Detection of conformational changes in human chorionic gonadotropin upon binding to rat gonadal receptors

After binding to rat testicular or ovarian luteinizing hormone (LH) receptors, human chorionic gonadotropin (hCG) and mammalian LH can be detected with monoclonal antibodies directed against a conserved epitope on the beta subunit of the hormones. Two such anti-hCG/anti-LH monoclonal antibodies, known as B105 and B110, compete with one another for binding to this epitope region on free and receptor-bound hormone. By comparing the affinities of B105 and B110 for these two forms of hCG, we have detected apparent changes in the structure of the hormone which develop subsequent to receptor binding. Whereas the affinity of B105 for receptor-bound hCG is approximately 10-fold lower than that for free hCG, the affinity of B110 for receptor-bound hCG is nearly 20-fold greater than that for free hCG. Both B105.hCG and B110.hCG complexes bind to the receptor; however, they have approximately 25 and 50% lower affinity than hCG. Thus, although B110 binds better to the form of hCG which is bound to receptors, binding of B110 to hCG does not appear to induce a conformational change in the hormone which facilitates hormone-receptor binding. Consequently, both B105 and B110 partially inhibit binding of hCG to its receptors. Fab fragments of B105 and B110 are as effective as intact B105 and B110 in inhibiting the binding of labeled B105 and B110 to hCG-receptor complexes, suggesting that circular complexes which might be formed by the interaction of divalent antibody, two molecules of hCG, and two membrane-bound receptors or one divalent receptor are not contributing to the affinity of the antibodies for receptor-bound hCG. Alternatively, formation of circular complexes can explain an increase in apparent affinity of B105 for ovine or bovine LH-receptor complexes. Data obtained with B105 suggest either that the structure of the epitope is altered following binding or that a portion of the epitope is partially obscured when hCG binds to the receptor. In contrast, the data obtained using B110 are not explained by models in which steric factors reduce the affinity of the antibody for the hormone-receptor complex. Therefore, as a minimal explanation for these observations, we postulate that the conformation of the B105/B110 epitope region is altered following binding of the hormone to receptors. The nature of the conformational change and its relationship to LH/hCG action is unknown.     

Characterization of New Alternaria alternata–Specific Rat Monoclonal Antibodies

In this study, three different rat hybridoma cell lines secreting monoclonal antibodies (mAbs) recognizing the spores from Alternaria alternata, a plant pathogenic fungus, contaminant of food products and important cause of both allergic rhinitis and asthma, have been characterized. These three mAbs are all of IgM isotype. Two antibodies, A1 and F10, were cross-reactive antibodies recognizing spores from Alternaria, Cladosporium, Penicillium, Aspergillus and Stachybotrys genera, but not the yeasts Saccharomyces cerevisiae or Candida albicans. Competitive and sandwich assays demonstrated that these two mAbs were directed against the same or very close repetitive(s) epitope(s). A1-based sandwich ELISA efficiently detected this epitope in various mould (but not yeast)-soluble extracts prepared from strains grown in the laboratory. Moreover, this A1-based sandwich ELISA detected its cognate epitope in air and dust samples obtained from dwellings. The third antibody, E5, recognized only the spores of Alternaria and the phylogenetically very close Ulocladium botrytis. This E5 antibody is directed against a repetitive epitope found in Alternaria and Ulocladium laboratory extracts and can be used in a sandwich assay for the quantification of these moulds. Therefore, E5 antibody is a promising tool for the development of Alternaria-Ulocladium-specific immunoassays, while A1 and F10 could be interesting tools for the quantification of the total mould biomass.     

Lateral diffusion of antigen receptors artificially incorporated onto B lymphocytes

