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J Sharon

Publications and source records attributed to J Sharon.

At least 37 records · Page 2Linked to original sources
PubMed1990

Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis.

Four anti-idiotopic mAB, 107, MB, AI, and AD8, react with mouse hybridoma protein 36-65 specific for the hapten p-azophenylarsonate. The four antiidiotypic antibodies do not react with hybridoma protein 36-71, a somatically mutated variant of 36-65 whose H and L chain V region sequence differs at 19 amino acid positions. To determine which regions of 36-65 are important for the interaction with each of the four anti-idiotypic antibodies, variants of 36-65 containing one or more of the 36-71 substitutions were generated by oligonucleotide-directed mutagenesis of the rearranged 36-65 H chain V region gene, followed by expression of mutant proteins containing either the 36-65 or the 36-71 L chain in transfected hybridoma cells. Idiotypic characterization of the mutant proteins showed that reactivity correlates with the 36-65 H chain, but some contributions from the 36-65 L chain come into play. In the 36-65 H chain V region, idiotopes were mapped to the first and third complementarity-determining regions for anti-idiotypic antibodies 107, MB, and AI, and to all three complementarity-determining regions for anti-idiotypic antibody AD8. The binding of all four anti-idiotypic antibodies to hybridoma protein 36-65 was hapten inhibitable. However, a comparison between the effect of individual 36-71 substitutions on idiotope expression and their effect on Ag-binding affinity suggests that none of the four anti-idiotypic antibodies bodies mimics the structure of Ag.

Amino Acid Sequence↗
PubMed1990

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies.

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.

Amino Acid Sequence↗
PubMed1990

Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

The basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence of the lower-affinity antibody into that of the higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data showed that out of the 19 amino acid differences between the two hybridoma antibodies, the affinity increase could be reproduced by three heavy-chain substitutions that are present in the high-affinity antibody. The combined effect on affinity of amino acid substitutions was generally found to reflect their individual effects. Although the light chain of the high-affinity antibody did not seem to play a major role in the affinity increase, its contribution varied with the kind and number of heavy-chain substitutions. The results hold promise for antibody engineering and are consistent with a stepwise acquisition of somatic hypermutations in which the existing structural context of an antibody most likely influences the affinity-based selection of later substitutions. They further suggest that many substitutions may be tolerated in vivo during the antigen-driven selection process, even though they confer on the antibody no affinity increase.

Amino Acid Sequence↗
PubMed1989

Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten.

Two mouse mAb specific for the hapten p-azophenylarsonate and encoded by the same combination of germ-line V, D, and J genes differ 200-fold in affinity for hapten. We determined the amino acid sequences of the V regions of the high affinity antibody and compared them to the published sequences of the low affinity antibody which is not somatically mutated. Of 19 amino acid substitutions, two, Ile57 and Thr58 in the H chain, also occur, either alone or together, in other somatically mutated antibodies specific for p-azophenylarsonate; these antibodies have been independently isolated. Introduction of either one of these mutations alone into the low affinity antibody by oligonucleotide-directed mutagenesis increased the antibody affinity for hapten three- to fourfold, whereas introduction of both mutations together conferred an eightfold increase in affinity. These results support the hypothesis that somatic mutations are selected on the basis of the affinity for antigen that they confer, and suggest that even relatively small increases in affinity may be selected, probably in a sequential manner.

Amino Acid Sequence↗
PubMed1988

The invariant tryptophan in an H chain V region is not essential to antibody binding.

The first amino acid residue of the second framework region in all antibody H and L chain V regions sequenced to date is invariably tryptophan. To test whether this invariance is essential to proper domain folding and generation of a functional antibody, the tryptophan residue in the heavy chain V region of a mouse anti-p-azophenylarsonate antibody was converted to an alanine residue by oligonucleotide-directed mutagenesis of the H chain gene. The mutant gene was transfected into mouse hybridoma cells that produce the homologous L chain, and the resulting mutant antibody was purified from the cell supernatant. It was shown to have essentially the same reactivity as wild type toward a series of anti-idiotypic antibodies and to bind Ag with a Ka similar to that of wild type.

Amino Acid Sequence↗
PubMed1986

Site-directed mutagenesis of an invariant amino acid residue at the variable-diversity segments junction of an antibody.

Structural analysis of 21 murine A/J antibodies specific for the hapten p-azobenzenearsonate (Ars), and bearing the major cross reactive idiotype (IdCRI), has revealed an invariant amino acid residue, serine, encoded by the variable-diversity gene segments junction of the heavy chain. To test whether this serine residue is essential for Ars binding, we changed it either to alanine or to threonine by oligonucleotide-directed mutagenesis of a heavy chain gene. Genes containing the mutations were separately introduced into mouse hybridoma cells producing the homologous light chain, and the resulting proteins were tested for antigen binding and idiotypic expression. Whereas the serine to threonine mutant retains full antigen binding activity, the serine to alanine mutant does not bind either to Ars-bovine serum albumin-Sepharose or to the Ars-tyrosine hapten. Both mutants show the same reactivity as wild type towards a series of anti-idiotypic antibodies. These results suggest that a hydroxyl group at the variable-diversity gene segments junction of A/J anti-Ars antibodies is essential for antigen binding.

