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PubMed1993

Epitope mapping of a monoclonal antibody which binds HIV-1 Gag and not the Gag-derived proteins.

Monoclonal antibody (MAb) 1G12 binds the uncleaved HIV-1 Gag polypeptide (p55), but fails to recognize the final products of the proteolytic processing [Sarubbi, E. et al. (1991) FEBS Lett. 279, 265-269]. In this report we show that binding of MAb 1G12 to a 110-residue Gag fragment containing the p17-p24 cleavage site prevents proteolysis of this site by the HIV-1 protease. Competition studies with synthetic peptides have been performed to map the binding site of MAb 1G12 on Gag. The antibody recognizes a sequential epitope that spans the HIV-1 protease cleavage site; determinants located on both p17 and p24 are required for antibody binding. MAb 1G12 is also shown to lack any cross-reactivity with other HIV-1 protease cleavage sites.

Amino Acid Sequence↗
PubMed1993

Epitope mapping and characterization of monoclonal antibodies to human protein C.

Six monoclonal antibodies specific to human protein C were characterized. Epitopes of these antibodies were determined on isolated proteolytic peptides of protein C by immunological methods. Three antibodies bound light chain of protein C: PC01 bound the gamma-carboxyglutamic acid domain calcium-dependently, while PC02 and PC08 bound the first epidermal growth factor-like domain in calcium-dependent and independent manners, respectively. The other three antibodies bound the heavy chain of protein C: PC13 bound activation peptide, PC04 recognized the activation site and PC09 bound the region close to a disulfide bond connecting light and heavy chains. Activation of protein C with thrombin-thrombomodulin complex was inhibited strongly by PC04 and moderately by PC08, PC09 and PC13. PC04 and PC13 may directly block the activation site. On the other hand, epitopes of PC08 and PC09 may be involved in interaction between protein C and thrombin-thrombomodulin complex, or locate close to activation site on the tertiary structure of protein C. Anticlotting activity of protein C was inhibited strongly by PC01 and moderately by PC02, PC08 and PC09, while amidolytic activity was inhibited only by PC09. The epitopes described here may constitute part of protein-C-specific sites, which are important for the function of protein C.

Amino Acid Sequence↗
PubMed1991

Epitope mapping and characterization of the infectious hematopoietic necrosis virus glycoprotein, using fusion proteins synthesized in Escherichia coli.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.

Antibodies, Monoclonal↗
PubMed1987

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus.

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.

Amino Acid Sequence↗
PubMed1990

Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.

Animals↗
PubMed1997

Epitope mapping of cytochrome P450cam (CYP101).

Eighteen linear antigenically active sites were revealed in cytochrome P450 from Pseudomonas putida (P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322-327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).

Amino Acid Sequence↗
PubMed1991

Epitope mapping of antibodies to acetylcholine receptor alpha subunits using peptides synthesized on polypropylene pegs.

Concurrent synthesis of overlapping octameric peptides corresponding to the sequence of the Torpedo acetylcholine receptor (AChR) alpha subunit has been carried out on polypropylene supports functionalized with primary amino groups according to a method developed by M. Geysen [(1987) J. Immunol. Methods 102, 259-274]. The peptides on the solid supports have been used in an enzyme-linked immunosorbent assay. Interactions of the synthetic peptides with antibodies are then detected without removing them from the solid support. By this procedure, epitopes of both antisera and monoclonal antibodies to the Torpedo acetylcholine receptor, its subunits, and synthetic peptide fragments have been mapped. Both rat and rabbit antisera to the alpha subunit show major epitopes spanning the residues 150-165, 338-345, and 355-366 on the Torpedo AChR alpha subunit. Epitopes of monoclonal antibodies to these major epitopes and to others have been rather precisely mapped by using this technique with peptides of varying lengths. The specificity of several of these mAbs are of interest because they have been used in mapping the transmembrane orientation of the AChR alpha-subunit polypeptide chain.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of the carcinoembryonic antigen with various related recombinant proteins expressed in Chinese hamster ovary cells and 25 distinct monoclonal antibodies.

