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M D Scharff

Publications and source records attributed to M D Scharff.

At least 91 records · Page 5Linked to original sources
PubMed1988

Identification of mutant monoclonal antibodies with increased antigen binding.

Sib selection and an ELISA have been used to isolate hybridoma subclones producing mutant antibodies that bind antigen better than the parental monoclonal antibody. Such mutants arise spontaneously in culture at frequencies of 2.5-5 X 10(-5). The sequences of the heavy and light chain variable regions of the mutant antibodies are identical to that of the parent and the Ka values of the mutants and the parent are the same. The increase in binding is associated with abnormalities of the constant region polypeptide and probably reflect changes in avidity of these antibodies.

Animals↗
PubMed1987

Pharmacokinetic analysis of biosynthetically radiolabeled mouse monoclonal antibodies in normal rhesus monkeys.

Two mouse monoclonal IgG2b antibodies that differ in their CH2 domains have been compared for their blood mean residence times (MRTs) in normal female Rhesus monkeys. A biosynthetically incorporated radiolabel (S-35 methionine) was used to follow blood elimination of the antibodies and urinary excretion. Results indicate that the blood (plasma) MRTs for the Ar13.4 and the ArM16.1 antibodies were remarkably similar in normal Rhesus monkeys. This observation varies greatly from those obtained with these same antibodies in separate studies in normal Balb/c mice. In Balb/c mice, the ArM16.1 antibody was found to be removed from the blood six times faster than the Ar13.4. The CONSAM program was used to fit a multiexponential function to the plasma data obtained from mice and monkeys. The sum of exponentials was then used to calculate the MRT values. These results demonstrate that mouse monoclonal antibody pharmacokinetics significantly differ between normal primates and mice. Presumably the Rhesus monkey better reflects the clinical behavior and pharmacokinetics of mouse monoclonal antibodies than do rodents. Therefore, the use of normal primates for the preclinical evaluation of monoclonal antibodies for in vivo use is suggested.

Animals↗
PubMed1987

Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region.

The S107 myeloma cell line expresses the germ-line sequence of the T15 antiphosphocholine (P-Cho) antibody, which is the major antibody made by BALB/c mice in response to P-Cho, either on a variety of bacterial polysaccharides or when attached to a protein carrier. We have previously reported that a somatic mutant of the S107 cell line produces an antibody that has lost the ability to bind P-Cho and has acquired binding for double-stranded DNA. This antibody has a substitution of an alanine for a glutamic acid at residue 35 in the heavy chain variable region. We now show that this amino acid substitution is due to a single A-C transversion, which is the only nucleotide change in the heavy and light chain variable regions. Further, it appears that this change is due to somatic mutation rather than to gene conversion.

Animals↗
PubMed1987

Studies on the somatic instability of immunoglobulin genes in vivo and in cultured cells.

We have examined the molecular mechanism and impact of somatic diversification on the T15 heavy chain variable region gene in vivo and in vitro. Somatic point mutation appears to be responsible for the changes we have observed in both hybridomas from early and late in the immune response and in the S107 myeloma cell line in culture. By identifying S107 mutants with decreases in antigen binding, we have shown that a single point mutation can cause the loss of binding to the eliciting antigen and the acquisition of binding to another antigen. Furthermore, in this case a point mutation of the T15 heavy chain variable region gene caused the conversion of an important protective antibody to an autoantibody. While the S107 cell line frequently generates both constant and variable region mutants, hybridomas appear to have relatively stable variable region genes and unstable constant region genes which in some cases result in mutants with increased binding.

Animals↗
PubMed1986

The role of monoclonal antibodies and the recombinant DNA technology in studying autoantibody production.

The hybridoma technology has made it possible to sample the B-cell repertoire and to generate monoclonal antibodies which can be analyzed for their specificity and idiotypy. Using the recombinant DNA technology, the structure of the genes which encode those antibodies can be analyzed. The knowledge gained from the application of these techniques has made it possible to pose specific questions about the origins of autoantibodies.

Animals↗
PubMed1986

Tailor-made monoclonal antibodies.

Monoclonal antibodies, potentially powerful diagnostic and therapeutic reagents, are having a significant impact in basic and clinical research. Some persisting problems must be solved, however, in order to realize the potential of this technology. We review the basic biology of antibody molecules and the genes that encode them, and discuss how this knowledge can be applied to make more effective monoclonal antibodies.

