Prolonged weightlessness and humoral immunity.

Preflight, inflight, and postflight serum samples obtained from crewmen aboard STS-9 were analyzed for immunoglobulin content. Control studies for circadian rhythm were conducted to further validate the analyses. Quantitation of immunoglobulins G, M, A, D, and E indicated relatively minor fluctuations in the concentration of each class of immunoglobulin during the experiment. Thus, microgravity effects on immunoglobulin levels during a 10-day flight were considered insignificant.

Relative binding properties of fluorescein and 9-hydroxyphenylfluoron (HPF) with murine monoclonal anti-fluorescein antibodies.

Contribution of the fluorescein (F1) carboxyl group to hapten binding by idiotypically related murine monoclonal anti-F1 antibodies 4-4-20, 9-40 and 12-40 was studied by comparing relative liganded active site properties with bound Fl or 9-hydroxyphenylfluoron (HPF). Kinetic studies revealed similar association rate constants between Fl and HPF to 4-4-20 (approximately 1.1 x 10(7) M-1 s-1); however, the 4-4-20 dissociation rate for Fl was approximately 200 times slower, relative to HPF, which resulted in relative intrinsic affinity values of 1.2 x 10(10) and 6.5 x 10(7) M-1, respectively. Mabs 9-40 and 12-40 also displayed a reduced affinity for HPF and affinity constants of 5.5 x 10(5) M-1 and 6.7 x 10(5) M-1 were obtained from a competitive ELISA. Additionally, previous studies revealed that upon binding Fl, Mabs 4-4-20 (92.1%), 9-40 (44.7%) and 12-40 (73.4%) quenched Fl fluorescence. Similar analyses with HPF resulted in 64.4% and 2.0% fluorescence quenching by 4-4-20 and 12-40, respectively; however, 9-40 increased HPF fluorescence by approximately 24%. Steady-state fluorescence polarization experiments revealed that in solution, Fl (P = 0.019) and HPF (P = 0.048) were polarized to different degrees. When bound, however, Fl and HPF expressed similar polarization values (P approximately 0.455), except 9-40 bound HPF which was significantly depolarized (P = 0.428). Fluorescence lifetime experiments revealed Fl to possess two discrete lifetimes: a 3.96 ns component (free Fl) and either a 0.52 ns (4-4-20), 2.23 ns (9-40) or 0.96 ns (12-40) short component that corresponded to bound Fl. HPF, however, when bound by 4-4-20 or 9-40, was best fit by three discrete exponentials: a relatively long 4.0 ns component, a 1.11 ns lifetime (free HPF) and either a 0.52 ns (4-4-20) or 2.23 ns (9-40) component. Finally, HPF bound by Mab 12-40 exhibited a single lorenzian distributed lifetime of 1.36 ns (+/- 0.43 ns). Results are discussed in terms of Mab active site structure and conformational state dynamics.

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein.

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.

Perturbation of antibody bound bifluorescent-ligand probe by polyclonal anti-metatype antibodies interacting with epitopes proximal to the liganded antibody active site.

General localization of metatypic determinants recognized by polyclonal anti-metatype antibodies relative to the antibody active site of the high-affinity anti-fluorescein monoclonal antibody 4-4-20 was achieved through use of a unique bifluorescent-ligand probe. The fluorescent probe possessed intrinsic energy-transfer properties with the fluorescein hapten serving as the energy acceptor. The donor group 5-(2-iodoacetyl) aminoethylaminonaphthalene-1-sulfonic acid (IAEDANS) proved environmentally sensitive both to binding of the FITC-cys-AEDANS ligand and to subsequent anti-metatype antibody interactions involving the antibody variable domains of 4-4-20. Spectral changes in ligand-conjugated AEDANS upon specific reactivity of the antibody with FITC suggested secondary interactions between AEDANS and the topological protein surface adjacent to the 4-4-20 active site. Results indicated that some anti-metatype antibodies (Fab fragments) within the polyclonal population bound to sites immediately surrounding the liganded active site and perturbed the interactions of AEDANS with topological sites. The results are discussed in terms of the types of interactions that may occur between the AEDANS moiety and the 4-4-20 antibody protein surface and subsequent perturbation of those interactions by anti-metatype antibodies.

Interaction of dual-specific autoantibodies with dsDNA and a synthetic dimer peptide simulating the hinge region of IgG2a molecules.

