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PubMed1997

Epitope mapping of murine monoclonal antibodies against human immunodeficiency virus type 1 Nef.

It has shown that the human immunodeficiency virus type 1 (HIV-1) Nef protein has the high antigenicity in HIV-1 seropostive individuals. We newly obtained seven monoclonal antibodies (mAbs). To identify the antigenic determinants of HIV-1 Nef protein against murine, epitope mapping of the mAbs was performed by enzyme-linked immunosorbent assay (ELISA) by using several recombinant truncated Nef fusion proteins, that were expressed in Esherichia coli, and synthetic peptides. The results showed that mAbs A6, A7, F2, F3, F4, F8 and E5 recognized epitopes on Nef protein located at amino acid residues 18-26, 28-45, 115-137, 128-137, 115-126, 128-137, and 170-181, respectively.

Animals↗
PubMed1991

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.

Amino Acid Sequence↗
PubMed1996

Epitope mapping, V-region DNA sequence, and neutralizing Fab fragments of two monoclonal antibodies against the HIV-1 V3 loop.

BACKGROUND: Experimental evidence suggests that neutralizing antibodies could constitute an important factor in the control of AIDS progression and that the V3 loop of gp120 constitutes the main target for such purposes. We have previously developed two neutralizing murine monoclonal antibodies (Mabs) against the V3 region of the HIV-1 MN strain. OBJECTIVES: To characterize those Mabs in terms of fine specificity and DNA sequence of their V regions and to study if Fab fragments retain their neutralizing potential in vitro. STUDY DESIGN: A set of 12-mer alanine substituted peptides were employed for epitope mapping using two ELISA procedures: (1) indirect, with each peptide bound to polystyrene plates, and (2) competitive, with co-incubation of peptides and Mabs in solution. The V regions of both Mabs were PCR amplified from cDNA and their nucleotide sequences were determined. Finally, Fab fragments of Mab 10F10 were generated and their neutralizing capacity against the MN isolated was assessed. RESULTS: We first restricted the minimal length of the epitopes recognized by 2C4 and 10F10 to the 12-mer peptide KRIHIGPGRAFY. The core of the epitopes recognized by Mabs 2C4 and 10F10 were IHIGP-R and IHIG-R, respectively. While substitution of proline in position 7 completely abolished the binding of 2C4, it only reduced that of 10F10 by 50%. Finally, Fab fragments of Mab 10F10 were still able to neutralize the HIV-1 MN strain in vitro. CONCLUSION: This subtle distinction in the fine mapping of the epitope recognized by Mabs 2C4 and 10F10 should correspond to three amino acid differences that we found in the heavy chain V-regions. The Fab fragments of Mab 10F10 retained the neutralizing capacity. This indicates that HIV neutralization by anti V3 Mabs is an Fc independent process.

Amino Acid Sequence↗
PubMed2006

Cloning, sequence analysis and confirmation of derived gene sequences for three epitope-mapped monoclonal antibodies against human phagocyte flavocytochrome b.

The integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the NADPH oxidase complex, a multicomponent enzyme system that initiates a cascade of reactive oxygen species that play a critical role in innate immunity and vascular physiology. Epitope-mapped, monoclonal antibodies (mAb) that recognize the large (gp91phox) and small (p22phox) subunits of Cyt b provide valuable reagents that have been used to examine structural and mechanistic aspects of oxidase function. In the present study, the heavy and light chain variable region genes of the Cyt b-specific mAbs 44.1, NS5, and NL7 have been amplified by RT-PCR, cloned and subject to DNA sequence analysis. Since the 5' degenerate primer sets used for mAb gene amplification were observed to introduce extensive heterogeneity into the heavy and light chain FR1 regions, N-terminal protein sequence analysis was also conducted to obtain the correct amino acid sequence of this region. In order to confirm the identity of the cloned genes, intact mAbs were resolved by two-dimensional electrophoresis and subject to in-gel tryptic digestion for analysis by both MALDI and nanospray LC-MS/MS. Databases searches using the derived mAb sequences predicted residues comprising CDR loops, identified candidate germline genes, and showed the respective germline genes to accurately predict the N-terminal amino acid residues for each variable region. The above studies report the amino acid sequence of Cyt b-specific mAb variable region genes with high confidence and provide essential information for future efforts at Cyt b structure analysis by resonance energy transfer and X-ray crystallography.