In the companion paper, we have shown that palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) incorporated onto B lymphocytes can bind DNP antigens and that this binding causes polyclonal B cell activation. In this study we use fluorescence photobleaching recovery (FPR) techniques to examine the lateral diffusion and mobile fractions of antigen-receptor complexes on receptor-decorated B cells as functions of antigen concentration and epitope density. Antigens used in this study are DNP conjugates of polymerized flagellin (DNP-POL) and linear dextran of 2 X 10(6) m.w. (DNP-DEX). The diffusion coefficient observed for antigen bound to artificial receptors decreases monotonically with increased antigen dose and epitope density. When the artificial receptor-bearing cells are labeled with either relatively high concentrations of medium epitope density antigen or high epitope density antigen, a large fraction of antigen-receptor complexes become immobile in the time scale of the experiment. We attribute this behavior to extensive receptor cross-linking by antigen. In parallel with these FPR experiments, we examined the effects of antigen concentration and epitope density on the polyclonal humoral response of receptor-decorated B cells. We found that the response is a function of both antigen concentration and epitope density similar to that seen in natural B cells. The combined results of these experiments show that cell activation results when the diffusion coefficient of the antigen-receptor complex ranges between 10 X 10(-11) cm2 sec-1 and 5 X 10(-11) cm2 sec-1. These values represent threefold and sixfold decreases from the diffusion coefficient of antigen-free receptors, respectively. However, when either a high antigen concentration or epitope density causes a large fraction of antigen-receptor complexes to become immobile, B cells become unresponsive not only to the bound antigen, but also to LPS. Results obtained in this study are very similar to those obtained in a study performed with natural antigen-specific B cells. Therefore, for the responding population of receptor-decorated B cells, it is possible that antigens activate and paralyze these B cells by mechanisms similar to those by which antigens regulate normal B cell responses.     

Reducing heterophilic antibody interference in immunoassays using single-chain antibodies

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.     

Characterization of monoclonal antibodies to human interferon-gamma and their application to enzyme-linked immunosorbent assay (ELISA)

The authors obtained two mouse monoclonal antibodies, G-208 and G-166, to recombinant human interferon-gamma (rH-IFN-gamma). Immunologically, they were classified as IgG1-K subclass. G-208 neutralized the antiviral activity of natural and recombinant human IFN-gamma, but did not bind to heat-denatured rH-IFN-gamma. G-166 was able to bind to rH-IFN-gamma as well as to heat-denatured rH-IFN-gamma, but it did not bind to natural human IFN-gamma (nH-IFN-gamma). A sandwich enzyme immunoassay specific to H-IFN-gamma molecule was developed using polyclonal rabbit anti-nH-IFN-gamma antibody and G-208. This assay monitors only biologically active H-IFN-gamma molecule. Thus, this method may be used for the direct determination of H-IFN-gamma instead of determination of antiviral activity of H-IFN-gamma.     

Transformation of the estrogen receptor detected by two monoclonal antibodies

The estrogen receptor from fetal guinea-pig uterus is recognised by two monoclonal antibodies (H222 and H226) developed against the human estrogen receptor but it interacts differently with each of them. The H222 antibody, whose epitope is located in the hormone-binding domain of the receptor, shifts the sedimentation coefficient of the nonactivated oligomeric receptor in low salt sucrose gradients from 9S to 11S. When this oligomeric receptor-H222 complex is centrifuged in high salt gradients, it dissociates to an 8S monomer-H222 complex, indicating that all the estradiol-binding units present in the nonactivated receptor can bind the H222 antibody. In contrast, the H226 antibody, whose epitope is located close to the DNA-binding domain, shifts the sedimentation coefficient of the nonactivated receptor only to 9.4S and when this complex sediments in high salt gradients, it dissociates to a 7S monomer-H226 complex plus a 4.5S monomeric receptor not bound to the antibody. This observation suggests that not all the H226 epitopes are accessible in the nonactivated receptor. On the other hand, the temperature-activated receptor reacts with the H226 antibody to form two complexes sedimenting at 7S and 9S in high salt gradients. This 9S complex indicates the formation of a homodimer that binds two molecules of the H226 antibody. However, only one H222 epitope seems to be accessible in this dimeric form of the receptor, since only one 8S complex is observed when the activated receptor reacts with the H222 antibody. In addition, binding to the H222 antibody before activation prevents the dimerisation. This suggests that the H222 epitope is near or directly involved in the dimerisation domain. Interaction of the H222 and H226 antibodies with the estrogen receptor reveals modifications of its structure during activation, and consequently of the exposure of its functional domains.     

Further characterization of cooperative interactions of monoclonal antibodies

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.     

Determination of crucial epitopes in the sperm protein calsperin employing synthetic peptides and monoclonal antibodies

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids ²²⁸WEKHFLDAS²³⁷ was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function.     

Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors

Inhibitors of receptor tyrosine kinases are implicated as therapeutic agents for the treatment of many human diseases including cancer, inflammation and diabetes. Cell-based assays to examine inhibition of receptor tyrosine kinase mediated intracellular signaling are often laborious and not amenable to high-throughput cell-based screening of compound libraries. Here we describe the development of a nonradioactive, sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the activation and inhibition of ligand-induced phosphorylation of the colony-stimulating factor-1 receptor (CSF-1R) in 96-well microtiter plate format. The assay involves the capture of the Triton X-100 solubilized human CSF-1R, from HEK293E cells overexpressing histidine epitope-tagged CSF-1R (CSF-1R/HEK293E), with immobilized CSF-1R antibody and detection of phosphosphorylation of the activated receptor with a phosphotyrosine specific antibody. The assay exhibited a 5-fold increase in phosphorylated CSF-1R signal from CSF-1R/HEK293E cells treated with colony-stimulating factor (CSF-1) relative to treated vector control cells. Additionally, using a histidine epitope-specific capture antibody, this method can also be adapted to quantify the phosphorylation state of any recombinantly expressed, histidine-tagged receptor tyrosine kinase. This method is a substantial improvement in throughput and quantitation of CSF-1R phosphorylation over conventional immunoblotting techniques.     

Structural evidence for recognition of a single epitope by two distinct antibodies

The structure of a complex between the hemagglutinin of influenza virus and the Fab of a neutralizing antibody was determined by X-ray crystallography at 2.8 A resolution. This antibody and another which has only 56% sequence identity bind to the same epitope with very similar affinities and in the same orientation. One third of the interactions is conserved in the two complexes; a significant proportion of the interactions that differ are established by residues of the H3 complementarity-determining regions (CDR) which adopt distinct conformations in the two antibodies. This demonstrates that there is a definite flexibility in the selection of antibodies that bind to a given epitope, despite the high affinity of their complexes. This flexibility allows the humoral immune response to be redundant, a feature that may be useful in achieving longer lasting protection against evolving viral pathogens.     

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies

A one-step two-site immunoradiometric assay for the measurement of free β subunit of human chorionic gonadotrophin (β-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free β subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free β subunit. Although the level of hCG “cross-reaction” was low when studied using conventional techniques, the apparent β-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of ¹²⁵I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.     

Development of an enzyme-linked receptor assay (ERA) for hCG.

An enzyme-linked receptor assay (ERA) for hCG was developed using horseradish peroxidase. The 1,500 g pellets, interstitial cell fraction, and solubilized homogenate from the rat testis linked to tanned sheep red blood cells (SRBC) were used as the binding fractions for hCG. In these ERA systems, the limit of detection for hCG was 30 IU/l (3-12 mIU/tube), which was almost equal to that of radio receptor assay (RRA). The ERA using SRBC linked solubilized receptor fraction showed the most satisfactory result in accuracy, reproducibility and easiness.     

Antigenic determinants on human choriogonadotropin alpha-subunit. II. Immunochemical mapping by a monoclonal antipeptide antibody

In order to study antigenic site(s) present in the carboxyl-terminal part of the alpha-subunit of human choriogonadotropin (hCG-alpha), we attempted to produce site-specific antibodies directed against a 34-residue synthetic peptide analogous to region 59-92 of hCG-alpha. From a fusion experiment performed with a mouse injected with hCG-alpha-(59-92)-peptide conjugated to tetanus toxoid as immunogen, we selected a monoclonal antipeptide antibody (designated FA36) which has high binding activity for 125I-hCG-alpha but not for 125I-hCG in a radioimmunoassay. This antibody is of the IgG1 subclass and displays an affinity constant for 125I-hCG-alpha of 3.1 x 10(8) M-1. Hapten inhibition experiments performed by either radioimmunoassay or enzyme-linked immunosorbent assay with synthetic peptides spanning different portions of the region (59-92) demonstrated that the binding site of FA36 resides on (minimally) the six COOH-terminal amino acids of hCG-alpha, namely Cys-Tyr-Tyr-His-Lys-Ser, and that FA36 binds preferentially to peptides containing a carboxyl group on the COOH-terminal residue. Monoclonal immunoradiometric assays were established to determine the location of antigenic regions recognized by FA36, by antibody AHT20 (which binds only to hCG-alpha), and by antibody HT13 (which binds to both hCG and hCG-alpha). FA36 has the capacity to bind to hCG-alpha bound to either AHT20 or HT13, demonstrating that both AHT20 and HT13 antibodies are directed against antigenic regions distinct from the epitope of FA36. Monoclonal immunoradiometric assays were also carried out to study the binding of FA36 to hCG, the ovine and equine lutropin alpha-subunit, or hCG-alpha minus the 5 COOH-terminal residues (hCG-alpha core). Whereas significant binding of 125I-FA36 was observed with the ovine lutropin alpha-subunit, no binding was found with the equine lutropin alpha-subunit. As expected, FA36 did not bind to hCG-alpha core. Binding was also not detected with hCG, confirming that FA36 is specific for free hCG-alpha and that the COOH-terminal part of hCG-alpha is either weakly or (more likely) not at all accessible in the alpha/beta-dimer for antibody binding. Finally, immunoblots performed on hCG-alpha-(59-62)-peptide and various denatured alpha-subunits indicated that, with the exception of the equine lutropin alpha-subunit, FA36 detected various denatured alpha-subunits and particularly the alpha-subunit of carp gonadotropin-thyrotropin. This latter observation suggests a high degree of homology between the COOH-terminal regions of the alpha-subunits of fish gonadotropin and analogous mammalian hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.     

