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PubMed1997

Characteristic epitope recognition pattern of autoantibodies against eukaryotic ribosomal protein L7 in systemic autoimmune diseases.

OBJECTIVE: To define the epitope-recognition pattern and the fine specificity of the autoantibody response to protein L7 in patients with rheumatic diseases. METHODS: The epitope-recognition pattern was studied by enzyme-linked immunosorbent assay utilizing overlapping fragments of L7. The fine specificity was examined by binding inhibition and isoelectric focusing. RESULTS: We observed a disease-specific epitope-recognition pattern of anti-L7 autoantibodies. There was one immunodominant epitope that was recognized by all anti-L7-positive sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and systemic sclerosis (SSc). Additional recognition of minor epitopes was observed; it arises by intramolecular epitope spreading and was correlated with disease activity in SLE patients. SSc patients differed from SLE and RA patients in that their sera did not recognize certain minor epitopes. The major epitope was recognized by high-affinity autoantibodies of limited heterogeneity. Minor epitopes were recognized by heterogeneous low-affinity autoantibodies. CONCLUSION: The anti-L7 autoantibody response is oligoclonal. Additional B cell clones are activated by antigen during active phases of disease.

Adolescent↗
PubMed1997

Combined structural and immunological refinement of HIV-1 HLA-B8-restricted cytotoxic T lymphocyte epitopes.

This study demonstrates that use of structural information improves the definition and optimization of cytotoxic T lymphocyte (CTL) epitopes. Epitope optimization usually requires numerous truncated peptides or a reverse immunogenetic approach, where the peptide binding motif is used to predict epitopes. These binding motifs do not reliably predict all peptides which are CTL epitopes. Comparison of 24 peptides eluted from HLA-B8 with 10 HLA-B8-restricted defined CTL epitopes demonstrated that known epitopes varied considerably at anchor positions. We used structural information based on determination of the crystal structure of the HLA-B8-GGKKKYKL complex to reassess previously described CTL epitopes, to predict new epitopes, and to predict the consequences of naturally occurring variation within epitopes. These predictions were confirmed by cytotoxicity and binding assays. Use of combined structural and immunological data more accurately defines the true peptide-binding motif of a restriction element than eluted peptide data allows.

Antigenic Variation↗
PubMed2004

Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy.

Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.

2S Albumins, Plant↗
PubMed1998

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Amino Acid Sequence↗
PubMed2001

Monoclonal antibodies against different epitopes of nonstructural protein sigmaNS of avian reovirus S1133.

Ten monoclonal antibodies (MAbs) were prepared against the nonstructural protein sigmaNS of avian reovirus S1133. Eight of them were selected for two-way competitive binding assay after coupling with horseradish peroxidase. The results allowed the definition of three epitopes, designated A, B, and C. Blocking assay of poly(A)-Sepharose binding activity of sigmaNS with MAbs indicated that MAb recognizing epitope B was able to block poly(A) oligomer binding, suggesting that epitope B is involved in ssRNA binding of sigmaNS. An immuno-dot binding assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to protein sigmaNS in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitopes B and C was not affected. The reactivity of MAbs recognizing epitope A was fully abolished by denaturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent; however, the recognition by MAbs of epitopes B and C is not conformation-dependent. In addition, the results from the cross-reactivity of MAbs to heterologous avian reovirus strains suggest that the three epitopes are highly conserved among these virus strains.

Animals↗
PubMed1996

Conformational display of two neutralizing epitopes of HIV-1 gp41 on the Flock House virus capsid protein.

We have developed an antigen presenting system based on the capsid protein of the Flock House virus (FHV) and used it to display, in different positions on its external surface, two neutralizing epitopes found at residues 735-752 of HIV-1 gp160. We have compared the immunoreactivity of these FHV chimeric proteins and of the corresponding synthetic peptide using a panel of neutralizing mouse monoclonal antibodies (mAbs) directed against two distinct sequences (IEEE and ERDRD) contained in this epitope of the gp41 region. We have observed that both the FHV chimeric protein and the synthetic peptide are clearly detected in ELISA procedures by the mAbs recognizing the sequence IEEE. The denaturation of these recombinant proteins had little effect on the recognition pattern of this group of monoclonals, suggesting minor conformational requirements for the display of this epitope. The FHV chimeric proteins were also recognized by the mAbs directed against the ERDRD epitope, whereas the corresponding synthetic peptide was not recognized. In this case, denaturation of these recombinant proteins completely abolished the reactivity of the second group of mAbs, arguing for the existence of strong conformational constraints. Additionally, we have investigated whether an isolated loop structure from the FHV protein was sufficient to provide the conformational requirements for the presentation of these epitopes. These experiments have shown that the stabilized loop structure, although improving the presentation of both epitopes, is not as efficient as the native loop in the intact FHV protein. The data obtained with these mAbs support the recently observed limitations in the use of synthetic peptides for the screening of the immune response against conformational epitopes. The establishment of appropriate tools able to present epitope sequences in a structure resembling the native conformation will be useful for accurate epidemiological studies and for the design of new epitope-specific vaccines.

