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Biomedical subjects

H M Geysen

Publications and source records attributed to H M Geysen.

At least 55 records · Page 3Linked to original sources
PubMed1990

Antibody activity in ankylosing spondylitis sera to two sites on HLA B27.1 at the MHC groove region (within sequence 65-85), and to a Klebsiella pneumoniae nitrogenase reductase peptide (within sequence 181-199).

74 overlapping peptides of varying lengths from Klebsiella pneumoniae nitrogenase reductase (residues 181-199) and from the HLA B27.1 molecule (residues 65-85) were synthesized and tested by ELISA against sera from HLA B27+ ankylosing spondylitis (AS) patients, and sera from HLA B27+ and HLA B27- healthy first-degree relatives. Antibody activity in AS sera to Klebsiella peptides of four to eight amino acids was maximal with the peptide NSRQTDR. Activity to HLA B27 peptides was maximal with the peptide KAKAQTDR (named epitope I). These peptides overlap with, but are proximal to the NH2 terminus from QTDRED, which is homologous in HLA B27.1 and K. pneumoniae nitrogenase reductase. A second weaker reactive site was noted in the HLA B27.1 peptides, proximal to the COOH terminus from the homologous sequence, namely peptide REDLRTLL (named epitope II). Little activity was seen against peptides that included the entire homologous sequence. Sera from 50 AS patients showed higher total Ig activity against peptides KAKAQTDR (p less than 0.001) and NSRQTDR (p less than 0.02) than did sera from 22 B27+ and 22 B27- healthy controls. These data indicate that AS patient sera contain antibodies that bind to K. pneumoniae nitrogenase peptides and HLA B27.1 peptides, and that there are at least two epitopes on HLA B27.1 in the alpha 1 domain, at the MHC groove region, that are autoantigenic in AS patients. Epitope I may be a site for crossreactivity between HLA B27 and Klebsiella.

Amino Acid Sequence↗
PubMed1990

Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein.

Human papillomavirus (HPV) type 16 is implicated in the aetiology of anogenital dysplasia which may progress to malignancy. HPV-16 DNA is actively transcribed in cervical carcinomas, the most abundant transcripts being from the E6 and E7 early open reading frames. The E7 protein has been shown to have transforming activity in vitro. In this report we define four immunodominant B epitopes within the protein corresponding to the E7 gene, using a panel of murine monoclonal antibodies. Three epitopes are linear and lie within the N-terminal region of the molecule, and are unique to the HPV-16 E7 protein. One epitope is non-linear and presumed to be conformational. At least three of the four epitopes of the E7 protein are detectable by immunoprecipitation from an HPV-16-infected cervical carcinoma cell line. The demonstrated immunogenicity of the E7 protein allows us to deduce that this molecule may be a potential candidate for incorporation in a vaccine against cervical cancer.

Amino Acid Sequence↗
PubMed1990

Epitope mapping of the Mycobacterium bovis secretory protein MPB70 using overlapping peptide analysis.

The gene encoding the major Mycobacterium bovis secreted protein MPB70 was cloned and isolated from a DNA library in lambda EMBL 3, and the restriction map of the area of chromosome containing the gene ascertained. After sub-cloning, the complete DNA sequence and predicted amino acid sequence were determined, and from this information a series of overlapping octapeptides encoding all possible linear epitopes of eight or less amino acids were synthesized. These peptides were probed with monoclonal antibodies specific for M. bovis and with sera from M. bovis-infected cattle. Epitopes defined by this technique were then examined using a substitution analysis that allowed the significance of each amino acid in the putative epitope to be quantified, and the exact specificity of the antibody response for the epitope determined.

Amino Acid Sequence↗
PubMed1990

Novel cross-reactive epitopes on asexual blood stage antigens of Plasmodium falciparum.