Antigenic epitopes of carcinoembryonic antigen (CEA) and non-specific cross-reacting antigen (NCA) were analysed in relation to their domain structures [domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M for CEA and domains N, I (A1-B1), and M for NCA]. We reconstructed cDNAs for CEA-N, CEA-N-I, CEA-N-I-II, CEA-N-I-II-III-M (CEA-whole), NCA-N, NCA-N-I and NCA-N-I-M (NCA-whole), which were expressed in Chinese Hamster Ovary (CHO) cells. The recombinant proteins were purified by immunoadsorption and gel filtration. Their mol. wts judged from Western blotting were 17,000-26,000 for CEA-N, 70,000 for CEA-N-I, 150,000 for CEA-N-I-II, 165,000 for s-CEA-whole which was spontaneously released from cells into culture medium, 180,000 for p-CEA-whole which was solubilized with phosphatidylinositol specific phospholipase C (PI-PLC) from cells, 18,000-25,000 for NCA-N, 63,000 for NCA-N-I, and 96,000 for p-NCA-whole which was solubilized with PI-PLC from cells. The divergence of the observed mol. wts from those calculated from cDNA sequences seems to indicate that these recombinant proteins are highly N-glycosylated. By enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 25 distinct anti-CEA monoclonal antibodies (MAbs), each representative of 25 different subgroups within five groups (Groups 1-5) previously classified by us in terms of the reactivity with CEA and CEA-related antigens. Twenty-one MAbs previously shown to react with different protein epitopes of the CEA molecule allow to define six groups (A-F) of epitopes according to their expression by different domains of the CEA and NCA molecules. Among four epitopes common to CEA and NCA, two were found to be present on domain N (Group A) and two on domain I (Group B). Among 15 epitopes absent from NCA but expressed by CEA and normal fecal antigens (NFAs), four were on domain N (Group C), five on domain I (Group D) and six on domain II (Group E). Two epitopes were previously described as "CEA distinctive", because they were recognized by MAbs reacting with CEA but not with the NFAs. These two epitopes (Group F) were found to be expressed by p-CEA-whole but not by s-CEA-whole. The latter results suggest that the Group F epitopes are located on a part of the domain III close to the anchoring device of the CEA molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Epitope mapping of hemagglutinating adhesion HA-Ag2 of Bacteroides (Porphyromonas) gingivalis.

Thirteen monoclonal antibodies (MAbs) directed against hemagglutinating adhesion HA-Ag2 of Bacteroides (Porphyromonas) gingivalis were produced by immunizing mice with the relevant immunoprecipitate from crossed immunoelectrophoresis (CIE). Crossed immuno-affinoelectrophoresis and hemagglutination experiments confirmed that our MAbs recognized a molecule able to bind erythrocytes and involved in the hemagglutination process. In immunoelectron microscopy, these MAbs labelled amorphous material as novel cell-bound appendages distinct from fimbriae. CIE experiments allowed differentiation of the MAbs according to reactivity with immunoprecipitates Ag2, Ag8a, and Ag8c, which define HA-Ag2. The epitopes recognized by nine MAbs were mapped on three main antigenic domains (I, II, and III) by competition experiments and further grouped according to chemical composition and distribution on CIE immunoprecipitate. Domain I, defined by two MAbs, comprises an epitope with protein and carbohydrate determinants and distributed on Ag2 only. Epitopes of domain IIA, defined by four MAbs, are distributed on Ag8a, Ag8c, and Ag2 and are essentially composed of protein determinants but also have carbohydrate determinants that enhance the binding of the MAbs but are not essential. Epitopes of domain IIB, defined by two MAbs, and of domain III, defined by a single MAb, have a composition similar to that of domain IIA epitopes but are distributed on Ag8a and Ag8c only. A competition enzyme-linked immunosorbent assay with serum from normal subjects and patients with periodontitis suggested that domain I is more immunogenic than domain II.

Adhesins, Bacterial↗
PubMed1994

Membrane topology of the GluR1 glutamate receptor subunit: epitope mapping by site-directed antipeptide antibodies.

In order to define the membrane topology of the GluR1 glutamate receptor subunit, we have examined the location of epitopes. Antibodies were produced against peptides corresponding to putative extracellular and intracellular segments of the rat brain GluR1 glutamate receptor subunit. Immunocytochemistry at the electron microscopic level in the dentate gyrus of the hippocampal formation showed that epitopes for the antiserum to the N-terminal part of the subunit are located at the extracellular face of the plasma membrane, whereas the antigenic determinants for the antiserum to the C-terminal part are found at the intracellular face of the postsynaptic membrane. Furthermore, antibodies to the N-terminal residues 253-267 reacted similarly with both intact and permeabilized synaptosomes, whereas the binding of antibodies to the C-terminal residues 877-889 increased about 1.6-fold following permeabilization. Our data suggest that the N- and C-terminal regions are located on the opposite side of the membrane and, therefore, the GluR1 subunit probably has an odd number of membrane spanning segments. The antibody cross-reactivities in different species and their effect on ligand binding activity were also established.

