scholarly journals Topography of N-CAM structural and functional determinants. II. Placement of monoclonal antibody epitopes.

1986 ◽  
Vol 103 (5) ◽  
pp. 1729-1737 ◽  
Author(s):  
A L Frelinger ◽  
U Rutishauser
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Biological Properties ◽  
Peptide Chain ◽  
Functional Domains ◽  
Peptide Fragments ◽  
Antigenic Sites ◽  
Cell Biol ◽  
Antibody Epitopes

The accompanying report (Watanabe, M., A. L. Frelinger III, and U. Rutishauser, 1986, J. Cell Biol., 103:1721-1727) describes a set of monoclonal antibodies (mAbs) directed against N-CAM epitopes representing the known major structural and functional domains of the molecule. In this study, we have generated and separated a variety of peptide fragments from N-CAM, and then used their size and reactivity with each antibody to position the antigenic sites along the peptide chain. This epitope map, together with the biological properties of the antibodies and previous studies on N-CAM, have been used to construct a topographical model for the molecule in the cell membrane.

scholarly journals Development of a New Highly Selective Monoclonal Antibody against Preferentially Expressed Antigen in Melanoma (PRAME) and Identification of the Target Epitope by Bio-Layer Interferometry

2021 ◽  
Vol 22 (6) ◽  
pp. 3166
Author(s):  
Jwala Priyadarsini Sivaccumar ◽  
Antonio Leonardi ◽  
Emanuela Iaccarino ◽  
Giusy Corvino ◽  
Luca Sanguigno ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cancer Biomarkers ◽  
Trypsin Digestion ◽  
Protein Fragments ◽  
Target Epitope ◽  
Potential Activity ◽  
Antibody Epitopes

Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.

Changes in expression of monoclonal antibody epitopes on laminin-5r induced by cell contact

1996 ◽  
Vol 109 (7) ◽  
pp. 1965-1973 ◽  
Author(s):  
G. Plopper ◽  
J. Falk-Marzillier ◽  
S. Glaser ◽  
M. Fitchmun ◽  
G. Giannelli ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Epithelial Cell ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Membrane Component ◽  
Human Epithelial Cell ◽  
Matrix Assembly ◽  
Basement Membrane Component ◽  
Antibody Epitopes

Laminin-5r is a basement membrane component that promotes rapid adhesion and hemidesmosome formation in epithelial cells. We raised monoclonal antibodies and identified their corresponding epitopes on the constituent chains of laminin-5r by western blotting. Using a combination of immunoprecipitation and ELISA assays, we determined that these epitopes are differentially exposed on two forms of the laminin-5r heterotrimer: soluble (passively adsorbed onto plastic) and cell-associated. Antibody 5C5 epitope is exposed on the cell-associated form, but not the soluble/passively adsorbed form of laminin-5r. Epitopes reactive with antibodies CM6, FM3, and TR1 are also preferentially exposed on cell-associated laminin-5r, such that reactivity of these antibodies with the cell-associated form is fourfold higher than with the soluble/passively adsorbed form in ELISA assays. Incubation of passively adsorbed laminin-5r with the human epithelial cell line SCC12 induced exposure of 5C5 and CM6, FM3, or TR1 epitopes. These data suggest that cells actively modify laminin-5r, perhaps during matrix assembly, and that the 5C5 epitope may serve as a marker for assembled laminin-5r matrix.

