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E W Voss

Publications and source records attributed to E W Voss.

At least 91 records · Page 5Linked to original sources
PubMed1989

Comparison of variable region primary structures within an anti-fluorescein idiotype family.

Previous reports described the properties of a high affinity (Ka = 1.7 X 10(10) M-1) prototype anti-fluorescein monoclonal antibody 4-4-20, an intermediate affinity (Ka = 3.7 X 10(7) M-1) prototype 9-40, and Ig members of the 9-40 idiotype family (comprised of 3-24, 5-14, 5-27, 10-25 and 12-40). Although the seven monoclonal anti-fluorescein antibodies expressed similar active site structural determinants (idiotypes) as determined serologically, each was characterized by different affinities for fluorescein and fine specificity binding patterns. Partial heavy (H)- and light (L)-chain N-terminal amino acid sequence analyses revealed all antibodies (except 5-27) were composed of highly homologous VHIII(C) and V kappa II subgroup genes, respectively. Antibody 5-27 utilized a VHIII(B) and a V kappa V subgroup genes and shared low V-region sequence homology with 4-4-20, 9-40 and the remaining 9-40 idiotype family. In addition, complete 4-4-20, VH- and VL-region primary structures were determined to better understand antibody-antigen interactions. Antibody 4-4-20 utilized a VHIII(C) subgroup VH-gene, a truncated Sp2 D gene segment, JH4, a V kappa II subgroup VL-gene, and J kappa 1. Antibody 4-4-20 VH and VL complementarity-determining regions contained many basic and aromatic amino acid residues capable of interaction with fluorescein. Results are discussed in terms of idiotypic and fluorescein-binding characteristics as well as antibody structural and functional diversity in the immune response.

Amino Acid Sequence↗
PubMed1989

Direct adsorption of ssDNA to polystyrene for characterization of the DNA/anti-DNA interaction, and immunoassay for anti-DNA autoantibody in New Zealand White mice.

DNA was adsorbed directly to polystyrene in concentrated NaCl (1.2 M) during simultaneous denaturation to single stranded form by high pH (12.0). Adsorption of single stranded DNA to polystyrene under these conditions (HsDNA) was complete within 15 min. DNA was not dissociated from polystyrene by NaCl concentrations as high as 5.0 M, nor by Tween 20 concentrations up to 50%. The method was ineffective for dT15 adsorption to polystyrene. HsDNA adsorption to polystyrene was compared to an indirect method in which methylated bovine serum albumin (mBSA) was adsorbed to polystyrene after which DNA was electrostatically bound to mBSA. DNA/mBSA interaction was affected by as low as 0.3 M NaCl. Using the DNA/mBSA assay, sera obtained from New Zealand White (NZW) mice showed IgG anti-DNA activity in approximately 50% of mice tested. HsDNA assay found greater than 62% of NZW mice have 10 micrograms anti-ssDNA or more per ml serum. Complex formation involving C1q complement and ssDNA in HsDNA assay were shown to be negligible. Anti-DNA autoantibody production by NZW implicated the NZW parental strain in autoimmunity of F1 progeny obtained from the New Zealand Black X NZW cross.

Adsorption↗
PubMed1989

Three-dimensional structure of a fluorescein-Fab complex crystallized in 2-methyl-2,4-pentanediol.

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

Antibodies, Monoclonal↗
PubMed1989

Structural properties of an anti-fluorescein monoclonal IgM cryoglobulin.

Prior studies with murine monoclonal anti-fluorescein IgM 18-2-3 indicated both a high affinity for Fl (Ka = 2.9 x 10(10)/M), a relatively lower affinity for phenyloxazolone and an active site mediated cryoprecipitability in the absence of bound ligand. Active site related electrostatic interactions appeared to correlate with the low temp insolubility of 18-2-3, since auto-aggregation was sensitive to pH, ionic strength, temp and protein concn. Results of solid phase binding assays indicated that 18-2-3 complexed with murine and human IgM molecules but not with murine anti-Fl Mab 4-4-20 (IgG2a, kappa). Hemagglutination studies showed that 18-2-3 was not a cold agglutinin. Pentameric monoclonal antibody 18-2-3 exhibited a slower association rate with fluorescyl ligand relative to the rate observed with non-cryoglobulin anti-Fl monoclonal antibodies. However, Fab fragments of 18-2-3 displayed relatively faster kinetics, normally observed with anti-Fl monoclonal antibodies. The slower association rate exhibited by pentameric 18-2-3 was attributed to competitive binding between the fluorescein ligand and 18-2-3 determinants. Derivation and characterization of 18-2-3 (Fc)5 fragments indicated co-purification of Fv fragments possessing functional antigen binding sites, providing further evidence for binding of 18-2-3 Fab fragments with isologous Fc. Since 18-2-3 bound other IgM molecules, the mechanism of cryoprecipitation appeared to be an interaction of the fluorescein antigen binding site with specific Fc epitope(s).

