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D C Benjamin

Publications and source records attributed to D C Benjamin.

At least 37 records · Page 2Linked to original sources
PubMed1994

Crystallization and preliminary X-ray analysis of an anti-staphylococcal nuclease-staphylococcal nuclease complex and of a second anti-staphylococcal nuclease antibody.

The Fab fragments of several monoclonal antibodies that bind Staphylococcal nuclease have been screened for crystallization conditions. Two of these, N10 and N25, have been crystallized in forms suitable for X-ray structural analysis. The anti-Staphylococcal nuclease antibody complex N10 Fab-nuclease crystallizes with symmetry consistent with space group C2 and cell parameters of a = 234.7 A; b = 43.5 A; c = 74.4 A; beta = 106.4 degrees. A second anti-Staphylococcal nuclease antibody, N25, although crystallized starting with the Fab-nuclease complex, apparently crystallizes as uncomplexed N25 Fab with symmetry consistent with space group P3(1)21 (or its enantiomorph P3(2)21) and cell parameters of a = b = 80.9 A; c = 138.4 A.

Antibodies, Bacterial↗
PubMed1992

Long-range changes in a protein antigen due to antigen-antibody interaction.

Amide exchange kinetics were used to probe the conformation of hen egg-white lysozyme complexed with the anti-lysozyme monoclonal antibody HyHEL-5. Following the technique developed by Paterson et al. [(1990) Science 249, 755-759] we used two-dimensional NMR to measure amide exchange kinetics of the lysozyme amide protons in the lysozyme-antibody complex. A total of 15 amide protons showed altered exchange kinetics in the presence of the complex. Five of these 15 protons reside on residues that are found within the epitope as defined by X-ray crystallography. Five residues are located at the perimeter of the epitope. The remaining five residues are removed from the epitope. The perturbation of amide exchange rates at sites distant from the epitope indicates that the formation of antigen-antibody complexes can produce changes in the antigen at sites that are quite distant from the structural epitope.

Amides↗
PubMed1992

Distances between the antigen-binding sites of three murine antibody subclasses measured using neutron and X-ray scattering.

For three different murine immunoglobulins (IgG subclasses 1, 2a, and 2b), the distances between their antigen-binding sites have been measured using neutron scattering from deuterated antigens complexed with proteated IgG. Neutron-scattering data were measured for each antibody-antigen complex in a 41% D2O solvent. Unlike the proteated antibody molecule, the deuterated antigens are strongly contrasted against the 41% D2O solvent and give rise to a scattering profile that contains an interference term related to the distance between the deuterated antigens. For all three subclasses, the damping of this interference term, which gives information on the relative flexibility of the antigen-binding sites, indicates that a single distance is inadequate to describe the observed scattering and a distribution of distances is needed. The scattering profile has been modeled for each subclass to give the mean distance between the antigens and the variance of this distance. For all three IgG subclasses, the mean distance is between 117 and 134 A, and the variance is large (approximately 40 A), indicating a high degree of flexibility of the Fab arms. Small-angle X-ray scattering measurements on the same samples are consistent with the neutron-scattering results.

Animals↗
PubMed1991

The antigenic surface of staphylococcal nuclease. I. Mapping epitopes by site-directed mutagenesis.

The analysis of the antigenic surface of staphylococcal nuclease was begun by generating and characterizing a panel of mAb. Twelve mAb were selected from a large number of anti-nuclease mAb and characterized for affinity and isotype, by their ability to block enzyme activity, and by complementation and competitive inhibition assays for the relative location of epitopes. The mAb were placed in complementation groups based on their distinct binding patterns. These groups define a series of eight overlapping epitopes that are estimated to cover a large portion of the nuclease surface. Four mAb blocked the enzyme activity of nuclease. The epitopes defined by two of these four mAb were localized on the surface of nuclease using single amino acid variant Ag generated by site-directed mutagenesis of the cloned nuclease coding sequence. mAb-25 maps to residue 46 which is located at the edge of the enzyme active site consistent with its ability to inhibit enzyme activity. mAb-19, which also blocks enzyme activity and belongs to the same complementation group as mAb-25, was unaffected by the substitution at position 46. This suggests that mAb-19 and mAb-25, if they do react with the same epitope, have differences in fine specificity. mAb-22 blocks enzyme activity and belongs to an overlapping complementation group. The fourth mAb, mAb-1, which belongs to a distinct, nonoverlapping, complementation group, does not blocks enzyme activity, and is directed to a region of nuclease that includes the amino acid at position 133. This residue is located a short distance from the active site in a region that has been suggested to participate in binding of DNA, a substrate for nuclease. Therefore, the four epitopes defined by these mAb are localized at or near the enzyme active site.