Amino Acid Sequence↗
PubMed1985

C57BL/6 x BALB/c hybridomas produce IgA which assembles into molecules with covalent bonds between heavy chains (H) and light chains (L), and into molecules lacking covalent bonds between H and L.

Examination of the gel electrophoresis patterns of 14C-biosynthetically labeled immunoglobulin from C57BL/6 X BALB/c IgA hybridomas reveals that each of the monoclonal cell populations produces two different forms of IgA: molecules with heavy chains (H) and light chains (L) joined by disulfide bonds, as well as molecules with H and L being noncovalently associated. The possible origin of this was explored: Southern blot analysis of the hybridoma DNA indicated that only one alpha gene is expressed by each cell line; hybridoma cells labeled in the presence of the N-glycosylation inhibitor tunicamycin exhibit both forms; and electrophoresis of biosynthetically labeled spleen cell IgA from C57BL/6, BALB/c and (C57BL/6 X BALB/c) F1 mice shows that BALB/c mice produce only the noncovalently associated form, while C57BL/6 and (C57BL/6 X BALB/c) F1 mice produce both. Possible mechanisms by which two types of IgA may be assembled by the same hybridoma cell are discussed.

Animals↗
PubMed1982

Immunochemical characterization of binding sites of hybridoma antibodies specific for alpha (1 leads to 6) linked dextran.

The combining sites of seven BALB/c IgM, four BALB/c IgA and one C57BL/6 IgA hybridoma antibodies specific for alpha (1 leads to 6) linked dextran were probed by precipitin and precipitin inhibition assays. The 12 antibodies are able to bind to linear determinants in the interior of the dextran molecule; some have sites complementary to six alpha (1 leads to 6) linked glucose residues and other have sites complementary to seven alpha (1 leads to 6) linked glucose residues. From the analysis of the precipitins and precipitin inhibitions, it is concluded that no two hybridoma proteins have identical binding sites.

Animals↗
PubMed1982

Association constants of hybridoma antibodies specific for alpha (1 leads to 6) linked dextran determined by affinity electrophoresis.

Binding constants of monomers of seven BALB/c IgM, four BALB/c IgA, and one C57BL/6 IgA anti-alpha (1 leads to 6) dextran hybridoma antibodies with dextran B512 and with isomaltoheptaose were determined by affinity electrophoresis. Bindings constants to dextran range from 1.52 X 10(5) to 4.43 X 10(5) ml/g for the five IgA monomers and from 1.70 X 10(3) to 6.10 X 10(4) ml/g for the seven IgM monomers. Antibody monomers containing both specific and nonspecific (derived from the myeloma cell that was used to generate the hybridomas) light chains are shown to have association constants with dextran 6 to 30-fold lower than monomers containing only specific light chain, suggesting that the association of specific heavy chain with nonspecific light chain does not result in an anti-dextran combining site. Binding constants with isomaltoheptaose range from 1.45 X 10(4) to 7.01 X 10(4)/M for the IgA proteins and from 6.46 X 10(3) to 7.70 X 10(4)/M for the IgM proteins. The binding constants with dextran and with isomaltoheptaose, and the electrophoretic, immunochemical and idiotypic characteristics of the hybridoma proteins are discussed.

Animals↗
PubMed1982

Diagnosis of cutaneous T cell lymphoma by use of monoclonal antibodies reactive with tumor-associated antigens.

