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.

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 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 Binding characteristics of antibodies to the TSH receptor

1998 ◽  
Vol 20 (2) ◽  
pp. 233-244 ◽  
Author(s):  
Y Oda ◽  
J Sanders ◽  
S Roberts ◽  
M Maruyama ◽  
R Kato ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Tsh Receptor ◽  
Affinity Constant ◽  
Translation System ◽  
Scatchard Analysis ◽  
Facs Analysis ◽  
Binding Characteristics ◽  
Fab Fragments

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

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.

scholarly journals Cytoplasmic and Nuclear Phospholipase C-β1 Relocation: Role in Resumption of Meiosis in the Mouse Oocyte

2000 ◽  
Vol 11 (12) ◽  
pp. 4369-4380 ◽  
Author(s):  
Nathalie Avazeri ◽  
Anne-Marie Courtot ◽  
Arlette Pesty ◽  
Clotilde Duquenne ◽  
Brigitte Lefèvre
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Electron Microscope ◽  
Phospholipase C ◽  
Western Blotting ◽  
Subcellular Distribution ◽  
Germinal Vesicle ◽  
Confocal Microscope ◽  
Mouse Oocyte

The location of the phospholipase C β1-isoform (PLC-β1) in the mouse oocyte and its role in the resumption of meiosis were examined. We used specific monoclonal antibodies to monitor the in vitro dynamics of the subcellular distribution of the enzyme from the release of the oocyte from the follicle until breakdown of the germinal vesicle (GVBD) by Western blotting, electron microscope immunohistochemistry, and confocal microscope immunofluorescence. PLC-β1 became relocated to the oocyte cortex and the nucleoplasm during the G2/M transition, mainly in the hour preceding GVBD. The enzyme was a 150-kDa protein, corresponding to PLC-β1a. Its synthesis in the cytoplasm increased during this period, and it accumulated in the nucleoplasm. GVBD was dramatically inhibited by the microinjection of anti-PLC-β1 monoclonal antibody into the germinal vesicle (GV) only when this accumulation was at its maximum. In contrast, PLC-γ1 was absent from the GV from the time of release from the follicle until 1 h later, and microinjection of anti-PLC-γ1 into the GV did not affect GVBD. Our results demonstrate a relationship between the relocation of PLC-β1 and its role in the first step of meiosis.

scholarly journals Production of Recombinant Antigens of Ureaplasma parvum Serotypes 3 and 6 for Development of a Serological Assay

2007 ◽  
Vol 15 (3) ◽  
pp. 447-451 ◽  
Author(s):  
E. Vancutsem ◽  
F. Echahidi ◽  
K. Van Geel ◽  
G. Muyldermans ◽  
O. Soetens ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Western Blotting ◽  
Cross Reaction ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Recombinant Antigens ◽  
Serological Assay ◽  
Ureaplasma Parvum ◽  
Human Sera

ABSTRACT Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serotypes 3 and 6. The antigens tested positive with serotype-specific monoclonal antibodies in Western blotting and in ELISA. Prominent reactions were detected with the rMBAs and their homologous monoclonal antibodies. Strong cross-reactions were visible in ELISA between rMBA 3 and the monoclonal antibodies from the other U. parvum serotypes. A weak cross-reaction was seen with rMBA 3 and the monoclonal antibody from serotype 4. rMBA 6 showed cross-reaction only with the monoclonal antibody from U. parvum serotype 1. Fifty-one percent of the sera obtained from culture-positive women reacted with one or both rMBAs. Only three (15%) of the sera from culture-negative women reacted with the rMBA. The positive reactions were observed only with rMBA 6. These preliminary tests showed the potential usefulness of the rMBAs produced for detecting an antibody response against Ureaplasma antigens.

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

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