Anti-dsDNA autoantibodies and immune complex formation are major factors in SLE pathogenesis. Understanding stable immune complex formation is critical in deciphering mechanisms of autoimmune pathogenesis. Previous studies identified a subpopulation of murine lupus monoclonal autoantibodies that exhibited dual specificity (anti-DNA and anti-IgG2a hinge) and formed stable immune complexes [J. Mol. Rec. 10(1997)225]. Two monoclonal autoantibodies, BV 17-45 and BV 16-13, were extensively studied because of their dual specificity. To quantitatively assess the role of each specificity in the formation of stable immune complexes, studies were performed to determine binding affinities for various sized dsDNA fragments (21, 43, 84, and 114 bp) and the covalent dimer of a nine amino acid hinge peptide. Results characterizing BV 17-45 showed that the affinity for dsDNA directly correlated with increased dsDNA size. Results with BV 16-13 revealed a generally lower affinity for the various dsDNA fragments. Binding inhibition studies, using a covalently linked dimer of a nine amino acid synthetic hinge peptide as an inhibitor of the antibody-43 bp dsDNA interaction, yielded relative affinities for the anti-hinge activity. Binding affinities for the synthetic hinge specificity were lower than affinities measured for the anti-dsDNA activity. Collectively, the binding and inhibition studies provided insight into the correlation between dual specificity and avid immune complex formation. A model was proposed based on the concept that large dsDNA fragments caused localization of the dual-specific antibodies through the anti-dsDNA activity, thereby facilitating subsequent binding and cross-linkage via the anti-hinge specificity. These synergistic interactions resulted in the formation of avid immune complexes.

A novel cellular interaction involving antigen-pulsed macrophage and antigen-specific B-lymphocytes.

Extensive documentation shows that macrophage efficiently present antigen to CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a novel fluorescent probe, FITC-BSA, was developed to analyze intracellular antigen processing and presentation pathways within viable peritoneal murine macrophage. The studies revealed fluorescein's accessibility to antibody binding when associated with peptides bound within the MHC II cleft. To determine if MHC II-fluoresceinated-peptide complexes on the surface of macrophage were also sufficient to stimulate antigen-specific B-cells, nylon wool-purified splenic B-cells from FITC-KLH injected BALB/c mice (H-2(d)) were co-cultured with antigen-pulsed macrophage. B-cell stimulation and antibody production was observed in the presence of FITC-BSA-pulsed macrophage, whereas, macrophage incubated in the presence of unlabeled BSA were not stimulated. Compared with control cells, similar levels of stimulation were detected following depletion of Thy 1.2(+) cells from nylon wool-based spleen cell preparations. Stimulation was inhibited upon preincubation with anti-fluorescein IgG antibodies. Stimulation was not measurable using B-cells derived from the naive mice. The interaction was inhibited upon addition of MHC II specific antibodies and leupeptin, a microbial product that inhibits MHC II-peptide complex formation. Importantly, antibody production was not observed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentration as well as the cell to cell ratio.

Autoimmune VH gene family: PCR-generated murine germline VH10 genes.

A relatively large number of variable region genes (V) contribute, via gene rearrangements with smaller numbers of additional gene elements (D and J), to generate diversity in the immune response. While some VH gene families are thought to contain 100- 1000 members, the VH10 family has only two known functioning members with 99% sequence homology. Both members (monoclonal antibodies) are capable of binding DNA, and since they were derived from inbred mice afflicted with the lupus syndrome they are considered autoimmune antibodies. Relative uniqueness of the VH10 primary nucleotide sequence presents a model system with which to examine unrearranged VH genes and attempt to identify germline genes eventually expressed as autoantibodies. PCR amplified germline sequences of the VH10 family are highly conserved, with few base substitutions evenly distributed between both framework and CDR regions. It was determined that the PCR amplified germline sequences are highly similar to the DNA sequences of the two monoclonal VH10 antibodies, and a non-functional psuedo-germline gene was found that is identical to a non-functional cDNA derived from a hybridoma cell line. These findings indicate that the use of unique CDR DNA sequences for the identification and amplification of specific germline V genes via PCR can yield vital information that may answer fundamental questions about the origins of autoimmune anti-DNA antibodies in afflicted individuals. The nature of the germline gene populations and the possible microheterogeniety of these genes may prove to be important in understanding the role of autoimmune antibodies in normal and diseased individuals.

