Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

Monoclonal antibodies to intra-acrosomal proteins inhibit gamete binding in vitro

The sperm-zona pellucida-binding assay in vitro was used as a functional test for zona pellucida-binding ability of boar spermatozoa after co-incubation with monoclonal antibodies against intra-acrosomal proteins. The effect of monoclonal antibodies ACR.2 against boar acrosin (55, 53, 45 and 38 kDa), and Hs-8 against boar intra-acrosomal protein (230, 110, 88, 60, 48 kDa) on boar spermatozoa-porcine oocyte binding was examined. The sperm-zona pellucida-binding was reduced when medium was supplemented with monoclonal antibodies during sperm-oocyte co-incubation, but not when capacitated spermatozoa were pretreated with monoclonal antibodies before incubation with oocytes. Our results show that the monoclonal antibodies (ACR.2, Hs-8) against intra-acrosomal proteins reduce the secondary sperm-zona pellucida-binding with statistically significant difference. This suggests the role of these proteins in the early phases of fertilization.     

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.     

Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity.

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.     

Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2.

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration-dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western-blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross-reacts with dog trypsinogen.     

Monoclonal antibodies to NTF2 inhibit nuclear protein import by preventing nuclear translocation of the GTPase Ran

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.     

Studies on the suppression of the immune response to a defined protein epitope by anti-idiotypic antibodies

We have previously reported that C3H.SW (CSW) and A/J mice immunized with the tobacco mosaic virus protein (TMVP) produce antibodies to a decapeptide epitope corresponding to amino acid residues 103-112 of the protein. In the C3H.SW (CSW) strain, the antibodies to the decapeptide contain major crossreactive idiotope, C10-IdX, which is found on a CSW-derived monoclonal antidecapeptide antibody, designated as C10. The in vivo administration of anti-C10 antibodies suppresses the response to the decapeptide epitope in CSW mice. The present communication describes experiments designed to elucidate several parameters responsible for the suppression produced by the in vivo administration of anti-C10. It was found that 50 micrograms of anti-C10 was required to suppress the response to the decapeptide in CSW mice when 2 weeks elapsed between administration of the anti-C10 and immunization with TMVP; however, only 10 ng was required when one injection of antigen instead of two was administered and when the interval between treatment with anti-C10 and immunization was extended to 6 weeks. This suggests that the anti-C10 induces alterations in the idiotypic network which are not yet fully developed after 2 weeks. Furthermore, experiments presented herein demonstrate that decapeptide-binding antibodies from A/J mice, which lack the C10-IdX, were also suppressed by pretreatment with anti-C10. Interestingly, unlike the case with the CSW strain, the titer to TMVP was decreased by the administration of anti-C10 to A/J mice which were subsequently immunized with TMVP. These findings suggest that the polyclonal anti-C10 contains antibodies to an idiotype which is a major component of the overall anti-TMVP response of A/J mice and may be important in the overall regulation of the anti-TMVP response.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization.

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.     

Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies

In an attempt to delineate spatial relationships between various allodeterminants of cell surface MHC antigens, competitive binding studies were performed using 4 different monoclonal antibodies, each of which reacted with the H-2Kk antigen but with different serologic specificities. Competition was studied by examining the effect of unlabeled antibodies on the binding of each 125I-labeled antibody to spleen cells of the H-2a haplotype. Mutual inhibition was observed between 2 of the antibodies, and a 3rd antibody of lower affinity was inhibited by the first 2 antibodies but did not itself inhibit the binding of these antibodies. The 4th antibody did not block the binding of the other 3 labeled antibodies, and binding of this 4th labeled antibody was only partially inhibitable by the other 3 antibodies. These results indicate the presence of at least 2 spatially distinct allodeterminants on H-2Kk molecules expressed on the cell surface.     

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. II. Monoclonal H-2-specific antibodies obtained from an unimmunized C57BL/KaLwRij (H-2b) mouse

A monoclonal 'natural' anti-H-2 IgM antibody produced by a hybridoma cell line OL-3.17 (H-2 m. 209) is described. The OL-3.17 monoclonal antibody was obtained by hybridization of spleen B cells from an unimmunized C57BL/Ka (H-2b) mouse in the serum of which simultaneously an IgM kappa paraprotein of high concentration and a natural H-2-specific antibody of high titer was detected. The monoclonal antibody OL-3.17 reacted strongly with H-2d and H-2s and weakly with H-2k,q,r lymphocytes, thereby detecting a hitherto unknown H-2 public determinant. The target molecules for OL-3.17 cocapped with class-I H-2 antigens, but immunoprecipitation of H-2 antigens was not achieved. This is the first monoclonal H-2-specific antibody obtained from a mouse without intentional immunization and, with high probability, was derived from a B-cell clone which produced natural H-2-specific antibodies detectable in the serum of the original mouse.     