Amino Acid Sequence↗
PubMed2006

Molecular dissection of an hCG-beta epitope using single-step solid phase radioimmunoassay.

BACKGROUND: Peptides and proteins have both sequence-specific (contiguous) and conformation-specific (discontiguous) epitopes. Sequence-specific epitopes are delineated by peptide approach and other robust methods like competition assays, gene expression assays, synthetic peptide library based assays etc. Available methods for delineation of conformation-specific epitopes are cumbersome (X-ray crystallography etc.), time-consuming and require costly sophisticated equipments. Hence, there is a need to develop a simple method for identification and mapping of conformation-specific epitopes. METHOD: In the single-step solid phase radioimmunoassay (SS-SPRIA), an immunochemical bridge of 'mouse IgG-anti-mouse IgG' was prepared in the polypropylene wells followed by adsorption with hCG specific monoclonal antibody (MAb) G(1)G(10).1. The extent of competitive inhibition in binding ability of (125)IhCG-beta with chemically or enzymatically modified hCG-beta to immobilized MAb G(1)G(10).1 in comparison to hCG-beta standards was utilized to identify the epitopic amino acid involved in epitope-paratope interaction. RESULTS: Data clearly suggest that the epitope under investigation consisted of Arg (94, 95) and Asp (99) at the core region with a Lys (104) and a His (106) in the proximity and absence of chymotrypsin susceptible Phe or Tyr in this region. CONCLUSION: The data of SS-SPRIA revealed the 93-100 loop of amino acid sequence, as the core region of conformation-specific epitope of hCG-beta at or near the receptor-binding region. Hence, SS-SPRIA seems to be a simple method for identification and mapping of conformation-specific epitopes.

Amino Acid Sequence↗
PubMed2005

Single B or T-cell epitope-based DNA vaccine using modified vector induces specific immune response against hepadnavirus.

Epitope-based DNA vaccine is an effective and powerful approach against a variety of pathogens or tumors. In present study, we reconstructed a vector that could effectively express short B and T-cell epitope of duck/hepatitis B virus, and investigated the role of the epitope-based DNA vaccination. The pUC19 was modified by inserting the compact transient framework (CTF), including HCMV IE1 promoter, enhancer, Kozak sequence, dual stop codon and 3' terminal bovine growth hormone terminal signal and so on. This modified vector was designated pEC(K) and supposed to effectively express short peptide. A well-defined single B-cell and T-cell epitope encoding gene of duck/hepatitis B virus has been synthesized as candidate epitope and cloned into pEC(K) plasmid, respectively. Transfection of the recombinant DNA into C(2)C(12) cell showed that modified plasmid could effectively express both the single B-cell and T-cell short epitope in the culture supernatant as confirmed by dot immunoblot assay (DIA). The recombinant single B and T-cell epitope-based DNA vaccine was administrated to C57BL/6 mice and could greatly induce specific humoral and CTL response. In addition, the specific antibody against B epitope could specifically bind to the DHBV particles. This report demonstrated that single epitope-based DNA vaccine using modified plasmid vector pEC(K) could induce effective specific immune responses and could be of great use for DNA vaccines.

Animals↗
PubMed2006

Monoclonal antibody 76F distinguishes IA-2 from IA-2beta and overlaps an autoantibody epitope.

IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized. 76F mAb recognized IA-2 of human, rat and mouse origin in native and denatured forms and had an epitope specificity for residues 626-630 (FEYQD) which are found in the juxtamembrane (JM) region of human and mouse IA-2, but not IA-2beta. This region overlaps with the autoantibody epitope JM2. Binding to the 76F monoclonal antibody was specifically inhibited by sera with antibodies to the JM2 epitope but not with antibodies to the adjacent JM1 epitope, indicating that unique epitopes can be distinguished by this approach. 76F mAb has the unique property to distinguish between the two closely related autoantigens IA-2 and IA-2beta by targeting an IA-2 specific epitope of the juxtamembrane region. The findings define an approach to develop assays for specific antibody epitope measurements which may be relevant for disease prognosis and monitoring intervention therapies.