Amino Acid Sequence↗
PubMed2001

Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production.

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.

Amino Acid Sequence↗
PubMed1998

Lectin-dependent modulation of interaction between human alpha-fetoprotein and its monoclonal antibodies. Epitope mapping.

The effect of lentil lectin (LCA) on the binding of mouse monoclonal antibody (MoAb) against human alpha-fetoprotein (AFP) to LCA-nonreactive AFP-L1 and LCA-reactive AFP-L3 was studied on a panel of 30 MoAbs provided by the TD-2 Workshop of ISOBM for epitope mapping. LCA inhibited the binding of MoAbs 93 and 98 to AFP-L3 but not to AFP-L1, indicating that there was a competition between the MoAbs and LCA for the AFP sugar chain. With MoAbs 100, 109, 118, and 120, LCA rather increased the binding to AFP-L3 over that to AFP-L1. These modulating effects of LCA on the MoAb binding to AFP-L3 were abolished by periodate treatment of the AFP preparations without affecting the binding of MoAb to AFP. Concanavalin A had similar inhibiting and enhancing effects on MoAb binding, but equally to AFP-L1 and AFP-L3, both of which are fully reactive with concanavalin A. The results suggested that MoAbs 93 and 98 recognized epitopes closely related to sugar chain, and their binding to AFP-L3 was inhibited by the bound LCA due to steric hindrance. The enhanced binding of some MoAbs to AFP-L3 over AFP-L1 with LCA, or both glycoforms of AFP with concanavalin A, may be explained by postulating an allosteric mechanism mediated by the oligosaccharide-lectin interaction.

Animals↗
PubMed2005

Epitope mapping of recombinant hepatitis B surface antigen by murine monoclonal antibodies.

Hepatitis B surface antigen (HBsAg) induces a potent protective antibody response in immunized healthy individuals. The antibody response in humans is largely directed to a restricted conformational immunodominant region of HBsAg, identified as "a" determinant. Our aim was generation and characterization of murine monoclonal antibodies (MAbs) against recombinant HBsAg and their use for epitope mapping of the antigen. Hybridoma cells were established from Balb/c mice immunized with recombinant HBsAg of the "adw" subtype and cloned by limiting dilution. Specificity of MAbs was studied by indirect ELISA and immunoblotting. Topology of the epitopes was analyzed by competitive and inhibition ELISA. Eight hybridoma clones producing MAbs specific for the immunogen were established. Five of the MAbs recognized overlapping conformational epitopes, whereas the remaining three MAbs were found to identify linear epitopes. Cross-inhibition studies suggest recognition of mutually exclusive epitopes by these MAbs. Our data suggest that, similar to the human system, the mouse antibody response is largely directed to restricted conformational overlapping epitopes of HBsAg.

Animals↗
PubMed1993

Epitope mapping studies with neutralizing and non-neutralizing monoclonal antibodies to the G1 and G2 envelope glycoproteins of Hantaan virus.

Epitopes recognized by three G1-specific and two G2-specific neutralizing monoclonal antibodies to Hantaan virus were mapped by sequence analyses of the complete M genome segments of neutralization escape variant viruses. For each variant, we detected nucleotide sequence substitutions which resulted in a single amino acid change in either the G1 or G2 protein. Serological properties of the variant viruses correlated with changes identified by nucleotide sequence analyses. To map epitopes recognized by three G1-specific and six G2-specific, non-neutralizing monoclonal antibodies, we prepared genes, truncated at the carboxy terminal coding regions of G1 or G2, and expressed them with baculovirus recombinants or transiently in a vaccinia/T7 RNA polymerase system. Reactivities of the monoclonal antibodies with the truncated proteins were monitored by immune precipitation of the radiolabeled, truncated glycoproteins. We determined that all three of the G1-specific antibodies reacted with truncated proteins, which retained the amino terminal one-third of G1, but lost reactivity with shorter G1 proteins. The G2-specific antibodies only recognized G2 proteins, which retained approximately 80% of the G2 gene.

Amino Acid Sequence↗
PubMed1996

Site-Directed Mutagenesis in Epitope Mapping

Site-directed mutagenesis is a very useful tool for mapping and defining epitopes on protein antigens. This review discusses the methods used in, and the results of, studies on four different protein antigen systems. In addition, computer analyses and molecular modeling were used in an attempt to better understand the structural and energetic mechanisms underlying the effects observed following mutagenesis. The advantages and limitations of site-directed mutagenesis as an experimental tool are also discussed.

