Specifications
Species Reactivity
Bacteriophage
Immunogen
M13 Bacteriophage
Applications
Application Notes
ELISA 0.05-0.1 ug/ml
Citations
Publication ()
Have you cited CAB-655M in a publication?
Let us know and earn a reward for your research.
A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders
Gao X, Kaluarachchi H, Zhang Y, Hwang S, Hannoush RN.
Applications: ELISA
Reactive species: Bacteriophage
"Abstract: Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance."
Article snippet: The plates were washed and incubate with anti-M13 antibody (1:10,000, HRP conjugated, Creative Diagnostics, cat# CAB-655M) for 30 min at room temperature, followed by washing, development and quenching (1 M H3PO4). The absorbance at 450 nm was recorded to calculate the IC50 values. 
Figure 1. Phage competition ELISA shows synthetic DCPs compete with DCPs displayed on phage surface for binding against human IgG Fc or serum albumin.
Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies
Krohn S, Holtrop T, Brandsma AM, Moerer P, Nederend M, Darzentas N, Brüggemann M, Klausz K, Leusen JHW, Peipp M
Applications: ELISA
Reactive species: M13 Bacteriophage
"Abstract: Antibodies that specifically bind to individual human fragment crystallizable γ receptors (FcγRs) are of interest as research tools in studying immune cell functions, as well as components in bispecific antibodies for immune cell engagement in cancer therapy. Monoclonal antibodies for human low-affinity FcγRs have been successfully generated by hybridoma technology and are widely used in pre-clinical research. However, the generation of monoclonal antibodies by hybridoma technology that specifically bind to the high-affinity receptor FcγRI is challenging. Monomeric mouse IgG2a, IgG2b, and IgG3 bind human FcγRI with high affinity via the Fc part, leading to an Fc-mediated rather than a fragment for antigen binding (Fab)-mediated selection of monoclonal antibodies. Blocking the Fc-binding site of FcγRI with an excess of human IgG or Fc during screening decreases the risk of Fc-mediated interactions but can also block the potential epitopes of new antibody candidates. Therefore, we replaced hybridoma technology with phage display of a single-chain fragment variable (scFv) antibody library that was generated from mice immunized with FcγRI-positive cells and screened it with a cellular panning approach assisted by next-generation sequencing (NGS). Seven new FcγRI-specific antibody sequences were selected with this methodology, which were produced as Fc-silent antibodies showing FcγRI-restricted specificity."
Article snippet: A 100 µL volume of bacterial supernatant containing monoclonal phage antibodies was added to the cells and incubated for 1 h on ice. Plates were washed three times with cold PBS supplemented with 0.1% BSA (680× g, 5 min, 4 °C). For phage detection, cells were incubated with mouse anti-M13 HRP-conjugated IgG1 antibody (Creative Diagnostics, Shirley, NY, USA; diluted 1:2000 in PBS supplemented with 4% BSA) for 1 h on ice. 
Figure 1. FcγRI-specific scFvs were enriched and selected from an antibody library by phage display.
Resurfaced ZIKV EDIII nanoparticle immunogens elicit neutralizing and protective responses in vivo
Georgiev GI, Malonis RJ, Wirchnianski AS, Wessel AW, Jung HS, Cahill SM, Nyakatura EK, Vergnolle O, Dowd KA, Cowburn D, Pierson TC, Diamond MS, Lai JR
Applications: Unspecified application
Reactive species: M13 Bacteriophage
"Abstract: Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop "resurfaced" (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV."
Article snippet: Horseradish peroxidase-conjugated anti-M13 antibody;Creative Diagnostics; Cat# CAB-655M 
Figure 1. Isolation of resurfaced ZIKV-EDIII variants (rsZDIIIs) by phage display
Evidence for deleterious effects of immunological history in SARS-CoV-2
Sanjana R Sen, Emily C Sanders, Alicia M Santos, Keertna Bhuvan, Derek Y Tang, Aidan A Gelston, Brian M Miller, Joni L Ricks-Oddie, Gregory A Weiss
Applications: ELISA
Reactive species: Human
"Abstract: A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous putative antigen, a sequence derived from the neuraminidase protein of H3N2 influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIN from a previous infection could underlie some cases of COVID-19 disease severity."
Article snippet: The peroxidase-conjugated detection antibody, αM13-HRP (Creative Diagnostics, Shirley, NY), was diluted 1,000-fold in Chonblock Secondary Antibody Dilution (*) buffer; 100 μl of this solution was added to each well before incubation for 30 min at RT with shaking at 150 rpm. 
Figure 1. ELISA examined binding of phage-displayed potential OAS epitopes to total Ig from three sets of pooled plasma from five αEp9(+) patients, or five αEp9(-) patients
Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display
Steffen Krohn, Ammelie Svea Boje, Carina Lynn Gehlert, Sebastian Lutz, Nikos Darzentas, Henrik Knecht, Dietrich Herrmann, Monika Brüggemann, Axel J Scheidig, Katja Weisel, Martin Gramatzki, Matthias Peipp, Katja Klausz
Applications: ELISA
Reactive species: Bacteriophage
"Abstract: To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies' enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified."
Article snippet: Plates were washed 3-times with cold PBS supplemented with 0.1% BSA and subsequently incubated for 1 h at 4°C with anti-M13-HRP antibody (Creative Diagnostics; cat. no. CAB-655M; diluted 1:2000 in PBS supplemented with 4% BSA). 
Figure 1. The 9 most promising candidates were further tested in whole cell ELISA experiments
Selective and Potent Peptide Binders of RNF43 for Wnt Signaling Inhibition
Hwang S, Salcedo PF, Korcari A, Nicoludis JM, Valdivia EM, Peng L, Balana AT, Mak J, Crittenden CM, Famili A, Liu P, Castillo-Azofeifa D, Hannoush RN, Miller SE, Schroeder CI, Gao X.
Applications: DCP Phage Library
Reactive species: M13
"Abstract: The Wnt/β-catenin pathway is critical in human tumor progression. Cell-surface transmembrane E3 ubiquitin ligase ring finger 43 negatively regulates Wnt signaling through ubiquitination of Wnt coreceptor Frizzled. Aberrant Wnt signaling through inactivating mutations of RNF43 has been identified in various forms of cancers, highlighting its significance in tumor biology. However, the precise mechanism underlying the function of RNF43 remains elusive, largely due to the absence of selective molecular tools allowing for detection or manipulation of endogenous RNF43. Here we present a series of disulfide-constrained peptides, including GUR-1.6.12.2, which exhibit high affinity and specificity against RNF43. GUR-1.6.12.2 can be used as a valuable research tool to delineate RNF43 activity in various contexts. We showcased its application in immunofluorescence, where RNF43 was detected in intestinal crypts using biotinylated GUR-1.6.12.2. We then combined experimental and computational structural approaches to propose a model of GUR-1.6.12.2 and its binding to RNF43. Importantly, we generated a functional RNF43-DCP by producing a hexavalent GUR-1.6.12.2 molecule, which exhibited inhibitory activity against Wnt signaling in cells by competing with R-spondin, a RNF43 ligand that potentiates signaling. The RNF43 binders presented here offer new opportunities for the research and development of anticancer therapies targeting Wnt signaling with improved selectivity."
Article snippet: The plate was then washed and incubated with a horseradish peroxidase (HRP)-conjugated anti-M13 antibody (CAB-655M, Creative Diagnostics) for 30 min at RT, followed by development and quenching with 1 M H3PO4. 
Figure 1. Identification and affinity maturation of DCPs against human RNF43.
COA
Loading ......
