摘要:

Dynamic protein interactions are thought to play an important role in regulating a wide variety of signal transduction pathways. Adapter molecules that contribute to the assembly and disassembly of these protein complexes are likely to play a critical role in the regulation of these pathways. The function of one such adapter molecule, p130CAS(CAS), has been implicated in signaling pathways involving cell growth, adhesion, and differentiation. We report here the isolation and characterization of a panel of monoclonal antibodies that specifically recognizeCAS. These antibodies are proving to be invaluable molecular reagents for defining the expression, phosphorylation, binding partners, and ultimately the function ofCASwith respect to cell signaling. In addition to their utility as conventional reagents for protein isolation, a subset of these antibodies has also proven to be a sensitive tool for distinguishing between different tyrosine-phosphorylated pools ofCASin the cell. Because tyrosine phosphorylation ofCASprovides a dynamic means with which to regulate protein—protein interactions, these antibodies may thus serve as molecular reagents that can discern the protein binding potential ofCAS. Collectively, the antibodies described in this report provide the means with which to define specific roles forCASin cell signaling that have been otherwise difficult to establis

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.403

年代:1997

数据来源: MAL

摘要:

Human monoclonal antibody (hMAb) cocktail SM-17220 (also known as BT-570), a heterofunctional antibody mixture of 3 human IgM MAbs (HI-223, MH-4H7, and IN-2A8; ratio of 1:10:10) directed againstPseudomonas aeruginosa, were administered to patients with pneumonia or burn wounds (or both) to assess the pharmacokinetics, safety, antigenicity, and preliminary efficacy. Twenty mg of SM-17220 was IV infused over 60 min once daily on 3 consecutive days. Twenty patients (8 pneumonia, 4 burns, and 8 both) completed the study. SM-17220 was safe and well tolerated, and no subjects developed antibodies to SM-17220 and mouse J-chain during the follow-up of 8 weeks. Each MAb of SM-17220 had a half-life ranging from 49 to 91 h, similar to native human IgM. Both MH-4H7 and IN-2A8 administration resulted in a high serum level for about 4 days over an effective concentration, whereas HI-223 showed a lower serum level than expected. Some indications of a potential efficacy were observed and are discussed here.

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.413

年代:1997

数据来源: MAL

摘要:

The variable regions of the murine monoclonal antibody A1, which effectively neutralizes the infection of susceptible cells by the murine hepatitis virus strain JHM, were cloned, sequenced, and expressed in mammalian cells as a functional recombinant antibody. To accomplish the concurrent synthesis of both antibody chains, the light- and heavy-chain-coding regions were inserted into a bicistronic expression cassette based on the encephalomyocarditis virus internal ribosomal entry site. The strategy of combining both coding regions in one bicistronic mRNA allows for the rapid isolation of cell clones expressing high levels of recombinant antibody.

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.421

年代:1997

数据来源: MAL

摘要:

The human monoclonal antibody GM4 was generated by fusing pooled lymphocytes from cancer patients with the lymphoblastoid cell line SHFP-1. Immunohistochemical staining of tumor and normal tissue indicated that this human IgG4 antibody preferentially reacted with melanomas and neuroblastomas. In this study, we demonstrate that GM4 recognizes a "vimentin-like" peptide sequence that we have termed AgGM4. To generate a recombinant derivative of this human antibody, we isolated and expressed the complete heavy and light chain genes. The entire coding sequence for both the heavy and light chains was isolated by RT—PCR using a set of degenerate 5′ signal sequence specific primers and a 3′ constant region primer. High level antibody synthesis and secretion was achieved in Chinese hamster ovary (CHO) cells using a vector designed to maximize expression. Western blot and FACS analysis indicated recombinant GM4 reacted with human tumor cell lines and AgGM4 in a manner similar to the antibody produced by the hybridoma cell line, demonstrating that the specificity of the antibody was not altered during molecular cl

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.427

年代:1997

数据来源: MAL

摘要:

A panel of monoclonal antibodies (8H10/D11, 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, 8A4/G12/F9, and 8H10/F12) was raised in mice against the recombinant 20-kDa extracellular part of the α-chain of the human IgE high affinity receptors (ecFc∈RIα) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc∈RIα, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc∈RIα covalently immobilized on the surface of a sensor chip. The generated hybridomas can be divided into three groups. Hybridoma supernatants 8A4/G12/F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc∈RIα in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/κ type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc∈RIα in ELISA with intensities comparable to each other. Isotype analysis of antibodies secreted by these hybridomas showed that the antibodies 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, and 8H10/D11 were IgG1/κ. The hybridoma supernatants were purified via protein A chromatography. In a SPR experiment, ecFc∈RIα, displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expected for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they do not inhibit the binding of human IgE to ecFc∈RIα. All purified monoclonal antibodies gave positive sign

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.441

年代:1997

数据来源: MAL

摘要:

