Topographic and functional assay of antigenic determinants of human prolactin with monoclonal antibodies

Six distinct antigenic determinants were identified on human prolactin (hPRL) by competition assays with murine monoclonal antibodies (MABs). The affinity of binding and the cross-reactivity of the antibodies with two non-primate prolactins was also determined. Binding of 125I-hPRL to MAB-coated microtitre plates in the presence of a second MAB resulted in either inhibition or enhancement of antigen binding to the plate. These results were interpreted in terms of conformational changes to epitopes, induced allosterically by the binding of a second MAB to the antigen. The topographic relationship of epitopes to the biologically active regions on the hormone was examined on the basis of the neutralizing potency of MABs in the proliferation of the prolactin-dependent cell line NB2. The NB2 growth-inhibitory activity was restricted to three distinct epitopes (NE02/6, 1208 and NE03) but absent from three other MABs tested (QB01, WC01/3 and 1200). On the basis of the competition and functional studies, an elemental scheme of the topographic localization of epitopes is presented. The experimental approach employed may contribute to studies on the allocation of biologically active sites on protein hormones.     

A rapid immunoperoxidase assay for determination of IgG antibodies to measles virus

A new indirect peroxidase antibody to membrane antigen (IPAMA) technique for the detection of IgG specific antibodies against measles virus is described. The technique utilizes as antigen measles-infected Vero cells dried on glass slides and stored at −70°C. Sera of 50 healthy medical students and laboratory workers and 24 sera of measles, encephalitis, and subacute sclerosing panencephalitis (SSPE) patients were checked by IPAMA and the results have been compared with the results obtained by the hemaglutination-inhibition (HI) test. There is good agreement between the results of both techniques as to the presence or absence of antibody in 48 out of the 50 tested. The advantages of the techniques are discussed.     

An enzyme-linked immunosorbent assay (ELISA) for quantification of antibodies to Streptococcus mutans surface antigens

An ELISA was developed to quantitate the level of antibodies to various cell surface antigens of the Gram positive bacterium. Streptococcus mutans. Whole cells and purified cell wall components of S. mutans, lipoteichoic acid (LTA) from Streptococcus pyogenes, and dextran T 2000 were employed as coating antigens in this study. Cell walls of S. mutans were purified by mechanical disruption of whole cells followed by differential centrifugation and proteolytic enzyme treatment. Serotype-specific carbohydrate was purified from an autoclaved, lyophilized S. mutans whole cell preparation by column chromatography. LTA was prepared by Sepharose 4B chromatography of a phenol-water extract of S. pyogenes and used for detection of anti-polyglycerophosphate (PGP) antibodies. A rabbit antiserum to S. mutans 6715 (serotype g), which precipitated with purified carbohydrate antigen (RR g), gave good reactions with purified cell walls and whole cells of S. mutans 6715, less activity with RR g and low activity to LTA and dextran when tested by ELISA. Adsorption of this antiserum with whole cells of S. pyogenes resulted in antibody activity with specificity only to the serotype carbohydrate. The specificity of the antibody for homologous coating antigen was RR g > cell wall > whole cells. An antiserum to S. mutans MT573 (serotype e) contained antibody predominantly to LTA, whereas, anti- S. mutans MT703 (serotype e) reacted with both dextran and LTA; however, the activity to LTA was removed by prior adsorption of the antiserum with S. pyogenes cells. This treatment did not alter the antibody activity to dextran. To establish the sensitivity of ELISA, a purified IgG anti-serotype carbohydrate antibody was prepared by adsorption of anti-S, mutans 6715 antiserum with a mutant of S. mutans which lacks serotype carbohydrate followed by adsorption and elution of specific antibodies from S. mutans 6715 whole cells. The minimum level of sensitivity of ELISA was 12.5 ng of IgG anti-serotype carbohydrate.     

