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Biomedical subjects

E W Voss

Publications and source records attributed to E W Voss.

At least 127 records · Page 7Linked to original sources
PubMed1982

Monoclonal murine anti-nucleic acid antibody with double-stranded specificity.

The NZB/NZW F1 murine model for the autoimmune disease systemic lupus erythematosus (SLE) has been employed in somatic cell hybridizations to develop hybridoma autoantibodies with double-stranded (ds) DNA specificity. Monoclonal anti-DNA antibodies from one hybridoma cell line were purified and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Results of comparative binding studies with tritiated [3H]-colicin E1 plasmid DNA probes suggested preferential binding for the native DNA conformation relative to single-stranded DNA. [3H]dsDNA binding was inhibited by several ribohomopolymers (poly G, U and I) but not by free nucleotides, indicating that the phosphodiester-ribose backbone may contribute to the binding specificity of the clonotype.

Animals↗
PubMed1982

Fluorescent nucleotides: application in a fluorescence immune assay.

A fluorescence immune assay designed to measure anti-nucleotide antibody activity is described based on the synthesis of a fluorescent nucleotide probe possessing a low fluorescence quantum yield when free in aqueous solution (neutral pH). The fluorophore, AmNS (1-naphthylamine-5-sulfonic acid), was covalently conjugated to various phosphate derivatives of nucleotides through carbodiimide activation to form the fluorescent probe. The quantum yield (phi) of the fluorescent nucleotide in solution (neutral pH) was approximately 0.025 based on an excitation maximum of 320 nm and an emission maximum of 460 nm. Anti-nucleotide antibodies elicited in rabbits and mice served as standard immunological reagents in development of the fluorescence assay. Upon binding of an AmNS-nucleotide conjugate with homologous anti-nucleotide antibodies, the fluorescence quantum yield of the conjugate was significantly enhanced (12-35 x). Fluorescence enhancement was not obtained upon incubation of the fluorescent probe with normal Ig, non-immune rabbit sera and murine ascites fluid, or bovine and rabbit serum albumin. Nucleotide inhibition reactions were quantitatively measured in the fluorescence assay. Nucleotide binding results obtained with the fluorescence assay were correlated with a modified radioimmune Farr assay.

Antibody Specificity↗
PubMed1982

Generation and characterization of three murine monoclonal nucleotide binding anti-ssDNA autoantibodies.

Three murine monoclonal anti-ssDNA autoantibodies have been produced by fusion of spleen cells from NZB/NZW F1 hybrid mice with Sp 2/0-Ag 14 murine myeloma cells. Specificity studies characterizing monoclonal anti-ssDNA autoantibodies revealed binding patterns for nucleotides. Clone E-4-1 showed preferential binding with mono-, di- and polyguanosine. Clones E-4-4 and E-11-1 specifically bound mono-, di- and polythymidine. Results of inhibition studies indicated an order of reactivity for each monoclonal autoantibody: polynucleotide greater than dinucleotide greater than mononucleotide. In a fluorescence immune assay, using aminonaphthyl derivatives of nucleotides, all three monoclonal antibodies exhibited a low affinity (5 x 10(4)-10(5) M-1) for the homologous mononucleotides. The affinity of clone E-4-1 increased approximately 10-fold when a dinucleotide derivative was used relative to a mononucleotide fluorescent ligand.

Animals↗
PubMed1982

Consequences of avidity in lymphocyte receptor-multivalent antigen binding in affinity maturation.

