Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies

High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.     

Production of monoclonal antibodies with preselected submolecular binding specificities to protein antigenic sites: antibodies to sperm whale myoglobin sites

The five synthetic antigenic sites of sperm whale myoglobin were used in their free form (i.e. not coupled to any carrier) to immunize separate groups of BALB/cByJ mice. The synthetic peptides corresponded to: site 1, residues 15-22; site 2, residues 56-62; site 3, residues 94-99; site 4, residues 113-119; site 5, residues 145-151. Serum samples obtained from each group of mice contained antibodies that bound specifically to myoglobin and exclusively to the immunizing antigenic site. Monoclonal antibodies to each of the five antigenic sites were subsequently obtained by hybridizing Fa/O mouse myeloma cells with spleen cells derived from each group of mice. These monoclonal antibodies were either IgM(kappa) or IgGl(kappa). They expressed the same isotypes as the antigen specific serum antibodies produced by the mice whose spleen cells were used for hybridization. Solid phase radioimmunoassay studies also indicated that each monoclonal antibody, like the immune serum of the parent animals, bound specifically to myoglobin and exclusively to the synthetic peptide used as an immunogen. These results suggested that the hybridoma antibodies expressed submolecular binding specificities that were the result of peptide immunization rather than hybrid selection. This strongly supports our previous findings that it is possible to produce monoclonal antibodies with preselected submolecular binding specificities to continuous protein determinants by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

Production of monoclonal antibodies with pre-selected submolecular binding specificities to protein determinants: Demonstration with sperm whale myoglobin

Two monoclonal antibodies with pre-selected submolecular binding specificities to sperm whale myoglobin (Mb) were obtained by hybridizing Fa/0 mouse myeloma cells with spleen cells derived from mice which had been immunized with free (not coupled to any carrier) Mb synthetic peptides 132–153 or 145–151 (antigenic site 5). Both monoclonal antibodies were IgG₁ (_k). Their homogeneity was confirmed by analytical isoelectric focusing electrophoresis. According to competitive inhibition studies in which Mb, the five synthetic antigenic sites of Mb, Mb peptide 132–153, bovine serum albumin (BSA), and lysozyme were used as inhibitors, the binding specificities of both monoclonal antibodies were restricted to determinants present in the peptides used for immunizations. The results of direct binding studies confirmed this conclusion and suggested that monoclonal antibodies with pre-selected submolecular binding specificities can be readily obtained by the techniques of somatic cell hybridization when the corresponding free synthetic determinants are used as immunogens.     

Structural differences among monoclonal antibodies with distinct fine specificities and kinetic properties

The mAbs HyHEL-8, HyHEL-26 (HH8, and HH26, respectively) recognize epitopes on hen egg-white lysozyme (HEL) highly overlapping with the structurally defined HH10 epitope, while the structurally related XRPC-25 is specific for DNP and does not bind HEL. All four Abs appear to use the same Vk23 germ line gene, and all but HH8 use the same VH36-60 germ line gene. Of the three anti-HEL Abs, the sequences of HH26 variable regions are closest to those encoded by the respective germ line sequences. HH8 utilizes a different member of the VH36-60 gene family. Thus, the same Vk and VH genes, combined with somatically derived sequence differences, are used to recognize the unrelated Ags HEL and DNP. In contrast, different VH36-60 germ line genes are used to bind the same antigen (e.g. HH8 vs HH10 and HH26). While the affinities of HH10, HH8, and HH26 for HEL vary by less than 10-fold, their affinities for mutated Ag vary over several orders of magnitude. Analyses of Fab binding kinetics with natural species variants and site-directed mutants of lysozyme indicate that these cross-reactivity differences reflect the relative sensitivities of both the association and dissociation rates to antigenic mutation: HH8 has relatively mutation-insensitive association and dissociation rates, HH10 has a relatively mutation-sensitive association rate but more variable dissociation rates, and HH26 has variable association and dissociation rates. Only a few amino acid differences among the antibodies produce the observed differences in the robustness of the association and dissociation rates. Our results suggest that association and dissociation rates and mutation sensitivity of these rates may be independently modulated during antibody repertoire development.     

Early screening for anti-plum pox virus monoclonal antibodies with different epitope specificities by means of gold-labelled immunosorbent electron microscopy

The technique of gold-labelled immunosorbent electron microscopy for the initial screening of monoclonal antibodies 10 days after cell fusion from 96-well culture plates is described. The technique is used to identify clones that secrete antibodies binding on the surface of the virion or to viral subunits, and compared to ELISA and Western blotting. High sensitivity was demonstrated.     