An IgM monoclonal antibody that reacts with the merozoite membrane and internal merozoite antigens was shown to recognize several previously characterized asexual blood stage antigens of Plasmodium falciparum as well as new antigens. Among the reactive antigens identified were FIRA, GYMSSA, RESA and the S-antigen. Analysis of the cross-reactions between FIRA and GYMSSA by epitope scanning was performed. The most reactive peptides in GYMSSA had the common sequence STNS. The cross-reactive epitopes in FIRA could, in many cases, be explained by the results of a replacement net analysis performed on the STNS epitope. It is proposed that the cross-reactive epitopes, which in several cases have no obvious linear homology but possess high S, T and N content, may be present as loops or coils on the surface of the molecules.

Amino Acid Sequence↗
PubMed1990

A monoclonal antibody raised against an undecapeptide sequence from human transforming growth factor alpha recognizes a hexapeptide epitope in mitotic centrosomes.

A monoclonal antibody (mAbIIa138) raised against the second-loop undecapeptide (residues [21-31]) of human transforming growth factor alpha, known to be involved in binding to its cell receptor, was found to define a hexapeptide epitope (RFLVQE, residues [22-27]). In enzyme-linked immunosorbent assay testing mAbIIa138 did not react with either native or reduced human transforming growth factor alpha, indicating that conformational restraints in this protein interfered with the antigenicity of the cognate sequence. Despite its failure to react with human transforming growth factor alpha, this monoclonal antibody proved very interesting when used to immunostain mammalian cells. mAbIIa138 was found to bind with great specificity to the centrosomes of late-interphase and mitotic cells. The staining of the centrosomes was most intense when the centrosomes were active in organizing the mitotic spindle and faded during telophase as the spindle was disaggregated. The epitope that mAbIIa138 recognizes is thus in a centrosomal protein, denoted CSP alpha, involved in some aspect of mitotic spindle organization or function. Analysis of the antigenic stringency of the epitope, using an amino acid replacement set, showed that 25 individual single amino acid replacements were possible without significant loss of antigenicity. Data bank searches for proteins of possible relevance were unsuccessful, so CSP alpha is an as yet unsequenced protein, specific to the centrosome.

Amino Acid Sequence↗
PubMed1990

Characterization of a Plasmodium falciparum epitope recognized by a monoclonal antibody with broad isolate and species specificity.

Monoclonal antibody (MAb) 7H8 raised against Plasmodium yoelii reacted with a series of proteins from P. falciparum that range in molecular weight from 46 to 194 kDa. By immunofluorescence assay, this MAb reacted with all isolates of P. falciparum tested. MAb 7H8 was used to screen a genomic expression library of asexual blood stage antigens of P. falciparum, Malayan Camp K+ and 7 independent clones were identified. These 7 clones were sequenced and the epitope recognized by MAb 7H8 in the recombinant protein of one of these clones was mapped. This epitope contained Lys Tyr Pro as core amino acids. However, similar sequences were not found in the other clones, indicating that this MAb binds to a structural epitope formed by different amino acids. The variable composition of the epitope may account for the number of P. falciparum malarial proteins recognized by MAb 7H8.

Amino Acid Sequence↗
PubMed1989

Bands, interbands and puffs in native Drosophila polytene chromosomes are recognized by a monoclonal antibody to an epitope in the carboxy-terminal tail of histone H1.

A monoclonal antibody was raised against Drosophila melanogaster histone H1. Immunoscreening of proteolytic cleavage fragments of H1 and of a set of all possible overlapping synthetic octapeptides corresponding to the amino acid sequence of H1, revealed that the antibody recognizes an epitope within the sequence 207VTAAKPKA214 near the centre of the carboxy-terminal tail. This antibody gives positive immunofluorescence over the entire length of native D. melanogaster polytene chromosomes isolated from salivary glands by microdissection at physiological pH and ionic strength. Bands, interbands and puffs are all seen to contain H1. The immunofluorescence over puffs, albeit lower than that over bands and interbands, indicates that chromatin decondensation can occur without complete loss of H1 in these structures. The reaction of the antibody with bands suggests that the segment of the C-terminal tail containing the epitope may be exposed in the condensed 30 nm chromatin filament.

Amino Acid Sequence↗
PubMed1989

Serologically defined linear epitopes in the envelope protein of dengue 2 (Jamaica strain 1409).

