathogenic autoantigen, retinal S-antigen (S-Ag). The epitopes were characterized for: (1) sequence diversity and cross-reactivity on S-antigens from several species; (2) conformational dependency; and (3) probability of their occurrence on the surface of S-antigen. A single C-terminal region containing sequence diversity was most frequently recognized, but no evidence for recognition of any other regions of sequence diversity was found. Thirteen of 16 monoclonal antibodies raised to native S-Ag bound epitopes strongly predicted to be on the surface of S-antigen. Conversely, only one of six antibody preparations raised to peptides or affinity-purified on peptides was found to recognize an epitope predicted to be on the surface, suggesting a good correlation between specificity for conformation-dependent sites and surface probability based on the surface prediction algorithm. Three of these six antibodies which preferred denatured epitopes bound sites which overlapped or coincided with T cell sites; two of these T cell sites are immunopathogenic. The epitopes recognized on denatured antigen and peptides were similar whether the antibodies were elicited with intact human or bovine S-antigen or with cyanogen bromide-cleaved peptides. Our data suggests that in the case of S-antigen, structural features are more significant factors in epitope selection than sequence diversity.

Amino Acid Sequence↗
PubMed1992

The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.

The T-helper response to the measles virus nucleoprotein (NP) has been studied in mice. The T-cell proliferative response was measured in lymphocytes from mice immunized with either a vaccinia measles-NP recombinant virus or a mouse neuro-adapted measles virus. A T-cell response was obtained with lymphocytes from H2d or H2k mice when stimulated with either measles virus or the NP expressed in bacteria. The response was CD4+ specific. The T-helper epitopes were mapped using truncated NP peptides. The major epitopes in both H2d and H2k mice were determined to be between amino acids 67-98. A further T-cell epitope (between amino acids 457-525) was identified when H2d mice were immunized with measles virus. Studies to quantitate the precursor cells for these epitopes confirmed that the region 67-98 of NP was immunodominant in both haplotypes immunized with the vaccinia-NP recombinant virus, whereas an additional major epitope was observed in the measles virus-infected H2d mice. The primary structure of the epitopes determined here are compared to predicted T-cell epitope motifs.

Amino Acid Sequence↗
PubMed1992

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

Amino Acids↗