Amino Acid Sequence↗
PubMed2004

Epitope analysis of GAD65Ab using fusion proteins and rFab.

The identification of disease-specific autoantibodies to the 65-kDa isoform of glutamate decarboxylase (GAD65Ab) epitopes in type 1 diabetes has been hampered by their conformational nature. Here, we compared two methods of GAD65Ab epitope analysis: GAD65/67 fusion proteins and competition assays using GAD65-specific recombinant fraction antigen binding (rFab). Sera from newly diagnosed type 1 diabetes patients (n=61) were studied using both approaches. Competition of GAD65 binding by an rFab to a specific epitope did not correlate with binding to the fusion protein that represented this epitope. Conversely, samples that bound to specific fusion proteins were not necessarily competed with rFab specific to determinants in the same region. We conclude that epitopes of different characteristics are detected by fusion proteins and by competition with rFab. Fusion proteins allow the definition of large epitope regions; however, some conformational GAD65Ab epitopes, especially those residing in the middle region, are destroyed or distorted in the fusion proteins. Competition studies using rFab allow the identification of conformational epitopes. However, monoclonal rFab may only reflect a limited proportion of the epitopes recognized by polyclonal sera. A combined analysis using both approaches may therefore be necessary to gain best understanding of autoantibody characteristics and affinity maturation.

Autoantibodies↗
PubMed2005

Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

Algorithms↗
PubMed2006

A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients.

BACKGROUND: A clinically relevant allergic reaction requires recognition of at least two different epitopes on the surface of the allergen by IgE. These epitopes may be specific or cross-reactive. Moreover, patterns of IgE reactivity may be patient-specific. The aim of our study was to compare specific and cross-reactive IgE epitopes and epitope patterns between individual patients. We used Bet v 1-related food allergy as a model. METHODS: Five patients were investigated by cross-competitive ELISA for specific and cross-reacting IgE to Bet v 1, and its homologues Gly m 4 (soybean), Ara h 8 (peanut), and Pru av 1 (cherry). Allergen-specific as well as cross-reactive IgE epitopes were assessed by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal purified IgE from individual sera. The resulting peptide mimics were mapped on the surface of the 3D-structure of the allergens using a computer-based algorithm. RESULTS: Competitive immunoscreening and epitope mapping identified patient-specific IgE epitope patterns. However, one IgE-binding surface area that was recognized by all patients and two recognized by three patients were identified on all four proteins. These results are consistent with the determination of IgE cross-reactivity of the individual patients' sera against the four recombinant allergens by cross-competitive ELISA. CONCLUSIONS: Selection of phage-displayed peptide mimics with serum IgE from allergic patients in combination with computer-based mapping of the peptide mimics onto the surface of the three-dimensional allergen structure is a promising novel tool to investigate IgE epitope specificity in individual patients. Such basic information on epitope structure may be used for prediction of cross-reactivity and potential allergenicity of novel foods.

2S Albumins, Plant↗
PubMed2006

Expression of the O-linked N-acetylglucosamine containing epitope H in normal myometrium and uterine smooth muscle cell tumors.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H (mAbH). We have previously shown that epitope H is present in more than one polypeptide and in various types of normal and pathological cells. In the present study, we focused on uterine smooth muscle cell tumors and their adjacent normal myometrium to gain further insight into the expression patterns of epitope H in human tissues. The indirect immunoperoxidase method was applied using the mAbH and the monoclonal anti-cytokeratin 8 antibody (AbCK8) in 50 cases of typical uterine leiomyomas and in five cases of uterine leiomyosarcomas, with four cases belonging to Group II A and one to Group III according to Bell et al. [6]. Western immunoblotting was applied using mAbH and AbCK8 in five cases of uterine leiomyomas and their adjacent myometrium. The main results were as follows: (1) epitope H showed intense immunohistochemical expression in 46% (23/50) and moderate expression in 54% (27/50) of uterine leiomyomas, (2) epitope H showed intense immunohistochemical expression in 40% (2/5) and moderate expression in 60% (3/5) of uterine leiomyosarcomas, (3) epitope H showed no difference in the immunohistochemical expression between leiomyomas and their adjacent myometrium and between leiomyosarcomas and their adjacent myometrium, (4) immunohistochemical expression of cytokeratin 8 was not detected in the normal and neoplastic smooth muscle cells, (5) Western immunoblotting showed that in the smooth muscle cells of the myometrium and leiomyomas, epitope H is localized in four polypeptides with molecular weights of 100, 61, 59, and 54 kDa, and (6) Western immunoblotting did not detect cytokeratin 8 in the normal and neoplastic smooth muscle cells. The present results indicate fluctuations of the epitope expression levels in uterine smooth muscle cell tumors and their adjacent myometrium. These fluctuations may be of interest for gaining insight into the pathogenesis of uterine smooth muscle cell tumors, since O-GlcNAc glycosylation is involved in cell cycle and apoptosis pathways and may modify proteins involved in oncogenesis (tumor suppressor proteins and oncoproteins) and proteins with important biological functions such as cytoskeletal proteins, transcription factors, and heat-shock proteins. Furthermore, the present results indicate that cytokeratin 8, without being present in the cells of the myometrium, leiomyomas and leiomyosarcomas, shares its epitope H, which contains its unique sugar O-N-acetylglucosamine residue, with four other unrelated polypeptides produced by the normal and neoplastic smooth muscle cells. This should be considered when using anti-cytokeratin 8 antibodies in immunohistochemistry against smooth muscle cell tumors to avoid false positive immunohistochemical results.