A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components

1993 ◽  
Vol 104 (2) ◽  
pp. 391-398
Author(s):  
A. Koutoulis ◽  
M. Ludwig ◽  
R. Wetherbee
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Endomembrane System ◽  
Thin Sections ◽  
Specific Location ◽  
Whole Cells

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.

scholarly journals Identification of an immunodominant functional domain on human CD36 antigen using human-mouse chimaeric proteins and homologue-replacement mutagenesis

1995 ◽  
Vol 305 (1) ◽  
pp. 221-224 ◽  
Author(s):  
L Daviet ◽  
R Buckland ◽  
M D Puente Navazo ◽  
J L McGregor
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Functional Domain ◽  
Oxidized Low Density Lipoprotein ◽  
Key Residues ◽  
Antibody Epitopes ◽  
Human And Mouse

The human CD36 antigen is a multifunctional membrane glycoprotein that acts as a receptor for thrombospondin, malaria-infected erythrocytes and oxidized low-density lipoprotein, as well as being implicated in the recognition of apoptotic neutrophils by macrophages. OKM5 and other anti-CD36 monoclonal antibodies have been shown to inhibit these CD36 adhesive functions, suggesting that the monoclonal-antibody epitopes and the domains that mediate these events are closely related. Analysis of a series of chimaeric exchanges between human and mouse CD36 shows that six anti-CD36 monoclonal antibodies (OKM5, FA6-152, L103, 5F1, SM phi and 10/5) recognize epitopes within the domain comprising amino acids 155-183. A seventh monoclonal antibody (13/10) binds to another domain that spans amino acids 30-76. Homologue-replacement mutagenesis performed within the human 155-183 immunodominant sequence identifies key residues for the binding of three functional monoclonal antibodies (OKM5, FA6-152 and L103). The fact that antibodies directed against the 155-183 domain can inhibit adhesion suggests that this domain is directly involved in CD36-ligand binding.

scholarly journals Anti-Lipid A Monoclonal Antibody Centoxin (HA-1A) Binds to a Wide Variety of Hydrophobic Ligands

1998 ◽  
Vol 66 (2) ◽  
pp. 870-873 ◽  
Author(s):  
E. J. Helmerhorst ◽  
J. J. Maaskant ◽  
B. J. Appelmelk
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Bovine Serum Albumin ◽  
Gel Electrophoresis ◽  
Lipid A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Properties ◽  
Antibody Epitopes

ABSTRACT This note describes the binding specificities of four lipid A monoclonal antibodies (MAbs) including Centoxin (HA-1A); these MAbs display similar binding properties. MAbs reacted with lipid A and heat-killed smooth bacteria, whereas no reactivity was observed with smooth lipopolysaccharide (LPS). Immunoblotting of bacterial extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the MAbs bound to many polypeptide bands including the molecular weight markers. Denaturation of bovine serum albumin (BSA) by boiling or dithiothreitol treatment unmasked antibody epitopes. In addition, binding both to a hydrophobic aliphatic C12 chain covalently coupled to BSA and to single-stranded DNA was observed. The polyreactivity of these clones is most likely mediated by a preferential reactivity with hydrophobic molecular patches.

scholarly journals Characterizing monoclonal antibody epitopes by filtered gene fragment phage display

2005 ◽  
Vol 388 (3) ◽  
pp. 889-894 ◽  
Author(s):  
Roberto DI NIRO ◽  
Fortunato FERRARA ◽  
Tarcisio NOT ◽  
Andrew R. M. BRADBURY ◽  
Fernando CHIRDO ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Antibiotic Resistance ◽  
Monoclonal Antibodies ◽  
Phage Display ◽  
Resistance Gene ◽  
Protein Interactions ◽  
Antibiotic Resistance Gene ◽  
Transglutaminase 2 ◽  
Phage Display Library ◽  
Antibody Epitopes

In the present paper, we describe a novel approach to map monoclonal antibody epitopes, using three new monoclonal antibodies that recognize h-TG2 (human transglutaminase 2) as an example. The target gene was fragmented and cloned upstream of an antibiotic-resistance gene, in the vector pPAO2, to select for in-frame polypeptides. After removal of the antibiotic-resistance gene by Cre/Lox recombination, an antigen fragment phage display library was created and selected against specific monoclonal antibodies. Using the h-TG2 fragment library, we were able to identify epitopes. This technique can also be broadly applied to the study of protein–protein interactions.