Animals↗
PubMed1989

Inter-relationship between immunoglobulin idiotype and metatype.

Allogenic anti-metatype (Met) and anti-idiotype (Id) reagents were elicited to the liganded and nonliganded states, respectively, of a high affinity murine monoclonal anti-fluorescein IgM antibody. Through comparisons of the relative immunogenicity and specificity patterns of the resulting antibody reagents, interpretations regarding the relationship between metatopes and idiotopes were rendered. Anti-Id specificity was measured in terms of the degree of ligand inhibition to two different forms of the fluorescyl hapten (i.e. free ligand and conjugated to a macromolecule). Idiotypic analysis of 18-2-3 H and L chains (immunoglobulin heavy and light chains) demonstrated that recognition of 18-2-3 Id determinants required recombination of H and L chains. Anti-Met reagents were evaluated relative to the liganded and nonliganded states of the IgM antibody. Binding studies indicated anti-Met specificity for liganded or affinity labeled Mab (monoclonal antibody) 18-2-3, but not for nonliganded 18-2-3 or the fluorescein ligand. The affinity labeled metatypic state provided the optimum immunogen yielding an antibody reagent which was rendered specific for the liganded state upon absorption with appropriate immunoglobulin reagents. Antibodies specific for affinity labeled 18-2-3 did not react with liganded 4-4-20, an IgG2a monoclonal anti-fluorescein antibody of similar high affinity but unrelated idiotypically. Results were discussed in terms of intrasite, proximal-site and distal-site epitopes.

Animals↗
PubMed1988

Differences in crystal properties and ligand affinities of an antifluorescyl Fab (4-4-20) in two solvent systems.

An antigen-binding fragment (Fab) from a murine monoclonal antibody (4-4-20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2-methyl-2,4-pentanediol (MPD) in forms suitable for X-ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 x 10(10) M-1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Fab during crystallization trials. In PEG, the ligand remained firmly bound to the protein. The liganded Fab crystallized in the monoclinic space group P2(1) in PEG, with a = 58.6, b = 97.2, c = 44.5 A and beta = 95.2 degrees. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 A, alpha = 83.9 degrees, beta = 87.6 degrees, and gamma = 84.5 degrees. X-ray diffraction data were collected for both forms to 2.5-A resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Fab had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04-01) with activity against single-stranded DNA.

Animals↗
PubMed1988

Cryoprecipitation properties of a high-affinity monoclonal IgM anti-fluorescyl antibody.

A high affinity (Ka = 2-3 x 10(10) M-1) murine monoclonal anti-fluorescein IgM antibody (18-2-3) exhibiting low temp. insolubility in the absence of bound ligand has served as a model to study cryoprecipitation. Insolubility of 18-2-3 at low temp. (4 degrees C) had been shown to be reversible at higher temp. and in the presence of fluorescyl ligand, indicating antigen binding site involvement. The primary objectives were to isolate and identify structural component(s) responsible for insolubility at low temp. Procedures developed for production and isolation of the monomeric subunit (IgMs) involved thiol reduction and gel filtration separation in the presence of the zwitterionic detergent, CHAPS. Fab and (Fc)5 fragments generated by papain digestion were purified sequentially by gel filtration (Sephadex G-200) and affinity chromatography (fluorescein-Sepharose). A protocol for covalent labeling of Fab fragments with the fluorescent chromophore 2,5 DNS-Cl was developed in order to measure steady-state fluorescence polarization. In kinetic and equilibrium cryoprecipitation assays, nonliganded 18-2-3 IgM and IgMs demonstrated insolubility. Bound fluorescyl ligand, increased ionic strength (0.5 M NaCl) or basic pH (greater than 8.0) abrogated cryoprecipitation. Isolated Fab or (Fc)5 fragments did not exhibit low temp. insolubility. Decreased cryoprecipitation occurred when (Fc)5 fragments were added to nonliganded 18-2-3 IgM. Fluorescence polarization of 2,5 DNS Fab 18-2-3 fragments indicated lack of Fab-Fab aggregation. Results suggested that electrostatic interactions involving 18-2-3 antibody combining sites with interactive sites in the Fc region of homologous IgM were responsible for the phenomenon of cryoprecipitation.