Active site contributions to fluorescein binding determined by in vitro heavy and light chain reassociations.

In addition to prior crystallographic studies that determined antigen contact residues for high affinity murine monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 1.7 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal anti-Fl antibody 9-40 (Ka = 5.7 x 10(7) M-1) possessed identical Fl contact residues with the exception of L34 His for Arg. Mab 9-40 L34 His was germ-line encoded and 4-4-20 L34 Arg correlated with increased 4-4-20 affinity and enhanced Fl quenching. To better define L34 Arg and L96 Trp contributions to antigen binding, in vitro H and L chain reassociation experiments were performed. Following reassociations, affinity purified and homologous chain reassociated proteins 4-4-20 (H4-4-20 L4-4-20) and 9-40 (H9-40 L9-40) yielded identical idiotypes (greater than 91% related), Qmax values (91% and 44.7%), affinity constants (approximately 2.0 x 10(10) M-1 and 5.5 x 10(7) M-1) and iodide quenching values (1.2% and 2.1%), respectively. Although heterologous reassociated proteins were idiotypically related to prototypic proteins (greater than or equal to 87.1%), differences in Fl-binding characteristics were observed. Recombinant H4-4-20 L9-40 expressed Ka and Qmax values similar to 9-40 and implicated L34 Arg in increased 4-4-20 Ka and Qmax. Residue L34 Arg replacement in the 9-40 active site (H9-40 L4-4-20). however, exhibited low Qmax (44.4%) and slightly increased affinity (3.7 x 10(8) M-1). In addition, L96 Leu substitution into 4-4-20 (H4-4-20 L-04-01) and 9-40 (H9-40 L04-01) resulted in lower Qmax values (15.1% and 20.5%, respectively) and significantly reduced Fl affinity (approximately 10(3)-fold). These results demonstrated: (1) L34 Arg was responsible for increased 4-4-20 affinity, (2) L96 Trp was critical for an intermediate Fl affinity (approximately 10(7) M-1) and (3) active site Fl contact residue orientations were potentially affected by differences in H chain complementarity-determining region 3 and CH 1 residues.

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.

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.

Effects of secondary forces on the ligand binding properties and variable domain conformations of a monoclonal anti-fluorescyl antibody.

Biochemical interactions occurring external to the antibody active site or pocket (i.e. secondary forces) that directly effect ligand binding efficiency, and the microenvironment-sensitive spectral properties of bound homologous ligand, residing within the active site of high affinity monoclonal antifluorescyl antibody (mAb) 4-4-20, have been previously reported. This study describes the synthesis and characterization of a series of specially designed and chemically distinct mono-fluoresceinated peptides of equal size (13-mer) as well as the changes in the spectral properties and free energy in the binding of each fluorescein derivatized peptide, upon interaction with mAb 4-4-20. Significant differences in binding efficiency and fluorescence quenching of the ligand, as well as the intrinsic tryptophan fluorescence, were observed for each monofluoresceinated peptide relative to one another and fluorescein ligand. In addition to the effects on the fluorescence quenching of fluorescein and intrinsic tryptophan residues, and the free energy of binding, the conformation of the variable domains of mAb 4-4-20 upon interaction with the fluoresceinated peptides was probed with polyclonal antimetatype (conformational dependent anti-liganded state) antibodies. Studies comparing the results of a solid-phase inhibition assay, with the binding of antimetatype antibodies in solution, suggested that variant metatypic states of mAb 4-4-20 resulted from binding of the various fluorescein derivatized peptides. Depiction of the mAb 4-4-20 active site as a series of thermally averaged substates is proposed as a model and framework to interpret further the results. It was concluded that secondary forces can dictate conformer selection from the various substates. thereby modulating the primary antibody ligand interaction.

Variable region primary structures of monoclonal anti-DNA autoantibodies from NZB/NZW F1 mice.