Acetylglucosamine↗
PubMed2005

Developing an epitope-driven tuberculosis (TB) vaccine.

Epitope-driven vaccines are created from selected sub-sequences of proteins, or epitopes, derived by scanning the protein sequences of pathogens for patterns of amino acids that permit binding to human MHC molecules. We developed a prototype tuberculosis (TB) vaccine that contains epitopes derived by (1) EpiMer mapping of previously published secreted proteins derived from Mycobacterium tuberculosis (Mtb), and (2) EpiMatrix mapping of selected Mtb genome open reading frames (ORFs). Each of the epitopes contains at least three distinct class II MHC binding motif matches. These Mtb epitope selections were validated by measuring T cell responses from peripheral blood mononuclear cells (PBMC) obtained from healthy, asymptomatic tuberculin skin test-positive donors. Twenty-four validated Mtb epitopes were selected for inclusion in a DNA plasmid vector. We immunized HLA-DR B*0101 transgenic mice with this vaccine prototype augmented by co-administration of rIL-15. Following administration of three immunizations at 14-day intervals in conjunction with rIL-15, epitope-specific T cell responses were observed to eight of the 24 epitopes contained in the DNA construct, one week following the last injection. The systematic application of bioinformatics tools to whole genomes, in combination with in vitro methods for screening and confirming epitopes, may lead to the development of novel vaccines for infectious diseases like TB.

Amino Acid Sequence↗
PubMed2004

HLA-A*0201-restricted cytolytic responses to the rtTA transactivator dominant and cryptic epitopes compromise transgene expression induced by the tetracycline on system.

The tetracycline-controlled transcription system (Tet-on) is widely used to regulate gene expression in mammalian cells. In gene therapy applications, immune responses to Tet-on proteins such as the rtTA transcription factor have been reported, raising concerns about their occurrence in humans. To monitor the HLA class I cytolytic responses against Tet-on regulators, we characterized the immunogenic CD8+ epitopes within rtTA and tTS regulators using HLA-A*0201 class I transgenic mice. Epitope prediction programs, HLA-A*0201 binding assays, and peptide immunization were used to select a set of immunogenic peptides within rtTA and tTS sequences. To identify further the rejection epitopes, we expressed Tet-on protein components in vivo and found a single dominant rtTA186 CTL epitope in the rtTA tetracycline repressor domain. Target cells expressing rtTA were susceptible to CTL lysis, and rtTA expression compromised muscle transgene engraftment. To reduce the occurrence of immune responses to rtTA protein, we mutated the dominant rtTA186 epitope and found that this leads to the appearance of subdominant epitopes. As a result, we think that an epitope modification strategy is not applicable to blunt the immune response in this model. Moreover, the identification of HLA-A*0201 rtTA epitopes allowed us to demonstrate here that the delivery of the Tet-on system with weakly immunogenic rAAV vectors does not trigger primary CTL responses in mice, in contrast to DNA transfer. Altogether, the existence of HLA-A*0201 rtTA epitopes may lead to the occurrence of immune responses depending on vectors and local inflammation in gene therapy applications involving rtTA-based regulatory systems.

Alkaline Phosphatase↗
PubMed2003

Human population-based identification of CD4(+) T-cell peptide epitope determinants.