Animals↗
PubMed1988

C1q binding by a high affinity anti-fluorescein murine monoclonal IgM antibody and monomeric subunits.

Comparative interactions of purified rabbit C1q with 18-2-3, a high affinity (2-3 X 10(10) M-1) anti-fluorescein (anti-F1) murine monoclonal IgM antibody (pentamer) and constitutive monomeric subunits (IgMs) were studied. Using a solid phase radioimmunoassay (SPRIA), based on immobilized polyvalent antigen, it was shown that the mechanism of C1q binding to IgM was characteristically multiphasic while IgMs yielded monophasic binding curves. The latter compared qualitatively and quantitatively with a monoclonal IgG2a anti-fluorescein antibody with the same intrinsic affinity of 2-3 X 10(10) M-1. C1q binding efficiency to antibodies was significantly enhanced when the immunoglobulins interacted with immobilized multivalent antigen. Monoclonal IgM antibody bind identically to six F1-carrier protein conjugates independent of epitope (F1) density. In contrast, the C1q-antibody interaction binding was dependent upon epitope density. An average distance between F1 epitopes of 80 A was optimal for C1q binding by IgM. At low concn of IgM, when fluorescein was bound by antigen-binding sites on adjacent subunits of an intact pentamer, C1q appeared to bind IgM intramolecularly.

Animals↗
PubMed1988

Anti-fluorescein antibody 3-13 VH gene rearrangement in idiotypically cross-reactive hybridomas.

The deduced amino acid sequence of anti-fluorescein (F1) antibody 3-13 VH region (residues 1-95) was 78% homologous to the alpha-1----3-dextran binding myeloma protein J558 VH region and was in the Wu-Kabat Subgroup II or Dildrop Group I classifications. The 3-13 VH region was rearranged to a D segment with only 8 of 30 bp in common with DFL16.1 germ line D gene and less homologous to all other previously identified D sequences. This sequence was joined to the third codon of JH4. The sequence encoding VH residues 5-91 was subcloned into pSP65 and used as a probe in Southern analyses to monitor 3-13 VH gene rearrangements in 12 other anti-F1 hybridomas differentially expressing (or not at all) the 3-13 idiotype. Three clones which inhibited the 3-13 idiotype-anti-idiotype interaction as effectively as 3-13 (3-12, 3-17 and 3-35), all had rearranged a gene which hybridized to the cloned 3-13 fragment, however, each was contained on a different size restriction fragment. Analyses of five other idiotypically related (but not identical) hybridomas indicated that four had rearranged a cross hybridizing VH gene while no such rearrangements were detected among four idiotypically negative cell lines. A restriction site assay indicated five clones examined had all rearranged a Vk gene to the Jk1 or Jk2 gene segment. The sequence of the antibody 3-13 VH gene and its use in hybridization studies represent the first molecular analysis of a recurrently expressed repertoire specific idiotype within an unrestricted immune response.

Amino Acid Sequence↗
PubMed1988

Polyclonal antibodies specific for liganded active site (metatype) of a high affinity anti-hapten monoclonal antibody.

Syngeneic polyclonal antibodies were elicited to an affinity labeled high affinity (2-3 X 10(10) M-1) anti-fluorescein murine IgG2a monoclonal antibody. Hyperimmune ascites fluid was tested for reactivity with homologous liganded, affinity labeled and non-liganded Fab fragments derived from the high affinity antibody. Binding results demonstrated antibody specificity for the liganded or affinity labeled site, but no reactivity with either the non-liganded form or the fluorescyl ligand. Kinetic analysis showed that the rate of dissociation of the fluorescein ligand was slowed down significantly upon binding of the anti-affinity labeled reagent to the liganded antibody. Antibodies specific for the affinity labeled prototype were not reactive with the liganded form of an IgM monoclonal anti-fluorescyl antibody of the same affinity but idiotypically unrelated. Results of the immunological studies suggested that the antibody active site stabilized by bound ligand differed from the idiotype of the antibody. The term "metatype" was proposed for the immunological definition of the liganded active site to distinguish it from idiotype (non-liganded). The general nature of metatopes is discussed in terms of conformational or sequential epitopes.

