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1.
Amyloid fibrils and partially unfolded intermediates can be distinguished serologically from native amyloidogenic precursor proteins or peptides. In this regard, we previously had reported that mAb 11-1F4, generated by immunizing mice with a thermally denatured variable domain (VL) fragment of the human kappa4 Bence Jones protein Len, bound to a non-native conformational epitope located within the N-terminal 18 residues of fibrillar, as well as partially denatured, Ig light chains (O'Nuallain, B., et al. (2006) Biochemistry 46, 1240-1247). To define further the antibody binding site, we used random peptide phage display and epitope mapping of VL Len using wild-type and alanine-mutated Len peptides where it was shown that the antibody epitope was reliant on up to 10 of the first 15 residues of protein Len. Comparison of Vkappa and Vlambda N-terminal germline consensus sequences with protein Len and 11-1F4-binding phages indicated that this antibody's cross-reactivity with light chains was related to an invariant proline at position(s) 7 and/or 8, bulky hydrophobic residues at positions 11 and 13, and additionally, to the ability to accommodate amino acid diversity at positions 1-4. Sequence alignments of the phage peptides revealed a central proline, often flanked by aromatic residues. Taken together, these results have provided evidence for the structural basis of the specificity of 11-1F4 for both kappa and lambda light chain fibrils. We posit that the associated binding site involves a rare type VI beta-turn or touch-turn that is anchored by a cis-proline residue. The identification of an 11-1F4-related mimotope should facilitate development of pan-light chain fibril-reactive antibodies that could be used in the diagnosis and treatment of patients with AL amyloidosis. 相似文献
2.
Danilov SM Watermeyer JM Balyasnikova IV Gordon K Kugaevskaya EV Elisseeva YE Albrecht RF Sturrock ED 《Biochemistry》2007,46(31):9019-9031
Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE. 相似文献
3.
Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3-4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography-mass spectrometry (LC-MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE-SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE-SDS assay. The amount of the pre-LC peak recovered from the CE-SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb. 相似文献
4.
CK-STAR, a monoclonal antibody against human brain creatine kinase (CK), can be shown by chemical cleavage mapping and peptide synthesis to recognize an epitope at the free N-terminus of the enzyme. The epitope could be largely reproduced by a synthetic peptide based on the first 18 amino acids and could be partly formed by the first 11 amino acids. The antibody did not bind to native CK, but it did bind to CK in various partially denatured forms and to an enzymically inactive intermediate in the refolding process. Competitive binding studies have shown that the N-terminal conformations of both the refolding intermediate and the free peptide resemble that of CK partially denatured by attachment to plastic. The results suggest that the final stages of CK refolding and reactivation involve a structural change at the N-terminus or its interaction with some other part of the CK molecule, thus masking the CK-STAR epitope. 相似文献
5.
Francesca Lavatelli Giulia Mazzini Stefano Ricagno Federica Iavarone Paola Rognoni Paolo Milani Mario Nuvolone Paolo Swuec Serena Caminito Masayoshi Tasaki Antonio Chaves-Sanjuan Andrea Urbani Giampaolo Merlini Giovanni Palladini 《The Journal of biological chemistry》2020,295(49):16572
Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils. 相似文献
6.