Characterization of monoclonal antibodies to cytochrome c: analysis of the antigenic structure of holo- and apo-cytochromes, and CNBr-peptide fragments of horse cytochrome c

1987 ◽  
Vol 65 (9) ◽  
pp. 783-789 ◽  
Author(s):  
In Cheol Kim ◽  
Hector Nolla ◽  
Suzette Priola
Keyword(s):  
Monoclonal Antibodies ◽  
Cytochrome C ◽  
Antigenic Site ◽  
Cross Reactivity ◽  
Peptide Fragmentation ◽  
Antigenic Specificity ◽  
Antigenic Structure ◽  
Peptide Fragments ◽  
Antigenic Sites ◽  
Hybridoma Cell Lines

Five mouse hybridoma cell lines secreting SA, SB, SC, SD, and SE monoclonal antibodies (McAb) to cytochrome c have been produced. From the cross-reactivities of these McAb with various vertebrate cytochromes c, the antigenic sites for SA and SB McAb were proposed to be at Thr(89)-Glu(92)-Ala(96) and Asn(103), respectively. The binding site for other McAb have not been determined. Cross-reactivity studies based on enzyme-linked immunosorbent assays and dot immunobinding assays indicated that SA, SB, and SC McAb did not bind to apo-cytochrome c nor to any of the three CNBr-peptide fragments. This observation suggests that (i) the antigenic specificity of these McAb is dependent on the conformatiuon of the antigenic site which is inherent to the native holoprotein molecule and (ii) the ordered conformation in the C-terminal regions of holo-cytochrome c is destroyed during CNBr-peptide fragmentation. On the other hand, the lack of binding of SD and SE McAb to apo-cytochrome c indicates that these McAb are also specific for conformational sites. The binding of SD and SE McAb to the heme-containing A-peptide fragment (residues 1–65) suggests that the conformation around the heme, as possible antigenic sites, are stable because of the thioether linkages by the Cys residues.

Two-monoclonal-antibody sandwich-type assay for thyrotropin, with use of an avidin-biotin separation technique.

1986 ◽  
Vol 32 (10) ◽  
pp. 1873-1878 ◽  
Author(s):  
W D Odell ◽  
J Griffin ◽  
R Zahradnik
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
The Other ◽  
Separation Technique ◽  
Simple Method ◽  
Polystyrene Beads ◽  
High Affinity ◽  
Antigenic Sites ◽  
Free Antibody ◽  
Sandwich Type

Abstract We have developed a sensitive, specific, noncompetitive, sandwich-type radioimmunoassay for human thyrotropin (hTSH), which can be performed in 30 min. The assay involves two monoclonal antibodies, selected for high affinity and specificity and also for reaction against antigenic sites on hTSH that are distal from each other. One of these antibodies is labeled with 125I; the other is conjugated covalently to biotin. Polystyrene beads were also conjugated covalently to biotin. After conjugation, the beads were incubated with avidin. These beads represent a rapid, simple method for separating hTSH-bound antibody from free antibody. The biotin-antibody-hTSH-125I-labeled antibody complexes bind to the beads and hTSH concentration is directly related to counts per minute. This assay can detect hTSH at a concentration of 0.06 milli-unit/L in serum.

scholarly journals Mapping two functional domains of clathrin light chains with monoclonal antibodies

1987 ◽  
Vol 104 (4) ◽  
pp. 897-903 ◽  
Author(s):  
DS Kohtz ◽  
V Georgieva-Hanson ◽  
JD Kohtz ◽  
WJ Schook ◽  
S Puszkin
Keyword(s):  
Monoclonal Antibodies ◽  
Heavy Chain ◽  
Casein Kinase ◽  
Light Chains ◽  
Functional Domains ◽  
Peptide Fragments ◽  
Structural Domains ◽  
Associated Proteins ◽  
Accessible Domain ◽  
Lower Form

The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.

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