Amino Acid Sequence↗
PubMed1985

Fc receptors on cultured myeloma and hybridoma cells.

The specificity of the Fc gamma receptors on the X63.Ag8.653 nonproducing myeloma cell line has been examined for binding to IgG1-, IgG2a-, and IgG2b-containing antigen-antibody complexes. Complexes containing each of these subclasses bind, and the binding of each is inhibited by the others. Trypsin treatment did not inhibit the binding of any of these subclasses. Furthermore, the monoclonal anti-Fc receptor antibody 2.4G2 inhibits the binding of all three subclasses. These results, together with those of other investigators, suggest that there is a single FcR for IgG1, IgG2a, and IgG2b on mouse B cells which differs in its specificity from the macrophage Fc gamma R. This is confirmed by the fact that a mutant IgG2b myeloma protein which binds to the macrophage Fc gamma 1/gamma 2b receptor does not bind to the Fc gamma R on X63.Ag8.653.

Animals↗
PubMed1984

The identification of monoclonal class switch variants by sib selection and an ELISA assay.

Monoclonal antibodies can now be generated against a wide variety of antigens. However, a potentially useful monoclonal antibody may be of the wrong class or subclass for a particular task. Antibodies of the desired class can be obtained by identifying rare subclones in which the variable region has been rearranged to a new constant region. We describe here the use of sib selection and an enzyme-linked immunoassay (ELISA) for isolating such class and subclass switch variants from 2 IgM and 1 IgG3 producing hybridoma. The relative simplicity of ELISA assays makes it feasible to apply this approach to many different hybridomas. Since commercial affinity purified class and subclass specific antibodies are now available, the method can be used by any laboratory. Furthermore, the technique does not require that the switch variants express surface immunoglobulin and enriches for hybridomas secreting higher amounts of antibody.

Animals↗
PubMed1984

Rat anti-T15 monoclonal antibodies with specificity for VH- and VH-VL epitopes.

BALB/c mice immunized with phosphorylcholine (PC) produce antibodies which are predominantly of the T15 idiotype. Monoclonal anti-T15 antibodies have been generated in a number of laboratories by allogenic or syngenic immunization. Most of these mouse monoclonal antibodies react with idiotopes that are in or near the PC-binding site and require the presence of both T15 heavy and T15 light chain variable regions. By immunizing rats with T15 immunoglobulins, we have obtained monoclonal antibodies that recognize idiotopes that are not near the antigen-binding site. Four of these rat anti-T15 monoclonal antibodies react with free T15 heavy chains and with T15 heavy chains associated with irrelevant light chains while four other rat monoclonal antibodies require the presence of both T15 heavy and T15 light chains. This battery of rat anti-T15 monoclonal antibodies is useful in searching for heterogeneity within the T15 antibodies and in following the expression of the T15 heavy chain variable region in different strains of mice.

Animals↗
PubMed1984

Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibody specificity.

The S107 IgA kappa-chain myeloma cell line makes an antiphosphocholine antibody of the T15 idiotype. A somatic mutant of this line, U4, makes an immunoglobulin with a single amino acid substitution of an alanine for a glutamic acid at residue 35. This single amino acid change results in a loss of phosphocholine binding activity. However, the U4 immunoglobulin has acquired reactivity with a variety of phosphorylated macromolecules, including double-stranded DNA, protamine, and cardiolipin. Thus, a single amino acid change in the T15 heavy chain can transform an antibacterial antibody into an antibody that resembles the autoantibodies seen in mice and man with autoimmune disease.

Animals↗
PubMed1983

Monoclonal antibodies reveal the structural basis of antibody diversity.

Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.

Amino Acid Sequence↗
PubMed1983

Immunoglobulin heavy chain class switch from IgM to IgG in a hybridoma.

A subclone of an IgM-producing hybridoma has been identified which has switched to producing an IgG1 antibody. The parent hybridoma, PC-140, produces an antibody which binds phosphorylcholine and reacts with monoclonal antibodies that recognize myelomas of the T-15 idiotype. The IgG1 antibody binds phosphorylcholine with the same affinity as the parental IgM and also reacts with the anti-T-15 monoclonal antibodies. While the IgM-producing parent hybridoma does not express detectable surface IgM, the IgG1-producing subclone produces both membrane and secreted IgG1.

Animals↗