A solid-phase assay of solubilized HeLa cell membrane receptors for binding group B coxsackieviruses and polioviruses

HeLa cell membranes were solubilized in sodium deoxycholate and adsorbed onto polystyrene microtiter plates to permit the measurement of receptors for binding group B Coxsackieviruses and polioviruses. Receptor activity was determined by measuring the amount of infectious virus attached to coated wells at 6°. Conditions for optimal binding of virus were determined. Receptor binding activity for group B Coxsackieviruses was detected from as few as 2.5 × 10⁴ cell equivalents/ml to provide a method which was over 400 times more sensitive than the virus eclipse assay used previously. The assay revealed a recovery of up to 7% of the receptor activity of whole cells based on virus saturation of receptors. The specificity of the binding activity of the immobilized receptors was demonstrated by virus attachment competition, as done previously for receptors on live HeLa cells. Application of this method to measure receptors in deoxycholate-solubilized fetal mouse brain revealed a 2500-fold increase in sensitivity from that obtained in a virus-binding assay used previously.     

Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1

Monoclonal antibodies directed against glycoprotein D of herpes simplex virus completely inhibited fusion of Vero cells infected with type 1 virus. In contrast, several monoclonal antibodies directed against other viral glycoproteins, including B, were ineffective or were only minimally inhibitory at the highest concentrations tested.     

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.     

Protein A-coated erythrocyte binding to cell surface antigens: Application to quantitate retrovirus infectivity in vitro

Fibroblasts infected with murine leukemia virus (MuLV) bind erythrocytes coated with protein A to form rosettes in the presence of MuLV-specific antisera. This method, which is potentially applicable to any retrovirus and susceptible cell, has been specifically adapted as a focus assay for quantitating both ecotropic and xenotropic MuLV.     

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.     

Methods for binding cells to plastic: application to a solid-phase radioimmunoassay for cell-surface antigens.

Two methods are described for attaching cells to plastic plates such that they may be used for antibody binding assays. In the first method, lymphoid cells or erythrocytes were attached to the wells of plastic plates using glutaraldehyde. This resulted in monolayers of fixed cells which retained surface antigens and were stable to storage. The second method involved binding of unfixed cells to the plastic surface by means of antibodies non-specifically adsorbed to the plate. Both methods resulted in cell layers which remained attached to the plate during the washing and incubation procedures of a radioimmunoassay. The cell layers were shown to be suitable for screening the product of hybrid cell lines for the presence of monoclonal antibodies to cell-surface antigens.     

Monoclonal IgA J539 binds galactopyranosyl antigens on its surface

Murine monoclonal IgA J539 binds to methyl beta-D-galactopyranoside. With nearly the same affinity, it binds to methyl 6-O-pivaloyl-beta-D-galactopyranoside (4) and to methyl 6-O-beta-D-gentiobiosyl-beta-D-galactopyranoside (7). These observations confirm that the combining area of J539 is of the surface-, and not of the cavity-type.     

Idiotypic studies of monoclonal anti-tobacco mosaic virus antibodies from one mouse

Monoclonal antibodies have been prepared from one BALB/c mouse immunized with tobacco mosaic virus. The monoclonal antibodies are distributed into three subgroups recognizing different epitopes on tobacco mosaic virus subunits. The idiotypic specificities of these monoclonal antibodies have been studied using syngeneic antiidiotypic sera. A sharing of idiotypic specificities has been observed between members of each subset. These idiotypes are not recurrent in BALB/c mice immunized with tobacco mosaic virus.     

Immunochemical studies of tobacco mosaic virus--VI. Attempts to localize viral epitopes with monoclonal antibodies

The specificity of 18 monoclonal antibodies directed to tobacco mosaic virus (TMV) was studied by measuring their ability to bind to viral mutants, to other tobamoviruses, to dissociated viral subunits and to peptide fragments of the viral coat protein. The apparent binding specificity of the antibodies was dependent on the type of enzyme-linked immunosorbent assay used, probably because the antigens were disrupted or denatured when attached to the plastic surface of microtiter wells. The capacity of different monoclonal antibodies to detect single substitutions in the viral coat protein was used to delineate some of the topographic epitopes of TMV. By means of computer-generated images of the surface residues of the viral subunit, it was possible to identify certain clusters of residues involved in binding to some of the monoclonal antibodies. The results clearly illustrate the operational limitations encountered when monoclonal antibodies are used for elucidating the antigenic structure of proteins.     