A mathematical model proposed by Crothers & Metzger (Immunochemistry 9, 341-357, 1972), describing the influence of multiple site interactions on the effective antigen binding property of antibodies was used to assess the possible role of lymphocyte receptor affinity in antibody affinity maturation. The model proposed that observed association constants (KobsS) were primarily dependent on the number of allowed active site-antigen interactions (n), the density and orientation of antigenic sites and the intrinsic association constant (K1) of an univalent antibody active site interacting with an univalent antigen. Because of the inherent uncertainty involved in the estimation of lymphocyte membrane structure and molecular parameters necessary to utilize other mathematical models, the Crothers and Metzger model was applied to the case of surface membrane bound immunoglobulin (smIg) on B-lymphocytes interacting with polyvalent antigens as a simple and direct concept. Consideration of red blood cells, dinitrophenyl16 guinea-pig albumin, and fluorescyl220 keyhole limpet hemocyanin as model antigens revealed that Kobs was many orders of magnitude higher than K1 when only a few interactions were allowed. A dissociation rate of several hours for release of polyvalent antigen from cooperating smIg was calculated for receptors with a K1 as low as 10(3) M-1. Assuming prolonged antigen contact as necessary to activate lymphocytes, cells bearing low-affinity receptors are functionally indistinguishable from those carrying higher-affinity smIg in their ability to retain antigen. The presumed role for antigen-driven selection of lymphocytes bearing increasingly higher-affinity smIg, which results in antibody affinity maturation, must be reexamined if the K1 of smIg does not confer a selective advantage to lymphocytes for retaining antigen.

Animals↗
PubMed1981

Kinetics and mechanism of deuterium oxide-induced fluorescence enhancement of fluorescyl ligand bound to specific heterogeneous and homogeneous antibodies.

Comparative kinetics studies of ligand dissociation and D2O enhancement were performed with both heterogeneous and homogeneous anti-fluorescyl immunoglobulin G antibodies. Heterogeneous rabbit and homogeneous mouse (monoclonal) antibody preparations were purified by immunoadsorption and found to be pure IgG by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoelectrophoresis. Relatively high affinities of all liganded antibody preparations were determined by dissociation rate studies, demonstrating comparatively long lifetimes for the dissociation of bound fluorescein. In addition, rabbit anti-fluorescyl preparations were found to display marked heterogeneity of off-rates while mouse monoclonal anti-fluorescyl preparations exhibited a single off-rate indicating homogeneity. D2O fluorescence enhancement studies showed that heterogeneous kinetics was observed with both heterogeneous and homogeneous antibody active sites. Temperature studies of ligand D2O enhancement and dissociation rates using homogeneous anti-fluorescyl antibodies revealed similar, yet different activation energies (22.7 +/- 0.8 cal and 20.2 +/- 0.3 cal, respectively) for both phenomena. The studies demonstrated that the anti-fluorescein antibody active site consists of both solvent accessible and relatively inaccessible components, and that the binding of ligand involves both exchangeable hydrogen atoms and other as yet unresolved interactions. The mechanism of D2O fluorescence enhancement is discussed in terms of its complexity involving heterogeneous rate mechanisms.

Animals↗
PubMed1981

Characterization of fluorescent 2-dimethylaminonaphthalene-5-sulfonyl-immunoglobulin G conjugates for application in fluorescence polarization studies.

Preparations of normal rabbit IgG antibody (NgG) and purified rabbit IgG antibody of anti-2,4-dinitrophenyl (Anti-Dnp) specificity were covalently labeled with the fluorescent probe 2-dimethylaminonaphthalene-5-sulfonyl chloride (2,5-Dns-Cl). 2,5-Dns-immunoglobulin G preparations were shown to have average fluorescence lifetimes of 29--32 ns by cross-correlated phase and modulation fluorescence lifetime measurements. Excitation maxima were observed at 275 and 368 nm, while the emission maximum occurred at 465 nm. With 390 nm excitation, an intrinsic fluorescence polarization of 0.43 was observed. Molar extinction coefficients of 36 000 M-1 . cm-1 (at 258 nm) and 2090 M-1 . cm-1 (at 352 nm) were determined by synthesis of 2,5-Dns-L-[4,5-3H]leucine, a tritiated sulfonamide of 2,5-Dns. Low labelling (1 mol 2,5-Dns/mol of antibody) of rabbit Anti-Dnp IgG antibody with 2,5-Dns-Cl did not significantly alter the antibody association constant with Dnp. Fluorescence polarization measurements were accurately measured (0.3% error) at antibody concentrations greater than 10(-7) M, using photon counting instrumentation. Rotational relaxation times of 128 and 131 ns were determined for 2,5-Dns-NgG and 2,5-Dns-Anti-Dnp, respectively.