Animals↗
PubMed1991

The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.

Animals↗
PubMed1991

Molecular approaches to the study of B cell epitopes.

For some years, a major goal of immunochemistry has been to determine the molecular architecture of the antibody-combining site and its cognate surface on the antigen, the antigenic determinant or epitope, and to determine the molecular basis of specificity and affinity. In recent years, the crystal structures of several antigen-antibody complexes have been determined. In addition, recombinant DNA technology is beginning to play an increasingly important role in analysis of protein-protein interaction including the study of antigen structure and its interaction with antibody. The purpose of this review is to briefly present some of the major and common properties of the antigen-antibody interface as it is known today and to demonstrate, using a few selected studies, the efficacy of using site-directed mutagenesis to study the nature of the antigenic surface of protein molecules and its interaction with antibody.

Animals↗
PubMed1988

Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations.

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

Animals↗
PubMed1987

Bone marrow irradiation chimeras in the BB rat: evidence suggesting two defects leading to diabetes and lymphopoenia.

A series of bone marrow irradiation chimeras were constructed in an attempt to determine the site of the defect(s) leading to diabetes and/or lymphopoenia in the BB rat. In BB rats that were lethally irradiated and reconstituted with T-cell-depleted Wistar-Furth (WF) rat bone marrow, the incidence of diabetes was reduced, and in animals treated with WF bone marrow at less than 44 days of age, the disease was completely prevented. Such animals demonstrated normal lymphocyte counts in peripheral blood, and normal lymphocyte function (as indicated by mixed lymphocyte response), but retained an abnormal T-cell subset distribution only partially improved above that of diabetes-prone BB rats. The incidence of diabetes in these irradiated chimeras was significantly reduced compared to the incidence in BB rats irradiated at the same age but reconstituted with bone marrow from BB rats. In WF rats that were lethally irradiated and reconstituted with T-cell-depleted bone marrow from overtly diabetic BB rats, no diabetes was induced. Such animals demonstrated normal lymphocyte counts in peripheral blood, normal lymphocyte function, and normal T-cell subset distributions. Overall, these results suggest two defects leading to diabetes and/or lymphopoenia in the BB rat. One of these occurs at the level of the bone marrow stem cell while the other resides in the T-cell differentiative environment.

Animals↗
PubMed1987

A unique epitope on human serum albumin recognized by monoclonal antibody HSA-1: a probe for identification of the human origin of blood or tissue.

A panel of monoclonal antibodies was raised against human serum albumin from fusions of BALB/c splenocytes and SP2/0-Ag14 murine myeloma cells. This panel was screened against purified albumins from 21 species including chimpanzee, gorilla, and orangutan. A monoclonal antibody (HSA-1) specific for human albumin was identified. The epitope recognized by HSA-1 was shown to be conserved in all human blood samples tested. A double antibody ELISA assay was developed using biotinylated HSA-1 as the specific probe for human albumin. This assay was capable of detecting as little as 30 nanograms or less albumin/ml. This assay was used to verify the presence of human albumin in blood, tissue extracts, and other body fluids. These results show that the HSA-1 monoclonal antibody can be used in determining the human origin of blood, tissue, and a variety of other body fluids.

Antibodies, Monoclonal↗
PubMed1987

Inhibition of the tyrosine kinase activity of v-src, v-fgr, and v-yes gene products by a monoclonal antibody which binds both amino and carboxy peptide fragments of pp60v-src.