Mouse monoclonal anti-Ia antibodies recognize cross-reacting determinants expressed on distinct subsets of human Ia-like cell-surface molecules

Human Ia-like cell-surface molecules from a homozygous HLA-DR (6/6) B lymphoblastoid cell line have been analyzed using five mouse anti-Ia m.Ab cross-reacting with HLA-DR antigens. The surface-iodinated molecules immunoprecipitated by these m.Ab were analyzed by SDS-PAGE under reducing conditions and by SDS-PAGE followed by isoelectrofocusing. As read from the different migration patterns, three distinct combinations of human Ia-like molecules were identified by these m.Ab. Three anti-I-E-reactive m.Ab immunoprecipitated two-chain molecules whose apparent mol. wt (32K, 29K) corresponded to those of the classical HLA-DR antigens. One m.Ab which on mouse cells recognized a determinant shared by the I-A and I-E molecules precipitated not only the 32-29K bands, but also a 26K band from human cell extracts. Finally, an I-A reactive m.Ab precipitated a complex set of polypeptides including in addition to the 32-29K bands, three additional chains of 30, 28 and 26K. Sequential immunoprecipitation demonstrated that removal of the classical 29-32K HLA-DR chains by an anti-I-E m.Ab did not affect the subsequent immunoprecipitation of the additional chains by the anti-I-A or the anti-I-A + I-E m.Abs. These patterns and those obtained by 2D-gels analysis which demonstrated the complexity of the 26K band are compatible with the coexpression of at least three different subsets of molecules: (1) Ia-like molecules of 29-32K, recognized by all the m.Ab used; (b) molecules of 28-30K recognized by the anti-I-A m.Ab and (c) molecules apparently constituted by 26K chains, precipitated by the anti-I-A m.Ab and by the anti-I-A + I-E m.Ab.     

Partial elucidation of an anti-hapten repertoire in BALB/c mice: Comparative characterization of several monoclonal anti-fluorescyl antibodies

The anti-fluorescyl repertoire in BALB/c mice was examined by producing nine hybridomas secreting antibodies with fluorescein specificity. All nine purified monoclonal anti-fluorescyl antibodies contained kappa light chains and gamma heavy chains (IgG1 and IgG2). Isoelectric focusing profiles of reduced and alkylated Ig preparations demonstrated restricted, yet relatively different spectrotypes. Collectively, the hybridomas provided a diverse range of antibody affinities as determined by several methods, including dissociation rate and ligand inhibition studies. Homogeneity of purified preparations was confirmed by dissociation rate experiments which showed that each monoclonal anti-fluorescyl preparation exhibited a single first-order off-rate. Competitive inhibition experiments with structurally related ligands indicated a high degree of fine specificity. Absorption profiles of bound fluorescein revealed distinct relative differences within the active sites of the molecules studied, and suggested the lack of any apparent correlation between affinity and wavelength maximum. Characteristics of the clones are discussed in terms of the range and prevalence of anti-fluorescyl antibody affinities and the diversity of possible binding mechanisms.     

Production of monoclonal antibodies to measles virus proteins by immunization of mice with heated and detergent-treated antigens

By use of the mouse hybridoma technique, monoclonal antibodies were obtained with specificity for the HA(79K), P(72K), and M(36K) polypeptides of measles virus. BALB/c mice were immunized with native measles virus and measles virus treated with detergents and heat. Clones obtained after immunization of mice with native measles virus showed specificity for the HA(79K) polypeptide only. After immunization with measles virus, treated with 1% sodium sarkosyl sulfate (SSS) at 20°, a clone was obtained producing antibodies to the M(36K) polypeptide. Heating of measles virus in the presence of 1% sodium dodecyl sulfate (SDS) under reducing conditions elicited a selective immune response to the P(72K) and the NP(60K) polypeptides. Thus, clones producing antibodies to the P(72K) polypeptides were isolated.     

Electron microscopic evidence for scorpion toxin binding to synapses of rat brain cortex

The venom from the scorpion Centruroides noxius Hoffman was fractionated by Sephadex G-50 gel filtration. The toxic fraction (n.II) was coupled to Sepharose-4B and used for purification of specific horse immunoglobulins. The purified immunoglobulins were linked to ferritin with glutaraldehyde and repurified in the same affinity column. This complex bound to the postsynaptic membrane of rat brain tissue previously exposed to toxin.     