VH and VL region primary structures of five NZB/NZW F1 derived monoclonal anti-DNA autoantibodies were determined from cloned cDNA. Comparative analysis of VH genes showed that except for two VH genes that shared complete identity the overall VH gene usage was diverse. Comparison of VH genes with those utilized in a variety of antibody responses showed they were generally unique to the autoanti-DNA response although framework homologies allowed assignment of four of five VH genes to existing murine heavy chain gene families. Only one out of five D segments shared homology to existing germline D segments, and all were rearranged to JH3. V kappa genes showed restriction for four of five light chains to the V kappa 1 subgroup. The V kappa 1 subgroup has been shown previously to be utilized in several anti-DNA autoantibodies as well as a variety of antibodies to exogenous antigens. H and L chain amino acid residues associated with the active site of a ssDNA specific autoantibody, 04-01, are discussed based on recently obtained crystallographic data.

Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies.

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins (Ka greater than 5.0 X 10(7) M-1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgGl or IgG2a possessing kappa light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (less than or equal to 3.0 X 10(6) M-1) and five possessing high affinities (greater than or equal to 5.3 X 10(8) M-1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3-13 and 3-17), showed that all the low-affinity clones (except 2-9 and 2-21) were capable of inhibiting (40-100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3-13 and 3-17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3-13 and anti-3-17 reagents detected on 3-13 and 3-17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889-898 (1981a)] identified a single clone [20-4-4 (Ka = 5.0 X 10(7) M-1)] significantly inhibiting the 3-13 and 3-17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3-13 and 3-17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3-13 and 3-17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.

Determination of dissociation constants and ligand specificity of detergent solubilized surface membrane immunoglobulin A from MOPC-315.

Surface membrane immunoglobulin from MOPC-315 plasmacytoma cells (smM315) was isolated by nonionic detergent lysis of radioiodinated cells and affinity chromatography on Dnp-aminohexyl-Sepharose 4B. Verification of the solubilized molecule as an integral membrane protein, distinct from secreted MOPC-315 IgA (M315) was accomplished by NaDodSO4-PAGE, charge-shift electrophoresis and molecular sieve gel filtration with NP-40 and deoxycholate. smM315 was compared to reduced and alkylated monomeric secreted immunoglobulins from MOPC-315, MOPC-460, and XRPC-25 by quantitative affinity chromatography (QAC) using two differently substituted Dnp-aminohexyl-Sepharose 4B resins. Unique patterns of cross-reactivity of all secreted myeloma proteins were independently established with a competitive hapten inhibition assay using 125I-Dnp26BSA as the precipitating probe. After derivation with dinitrobenzylsulfonate, Dnp-aminohexyl-Sepharose 4B was modified with succinic anhydride which, with the inclusion of 0.03% Doc in a PBS and 0.1% NP-40 buffer, prevented nonhapten specific protein-matrix interactions during QAC. Dissociation constants determined by QAC for three ligands, (dinitrophenyl-glycine, trinitrophenyl-amino-caproate and tetramethylrhodamine) were essentially the same for smM315 and M315. Both of the other nitrophenyl binding IgA myelomas had distinct and significant differences in dissociation constants. Thus, for a differentiated antibody secreting cell which has undergone a heavy chain class switch, such as MOPC-315, the cell surface immunoglobulin has an identical ligand binding active-site as the secreted immunoglobulin.

Specificity of anti-DNA antibodies in SLE-I. Definition and gross specificity of antibody populations in human SLE plasma.

Populations of anti-DNA antibodies in two SLE plasma were defined based on their patterns of reactivity in inhibition assays with single and double-stranded DNA as well as mono- and oligonucleotides. Two populations of anti-DNA antibodies were seen in both plasma tested. The first population reacted specifically with ssDNA and was inhibited by relatively low concentrations of free nucleotides indicating that it recognized the nucleotide bases in ssDNA. The second population bound both ss and ds calf thymus DNA with apparent equal affinity. The cross-reactive anti-DNA antibodies were inhibited by mononucleotides (at high concentrations) and by single-stranded oligonucleotides (average length tetranucleotides). For one of the plasma tested (PS), pBR322 plasmid DNA (54% G + C) was a significantly more effective inhibitor than calf thymus DNA (39% G + C). These results suggested that nucleotide bases contributed to dsDNA binding by cross-reactive anti-DNA antibodies.

Analysis of heterogeneous dissociation kinetics in polyclonal populations of rabbit anti-fluorescyl-IgG antibodies.