Antibody colocalization microarray: a scalable technology for multiplex protein analysis in complex samples

DNA microarrays were rapidly scaled up from 256 to 6.5 million targets, and although antibody microarrays were proposed earlier, sensitive multiplex sandwich assays have only been scaled up to a few tens of targets. Cross-reactivity, arising because detection antibodies are mixed, is a known weakness of multiplex sandwich assays that is mitigated by lengthy optimization. Here, we introduce (1) vulnerability as a metric for assays. The vulnerability of multiplex sandwich assays to cross-reactivity increases quadratically with the number of targets, and together with experimental results, substantiates that scaling up of multiplex sandwich assays is unfeasible. We propose (2) a novel concept for multiplexing without mixing named antibody colocalization microarray (ACM). In ACMs, both capture and detection antibodies are physically colocalized by spotting to the same two-dimensional coordinate. Following spotting of the capture antibodies, the chip is removed from the arrayer, incubated with the sample, placed back onto the arrayer and then spotted with the detection antibodies. ACMs with up to 50 targets were produced, along with a binding curve for each protein. The ACM was validated by comparing it to ELISA and to a small-scale, conventional multiplex sandwich assay (MSA). Using ACMs, proteins in the serum of breast cancer patients and healthy controls were quantified, and six candidate biomarkers identified. Our results indicate that ACMs are sensitive, robust, and scalable.     

Identification of epitopes associated with hCG and the beta hCG carboxyl terminus by monoclonal antibodies produced against a synthetic peptide

We have produced a library of monoclonal antibodies directed against a 37-amino acid synthetic polypeptide analogous to the carboxyl terminus of hCG. Five antibodies, designated FB01, FB02, FB03, FB04, and FB00, were developed and analyzed with respect to affinity and specificity for epitopes on human chorionic gonadotropin (hCG) and beta hCG by enzyme-linked immunoabsorbent and radioimmunoassays (RIA). All monoclonal antibodies demonstrated low affinity constants (approximately 10(-7) liters/mol) compared with those obtained by immunization with native beta hCG. One antibody, namely FB00, bound only to the synthetic peptide, whereas all other monoclonal antibodies recognized either free native beta hCG or both beta hCG and HCG. Antibodies produced against the synthetic peptide did not cross-react with other glycoprotein hormones such as LH, TSH, and FSH. Characterization of the monoclonal antibody-binding sites revealed the presence of at least four separate and distinct epitopes on the last 35 amino acids of beta hCG. Indeed, one epitope recognized by FB01 is located between residues 109 and 118, whereas another antigenic region recognized by FB04 appears to be present on the 109-121 portion of the molecule near or at position 118. One additional antigenic site was localized between residues 118 and 136. Finally, FB00 recognized an epitope located on the last 10 amino acids (136-145) of beta hCG. With the use of such antibodies, two- and three-site monoclonal RIA were developed and employed to detect free beta hCG and hCG in sera of patients with choriocarcinoma. These assays may be useful in the detection of beta hCG- and hCG-producing tumors and subsequent monitoring of patients in response to surgery and/or chemotherapy.     

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.     