Journal Article↗
PubMed1989

Human T cell clones define S1 subunit as the most immunogenic moiety of pertussis toxin and determine its epitope map.

Human T lymphocyte clones specific for pertussis toxin (PT) were used to analyze the fine specificity of the response to PT, the basic component of new acellular vaccines against whooping cough. The majority (83%) of the clones specific for PT recognized S1, the subunit that in animal models has been shown to be highly immunogenic. To map T cell epitopes on S1, 18 S1-specific clones were tested for recognition of recombinant fragments representing NH2-terminal and COOH-terminal deletions of S1 and two recombinant S1 subunits containing amino acid substitutions. This approach led to the identification of three regions of the protein as the sequences containing T cell antigenic sites: 1-42, 181-211, and 212-235. Synthetic peptides were eventually used for a finer localization of the T cell epitopes. Two peptides, one of 13 residues (27-39) at the NH2 terminus and one of 24 residues (171-194) at the COOH terminus, stimulated proliferation of three and four clones, respectively. Both peptides are recognized in association with HLA DR1 molecules. These results stress the role of S1 in the immune response to PT and provide data useful for the development of a recombinant or synthetic antipertussis vaccine containing T cell epitopes from S1.

Amino Acid Sequence↗
PubMed1993

Epitope mapping identifies an exposed loop between the unique amino- and conserved carboxy-domains of the large subunit of herpes simplex virus type 1 ribonucleotide reductase.

The large subunits of herpes simplex virus types 1 and 2 ribonucleotide reductases contain unique amino-terminal regions comprising 311 and 318 residues respectively, which are not found in ribonucleotide reductases from other sources. We report the mapping of the epitope recognized by monoclonal antibody 1026, which is specific for the large subunit (R1) of HSV-1, and then deduce the structural relationship of the amino-terminal region of R1 with the rest of the protein. A panel of 10 fusion proteins containing sequences spanning the entire R1 subunit were constructed. They were used together with proteolytic fragments of R1 and several synthetic peptides to show that the epitope is discontinuous and appears to be a loop structure centered on a previously located trypsin-sensitive site at residue 305. The existence of the loop was suggested by the observation that reactivity of the antibody with R1 could be blocked by peptides corresponding to residues 289 to 303 and 308 to 313 which flank the trypsin-sensitive site. Our results suggest that the unique amino-terminal region of R1 consists of a structurally distinct domain which is linked to the conserved carboxy region by an exposed loop.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of cytochrome P4502D6 autoantigen in patients with chronic hepatitis C during alpha-interferon treatment.

BACKGROUND/AIMS: Cytochrome P450 2D6 (CYP2D6) has been documented as the major target antigen of liver kidney microsomal autoantibodies type-1 (anti-LKM-1) in both autoimmune hepatitis type-2 (AIH-2) and hepatitis C (HCV). In HCV/anti-LKM-1-positive patients, the choice between alpha-interferon (alpha-IFN) or immunosuppression may be difficult. This study was conducted to evaluate the course and outcome of alpha-IFN therapy in HCV/anti-LKM-1-positive and -negative patients and the alterations in these autoantibody titers by the indirect immunofluorescence and a novel radioligand assay. Epitope mapping was also performed to screen for a potential shift in anti-LKM-1 binding towards small linear epitopes, which are more often detected in AIH-2 patients. METHODS: Twenty-one patients with HCV infection received alpha-IFN. Seven patients were anti-LKM-1 positive (study group) and 14 patients were anti-LKM-1 negative (disease control group). Anti-CYP2D6 detection was based on immunoprecipitation of [35S]-methionine-labeled CYP2D6 recombinant protein (rCYP2D6) produced by in vitro transcription/translation. RESULTS: Four out of seven (57%) patients in the study group and 5/14 (36%) in the disease control group initially responded, but subsequently relapsed. During follow-up, alanine aminotransferase significantly increased in the study group compared to the disease control group (p<0.01). A slight increase, followed by a plateau of autoantibody titers was recorded by the radioligand assay and by indirect immunofluorescence during therapy and follow-up in most cases. In one patient, however, gamma-globulins and anti-LKM-1 titers increased, reaching very high levels (1:40 960). alpha-IFN was interrupted and immunosuppression was started. HCV/anti-CYP2D6 positive sera recognized CYP2D6 expressed in E. coli and two truncated proteins (aa 250-494 and 321-494). Two out of seven sera, in addition reacted with a small linear epitope of aa 257-269 (one of which also reacted with a C-terminal domain of aa 350-494). CONCLUSIONS: A rather mild deterioration in liver disease was observed in only 1/7 HCV/anti-LKM-1-positive patients during alpha-IFN treatment. This patient showed high anti-CYP2D6 titers before the initiation of therapy, a sharp increase in anti-LKM-1 titers during treatment, and reactivities to a small linear epitope and an infrequently recognized C-terminal domain of CYP2D6. After switching to immunosuppressive treatment, a complete and sustained response was recorded. Further prospective studies from many centers are needed to define whether these features have general, clinical significance or not.