A human cell-based method to identify functional CD4(+) T-cell epitopes in any protein has been developed. Proteins are tested as synthetic 15-mer peptides offset by three amino acids. Percent responses within a large donor population are tabulated for each peptide in the set. Peptide epitope regions are designated by difference in response frequency from the overall background response rate for the compiled dataset. Epitope peptide responses are reproducible, with a median coefficient of variance of 21% when tested on multiple random-donor sets. The overall average response rate within the dataset increases with increasing putative human population antigenic exposure to a given protein. The background rate was high for HPV16 E6, and was low for human-derived cytokine proteins. The assay identified recall epitope regions within the donor population for the protein staphylokinase. For an industrial protease with minimal presumed population exposure, immunodominant epitope peptides were identified that were found to bind promiscuously to many HLA class II molecules in vitro. The peptide epitope regions identified in presumably unexposed donors represent a subset of the total recall epitopes. Finally, as a negative control, the assay found no peptide epitope regions in human beta2-microglobulin. This method identifies functional CD4(+) T-cell epitopes in any protein without pre-selection for HLA class II, suggests whether a donor population is pre-exposed to a protein of interest, and does not require sensitized donors for in vitro testing.

Blood Donors↗
PubMed1997

Epitope mapping by phage display: random versus gene-fragment libraries.

We present a comparative study on epitope mapping of four monoclonal antibodies directed against four different antigens using alternative phage display techniques and peptide scanning: mAb215 reacts with the largest subunit of RNA polymerase II, mAbBp53-11 with the tumor suppressor protein p53, mAbGDO5 with the Hantaan virus glycoprotein G2 and mAbL13F3 with the Hantaan virus nucleocapsid protein. Epitopes were determined (i) by gene-fragment phage display libraries, constructed by DNaseI digested random gene fragments cloned into the 5' terminus of the pIII-gene of fd phage and (ii) by random peptide phage libraries displaying 6mer and 15mer peptides at the N-terminus of the pIII protein. Using the gene-fragment phage display libraries a single round of affinity selection resulted in the determination of the corresponding epitopes for all monoclonal antibodies tested. In contrast, biopanning of 6mer and 15mer random peptide libraries was only successful for two of the antibodies (mAbBp53-11 and mAbGDO5) after three or four rounds of selection. For the anti-p53 antibody we recovered the epitope from both the 6mer and 15mer library, for mAbGDO5 only the 6mer library displayed the epitope sequence. However, screening of the random peptide libraries with mAb215 and mAbL13F3 failed to yield immunopositive clones. Fine mapping of the epitopes by peptide scan revealed that the minimal epitopes recognized by mAbBp53-11 and mAbGDO5 consist of four and five amino acids, respectively, whereas mAb215 requires a minimal epitope of 11 amino acids for antigen recognition. In contrast, mAbL13F3 did not react with any of the synthesized 15mer peptides. The limits of the different methods of epitope mapping tested in this study are compared and the advantages of the gene-fragment phage display system are discussed.

Amino Acid Sequence↗
PubMed1998

Identification of epitopes within beta lactoglobulin recognised by polyclonal antibodies using phage display and PEPSCAN.

Two different epitope mapping techniques were used to identify linear epitopes recognised by polyclonal IgG antibodies from rabbits immunised with bovine beta lactoglobulin (BLG), which is generally regarded as a major allergen in milk. The first, PEPSCAN, was used to investigate the binding of several rabbit polyclonal antisera to sequential overlapping peptides (12-mers) across the sequence of BLG. Each peptide was synthesized on a different polypropylene PIN, and a standard ELISA procedure was used to locate which of these peptides bound the antibodies under investigation. Comparisons of PEPSCANs for antisera from six different rabbits showed that each rabbit recognized a similar set of epitopes within BLG. PEPSCAN analysis also showed that polyclonal antibodies from the mouse recognize a set of epitopes similar to those recognized by the rabbit. The second epitope mapping technique is known as phage display and utilizes libraries of randomized short peptides fused to the coat proteins of filamentous phage as a source of epitopes for analysis. A gene VIII phage display library was used in this study with constrained nonapeptides, which were screened for epitopes recognized by affinity purified rabbit anti-BLG IgG. Immobilised rabbit anti-BLG IgG was screened in two separate experiments, each consisting of three rounds of panning. For each separate experiment, a sensitive phage ELISA was used to screen several hundred single phage clones for binding to anti-BLG IgG immobilised on microtiter plates. As a result, a number of positive phage were identified from the two separate screens of the library (19 different peptides were isolated, which resembled four different regions of BLG). The identified sequences were found to constitute a subset of the linear epitopes recognized by the PEPSCAN technique. The coordinates of the crystal structure of BLG were used to display mapped epitopes on its structure. This study has permitted detailed mapping of the major linear antigenic regions within BLG recognised by IgG antibodies from immunised rabbits and mice.

Amino Acid Sequence↗