Animals↗
PubMed1988

Monoclonal antibodies specific for rat relaxin. I. Production and characterization of monoclonal antibodies that neutralize rat relaxin's bioactivity in vivo.

The physiological role of relaxin during pregnancy and at parturition in the rat is not absolutely established. There are limitations to the experimental approach used in the few studies that examined the influence of relaxin in the pregnant rat. These studies were unphysiological, since they involved administration of porcine relaxin as well as progesterone and estrogen to ovariectomized pregnant rats. A more physiological approach is to use antibodies to neutralize the biological actions of endogenous relaxin in the intact pregnant rat. The purpose of the present study was to produce and characterize monoclonal antibodies suitable for this approach. Six stable and rapidly growing hybridoma clones which produced monoclonal antibodies specific for rat relaxin (MCA-rR) were obtained after the fusion of NSO mouse myeloma cells with lymphocytes from the spleen of a BALB/c mouse immunized with rat relaxin. Five MCA-rR (MCA1-5; all immunoglobulin G1 kappa) inhibited the ability of exogenously administered rat relaxin to increase the interpubic ligament length in estrogen-primed mice. Of the five MCA-rR that neutralized rat relaxin's bioactivity in vivo, MCA1 exhibited the highest relative affinity for rat relaxin. MCA1 was also highly specific for rat relaxin. MCA1 demonstrated no cross-reactivity with rat insulin, rat insulin-like growth factor I and II, or porcine relaxin-proteins that are structurally related to rat relaxin. In view of its high affinity and high specificity for rat relaxin as well as its ability to neutralize rat relaxin's bioactivity in vivo, MCA1 was selected for use in subsequent studies aimed at the neutralization of endogenous relaxin in intact pregnant rats.

Animals↗
PubMed1988

Functional and structural implications of variable region immunoglobulin dynamic states.

The consequence of multiple conformational states in the immunoglobulin variable region are considered. Bound ligand is viewed as a stabilizer of one conformer from a series of non-liganded conformers. Kinetic, equilibrium, thermodynamic and crystal formation information are used as supporting evidence for a unique conformer which is the crystallizable liganded state. The multi-state model is discussed in terms of certain biological and biochemical properties exhibited by the immunoglobulin molecule.

Antigen-Antibody Reactions↗
PubMed1986

Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies.

The effects of temperature on the binding of fluorescein by three monoclonal anti-fluorescyl antibodies (4-4-20, 20-19-1, and 20-20-3) were assessed by measurements of affinity constants (Ka) over a temperature range of 2-70 degrees C. Values for Ka were determined from the degree of ligand association by using fluorescence methodology. Curvilinear van't Hoff plots (ln Ka vs. T-1) were observed for all three antibodies, indicating that their standard enthalpy changes (delta Ho) were temperature dependent. This phenomenon was further investigated by plotting the changes in unitary free energy (delta Gu), standard enthalpy (delta Ho), and unitary entropy (delta Su) vs. temperature. Strong temperature dependencies were observed for enthalpy and entropy values, while free energy plots were only weakly dependent on temperature. At low temperatures (4 degrees C), entropy played a major role in the binding of fluorescein by all three antibodies, while enthalpy dominated at higher temperatures. This was a consequence of the negative heat capacity changes (delta Cpo approximately equal to -320 cal K-1 mol-1) observed for these antibodies, which produced a negative trend in both enthalpy and entropy values with increasing temperature. The negative heat capacity values also indicated that the hydrophobic effect was instrumental in the binding of fluorescein. Entropy changes were lower than expected for hydrophobic binding alone, suggesting that other forces were acting to mitigate the hydrophobic effect. One possibility was that the binding of fluorescein acted to restrain vibrational fluctuations in the active-site region, producing negative changes in both heat capacity and entropy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells.

Immunized BALB/c mice were injected with 10(6) viable Sp2/0-Ag14 cells to induce ascites fluid concurrently with maximum serum antibody activity. Ascites fluids developed in a systematic manner, yielded large amounts of specific polyclonal antibody even when the mice were injected with relatively small amounts of immunogen. A combination of an initial pristane injection prior to 3 intraperitoneal immunizations of immunogen emulsified in Freund's complete adjuvant (total volume of 0.1-0.2 ml per injection), followed 4 days later by injection of 10(6) viable Sp2/0-Ag14 cells, resulted in a consistent production of ascites fluid with substantial specific antibody activity.