Use of α- and β-subunit specific antibodies in studying interaction of hCG with Leydig cell receptors

Antisera (a/s) raised to individual α- and β-subunits of human chorionic gonadotropin (hCG) have been characterized for specificity using immunoaffinity procedures and used to study the disposition of the two subunits when intact hCG is complexed with luteinizing hormone (LH) receptor of the Leydig cells. Three kinds of experiments were done. (a) The ability of the preformed hormone-antibody (H-Ab) complex to bind to receptor and stimulate a response; (b) the ability of the a/s to dissociate hCG from its complex with the receptor and thereby terminate response; and (c) the ability of the premixed antibody and receptor to compete for binding of labeled hCG. Although the subunit specific a/s used here were equipotent in binding hCG (capacity to bind and K_a being very similar), their behavior once the receptor preparation or Leydig cell is introduced into the system was drastically different. The β-subunit antibody relative to the α-subunit antibody, appeared to be poorly effective in preventing hCG from either binding to the receptor or inhibiting the continuation of response. The results suggest that hCG upon interaction with the receptor loses the determinants specific to the β-region more rapidly compared to those specific to the α-region suggesting thereby that the initial interaction of hCG with the receptor should be occurring through sites in the β-subunit. Although the α-subunit portion of the hCG molecule is available for binding to the antibody for a relatively longer time, the biological response of the cell seems very sensitive to such binding with the antibody as it invariably results in loss of response. In the Leydig cell system, the ability of the a/s to bind hCG that is already complexed to the receptor appears to be dependent upon the time of addition of the antibody to the incubation medium. The antisera were totally ineffective in inhibiting steroidogenic response to hCG if added 60 min after addition of hCG. This would suggest that the hormone-receptor complex once formed perhaps continues to change its orientation with the result that with time relatively less and less of antigenic determinants become available for antibody binding.     

Generation and epitope analysis of thyroid stimulating hormone-specific monoclonal antibodies for enzyme immunoassays

By using an improved hybridoma technique with a semisolid medium of methylcellulose for initial cloning, numerous high affinity monoclonal antibodies against human thyroid stimulating hormone (TSH) were generated. These antibodies were characterized with respect to their subunit and epitope specificity. Epitope analysis of antibodies specific to the beta-subunit of TSH was performed by a sandwich pairing procedure. Based on the results of this analysis, it was concluded that there are four distinct TSH-specific epitopes on the beta-subunit of TSH; these are designated a, b, c, and ab. The five antibodies binding to epitopes a, b, and c are not mutually exclusive. However, the antibody binding to epitope ab prevents further binding of other antibodies to epitope a or b, but not to epitope c. This epitope analysis enabled us to combine three high affinity monoclonal antibodies, each of which reacts with epitopes a, b, and c, respectively, in a typical sandwich enzyme immunoassay. One was immobilized on polystyrene beads and the other two were conjugated with horseradish peroxidase and served as the second antibodies. This enzyme immunoassay can be performed within 90 min and with a minimum sensitivity of 0.2-0.3 microIU/ml.     

Epitope mapping of the receptor-bound agonistic form of human chorionic gonadotropin (hCG) in comparison to the antagonistic form (deglycosylated hCG).

On the surface of free human chorionic gonadotropin (hCG), we can distinguish with our panel of monoclonal antibodies (MCA) 14 topographically distinct epitopes (designated alpha 1 - alpha 5, beta 1 - beta 5, alpha beta 1 - alpha beta 4, depending on the subunit they are attached to). Only 2, i.e. the adjacent beta 3 and beta 5 epitopes, of these 14 are accessible to 125I-labeled MCA binding, when hCG is first allowed to bind to the rat testis hCG receptor. This result indicates that the agonist hCG assumes a defined orientation in its receptor-bound state and that, except for that small area comprising the beta 3 and beta 5 epitopes, most of its surface is masked by the hCG receptor. We therefore asked whether the competitive antagonist deglycosylated hCG (degly-hCG), which, when free, is antigenically (as to number and topography of epitopes) indistinguishable from native hCG, would interact with the receptor differently, that is, in a way that can be discerned by this epitope accessibility paradigm. Here we describe that on receptor-bound degly-hCG the beta 3 and beta 5 epitopes were concealed as were all other epitopes. This observation, together with finding the receptor affinity of degly-hCG to be 4 times higher than that of native hCG, suggests that degly-hCG assumes a signal transduction-incompetent ligand orientation and at the same time interacts with the receptor more intensively, i.e. establishes additional ("antagonist accessory") protein-protein contacts besides those involved in agonist binding. It thus appears that the carbohydrate moieties function to prevent formation of such accessory contacts.