Animals↗
PubMed1981

Restricted reassociation of heavy and light chains from hapten-specific monoclonal antibodies.

Six murine monoclonal antifluorescyl antibody clones encompassing a defined range of affinities and containing kappa light chains with IgG1 or IgG2 heavy chains were examined. As the fluorescence of the ligand is quenched greater than 90% when fluorescein is bound by antifluorescyl antibodies, fluorescence quenching was assayed to monitor polypeptide reconstitution and active site formation on mixing of resolved heavy (H) and light (L) chains. Of 36 possible experimental combinations of H- and L-chain reaction mixtures, only homologous H and L chains (derived from the same parental immunoglobulin molecule) bound fluorescein. Results from fluorescence polarization studies, conducted independently of fluorescence quenching, confirmed the findings. Competitive inhibition and molecular sieve experiments showed that, despite preferential association of homologous H and L chains, several heterologous H and L chains associated to form intact 7S molecules, although no active site was constituted. Thus, polypeptide recombination and formation of functional antigen binding sites are two processes that immunocytes must regulate during cell differentiation and generation of diversity. A mechanism and underlying the observed preferential reassociation of specific H and L chains and a means of generating affinity maturation, as exhibited by the antifluorescein system, is proposed.

Antibodies, Monoclonal↗
PubMed1980

Colicin E1 plasmid as a probe for detection and study of anti-dna activity in SLE sera.

Tritiated colicin E1 plasmid DNA ([3H]DNA) was purified, characterized and developed as a test antigen for study of anti-DNA antibody activity in a modified Farr assay. The homogeneous molecular weight (4.2 X 10(6) daltons), physical state (intact covalently closed circles), and capacity for intrinsic radioactive labeling to a suitable specific activity were all important properties of the plasmid probe. In addition, the ability of the Farr assay to measure primary antigen/antibody interactions made quantitative determinations of anti-double-stranded DNA (dsDNA) antibody activity possible. Sonication of the plasmid resulted in definable fragments which were thermally denatured and used in the measurement of anti-single-stranded DNA (ssDNA) antibody activity. Results from studies in which unlabeled dsDNA, ssDNA, amd free 2'-deoxyribonucleotides were employed to inhibit binding of [3H]DNA by anti-DNA antibodies indicated the presence of 3 distinct anti-DNA specificities in SLE sera.

Antibodies, Antinuclear↗
PubMed1980

Modified hybridoma methodology: antigen-directed chemically mediated cell fusion.

The concept of antigen-directed cell fusions to increase the yield of hybridomas was investigated. To facilitate cell-cell contact, antigen conjugated cells were used in cell fusion studies. Specifically, fluorescyl conjugated murine myeloma cells (Sp 2/0-Ag14) incubated with murine immune (anti-fluorescyl) splenocytes formed aggregates containing fluorescent and non-fluorescent cells. Fusion of these populations with polyethylene glycol resulted in a greater number of anti-fluorescyl hybridomas relative to normal fusions under non-antigen directed condition. Ligand binding data indicated that despite the multi-cellular aggregates the hybridomas resulting from chemically mediated fusions produced only one monoclonal Ig.

Animals↗
PubMed1979

Recombination of heavy and light chains of immunoglobulins. Use of specific variable and constant region probes to monitor reassociation reactions.

The reassociation of antibody (IgG)-derived heavy (gamma) and light (L) chains was studied by utilizing two specifically localized fluorescent probes. L chains labeled in either the variable domain with fluorescein (specific antigen) or the constant domain with N-(iodoacetamidoethyl)-5-naphthylamine-1-sulfonic acid were prepared. Reassociation of these chains with isolated, unlabeled, gamma chains was monitored by following an increase in the fluorescence polarization of fluorophores at 4 degrees C. Results obtained indicate that the reassociation reaction could be resolved into two distinct steps; the primary interaction of the reactants occurring rapidly, followed by a slower secondary phase possibly representing conformational changes in the variable domains of the reactants after reassociation.

Fluoresceins↗