Antisera from dengue patients and dengue virus infected rabbits recognized octapeptides corresponding to linear amino acid sequences in the envelope protein of dengue 2 (Jamaica 1409). Although no peptide was recognized by sera from all dengue infected hosts, two peptides (216LPLPWLPG223 and 448FSGVSWTM455) were recognized by sera from all dengue 2 infected rabbits. One of these 448FSGVSWTM455 was also recognized by sera from both the dengue 2 patients tested. No peptides were identified which reacted exclusively with all dengue 2 infected animals. Use of a mouse monoclonal antibody (1B7) enabled identification of two regions (50AKQPATLR57 and 127GKVVLPEN134) and possibly a third (349GRLITVNP356) in the envelope protein of dengue 2 likely to be involved in haemagglutination inhibition and virus neutralization in vitro.

Amino Acid Sequence↗
PubMed1989

Similar binding properties of peptide ligands for a human immunoglobulin and its light chain dimer.

The urinary light chain dimer and serum monoclonal IgG1 protein from a patient (Mcg) with multiple myeloma and amyloidosis were systematically tested for their binding activities to peptides presented on solid supports. The system was validated using a series of enkephalins, beta-casomorphins and DNP-lysine derivatives which were known to complex with the dimer. Sets of peptide ligands binding to the proteins were constructed by incremental additions of amino acid residues to minimal binding units [Geysen et al., J. Immun. Meth. 102, 259-274 (1987)]. Both the amino acid sequences and the combinations of optical isomers were optimized at each stage of the syntheses. Binding could be demonstrated for ligands ranging in size from a tethered single amino acid to pentapeptides. At the dipeptide levels, the dimer and the IgG1 protein showed different preferences (Hp versus qf, where lower case letters designate D-amino acid residues). However, in a tetrapeptide ligand (qfHp) for the dimer, both of these initial preferences had converged. With few exceptions, the IgG1 molecule showed binding activity for the ligands developed for the dimer. Two sets of selected peptides, one based on Hp and the other on mW, were synthesized for diffusion into crystals of the dimer. X-ray analyses showed that these peptides bound exclusively in the main binding cavity between the "variable" domains of the dimer. As predicted from the ELISA results with tethered ligands, the relative occupancies in the crystals followed the order of tetrapeptide greater than tripeptide much greater than dipeptide. The crystallographic studies confirmed that peptides with very different sequences can bind in the same cavity.

Bence Jones Protein↗
PubMed1989

Localization of virus-specific and group-specific epitopes of plant potyviruses by systematic immunochemical analysis of overlapping peptide fragments.

Virus-specific or group-specific antibody probes to potyviruses can be produced by targeting the immune response to the virus-specific, N-terminal region of the capsid protein (29-95 amino acids depending on the virus) or to the conserved core region (216 amino acids) of the capsid protein, respectively. Immunochemical analysis of overlapping, synthetic octapeptides covering the capsid protein of the Johnsongrass strain of Johnsongrass mosaic virus (JGMV-JG) has delineated the peptide sequences recognized by five polyclonal rabbit antisera and two mouse monoclonal antibodies (mAbs). The antibodies characterized were (i) three virus-specific rabbit polyclonal antisera and one virus-specific mouse mAb (1/25) raised against native virus particles, (ii) one polyclonal antiserum raised against trypsin-derived core particles of JGMV-JG, (iii) one group-specific polyclonal antiserum raised against the denatured, truncated coat protein from trypsin-derived core particles of JGMV-JG, and (iv) one group-specific mouse mAb (1/16) raised against native virus particles. The two epitopes seen by mAb 1/25 occurred at residues 18-27 and 43-52 and overlapped with the two major epitopes seen by the virus-specific polyclonal antiserum. The group-specific epitope seen in JGMV-JG by mAb 1/16 was also recognized strongly in potato virus Y, the type member of the potyvirus group. The multiple epitopes seen by the cross-reactive polyclonal antisera were distributed across the entire core region of the coat protein and their relative antibody binding responses varied between JGMV-JG, potato virus Y, and six other distinct potyviruses.