Two murine monoclonal antibodies (BE1 and BE2), produced by using leukemic helper T cells from a patient with cutaneous T-cell lymphoma (CTCL) as immunogens, reacted selectively with CTCL lymphocytes and some transformed cultured lymphocytes, as determined by radioimmunoassay (RIA) and indirect immunofluorescence (IIF). BE1 reacted significantly (P less than or equal to 0.001) with leukemic CTCL lymphocytes and with CTCL cells from infiltrated lymph nodes (RIA, mean +/- SD = 776 +/- 275 cpm), as compared with background counts (263 +/- 68). BE1 binding to normal blood mononuclear cells (RIA, mean +/- SD = 283 +/- 58 cpm) was indistinguishable from background. BE1 also reacted with Epstein-Barr virus (EBV)-transformed B-cell lines (RIA, mean +/- SD = 794 +/- 230) and some long-term T-cell lines. BE1 did not react with the majority of lymphoid cell lines or tumor cell lines tested. BE1 also did not react with any normal tissues screened by IIF. BE1 precipitated a molecule from CTCL cells that, under reducing conditions, has two components with molecular mass of 27,200 and 25,800 D. BE2 also reacted significantly (P less than or equal to 0.001) with CTCL cells from two of four patients (RIA, mean +/- SD = 519 +/- 113 cpm). The binding of BE2 to normal mononuclear cells was indistinguishable from background (309 +/- 38 cpm). BE2 also reacted with an antigen present on EBV-B-cell lines (RIA, mean +/- SD = 654 +/- 194) and MOLT 3 and HUT 78 T-cell lines. BE2 reacted with an antigen expressed on a subpopulation of lymphocytes from five of eight patients with B-cell CLL studied by IIF (mean +/- SD = 18 +/- 6). Other long-term T-cell lines and tumor cell lines studied by IIF were unreactive with BE2. BE2 did not react with any of the normal tissues studied. BE2 precipitated a molecule (78,000 D) from CTCL cells and EBV-B cells with a single component under reducing conditions. Immunoperoxidase-labeled BE1 and BE2 reacted with CTCL cells in frozen sections of infiltrated lymph nodes and skin. In addition, BE1 and BE2 reacted with blood lymphocytes from 16 of 21 patients whose CTCL had otherwise been considered localized to skin. These two monoclonal antibodies react with tumor antigens associated with CTCL and appear to be useful in the diagnosis of this disorder.

Antibodies, Monoclonal↗
PubMed1982

A cross-reactive idiotype, QUPC52 IdX, present on most but not all anti-alpha (1 replaced by 6) dextran-specific IgM and IgA hybridoma antibodies with combining sites of different sizes.

Seven BALB/c IgM, 4 BALB/c IgA, and 1 C57BL/6 IgA anti-alpha (1 replaced by 6) dextran hybridoma antibodies were characterized idiotypically. Five of the 7 IgM and all 4 BALB/c IgA proteins bear a cross-reactive idiotype present on the anti-alpha (1 replaced by 6) dextran BALB/c myeloma protein QUPC52 and on a majority of anti-alpha (1 replaced by 6) dextran antibodies in BALB/c mice. Of these 9 monoclonal antibodies, some have combining sites as large as 6 glucose residues, and some have combining sites as large as 7 glucose residues. Individual idiotypes present on QUPC52 are differentially expressed on the 9 hybridoma proteins that bear the cross-reactive idiotype. One BALB/c IgM hybridoma protein and the C57BL/6 IgA hybridoma protein did not react with anti-QUPC52 idiotypic antibodies; another BALB/c IgM hybridoma antibody showed only marginal reactivity.

Animals↗
PubMed1980

Formation of hybridoma clones in soft agarose: effect of pH and of medium.

Optimal conditions for the formation of hybridoma clones in soft agarose are described. The hybridization frequency is shown to be highly dependent on the pH of the polyethylene glycol (PEG) solution used for fusion and on the cloning medium. Maximal numbers of clones are obtained when the PEG solution used for fusion is at pH 8.0-8.2.

Animals↗
PubMed1979

Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies.

A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after a week. Nitro-cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is removed and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with radiolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with alpha-(1 leads to 3)-specific antidextran myeloma J558, and the method's applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for alpha-(1 leads to 3) dextran.

Animals↗
PubMed1976

Preparation of Fv fragment from the mouse myeloma XRPC-25 immunoglobulin possessing anti-dinitrophenyl activity.

The myeloma IgA protein produced by plasmacytoma XRPC-25, was isolated by affinity chromatography on dinitrophenyllysine-Sepharose. The affinity constant of the intact protein or its Fab' toward 2,4-dinitrophenyl-L-lysine (Dnp) was found to be 2.6 X 10(5) M-1. In order to prepare an Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1973), Biochemistry 12, 1130) from this protein, the heavy and light chains were separated and the light chain was digested with trypsin at pH 8.2 to yield half a light chain. This digest was reassociated with the heavy chain and the recombinant was digested with papain at pH 5.7. Fractionation of this digest on a Sephadex G-75 column and Dnp-lysine-Sepharose resulted in the isolation of an Fv fragment which possesses one binding site for Dnplysine (Ka = 2.0 X 10(5) M-1). The active Fv fragment has a molecular weight of 23,400 and is composed of two peptide chains, each having a molecular weight of approximately 12,000. The N-terminal residues of these chains are aspartic and glutamic acids, which are also N-terminal in the heavy and light chains, indicating that the Fv is composed of VL and VH.

Binding Sites, Antibody↗
PubMedDate not supplied

Expression of a VHC kappa chimaeric protein in mouse myeloma cells.

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.

Animals↗