Animals↗
PubMed1986

Linkage of low and high affinity anti-fluorescein idiotype families.

Data presented in this study describes the isolation and characterization of two anti-fluorescein (F1) hybridoma proteins 3-24 and 12-40, both IgG1, kappa with a Ka = 2.8 and 3.4 X 10(6) M-1, respectively, at 37 degrees C. These clones inhibited (6.8 +/- 2.8 -20.8 +/- 0.6% at 1 microgram/well) the idiotype-anti-idiotype interactions (IAII) of anti-F1 clones 3-13 and 3-17, which define a previously described low affinity idiotype family. Antibodies 3-24 and 12-40 also inhibited (45.0 +/- 3.0 and 61.3 +/- 5.6%, respectively, at 1 microgram/well) an IAII defined by a high affinity (Ka = 5.2 +/- 1.5 X 10(9) M-1 at 37 degrees C) anti-F1 clone, 4-4. Hybridoma proteins 3-13 and 3-17 possess similar affinities for F1 (Ka = 3.8 +/- 5.1 and 5.9 +/- 4.0 X 10(4) M-1) and are known to be idiotypically unrelated to clone 4-4. While 3-24 and 12-40 appeared very similar, non-identity of their active sites was established by heterologous idiotypic inhibitions, fine specificity of binding and spectral measurements (Qmax and lambda max) of bound F1. All IAII (3-13, 3-17, 9-40 and 4-4) were inhibited greater than 80% by the presence of 10(-4) M F1 or F1-BSA. In addition, four intermediate affinity (6.0 X 10(6) less than or equal to Ka less than or equal to 5.3 X 10(8) M-1) anti-F1 clones, comprising a second previously described idiotype family (designated the 9-40 family) were further analyzed. Inhibition of the 9-40 IAII by all heterologous proteins in the 9-40 family (except clone 5-27), and clones 3-24, 12-40 and 4-4 ranged from 87.7 +/- 1.3 to 95.4 +/- 1.0% at 1 microgram/well. Titration of the 9-40 IAII inhibition by antibodies 9-40, 3-24, 12-40 or 4-4 generated essentially superimposable profiles. In reciprocal inhibition experiments, using the 4-4 IAII, clones 3-24, 12-40, 9-40 and 4-4 gave distinct idiotypic titration patterns. Thus, members of the 9-40 family, 3-24 and 12-40 were more closely related to intermediate affinity clone 9-40 than high affinity clone 4-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Comparative properties of monoclonal antibodies comprising a high-affinity anti-fluorescyl idiotype family.

The T-dependent BALB/c murine immune response to fluorescein (F1) is characterized by structural heterogeneity at the protein level exemplified in part by a significantly wide range of affinities (Ka), and apparent lack of dominant idiotypes. In order to generate an idiotype family, xenogenic anti-idiotype (anti-ID) antibodies raised against anti-F1 monoclonal antibody (MCA) 4-4 (Ka = 1.7 X 10(10) M-1) were used in a solid-phase radioimmunoassay (SPRIA) to screen 68 anti-F1 hybridomas generated from multiple cell fusions for idiotypically related immunoglobulins. Four affinity-purified MCAs (designated 9-40, 10-25, 5-14 and 5-27) bearing 4-4 idiotypic determinants (ID 4-4) exhibited discrete isoelectric focusing spectrotypes (pI range = 6.8-7.7), significantly different fluorescence quenching values (38-95%) of bound ligand, binding affinities ranging from 3.3 X 10(7) to 5.3 X 10(8) M-1, similar active site inaccessibility to iodide, and closely related fine-specificity patterns for fluorescyl analogues. Idiotypic relatedness of each MCA to prototype 4-4 was quantitated by SPRIA, the results demonstrating that: each 125I-labeled MCA bound significantly to solid-phase anti-ID 4-4, and the concns of heterologous MCAs 9-40, 10-25 and 5-14 required for 50% inhibition of 125I-4-4/anti-ID 4-4 binding were comparable to homologous Ig protein. The finding that ID 4-4 bearing anti-F1 MCAs exhibit various binding properties and affinities is consistent with variable-region somatic diversification in anti-F1 affinity maturation.

Animals↗