A monoclonal antibody, R2D2, raised to the src gene product of Rous sarcoma virus was found to inhibit the tyrosine protein kinase activity of pp60v-src in autophosphorylation reactions and in reactions involving exogenously added substrates, such as casein and histone. R2D2 also inhibited the enzymatic activity of two related viral transforming proteins, pp70gag-fgr and pp90gag-yes. The inhibitory ability of R2D2 was dependent upon immunoglobulin concentration and could be demonstrated in both immune complexes formed directly with R2D2 and preformed immune complexes to which R2D2 was added. Binding sites in both the amino-terminal 110 amino acid residues and the carboxy-terminal 240 amino acids of pp60v-src were identified for R2D2. These results indicate that at least part of the epitope recognized by R2D2 resides within a region of the src protein which is required for protein kinase activity. The localization of the R2D2 epitope to the amino- as well as to the carboxy-terminal portions of pp60v-src, together with results of studies analyzing the relative binding efficiencies of R2D2 to the intact protein and to V-8 proteolytic fragments of pp60v-src, are consistent with the view that the R2D2 epitope is conformational in nature and that it is assembled from residues contained within both N-terminal and C-terminal regions of the molecule.

Antibodies, Monoclonal↗
PubMed1986

Prevention of diabetes in BB rats. I. Evidence suggesting a requirement for mature T cells in bone marrow inoculum of neonatally injected rats.

Injection of major histocompatibility complex (MHC)-compatible bone marrow cells from normal animals into neonatal BB rats resulted in a striking decrease in incidence of diabetes and restoration of concanavalin A (ConA) and mixed lymphocyte responses. However, injection of bone marrow cells pretreated with anti-rat thymocyte antiserum plus complement to remove mature T cells had no effect on incidence of disease, suggesting that mature T cells in the bone marrow inoculum were responsible for prevention of diabetes. Because the decreased incidence of diabetes in rats injected with untreated bone marrow appeared to be unrelated to the extent of lymphopenia in these animals, the involvement of T cells in the onset of diabetes must reflect a defect in the normal function of these cells rather than their absolute number. Approximately 50% of the W3/13+ cells in the spleens of BB rats lacked the OX-8 and W3/25 T cell subset markers. The identity of this W3/13+, OX-8-, W3/25- blank subset remains to be established. Our results, interpreted in light of studies from the other laboratories, suggest the existence of multiple abnormalities in the BB rat, including the presence of T cells as effector or helper cells that augment onset of disease and the absence of a regulatory T cell circuit that could prevent the disease.

Animals↗
PubMed1985

Tolerance to azobenzenearsonate: idiotype-specific suppression, B cell dominance, and clonal elimination.

Twenty to 70% of the antibody molecules produced by individual A/J mice in response to azobenzenearsonate (ABA) bear a particular idiotype termed the major cross-reactive idiotype (CRI). Mice that were made tolerant to ABA by injection of ABA coupled to human gamma-globulin show a decrease in production of ABA-specific antibody and a preferential loss of the major CRI. In the experiments reported here, we have used adoptive cell transfers and splenic fragment culture assays to study the mechanism(s) involved in the tolerance to ABA, with emphasis on the preferential loss of the CRI. These studies show that the decrease in total anti-ABA after the induction of tolerance is the result of a decrease in the number of ABA-responsive B cells independent of CRI expression. The preferential loss of the CRI is due to idiotype-specific T cell suppression and/or B cell dominance. In addition, it is demonstrated that immunization in the presence of idiotype-specific suppression converts a normally immunogenic stimulus into a tolerogenic signal, resulting in a decrease in the absolute number of CRI+ B cell precursors.

Animals↗
PubMed1985

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody.

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.

Antibodies, Monoclonal↗
PubMed1984

Monoclonal antibodies specific for the virion polypeptide, p27, of avian retroviruses.

The major component of the core structure of avian sarcoma leukosis viruses is a 27 kD molecular weight polypeptide, p27. Spleen cells from mice immunized with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) were fused with mouse myeloma cells (SP2/0), and hybridoma cell lines producing monoclonal antibodies to p27 were isolated. The monoclonal antibodies were all of the IgG1 subclass with kappa light chains. These antibodies immunoprecipitated p27 and its precursor proteins from extracts of RSV-transformed cells. Reciprocal competitive binding experiments defined five nonoverlapping antigenic determinants within p27. The monoclonal antibodies also immunoprecipitated the transforming protein, p110gag-myc, from avian myelocytomatosis virus transformed cells. Their usefulness in studies of virion maturation and viral oncogenesis is discussed.

Alpharetrovirus↗