R Pulido M J Elices M R Campanero L Osborn S Schiffer A García-Pardo R Lobb M E Hemler F Sánchez-Madrid 《The Journal of biological chemistry》1991,266(16):10241-10245
The human integrin VLA (very late activation antigens)-4 (CD49d/CD29), the leukocyte receptor for both the CS-1 region of plasma fibronectin (Fn) and the vascular cell surface adhesion molecule-1 (VCAM-1), also mediates homotypic aggregation upon triggering with specific anti-VLA-4 monoclonal antibody (mAb). Epitope mapping of this integrin on the human B-cell line Ramos, performed with a wide panel of anti-VLA-4 mAb by both cross-competitive cell binding and protease sensitivity assays, revealed the existence of three topographically distinct epitopes on the alpha 4 chain, referred to as epitopes A-C. By testing this panel of anti-VLA-4 mAb for inhibition of cell binding to both a 38-kDa Fn fragment containing CS-1 and to VCAM-1, as well as for induction and inhibition of VLA-4 mediated homotypic cell adhesion, we have found overlapping but different functional properties associated with each epitope. Anti-alpha 4 mAb recognizing epitope B inhibited cell attachment to both Fn and VCAM-1, whereas mAb against epitope A did not block VCAM-1 binding and only partially inhibited binding to Fn. In contrast, mAb directed to epitope C did not affect cell adhesion to either of the two VLA-4 ligands. All mAb directed to site A, as well as a subgroup of mAb recognizing epitope B (called B2), were able to induce cell aggregation, but this effect was not exerted by mAb specific to site C and by a subgroup against epitope B (called B1). Moreover, although anti-epitope C and anti-epitope B1 mAb did not trigger aggregation, those mAb blocked aggregation induced by anti-epitope A or B2 mAb. In addition, anti-epitope A mAb blocked B2-induced aggregation, and conversely, anti-epitope B2 mAb blocked A-induced aggregation. Further evidence for multiple VLA-4 functions is that anti-Fn and anti-VCAM-1 antibodies inhibited binding to Fn or to VCAM-1, respectively, but did not affect VLA-4-mediated aggregation. In summary, we have demonstrated that there are at least three different VLA-4-mediated adhesion functions, we have defined three distinct VLA-4 epitopes, and we have correlated these epitopes with the different functions of VLA-4. 相似文献
7.
We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NC-like structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'- and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein. 相似文献
8.
Teh EM Hewitt J Ung KC Griffiths TA Nguyen V Briggs SK Mason AB MacGillivray RT 《The FEBS journal》2005,272(24):6344-6353
The molecular basis of the transferrin (TF)-transferrin receptor (TFR) interaction is not known. The C-lobe of TF is required to facilitate binding to the TFR and both the N- and C-lobes are necessary for maximal binding. Several mAb have been raised against human transferrin (hTF). One of these, designated F11, is specific to the C-lobe of hTF and does not recognize mouse or pig TF. Furthermore, mAb F11 inhibits the binding of TF to TFR on HeLa cells. To map the epitope for mAb F11, constructs spanning various regions of hTF were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The recombinant fusion proteins were analysed in an iterative fashion by immunoblotting using mAb F11 as the probe. This process resulted in the localization of the F11 epitope to the C1 domain (residues 365-401) of hTF. Subsequent computer modelling suggested that the epitope is probably restricted to a surface patch of hTF consisting of residues 365-385. Mutagenesis of the F11 epitope of hTF to the sequence of either mouse or pig TF confirmed the identity of the epitope as immunoreactivity was diminished or lost. In agreement with other studies, these epitope mapping studies support a role for residues in the C1 domain of hTF in receptor binding. 相似文献
9.
构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1~30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1~30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。 相似文献
10.
Conventional capture ("Sandwich") ELISAs equally detect denatured inactive and native active botulinum type A toxin. Light chain endoprotease activity assays also fail to distinguish between various inactive molecules including partially denatured and fragmented material still retaining this protease activity. By co-coating microtiter plates with SNAP25 substrate and a monoclonal antibody specific for a conformational epitope of the toxin's Hc domain, it was possible to develop a highly sensitive (130 aM LoD), precise (1.4% GCV) new assay specific for the biologically active toxin molecule. Capture was performed in phosphate buffer with a fixed optimal concentration of chaotropic agent (e.g., 1.2 M urea) to differentially isolate functional toxin molecules. Addition of enzymatically favorable buffer containing zinc and DTT reduced the interchain disulfide bond releasing and activating the captured L-chain with subsequent specific cleavage of the SNAP25(1-206) substrate. A neoepitope antibody specific for the newly exposed Q(197) epitope was used to quantify the cleaved SNAP25(1-197). The assay's requirement for the intact toxin molecule was demonstrated with pre-reduced toxin (heavy and light chains), recombinant LHn fragments, and stressed samples containing partially or fully denatured material. This is the first known immunobiochemical assay that correlates with in vivo potency and provides a realistic alternative. 相似文献
11.
Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies. 总被引:3,自引:2,他引:1
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M. A. Speed T. Morshead D. I. Wang J. King 《Protein science : a publication of the Protein Society》1997,6(1):99-108
The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway. 相似文献
12.
Prachi Bhoskar Brett Belongia Robert Smith Seongkyu Yoon Tyler Carter Jin Xu 《Biotechnology progress》2013,29(5):1131-1139
Monoclonal antibodies (mAbs) are currently the dominant class of biopharmaceuticals. Due to the high dosage requirements of most mAb therapeutics, high productivity and low aggregation are prevailing criteria during cell line generation and process development. Given that light chains (LCs) play an important role in antibody folding and assembly, and that most mAb producing cell lines also manufacture free LCs, we sought to investigate whether there was a relationship between free LC levels in cell culture media and mAb productivity/quality. To this end, a series of analytical methods were developed in order to quantify free LC content in cell culture media and assess mAb productivity and aggregation levels. Afterwards, conditioned media samples from different cell lines at identical culturing conditions and a single clone under varying culturing conditions were analyzed. Higher LC expression was found to correlate with higher cell viability, higher mAb productivity, and lower aggregation. While LC expression cannot yet be definitively considered the root cause of these phenomena, these results are consistent with the role of LCs in mAb production, suggesting that free LC expression levels may potentially serve as a parameter for cell line generation and cell culture process optimization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1131–1139, 2013 相似文献
13.
We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass IgG1, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide. mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72.11 is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72.11 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column. 相似文献
14.
Connors LH Jiang Y Budnik M Théberge R Prokaeva T Bodi KL Seldin DC Costello CE Skinner M 《Biochemistry》2007,46(49):14259-14271
Immunoglobulin light chain amyloidosis is a protein misfolding disease in which a monoclonal immunoglobulin (Ig) light chain (LC) with a critically folded beta-conformation self-aggregates to form highly ordered, nonbranching amyloid fibrils. The insoluble nature of amyloid fibrils ultimately results in the extracellular deposition of the LC in tissues and organs throughout the body. Structural features that confer amyloidogenic properties on an Ig LC likely include amino acid sequence variations and post-translational modifications, but the specific natures of these changes remain to be defined. As part of an exploration of the effective range of amyloidogenic modifications, this study details the structural and genetic analyses of nine kappa1 LC proteins. Urinary LCs were purified by size exclusion chromatography using FPLC, and structural analyses were performed by electrospray ionization, matrix-assisted laser desorption/ionization, and tandem mass spectrometry. RT-PCR amplification, cloning, and sequencing of the monoclonal LC genes were accomplished using bone marrow-derived mRNA. Clinical data were reviewed retrospectively. Characterization of the urinary kappa1 LCs revealed extensive post-translational modification in all proteins, in addition to somatic mutations expected on the basis of results from genetic analyses. Post-translational modifications included disulfide-linked dimerization, S-cysteinylation, glycosylation, fragmentation, S-sulfonation, and 3-chlorotyrosine formation. Genetic analyses showed that several LC variable region germline gene donors were represented including O18/O8, O12/O2, L15, and L5. Clinical features included soft tissue, cardiac, renal, and hepatic involvement. This study demonstrated the extensive heterogeneity in primary structure, post-translational modifications, and germline gene usage that occurred in nine amyloidogenic kappa1 LC proteins. 相似文献
15.
Fukunaga A Nagai H Noguchi T Okazawa H Matozaki T Yu X Lagenaur CF Honma N Ichihashi M Kasuga M Nishigori C Horikawa T 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(7):4091-4099
Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-alpha-induced migration of LCs. The emigration of dendritic cells expressing I-A(b+) from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3beta was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration. 相似文献
16.
Naperova IA Balyasnikova IV Schwartz DE Watermeyer J Sturrock ED Kost OA Danilov SM 《Journal of proteome research》2008,7(8):3396-3411
Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology. 相似文献
17.