Detection of antigen-specific suppressor T cell factor by sandwich radioimmunoassay using two monoclonal antibodies with different specificities

We established a sandwich radioimmunoassay using two monoclonal antibodies specific for the distinct allotypic determinants on heavy and light chains of antigen-specific suppressor T cell factors (TsF). By use of this assay system, we confirmed our previous findings that the allotypic determinants (Ct) detected by BALB/c anti-CB-20 (7C5 and 7D1) were shared in various TsF with different antigen specificities, and also that the genes coding for the Ct determinants were indicated to be located on the right side of the Igh-V gene cluster, because the assay system equally detected either KLH-TsF or OVA-TsF from Ighb mice, such as C57BL/6, CB-20 and BAB-14, but not from BALB/c or C3H with other Igh allotypes. Furthermore, the radioimmunoassay, when used with two different anti-Ct (7C5 and 7D1), preferentially detected the extracted form of TsF, whereas the secreted TsF was easily detected by the combination of anti-Ct (7D1) and anti-I-Jb. In fact, the assay system defined two types of Ts hybridomas: one producing TsF in cytoplasm or on the membrane but not secreting and another secreting in the culture supernates. We also demonstrated that materials bound to the plate coated with anti-Ct antibody that had been incubated with different concentrations of cell-free extracts exhibited the dose-dependent suppression of antibody responses.     

Human monoclonal antibodies with different fine specificity for digoxin derivatives: cloning of heavy and light chain variable region sequences.

Human-mouse hybridoma cell lines producing human monoclonal antibodies against the cardiac glycoside digoxin were established after in vitro immunization or direct immortalization of human peripheral blood lymphocytes with digoxin. Three antibodies, designated MO6, LH92 and LH1114, displayed different patterns of fine specificity against digoxin and several digoxin analogues, as elucidated by inhibition ELISA. All three monoclonal antibodies had mu heavy chains, two of them (MO6 and LH114) had kappa light chains and one (LH92) lambda light chains. DNA encoding the variable regions of both heavy and light chains of the three antibodies were amplified from cDNA using the polymerase chain reaction (PCR). The nucleotide sequences of the amplified DNA were determined after subcloning of PCR fragments in M13 vectors. The deduced amino acid sequences revealed considerable sequence differences in the complementarity determining regions between the three antibodies.     

Characterization of three monoclonal antibodies against C3 with selective specificities.

We raised 15 mouse monoclonal antibodies against human C3 from two separate fusions. Mouse plasmacytoma cells were fused with spleen cells from mice immunized either with a mixture of C3, C3b and C3c, or with a mixture of C3dg and C3d. Three of the 15 monoclonals were characterized in detail. N-7A reacted with native C3 as well as C3b and C3c. Two other monoclonals, C-5G and G-3E, recognized neoantigenic determinants on C3c and C3dg, respectively. In ELISA, C-5G reacted with C3b and C3c but not with native C3 nor with C3dg; and on the other hand G-3E reacted only with C3dg. The selective specificities of these monoclonals were further confirmed in a binding assay to C3 fragments formed on cellular intermediates. C-5G bound exclusively to EC3b, and G-3E bound to EiC3b, EC3dg and EC3d. N-7A bound only very poorly to EC3b. C-5G inhibited both the haemolytic activity of C5 convertase and also the CR1-mediated rosette formation of B-enriched peripheral mononuclear cells with EC3b. G-3E inhibited the CR2-mediated EC3dg-rosette formation of Raji cells. These monoclonals, with selective specificities, can distinguish the state of activation and degradation of the C3 molecule.     

Two simple and rapid methods to detect monoclonal antibodies with identical epitope specificities in a large population of monoclonal antibodies.

Two novel and simple methods have been developed for detecting monoclonal antibodies (MAbs) with identical epitope specificities from a large population of MAbs against human chorionic gonadotropin (hCG). The first method was based on the observation that following radioiodination many molecules of an antigen are altered in such a manner that they cannot be recognized by MAbs. The proportion of radiolabelled antigen (Bmax) able to bind to a MAb was characteristic of that MAb and MAbs having identical specificities showed identical Bmax values. Using this principle it was possible to identify MAbs having identical epitope specificities within a large population of MAbs against hCG. In the second method one test MAb was immobilized on a plastic surface through an immunochemical bridge. This MAb was then incubated with 125I-hCG previously complexed with a second MAb. Such a complex could bind to the solid phase MAb only if the two MAbs were not identical. The results obtained with both methods were concordant. With such methods it is possible to identify MAbs with identical epitope specificities immediately after the initial screening of the fusion. These methods do not require subcloning, ascites production, or the purification and iodination of large number of MAbs.     

Comparative studies of the protein binding of digoxin and its metabolites

The protein binding of Digoxin (D), Dihydrodigoxin (DH), Digoxigenin Bisdigitoxoside (DB) and Digoxigenin Monodigitoxoside (DM) was concentration independent and unaltered by the presence of high concentrations of the analogous compounds. The respective percentage of protein binding for D, DH, DB and DM were 22%, 22%, 23% and 27%.Dedicated to K. Bucher on the occasion of his 65th birthday.     