Amino Acid Sequence↗
PubMed1989

Cross-reactivity of antibody against an epitope of the Plasmodium falciparum second merozoite surface antigen.

Monoclonal antibodies directed against the 51 kD merozoite surface antigen of Plasmodium falciparum also bind to other antigens within the infected cell. The sizes of these cross-reacting antigens have been characterized. Immunofluorescence due to the reaction of one of the monoclonal antibodies with these cross-reacting antigens was localized in the intra-erythrocytic parasite and in granules in the infected red cell cytoplasm. This immunofluorescence could be distinguished from the merozoite surface antigen in parasite lines with a variant serotype of the merozoite surface antigen which fails to react with the monoclonal antibodies. It was found that the in-vitro growth inhibition caused by the presence of one of the monoclonal antibodies, 8G10/48, was dependent on the expression of the corresponding serotype of merozoite surface antigen, a finding consistent with the inhibitory effect of this antibody being primarily directed against the merozoite surface antigen and not the cross-reacting antigens. Analysis of the frequency at which epitopes occur suggests that such cross-reacting proteins will be commonly seen in malaria, without the need to postulate a selective advantage for such cross-reacting specificities.

Animals↗
PubMed1988

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution.

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.

Amino Acid Sequence↗
PubMed1988

Cognitive features of continuous antigenic determinants.

We sought to identify the features controlling the specificity of antibody recognition and thus gain insights into molecular recognition between proteins in general. A total of 103 epitopes within 63 well-defined antigenic peptides homologous with the relevant antigen sequence were identified. The contribution of each amino acid residue to the antibody binding activity of each epitope was investigated by ELISA testing of complete sets of peptide analogs containing single amino acid replacements. The data are summarized in a replaceability matrix. Some of the high frequency replaceabilities were expected, such as aspartate for glutamate, serine for threonine, etc., but unexpected relationships were also found, such as a high degree of acceptability of methionine as a replacement. Replaceability with a residue of opposite charge was rare. Glycine and tyrosine were frequently of low acceptability, except for glycine as a replacement for alanine. It was found that on average only about four to five amino acid residues in epitopes were required to determine specificity and provide binding energy. Specificity and binding energy were attributed to amino acid side chains rather than main chain atoms. Propensity factors for occurrence of amino acids in antigenic determinants were calculated. The prominence of certain hydrophobic residues as residues critical to recognition by antibody suggests that the molecular surface of an antigen in its combined form with antibody is altered from that occurring in the absence of antibody. Thus, antigenicity is not a static surface phenomenon but depends on the ability of the antigen to undergo rearrangement, supporting the induced fit concept.

Amino Acids↗
PubMed1988

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum.

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.

Amino Acid Sequence↗
PubMed1987

Strategies for epitope analysis using peptide synthesis.

A recently developed approach to the synthesis and ELISA screening of large numbers of peptides is described. The method has created the opportunity to tackle questions about the sites and specificity of antigenic determinants which were formerly thought to be too difficult to answer. The various strategies for application of this method are described along with examples of their successful use. They include a procedure for locating all the continuous antigenic peptides of a protein antigen, and the identification of non-replaceable amino acid residues within an antigenic peptide. An approach to the determination of amino acid residues involved in the epitope for any monoclonal antibody is also described. These strategies open up the prospect of rapid mapping of the antigenic properties of hitherto poorly understood antigens.

Antibody Affinity↗
PubMed1987

Chemistry of antibody binding to a protein.

The chemistry of antibody recognition was studied by mapping the antigenicity of the protein myohemerythrin with peptide homologs of the protein sequence. The results suggest that the entire protein surface is antigenic, but the probability of there being antibodies to a given site is influenced by local stereochemistry. Although accessible to an antibody binding domain, the least reactive positions cluster in the most tightly packed and least mobile regions and are closely associated with narrow, concave grooves in the molecular surface containing bound water molecules. The most frequently recognized sites form three-dimensional superassemblies characterized by high local mobility, convex surface shape, and often by negative electrostatic potential.

Antibodies↗