Clostridium botulinummay produce any of seven known serotypes of neurotoxin (BoNT/A-/G), which are the most toxic bacterial proteins known. Efforts to develop a second-generation vaccine to these toxins would benefit from the isolation of hybridomas producing neutralizing monoclonal antibodies (MAbs). We hypothesized that previous efforts to isolate neutralizing MAbs against various BoNTs failed due to use of toxoided, chemically altered antigens. We employed a novel vaccination regimen employing native, active, single-chain BoNT/E (scBoNT/E). A number of the BoNT/E immunized mice were further vaccinated with lethal doses of fully active BoNT/F. MAb 7F8 consistently neutralized BoNT/F in three different assays:in vivoneutralization, passive neutralization, and neutralization of regional paralysis. There was no detectable recognition and essentially no neutralization of scBoNT/E. The epitope recognized by this MAb was denatured when treated with formalin, urea, guanidine chloride, or sodium dodecyl sulfate. Preliminary epitope mapping studies indicate that the MAb bound to a conformational epitope.

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.447

年代:1997

数据来源: MAL

摘要:

Mouse monoclonal antibodies were raised against the C-terminal domain of human FLI-1, a member of the ETS family of transcription factors which is involved in various murine and human malignancies. This FLI-1 specific domain is included in the fusion product EWS-FLI-1, an oncogenic variant of FLI-1 expressed in Ewing tumors. Antibodies were screened first by enzyme-linked immunosorbent assay onto recombinant FLI-1-coated plates. Positive clones were then tested for their ability to immunoprecipitate over-expressed EWS-FLI-1 protein. Three monoclonal antibodies were selected and further characterized. One of them, termed 7.3 MoAb, was shown to react with FLI-1 and EWS-FLI-1 in immunoblotting, immunoprecipitation, and immunofluorescence experiments. With all three methods, this antibody not only enabled the detection of over-expressed proteins but also more interestingly, that of endogenously expressed proteins. Furthermore, the 7.3 MoAb can specifically inhibit the interaction of FLI-1 with its DNA-binding site as shown by electrophoretic mobility shift assay. The 7.3 MoAb appears to be specific for FLI-1 because it does not react with ERG, the ETS family member most closely related to FLI-1. This antibody should be a useful tool in the diagnostic evaluation of Ewing tumors and should permit biochemical analyses to study the function of the wild-type FLI-1 protein and of the EWS-FLI-1 fusion protein.

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.457

年代:1997

数据来源: MAL

摘要:

Vascular endothelial cell growth factor (VEGF) plays a pivotal role in the regulation of angiogenesis by binding to its cognate receptor molecule type II (VEGFr-II, KDR). VEGFr-II is an endothelial cell-specific transmembrane tyrosine kinase important for vascular endothelial cell development and differentiation during embryogenesis, angiogenic processes under physiological conditions, and various diseases. An increasing number of reports indicate that VEGF/VEGFr-II also play a fundamental role for tumor angiogenesis. We present the generation and in vitro characterization of the monoclonal antibodies 2–7–9 and 2–10–1. Both antibodies are highly specific for VEGFr-II as demonstrated by Western blotting and immunoprecipitation. MAbs 2–10–1 and 2–7–9 bind to a disulphide bridge-stabilized epitope within domains 6 and 7 of the human VEGFr-II with an affinity of 8 and 80 nM, respectively. Furthermore, the antibodies are suitable for immunohistochemistry and ELISA techniques. Because both antibodies recognize their epitope on living cells, they also have the potential for drug targeting and diag

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.465

年代:1997

数据来源: MAL

摘要:

Recombinant mouse/sheep IgE was used in the production of an anti-IgE monoclonal using conventional hybridoma techniques. The specificity of hybridomas secreting anti sheep IgE monoclonal antibodies was verified using a number of assays including competitive ELISAs, ability to induce mediator release from mast cells, and IgE binding using western blotting. Immunohistochemical staining demonstrated the binding of putative anti-IgE monoclonals to the sheep mast cell surface. The isotype of the antibody was IgG1:K.

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.473

年代:1997

数据来源: MAL

摘要:

Three monoclonal antibodies (all IgG1) were prepared against the plant cytokinin dihydrozeatin riboside (diH[9R]Z) and characterized for suitability in the isolation and quantification of dihydrozeatin-type phytohormones. These antibodies detected as little as 5–10 pg of the homologous cytokinin when used in both competitive and noncompetitive immunoassays and each had characteristic crossreactivity with structurally-related cytokinins. One antibody was used to quantify HPLC-purified diH[9R]Z recovered from wheat tissue. The same antibody also was linked to an insoluble support for use in affinity purification of a mixture of cytokinins. These antibodies will be useful for purification and quantification of dihydrozeatin and its 9-riboside and 9-glucoside in plant tissu

ISSN:0272-457X    

DOI:10.1089/hyb.1997.16.479

年代:1997

数据来源: MAL