A monoclonal antibody (mAb), designated 15D8, was produced from BALB/c splenocytes of mice injected with Escherichia coli flagella. ELISA of motile cells, non-motile cells and partially purified flagellin proteins showed that the mAb reacted specifically with flagella of E. coli and with other members of the family Enterobacteriaceae. Western immunoblot analyses of enterobacterial flagella or cell extracts demonstrated that the antibody reacted with a single protein species in the extracts which was identical in size to purified flagellin. The antigenic determinant for this antibody appears to be surface exposed and linear in configuration, since the antibody reacted with native flagella and flagella which had been denatured. This antibody was also used to demonstrate that although the flagella proteins are heterogeneous in size, at least one epitope is highly conserved. 相似文献
18.
P Thullier P Lafaye F Mégret V Deubel A Jouan J C Mazié 《Journal of biotechnology》1999,69(2-3):183-190
A recombinant Fab that recognizes a neutralizing epitope located in the (296-400) region of protein E of dengue virus was obtained from cloned hybridoma cells secreting the mouse monoclonal antibody (mAb) 4E11. The Fd and light chain antibody genes were amplified by polymerase chain reaction, cloned into the phagemid vector pMad, expressed in bacteria to produce Fab fragments and sequenced. The mAb 4E11, in particular its light chain complementary-determining regions, shared homologies with two other anti-viral mAbs. The affinity of the parental mAb and the cloned Fab to the MalE-E(296-400) fusion protein were shown to be of the same magnitude, i.e. nanomolar. Fab 4E11 neutralization capacity was found between 8 and 4-times or less lower than that of mAb 4E11, depending on serotypes, thus the Fab could have a smaller antiviral activity than the mAb in vitro. 相似文献
19.
G C Spagnoli C Ausiello C Palma G Bellone G Ippoliti M Letarte F Malavasi 《Cellular immunology》1991,136(1):208-218
The 4F2 antigenic complex is expressed on most human cell lines in culture, on monocytes and activated lymphocytes, but not on resting T and B lymphocytes. Monoclonal antibody (mAb) CB43 recognizes an epitope of the 4F2 heterodimer either located on the light chain or dependent on the conformation of the molecule. The binding of CB43 mAb to peripheral blood mononuclear cells (PBMC) induced a dose-dependent comitogenic effect in the presence of submitogenic concentrations of anti-CD3 mAb. Significant amounts of interleukin (IL)-1 beta but not IL-2 or interferon-gamma were released in the supernatant. Pretreatment of monocytes with CB43 mAb increased the phytohemagglutinin-induced T lymphocyte proliferation. However, CB43 mAb did not exert agonistic effects on activated T lymphocytes. Depletion of CB43+ cells from PBMC decreased the proliferation and generation of cytotoxic effector cells induced by a mannoprotein (MP) derived from Candida albicans cell wall but not by recombinant IL-2. Furthermore, depletion of CB43+ cells from PBMC preactivated with MP or rIL-2 led to a significant decrease in their cytotoxic activity. CB43 mAb did not inhibit the growth of cell lines nor the proliferation of T cells. Thus CB43 mAb identifies a distinct functional epitope on the 4F2 molecular complex and might be useful in further studying the role of this molecule in cellular activation. 相似文献
20.
E Puré M D Witmer J B Lum I Mellman J C Unkeless 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(12):4152-4158
The murine macrophage and lymphocyte Fc receptor for aggregated IgG (Fc gamma R) has previously been characterized by using the anti-Fc gamma R monoclonal antibody (mAb), 2.4G2. In the studies presented here, we describe a new mAb, 6B7C, that defines a second epitope of the Fc gamma R. The tissue distribution of the 6B7C epitope is coincident with the 2.4G2 epitope. However, only the 2.4G2 epitope is accessible to mAb binding on intact primary macrophages or lymphocytes. The 6B7C epitope is not detectable on primary macrophages or lymphocytes but is exposed on a portion of B lymphocyte Fc gamma R after activation by lipopolysaccharide and on some tumor cell lines. The expression of the 6B7C epitope on the surface of B lymphoblasts and tumor cell lines seems to correlate with their ability to release soluble Fc gamma R. The 6B7C mAb has the advantage that it reacts with native as well as denatured receptor and therefore can be used for techniques such as immunoblotting. 相似文献