Hapten dissociation kinetics were used to characterize the expression of hyperimmune anti-fluorescyl-IgG antibodies within a population of 14 New Zealand White (NZW) rabbits. Anti-fluorescyl-IgG antibody preparations, obtained from individual hyperimmune NZW rabbits, exhibited either three or four discrete dissociative components. The fastest component was typically heterogeneous with respect to dissociation rate, exhibiting an average dissociative lifetime of 8 sec, where dissociative lifetime was the reciprocal dissociation rate constant. Longer-lifetime components exhibited lifetimes of several hundred seconds, and became progressively more homogeneous with increasing lifetime. Comparison of dissociative components, within the NZW rabbit population, revealed the presence of four distinct groupings of dissociative lifetimes, which were approximately Gaussian in their distribution. Average lifetimes of 8, 52.6, 313 and 1417 sec were observed for the four groupings, which corresponded to free-energy (delta G0) values of -9.6, -10.6, -11.6 and -12.42 kcal/mole, respectively (using an association rate value of 5 X 10(6) M-1 sec-1). These findings implied that affinity maturation of the anti-fluorescyl-IgG antibody response involved the generation of discrete high-affinity subpopulations of antibodies rather than a continuous spectrum of high-affinity antibodies.

Specificity of anti-DNA antibodies in SLE--II. Relative contribution of backbone, secondary structure and nucleotide sequence to DNA binding.

Fine specificity of a population of anti-DNA antibodies which bound both ssDNA and dsDNA with apparently equal affinity was studied in two SLE plasma. Sensitivity of DNA binding to increasing sodium chloride concentration indicated that electrostatic interactions occurred between antibody and phosphate moieties of DNA. Secondary nucleic acid structure was important to DNA binding as double-stranded synthetic deoxynucleotide polymers were more effective inhibitors than their substituent single-stranded polymers. Nucleotide bases were also found to play a role in recognition of DNA by these cross-reactive antibodies, as ssDNA binding was sensitive to increasing temperature which caused unstacking of the nucleotide bases. Differing patterns of reactivity with synthetic deoxynucleotide polymers with similar secondary structures but different nucleotide compositions further indicated the importance of nucleotide bases to dsDNA binding by cross-reactive anti-DNA antibodies in SLE plasma.

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.

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: localization and characterization of idiotypic determinants.

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (Ka values ranging from 5 X 10(6) to 10(10) M-1) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (gamma, kappa) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15-20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained gamma 1 heavy (H) chains and 4-6-10 contained both gamma 1 and gamma 2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which defined binding mechanism diversity. The location of determinants recognized by anti-idiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotype by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (approximately 25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the VH- and VL-chain interaction must exist for the expression of these idiotypes.

Characterization of interactions involving anti-metatype antibodies and immune complexes.

Immunizations of high affinity anti-fluorescein monoclonal antibody 4-4-20 affinity labeled with fluorescein 5-isothiocyanate into a rabbit elicited antibodies specific for the liganded conformation of 4-4-20 (termed "anti-metatype" antibodies). Reaction of liganded 4-4-20 with anti-metatype antibodies caused significant delay (up to 23-fold) in the rate of dissociation of fluorescein ligand from the active site. In this study, structural analogues of fluorescein, including fluorescein 5-isothiocyanate, fluorescein 6-isothiocyanate, 5-dichlorotriazinyl aminofluorescein and 5-carboxyfluorescein, were bound by monoclonal antibody 4-4-20 and anti-metatype antibody reactivity was observed through delay in the dissociation rate of ligand from Mab 4-4-20. Significant delays (ranging from 5- to 242-fold) were observed for all structural analogues examined indicating that 4-4-20 maintained similar but not necessarily identical conformations upon binding fluorescein structural analogues. Additionally, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate were conjugated to carrier molecules of increasing mol. wt (ranging from 225 to 14,600 D) in an attempt to sterically interfere with "metatopes" at the mouth of the active site and localize regions of anti-metatype antibody binding. These fluorescein-conjugated compounds were reacted with 4-4-20, and binding of anti-metatype antibodies delayed dissociation rates from 24- to greater than 1500-fold. These results indicated that the mechanism whereby anti-metatype antibodies delay the release of fluorescyl ligands from the active site probably does not solely involve steric hindrance of the ligand due to binding of anti-metatype antibodies at the mouth of the active site. Studies with 4-4-20 Fab fragments and a single chain derivative of 4-4-20 (consisting of the variable regions tethered by a 14 amino acid linker) indicated that anti-metatype reactivity was specific for the immunoglobulin variable region.