Specificity and binding kinetics of murine lupus anti-DNA monoclonal antibodies implicate different stimuli for their production

The origin and relative biological importance of the many different DNA-reactive antibodies that appear in systemic lupus erythematosus are not well understood. A detailed analysis of their fine specificity and binding characteristics with DNA is a necessary step in understanding their biology. We have examined here two monoclonal antibodies (mAb) IV-228 and V-88 that are, respectively, characteristic of antibodies, which bind exclusively to single-stranded (ss) DNA and to both double-stranded (ds) DNA and ssDNA. By surface plasmon resonance (SPR) on BIAcore, we characterized the kinetics of binding of each antibody to synthetic ss and ds oligonucleotides. Antibody V-88 and IV-228 showed different patterns of reactivity for both ss and ds oligonucleotides, characterized by distinctly different kinetic parameters. Analysis of their binding kinetics indicates the importance of base composition in defining DNA epitopes, and shows that some epitopes, such as that recognized by mAb V-88, are expressed on dsDNA and ssDNA, whereas others, as recognized by IV-228, are not. The base preferences of V-88 for ds GC-rich structures over AT-rich, and of IV-228 for ss T-rich structures, also reveal distinct differences between these antibodies. We conclude that the different binding properties of the antibodies will relate to their biological activities. The base preferences of the antibodies suggest that they might be induced by different immunological stimuli, such as those that could be provided by the various DNA fragments and structures released during programmed cell death.     

Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles

The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and kappa) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses V(H)3-64 in the germ line configuration. G14 and G19 use V(H)6-1, which has somatic mutations. All of the MAbs use V kappa DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that V(H) gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V(H) gene use may have in resistance and susceptibility to cryptococcosis.     

Development and characterization of monoclonal antibodies with specificity for metallic radioisotope chelators linked to antibodies and other proteins

Monoclonal antibodies with specificity for the diethylenetriaminepentaacetic acid (DTPA) portion of the GYK-DTPA (glycyl-tyrosyl-lysine-DTPA) linker structure used to chelate metals to immunoglobulins have been prepared. A significant proportion of these antibodies were lambda light chain isotype. Competition assays demonstrated that DTPA, rather than GYK, was the binding site of the antibodies tested. These monoclonal antibodies should be useful reagents for use in assays specific for the presence of the common linker structure used to chelate metallic radioisotopes to monoclonal antibodies. The antibodies tested did not discriminate between chelated and unchelated DTPA.     

Heteroclitic antibodies: differences in fine specificities between monoclonal antibodies directed against dinitrophenyl and trinitrophenyl haptens

The inhibition of binding of monoclonal antibodies by different haptens was studied using the 50% antibody binding assay. The binding of antidinitrophenyl and antitrinitrophenyl antibodies to dinitrophenylated or trinitrophenylated bovine serum albumin could be inhibited by monovalent dinitrophenyl or trinitrophenyl epsilon-aminocaproic acid. Some of the antibodies could be inhibited to a greater degree with the cross-reacting haptens than with the haptens homologous to the immunizing antigen, therefore these antibodies were heteroclitic.     

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum, hippocampus and spinal cord using monoclonal antibodies with different epitope specificities

The HNK-1 carbohydrate, an unusual 3-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated 2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.     

Functional and binding activity of monoclonal anti-Thy-1 antibodies: evidence for different expression of the two alleles

Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antisera selected for complement-dependent cytotoxicity have high cytotoxic and binding titers on thymocytes and peripheral T cells of mouse strains bearing the appropriate Thy-1 allele. The effect of both anti-Thy-1.1 and anti-Thy-1.2 monoclonal antisera plus complement on cytotoxic T cell effectors is to abrogate their activity. On the functional activity of precursor cytotoxic T cells, monoclonal antisera against the two alleles have different effects: anti-Thy-1.2 plus complement removes precursor activity of Thy-1.2-bearing strains, including (Thy-1.1 × Thy-1.2)F₁ heterozygotes. In contrast, six different anti-Thy-1.1 monoclonals, including four of the IgM class and two of the IgG class, failed to remove cytotoxic precursor activity from the splenic T cells of AKR, A. Thy-1.1 or (CBA × AKR)F₁ mice. Analysis by fluorescence-activated cell sorting of in vitro cultured AKR spleen cells shows that Thy-1.1 antigen appears on the cell surface during the five-day culture period.     

Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies

The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.     

Monoclonal antibodies of four different specificities for neutralization of type 1 polioviruses.

Four independent hybridoma clones have been established that produce neutralizing antibodies specific to type 1 poliovirus. Each clone produced antibody which neutralized a distinct set of type 1 test strains: (i) all 15 strains tested; (ii) the inducer strain only; (iii) predominantly wild strains; or (iv) all vaccine-related and some wild strains.