Amino Acid Sequence↗
PubMed1993

Epitope mapping of histone 5 (H5) with systemic lupus erythematosus, procainamide-induced lupus and hydralazine-induced lupus sera.

To define the linear epitopes on H5 that react with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) sera, concurrent overlapping hexameric peptides corresponding to the sequence of H5 were synthesized by stepwise elongation of the polypeptide chains on polyethylene supports. The hexapeptides were tested for reactivity with 8 SLE and 8 DIL sera using an enzyme linked immunosorbent assay (ELISA). SLE and hydralazine-induced lupus (HIL) antibodies were most reactive with peptide 45 (SSRQSI) and patients with procainamide-induced lupus (PIL) were most reactive with peptide 24 (SHPTYS). The epitopes of highest reactivity were in the globular domain of H5. Low reactivity was observed with carboxyl terminal peptides. These findings differ from immunoblotting studies of protease cleaved peptides which have previously shown that the H5 determinants are in the carboxyl terminus.

Amino Acid Sequence↗
PubMedDate not supplied

Epitope mapping of the 273-284 region of HSV glycoprotein D by synthetic branched polypeptide carrier conjugates.

To investigate the antigenicity of a predicted epitope region of herpes simplex virus gD, peptides comprising the 273-284 sequence have been synthesized and conjugated to a branched polypeptide with polylysine backbone (poly[L-Lys-(DL-Alam)], AK). In order to analyze the effect of the carrier on the solution conformation of the potential peptide-epitopes, three peptides (273-284, 273-281 and 276-284) and their polypeptide conjugates were studied by CD spectroscopy in PBS or in TFE. In immunized BALB/c and CBA mice, the level of peptide-, conjugate- and carrier-specific antibody responses were measured. Conjugates with synthetic polypeptide carrier AK induced epitope-specific IgG responses, accompanied by the appearance of a low level of carrier-specific antibodies. The cross-reactivity pattern of induced antibodies revealed the presence of at least two functionally distinct, overlapping epitopes, the availability of which was influenced by flanking residues at the N-terminus. Preimmunization of BALB/c or CBA mice with the [276-284]-AK conjugate resulted in the production of HSV-specific antibodies and in prolonged survival of animals infected with a lethal dose of herpes simplex virus. The degree of protection was comparable to that of [1-23]-AK conjugate (30).

Alanine↗
PubMed1994

Limitations of predictive motifs revealed by cytotoxic T lymphocyte epitope mapping of the human papilloma virus E7 protein.

Human papilloma virus (HPV) type 16 is found in the majority of cervical cancer patients and the transforming protein E7 is consistently expressed in cancer cells, making it a potential target for immune attack. In this study we have investigated whether E7 gains access to the MHC class I processing pathway and provides cytotoxic T lymphocyte (CTL) stimulating peptide epitopes. CTL were induced in H-2b mice by immunization with recombinant vaccinia virus expressing E7 (Vac-E7). To map CTL recognition, natural peptides were purified from cells expressing either intact or truncated E7 protein. Following peptide separation by HPLC one major CTL epitope was detected and truncated constructs localized this epitope to the C-terminal region. Mapping with synthetic peptides indicated that residues 49-57 (RAHYNIVTF) were recognised by anti-E7 CTL. Synthetic 49-57 peptide was used to induce CTL, which recognized the same HPLC purified natural peptide fractions as anti-E7 CTL. Binding motifs for H-2b class I molecules did not predict residues 49-57 to be a CTL epitope, but instead the sequence 21-28 (DLYCYEQL) which contains a Kb anchor motif. Synthetic 21-28 peptide was found to bind to Kb class I molecules and readily induced CTL, indicating that the T cell repertoire of H-2b mice can recognize this epitope. However, these CTL did not recognize peptides isolated from E7 expressing cells, showing that natural processing did not produce detectable levels of the 21-28 epitope. Together, the data demonstrate that an unexpected E7 peptide can function as a major CTL epitope.

Amino Acid Sequence↗
PubMed1991

Epitope mapping of two immunodominant domains of gp41, the transmembrane protein of human immunodeficiency virus type 1, using ten human monoclonal antibodies.

