scholarly journals Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display

2021 ◽  
Vol 11 (17) ◽  
pp. 7910
Author(s):  
Laura M. De Plano ◽  
Domenico Franco ◽  
Martina Bonsignore ◽  
Enza Fazio ◽  
Sebastiano Trusso ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Phage Display ◽  
Plasma Cells ◽  
Fluorescence Probe ◽  
Residual Disease ◽  
Ex Vivo ◽  
Disease Status ◽  
Phage Display Library ◽  
Amino Acid Sequences ◽  
Clusters Of Differentiation

Multiple myeloma (MM) is a malignant disease based on differentiated plasma cells (PCs) in the bone marrow (BM). Flow cytometry and fluorescence microscopy, used to identify a large combination of clusters of differentiation (CDs), are applied for MM immunophenotyping. However, due to the heterogeneous MM immunophenotypes, more antibody panels are necessary for a preliminary diagnosis and for the monitoring of minimal residual disease (MRD). In this study, we evaluated the use of phage clones as probes for the identification of several PCs immunophenotypes from MM patients. First, A 9-mer M13-pVIII phage display library was screened against an MM.1 cells line to identify peptides that selectively recognize MM.1 cells. Then, the most representative phage clones, with amino acid sequences of foreign peptides closer to the consensus, were labelled with isothiocyanate of fluorescein (FITC) and were used to obtain a fluorescent signal on cells in ex-vivo samples by fluorescence microscopy. Selected phage clones were able to discriminate different MM immunophenotypes from patients related to CD45, CD38, CD56, and CD138. Our results highlight the possibility of using a phage-fluorescence probe for the simultaneous examination of the presence/absence of CDs associated with disease usually detected by combination of anti-CD antibodies. The design of a multi-phage imaging panel could represent a highly sensitive approach for the rapid detection of immunophenotype subtypes and the subsequent characterization of patient disease status.

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy

2004 ◽  
Vol 295 (1-2) ◽  
pp. 119-127 ◽  
Author(s):  
David L. Jaye ◽  
Cissy M. Geigerman ◽  
Ross E. Fuller ◽  
Adil Akyildiz ◽  
Charles A. Parkos
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Microscopy ◽  
Phage Display ◽  
Phage Display Library

scholarly journals Convergent Evolution of Neutralizing Antibodies to Staphylococcus aureus γ-Hemolysin C That Recognize an Immunodominant Primary Sequence-Dependent B-Cell Epitope

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
David N. Hernandez ◽  
Kayan Tam ◽  
Bo Shopsin ◽  
Emily E. Radke ◽  
Karen Law ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Phage Display ◽  
B Cell ◽  
Neutralizing Antibodies ◽  
Ex Vivo ◽  
Cell Epitope ◽  
Phage Display Library ◽  
Structural Basis ◽  
Peptide Fragments ◽  
Content Type

ABSTRACT Staphylococcus aureus infection is a major public health threat in part due to the spread of antibiotic resistance and repeated failures to develop a protective vaccine. Infection is associated with production of virulence factors that include exotoxins that attack host barriers and cellular defenses, such as the leukocidin (Luk) family of bicomponent pore-forming toxins. To investigate the structural basis of antibody-mediated functional inactivation of Luk toxins, we generated a panel of murine monoclonal antibodies (MAbs) that neutralize host cell killing by the γ-hemolysin HlgCB. By biopanning these MAbs against a phage-display library of random Luk peptide fragments, we identified a small subregion within the rim domain of HlgC as the epitope for all the MAbs. Within the native holotoxin, this subregion folds into a conserved β-hairpin structure, with exposed key residues, His252 and Tyr253, required for antibody binding. On the basis of the phage-display results and molecular modeling, a 15-amino-acid synthetic peptide representing the minimal epitope on HlgC (HlgC241-255) was designed, and preincubation with this peptide blocked antibody-mediated HIgCB neutralization. Immunization of mice with HlgC241-255 or the homologous LukS246-260 subregion peptide elicited serum antibodies that specifically recognized the native holotoxin subunits. Furthermore, serum IgG from patients who were convalescent for invasive S. aureus infection showed neutralization of HlgCB toxin activity ex vivo, which recognized the immunodominant HlgC241-255 peptide and was dependent on His252 and Tyr253 residues. We have thus validated an efficient, rapid, and scalable experimental workflow for identification of immunodominant and immunogenic leukotoxin-neutralizing B-cell epitopes that can be exploited for new S. aureus-protective vaccines and immunotherapies.

Sorting a Staphylococcus aureus phage display library against ex vivo biomaterial

2004 ◽  
Vol 53 (10) ◽  
pp. 945-951 ◽  
Author(s):  
Joakim Bjerketorp ◽  
Anna Rosander ◽  
Martin Nilsson ◽  
Karin Jacobsson ◽  
Lars Frykberg
Keyword(s):  
Staphylococcus Aureus ◽  
Phage Display ◽  
Ex Vivo ◽  
Phage Display Library

scholarly journals Canine CD117-Specific Antibodies with Diverse Binding Properties Isolated from a Phage Display Library Using Cell-Based Biopanning

Antibodies ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 15 ◽  
Author(s):  
Mohamed Alfaleh ◽  
Neetika Arora ◽  
Michael Yeh ◽  
Christopher de Bakker ◽  
Christopher Howard ◽  
...  
Keyword(s):  
Phage Display ◽  
Ex Vivo ◽  
Phage Display Library ◽  
Cross Reactivity ◽  
Tyrosine Kinase Receptor ◽  
Binding Assays ◽  
Comparative Oncology ◽  
Whole Cells ◽  
Exposed Region

CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.

scholarly journals Specific detection of myeloma plasma cells using anti-idiotypic single chain antibody fragments selected from a phage display library

Leukemia ◽  
1998 ◽  
Vol 12 (8) ◽  
pp. 1295-1302 ◽  
Author(s):  
PMW Willems ◽  
RMA Hoet ◽  
ELPG Huys ◽  
JMH Raats ◽  
EJBM Mensink ◽  
...  
Keyword(s):  
Phage Display ◽  
Plasma Cells ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Single Chain ◽  
Specific Detection ◽  
Single Chain Antibody

BCRs of Mutated and Unmutated CLL Bind Peptides with Distinct Sequence Features: Evidence from Phage Display Libraries.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1118-1118
Author(s):  
Manuela Woelfle ◽  
Till Seiler ◽  
Rosa Catera ◽  
Hartmut Dohner ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phage Display ◽  
Synthetic Peptides ◽  
Specific Binding ◽  
Phage Display Library ◽  
Amino Acid Sequences ◽  
Phage Display Technology ◽  
Ligand Interactions ◽  
Distinct Sequence ◽  
Peptide Phage Display

Abstract In CLL, the use of specific IgV genes to code the clone’s BCR is non-random and there is an apparent selection for particular genetic and amino acid structures that can be shared by different patients, supporting the hypothesis that antigenic stimulation influences the development and course of CLL. As the binding specificities of the BCR are largely unknown, a vast variety of antigens may affect the BCRs and defined antigens have yet to be identified. Therefore, we used peptide phage display technology to identify ligands for CLL BCRs. BCRs from 2 IgVH unmutated (U-CLL) and 3 mutated (M-CLL) patients were expressed as IgG1 mAbs and used to probe a 12-mer peptide phage display library. In each case, after 3 rounds of selection, we isolated ligands reactive with the CLL mAbs. For the 3 M-CLL mAbs, phage clones carrying peptide inserts with conserved consensus motifs were found. Specificity of the BCR-ligand interactions was demonstrated in direct and indirect ELISA, since selected phage clones and synthetic peptides bound to their respective M-CLL mAb but not to other M-CLL mAbs. Variation of the amino acid sequence of the synthetic peptides significantly altered their reactivity with the corresponding M-CLL mAb. Furthermore, synthetic peptides were bound only by the proper mAb/BCR, but not by mAbs of other M-CLL or U-CLL patients with BCRs comprised of different IgVH genes, supporting the hypothesis that BCRs of M-CLL recognize a defined epitope. In contrast, the mAbs from 2 U-CLL cases did not select phages bearing a consensus motif. Rather these U-CLL mAbs bound multiple phages expressing the same 12-mer peptides, although these differed in sequence between the two U-CLL cases tested. Furthermore, 2 separate selection procedures using 1 U-CLL mAb isolated multiple phage bearing the same 12-mer sequence on each occasion as well as another set of phages with a completely distinct sequence in 1 of 2 selections. ELISAs demonstrated specific binding of all phage clones and of the synthetic peptides by the U-CLL mAbs. Despite this level of specificity, the 2 U-CLL mAbs also reacted with peptides isolated from panning with other CLL mAbs, thereby displaying considerable polyreactivity. Rather than binding only one distinct epitope, mAbs from U-CLL appear capable of interacting with multiple, unrelated structures. Finally, one of the peptides isolated with an U-CLL mAb was bound by all of the CLL mAbs tested, including those from M-CLL cases; therefore this target is antigenically “polyreactive”. Thus, phage display is a feasible approach to identify specific ligands for CLL BCRs. The two classes of BCRs in M-CLL and U-CLL show substantially different binding properties - the former binding shared amino acid motifs and the latter binding multiple ligands of distinct and identical 12-mer amino acid sequences. These peptides can be used to analyze more precisely the binding sites of CLL BCRs as well as the consequences that ensue after BCR crosslinking, and they might help develop BCR-specific therapeutic agents.

scholarly journals Detection of Cystic Fibrosis Serological Biomarkers Using a T7 Phage Display Library

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Harvinder Talwar ◽  
Samer Najeeb Hanoudi ◽  
Andreea Geamanu ◽  
Dana Kissner ◽  
Sorin Draghici ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Phage Display ◽  
Phage Display Library ◽  
T7 Phage Display ◽  
Serological Biomarkers ◽  
T7 Phage

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage

Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library

1997 ◽  
Vol 270 (1) ◽  
pp. 26-35 ◽  
Author(s):  
Shane Atwell ◽  
John B.B Ridgway ◽  
James A Wells ◽  
Paul Carter
Keyword(s):  
Phage Display ◽  
Phage Display Library

scholarly journals Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

1994 ◽  
Vol 124 (3) ◽  
pp. 373-380 ◽  
Author(s):  
E Koivunen ◽  
B Wang ◽  
E Ruoslahti
Keyword(s):  
Phage Display ◽  
Cyclic Peptide ◽  
Cell Attachment ◽  
Phage Display Library ◽  
Cell Binding ◽  
Rgd Peptides ◽  
Beta 1 Integrin ◽  
Alpha V Beta 3 ◽  
Integrin Ligand ◽  
Specific Ligand

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.

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