Preparation and properties of monoclonal antibodies against murine interferon-beta

Wistar rats were immunized with total crude, poly(I) poly(C)-induced mouse L cell interferon. Spleen cells of one rat producing serum antibodies against Mu IFNβ were fused with the NS-1 mouse myeloma cell line using standard procedures. Two hybridomas secreting IgG antibodies to Mu IFNβ were selected using a direct neutralization assay. The hybridomas were cloned by limiting dilution. One clone (2E8B3) was propagated in nude mice. The immunoglobulin fraction was isolated from sera of mice transplanted with this clone and immobilized by coupling to CNBr-activated Sepharose. Affinity chromatography analysis showed that a column prepared from this material was capable of complete binding of Mu IFNβ All Mu IFNa activity was present in the flow through fraction. Analysis of a [³⁵S]methionine-radiolabeled L cell interferon preparation over the monoclonal anti-Mu IFNβ agarose column resulted in a complete purification of Mu IFNβ SDS-polyacrylamide gel electrophoresis of the purified material showed that poly(I) · poly(C)-induced mouse L cell interferon contains one Mu IFNβ species with a molecular weight of 32,000 to 34,000.     

Specific DNA binding activity of T antigen subclasses varies among different SV40-transformed cell lines

Large tumor antigen (T antigen) occurs in at least three different oligomeric subclasses in cells infected or transformed by simian virus 40 (SV40): 5-7 S, 14-16 S, and 23-25 S. The 23-25 S form is complexed with a host phosphoprotein (p53). The DNA binding properties of these three subclasses of T antigen from nine different cell lines and free p53 protein were compared using an immunoprecipitation assay. All three subclasses of T antigen bound specifically to SV40 DNA sequences near the origin of replication. However, the DNA binding activity varied between different cell lines over a 40- to 50-fold range. The 23-25 S and 14-16 S forms from most of the cell lines tested bound much less SV40 origin DNA than 5-7 S T antigen. The free p53 phosphoprotein did not bind specifically to any SV40 DNA sequences.     

beta-Carboline binding to deoxycholate solubilized benzodiazepine receptors from calf cerebral cortex

The beta-carbolines harmane and norharmane competitively inhibit [3H]flunitrazepam ([3H]FNZ) binding to deoxycholate-solubilized benzodiazepine receptors from calf cerebral cortex, with Ki in the micromolar range [3H]Propyl-beta-carboline-3-carboxylate ([3H]PrCC) binds to the soluble receptors with an affinity similar to its binding to particulate receptors (0.41 nM vs 0.48 nM, respectively). The component that binds [3H]PrCC displays a sedimentation profile on sucrose gradient centrifugation similar to that of [3H]FNZ binding component (sedimentation coefficient about 11S).     

Effector functions of a monoclonal aglycosylated mouse IgG2a: binding and activation of complement component C1 and interaction with human monocyte Fc receptor

Aglycosylated monoclonal anti-DNP mouse IgG2a produced in the presence of tunicamycin was compared with the native monoclonal IgG2a with respect to its ability to interact with the first component of complement, C1, and to compete with human IgG for binding to human monocyte Fc receptors. The aglycosylated IgG2a was found to bind subcomponent C1q with an equivalent capacity to the native IgG2a, but the dissociation constant was found to be increased three-fold. When activation of C1 by the glycosylated and aglycosylated IgG2a was compared, the rate of C1 activation by the aglycosylated IgG2a was reduced approximately three-fold. In contrast aglycosylation was accompanied by a large decrease (greater than or equal to 50-fold) in the apparent binding constant of monomeric IgG2a to human monocytes. The data suggest that the aglycosylated IgG2a has a structure which differs in the CH2 domain from the native IgG2a, and that the heterogeneous N-linked oligosaccharides of this monoclonal IgG2a which occur at a conserved position in the CH2 domain play a role in maintaining the integrity of its monocyte-binding site. This lack of monocyte binding may result either from a localized conformational change occurring in a single CH2 domain or from an alteration in the CH2-CH2 cross-domain architecture which is normally structured by a pair of opposing and interacting oligosaccharides. The minimal changes in C1q binding and C1 activation suggest that the oligosaccharides are, at most, indirectly involved in these events.