Immunogenic regions of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) were previously mapped by examining polyclonal sera from HIV-infected patients and rodent polyclonal and monoclonal antibodies (MAbs) to peptides of gp41. To define the epitopes within these regions to which infected humans respond during the course of infection, the specificity of human MAbs to these regions had to be studied. Using 10 human MAbs identified initially by their reactivity to whole gp41 in HIV-1 lysates, the epitopes within the immunodominant region of gp41 and within a second immunogenic region of gp41 have been mapped. Thus, five MAbs (from five different patients) to the immunodominant domain of gp41 in the vicinity of the cysteines at positions 598 and 604 (hereinafter designated cluster I) reacted with a stretch of 11 amino acids from positions 590 to 600. Four of these five MAbs were reactive with linear epitopes, while one MAb required the conformation conferred by the disulfide bridge between the aforementioned cysteines. Three MAbs to cluster I revealed dissociation constants ranging from 10(-6) to 10(-8) M, depending on the MAb tested and the size of the synthetic or recombinant peptide used in the assay. Five additional MAbs reacted with a second immunogenic region between positions 644 and 663 (designated cluster II). Four of these five MAbs were specific for conformational determinants. Titration of sera from HIV-infected patients showed that there was about 100-fold more antibody to cluster I than to cluster II in patients' sera, confirming the immunodominance of cluster I.

Amino Acid Sequence↗
PubMed1983

Mapping epitopes on the insulin molecule using monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.

Animals↗
PubMed1993

Epitope mapping of anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation of the Transmembrane 4 Superfamily.

CD53 is a pan-leukocyte glycoprotein which is a member of the recently described Transmembrane 4 Superfamily (TM4SF) of membrane proteins that are predicted to span the lipid bilayer four times. The major hydrophilic region of murine CD53 was expressed as a glutathione-S-transferase fusion protein, and the epitopes of four mouse anti-rat CD53 monoclonal antibodies (mAb) (OX-44, 2D1, 6E2 and 7D2) were mapped to this region using mouse/rat chimeric fusion proteins. The epitopes of OX-44, 6E2 and 7D2 are restored by the substitution of a single isoleucine residue for threonine at position 154 in the mouse protein. The 2D1 epitope is non-linear and appears to require the juxtaposition of isoleucine at position 154 with one or more of the amino acids arginine (132), methionine (133) and serine (140). All of these epitopes are shown to be sensitive to reduction, thus indicating the importance of disulfide bonding in the correct folding of the CD53 hydrophilic domain. Moreover, as these four mAb recognize CD53 at the cell surface, the data provide direct molecular evidence for the proposed membrane orientation of the TM4SF.

Amino Acid Sequence↗
PubMed1992

Characterization of a functionally important and evolutionarily well-conserved epitope mapped to the short consensus repeats of E-selectin and L-selectin.

Selectins represent a new family of adhesion molecules, expressed by leukocytes and endothelial cells, that are involved in the regulation of leukocyte traffic. Here we have characterized a new monoclonal antibody (mAb) (EL-246) that recognizes both human leukocyte L-selectin (previously called LAM-1, LECAM-1, or gp90MEL-14) and endothelial cell E-selectin (previously called ELAM-1). EL-246 recognized a 110-kD protein expressed on cells transfected with E-selectin cDNA and stained many postcapillary venules in inflamed human tonsil. EL-246 also stained human peripheral blood leukocytes and showed identity with anti-L-selectin mAb in two-color flow cytometric analysis. The expression of the leukocyte EL-246 antigen was regulated in the same manner as L-selectin and EL-246 recognized anti-L-selectin mAb affinity-purified antigen in SDS/PAGE Western blot analysis. Further, L-selectin cDNA transfectants were specifically stained by EL-246. EL-246 blocked greater than 95% of lymphocyte adhesion to peripheral lymph node high endothelial venules and greater than 90% of neutrophil adhesion to E-selectin transfectants. In addition to the EL-246 epitope being expressed on two different human selectins, it was detected on L-selectin from a variety of different animals. Interestingly, domain mapping studies localized the EL-246 epitope to the short consensus repeat (SCR) domains of L-selectin. EL-246 is the first mAb that recognizes two different selectins and potentially defines a functional epitope encoded by the SCR domains. Inhibitors of selectin function targeted to this region would be expected to have the added advantage of simultaneously blocking the activity of two distinct adhesion proteins involved in inflammation.

Animals↗