Monoclonal antibodies to adenosine receptor by an auto-anti-idiotypic approach

Monoclonal antiadenosine receptor antibodies have been raised by an auto-anti-idiotypic approach. BALB/c mice were immunized with adenosine 6-aminocaproyl-bovine serum albumin. Hybridoma cell lines were raised and lines that secreted antibodies that bound to rabbit antiadenosine antibodies were obtained. Two such monoclonal antibodies, AA18 and AA21, were studied in detail and found to be directed at adenosine receptors by the following criteria. They inhibited the binding of [3H] adenosine to rabbit antiadenosine antibodies that had binding characteristics similar to those of adenosine receptors. They bound to rat brain membranes and binding could be inhibited by N6-cyclohexyladenosine and L-N6-phenylisopropyladenosine, both adenosine receptor agonists. They also inhibited the binding of [3H]L-N6-phenylisopropyladenosine to rat brain membranes. In functional assays, they inhibited adenylate cyclase of rat brain membranes, but had no effect on adenylate cyclase of rat hepatic membranes, indicating that they mimic agonists of the A1 receptor, therefore, carrying an "internal image" of the adenosine molecule. When adenosine receptors of rat brain membranes were solubilized with 1% cholic acid, partially purified on an adenosine 6-aminocaproyl AH-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, both AA18 and AA21 recognized a 62,000 band under nonreducing conditions, and a major band of 36,000 under reducing conditions. We conclude that the auto-anti-idiotypic route has yielded specific antibodies that recognize the A1 adenosine receptor.     

Monoclonal antibodies as an instrument to study fibrin polymerization

Examples of using monoclonal antibodies (MAb) for studying the fibrin polymerization mechanism are considered. MAb with epitopes situated in the fibrin polymerization sites or in the recognition sites of enzymes thrombin, plasminogen, and factor XIII, which are the functional partners of fibrin, are primarily discussed. The MAb to epitopes in various regions of A alpha, B beta, and gamma polypeptide chains of the functionally important E, D, and alpha C domains of fibrin are successively described.     

Inability of mice to generate cytotoxic T lymphocytes to vesicular stomatitis virus restricted to H-2Kk or H-2Dk

H-2k mice are unable to generate cytotoxic T lymphocytes (CTL) to vesicular stomatitis virus (VSV). This apparent unresponsiveness is found for both major serotypes of VSV, VSV-Indiana and VSV-New Jersey. CTL unresponsiveness occurs despite the ability of H-2k mice to generate a humoral immune response against VSV that is comparable to that found in responder (H-2b and H-2a) strains. All H-2k mice regardless of background genes, including various Ig allotypes, were found to be nonresponders. H-2k-linked unresponsiveness mapped to both H-2Kk and H-2Dk and occurred despite the presence of responder alleles in (responder x nonresponder)F1 mice. The unresponsiveness cannot be attributed to an inability of VSV-infected H-2k target cells to express viral surface antigens of H-2 molecules. Further, unresponsiveness cannot be overcome by using secondary stimulation in vivo or in vitro. H-2k-linked unresponsiveness does not appear to be due to suppression, and no complementation has been found in various (nonresponder x nonresponder)F1 mice. Thus unresponsiveness to VSV in association with H-2Kk or H-2Dk appears to represent an extensive defect of immune responsiveness that probably occurs because VSV is not a natural mouse pathogen.     

Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells

Monoclonal neutralizing antibodies against DNA polymerase-alpha substantially inhibit nuclear DNA replication in lysolecithin-permeabilized cultured human fibroblasts. The degree of inhibition of DNA synthesis is proportional to antibody concentration, and the effect is specific in that RNA synthesis measured under the same experimental conditions is unperturbed. Autoradiographic data demonstrate that the magnitude of the inhibition measured in the mass culture reflects the uniform response of all the constituent cells in the target population. These observations confirm the participation of DNA polymerase-alpha in replicative DNA synthesis and identify a versatile, novel approach to the dissection of mammalian processes of DNA replication and repair.