Adolescent↗
PubMed1991

Epitope mapping of the Dermatophagoides pteronyssinus house dust mite major allergen Der p II using overlapping synthetic peptides.

Fourteen synthetic peptides of 15 amino acid residues length, overlapping by five residues and spanning the entire sequence of the major allergen Der p II from the house dust mite Dermatophagoides pteronyssinus were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope mapping studies using human IgE antisera. These antibodies bound predominantly to the peptide comprising residues 65-78, the binding of which was inhibited by native Der p II. In addition these antisera bound, to a lesser extent, to the peptide that comprised residues 1-15, which binding was not inhibited by native Der p II. Thus, we found one sequential epitope for a number of IgE sera.

Allergens↗
PubMed2005

Fine epitope mapping of monoclonal antibodies specific to human alpha-synuclein.

The neuronal phosphoprotein alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases; however, the exact function of alpha-synuclein still remains illusive. Suitable antibodies (Abs) specific for the gene of interest are indispensable for studying biological and immunological properties of the target gene. Here, we report not only the generation and characterization of monoclonal Abs, Syn-1 and Syn-17, against human alpha-synuclein, but also the epitope mapping by using recombinant synuclein family proteins and various GST fusion proteins of human alpha-synuclein domains. Syn-17 recognizes human and rodent alpha-synuclein, and its epitope is localized within residues 97-99 and 101 of alpha-synuclein. In contrast, the Syn-1 epitope is localized in residues 121 and 122 of human alpha-synuclein, and Syn-1 recognizes only human but not rodent alpha-synuclein, indicating that it can be utilized as a useful reagent for studying human alpha-synuclein transgenic mouse and zebrafish lines.

Animals↗
PubMed1993

Nucleotide sequence analysis and epitope mapping of the merozoite surface protein 1 from Plasmodium chabaudi chabaudi AS.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

Amino Acid Sequence↗
PubMed2003

Epitope mapping of the Brucella melitensis BP26 immunogenic protein: usefulness for diagnosis of sheep brucellosis.

Sequencing of bp26, the gene encoding the Brucella sp. immunogenic BP26 periplasmic protein, was performed in the reference strains of Brucella abortus, B. suis, and B. ovis. The three bp26 sequences were almost identical to that published for B. melitensis 16M bp26, and only minor nucleotide substitutions, without modifying the amino acid sequence, were observed between species. The bp26 genes of the seven B. abortus biovar reference strains and B. abortus S19 and RB51 vaccine strains were also sequenced. Again, only minor differences were found. Surprisingly, the bp26 nucleotide sequence for B. abortus S19 was almost identical to that found for B. melitensis 16M and differed from the sequence described previously by others (O. L. Rossetti, A. I. Arese, M. L. Boschiroli, and S. L. Cravero, J. Clin. Microbiol. 34:165-169, 1996) for the same B. abortus strain. The epitope mapping of BP26, performed by using a panel of monoclonal antibodies and recombinant DNA techniques, allowed the identification of an immunodominant region of the protein interesting for the diagnosis of B. melitensis and B. ovis infection in sheep. A recombinant fusion protein containing this region of BP26 reacted indeed, in Western blotting, as the entire recombinant BP26 against sera from B. melitensis- or B. ovis-infected sheep while it avoided false-positive reactions observed with sera from Brucella-free sheep when using the entire recombinant BP26. Thus, use of this recombinant fusion protein instead the entire recombinant BP26 could improve the specific serological diagnosis of B. melitensis or B. ovis infection in sheep.

Amino Acid Sequence↗