Hrp conjugated anti m13 antibody

Manufactured by GE Healthcare

The HRP-conjugated anti-M13 antibody is a laboratory reagent used in various immunoassay techniques. It is a monoclonal antibody that specifically binds to the M13 bacteriophage, and is conjugated with the enzyme horseradish peroxidase (HRP). This antibody-enzyme conjugate can be utilized for the detection and quantification of M13 phage in samples.

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Lab products found in correlation

Human cord serum, by Lee Biosolutions (1 mentions) Spectramax 190 microplate reader, by Molecular Devices (1 mentions) Maxisorp immunotubes, by Thermo Fisher Scientific (1 mentions) T4 ligase, by Thermo Fisher Scientific (1 mentions) Taq dna ligase, by New England Biolabs (1 mentions) Hyperphage, by Progen Biotechnik (1 mentions) Anti flag mouse antibody, by Thermo Fisher Scientific (1 mentions) T5 exonuclease, by New England Biolabs (1 mentions) Trypsin, by Merck Group (1 mentions) Alkaline phosphatase, by Merck Group (1 mentions) Kod polymerase, by Merck Group (1 mentions) Restriction enzyme, by New England Biolabs (1 mentions) 2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions) Superblock buffer, by Thermo Fisher Scientific (1 mentions) 3 3 5 5 tetramethylbenzidine (tmb), by Promega (1 mentions)

Close spelling variants of this product

HRP-conjugated anti-M13 monoclonal antibody (14 citations) Anti-M13-HRP antibody (12 citations) Horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (7 citations) Anti-M13-HRP (6 citations) Horseradish peroxidase (HRP)-conjugated anti-M13 antibody (6 citations) Anti-M13 antibody (5 citations) HRP-conjugated anti-M13 (3 citations) Horseradish peroxidase (HRP)-conjugated mouse anti-M13 monoclonal antibody (3 citations)

7 protocols using hrp conjugated anti m13 antibody

Rabbit Antibody Phage Display for Anti-AFP ELISA

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In order to construct a highly sensitive sandwich ELISA with FasMab, an anti-AFP antibody was developed using a rabbit antibody phage display system. Rabbit antibody cDNAs were derived from the spleen cells of two New Zealand White rabbits immunized with AFP protein from human cord serum (Lee Biosolutions). Rabbit antigen binding fragment (Fab) libraries in the phagemid vector were generated from the cDNAs as described^{20 (link),21 (link)}. Phage clones specific to AFP were obtained through two round panning using AFP-immobilized plates. Phage ELISA was performed, using an ELISA plate coated with 200 ng AFP protein or 500 ng mouse IgG (as a negative control). E. coli culture supernatants or purified phages diluted with 2% skim milk were added to the plates and incubated at RT for 1 h. After the plates were washed with PBS-T, an HRP-conjugated anti-M13 antibody (GE Healthcare) was added to the plates and incubated at RT for 1 h. The enzymatic reaction was conducted using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and optical density was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices). The positive clones were subjected to sequence analysis.

Egashira Y., Suganuma M., Kataoka Y., Higa Y., Ide N., Morishita K., Kamada Y., Gu J., Fukagawa K, & Miyoshi E. (2019). Establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research. Scientific Reports, 9, 12359.

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High-Throughput VNAR Library Screening

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All clones were isolated from a synthetic VNAR library containing 100 billion unique clones. Solid-phase, phage display library antigen selections were carried out as detailed previously [49 (link)] using MaxiSorp immunotubes (Nunc, 444474) coated with 1–0.1 μg/ml antigen in PBS pH 7.4. Predecorated biotinylated antigen bead selection protocols were adopted from our previous work [42 (link)]. Outputs from each selection round were screened for antigen-specific binders by monoclonal phage and periplasmic extract ELISAs against human or mouse ICOSL and unrelated protein controls at 1 μg/ml in PBS coating concentration. Phage binders were detected using HRP-conjugated anti-M13 antibody (GE Healthcare, 27942101), and periplasmic protein was detected using HRP-conjugated to an anti-c-Myc antibody (Roche, 118 141 50 001).

O'Dwyer R., Kovaleva M., Zhang J., Steven J., Cummins E., Luxenberg D., Darmanin-Sheehan A., Carvalho M.F., Whitters M., Saunders K, & Barelle C.J. (2018). Anti-ICOSL New Antigen Receptor Domains Inhibit T Cell Proliferation and Reduce the Development of Inflammation in the Collagen-Induced Mouse Model of Rheumatoid Arthritis. Journal of Immunology Research, 2018, 4089459.

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Phage Display Peptide Screening

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Standard molecular biology techniques were used throughout. Biotinylated and fluorescein isothiocyanate peptide probes were purchased from CHI Scientific. Hyperphage were purchased from Progen Biotechnik GmbH. Neutraviden 96-well plates and T4 ligase were purchased from Thermo Scientific. KOD polymerase and anti-sulfotyrosine monoclonal antibody were purchased from EMD Millipore. Restriction enzymes, Taq DNA ligase and T5 exonuclease were purchased from New England Biolab. Trypsin, alkaline phosphatase and BSA-P were purchased from Sigma Aldrich. Potassium phenyl sulfate was purchased from Tokyo Chemical Industry. HRP conjugated anti-M13 antibody was purchased from GE Health Care Life Science. Anti-FLAG mouse antibody was purchased from Invitrogen. Anti-mouse goat antibody was purchased from BioRad. E. coli TOP10F’ were used for all cloning and phage propagation experiments. E. coli BL21(DE3) were used for all protein expression experiments. All solutions were prepared using deionized water passed through the Barnstead Nanopure ultrapure water filtration system. Standard antibiotic concentrations used were 100 μg/mL ampicillin, 50 μg/mL kanamycin and 5 μg/mL tetracycline.

Lawrie J., Waldrop S., Morozov A., Niu W, & Guo J. (2021). Engineering of a small protein scaffold to recognize sulfotyrosine with high specificity. ACS chemical biology, 16(8), 1508-1517.

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Phage ELISA Screening for Anti-MET Antibodies

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Phage ELISA was performed as previously described (28 ) to screen for binding clones. Briefly, antibody-displaying phage were expressed from 93 clones from the 3rd round of selection to detect human MET protein immobilized on a 96-well. HRP conjugated anti-M13 antibody (GE Healthcare, Cat#: 27-9421-01) was used to detect the bound phage. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma, A9941) was used as the HRP substrate. The 64 clones with the highest positive signals (A₄₀₅) were subjected to DNA sequencing.
To determine cross reactivity to mouse MET protein, purified anti-MET cys-diabodies were used to coat a ELISA plate (anti-EpCAM cys-diabody or 2% milk-PBS were used for negative controls). 0.1 μg human or mouse MET-hIgG Fc fusion protein (Sino Biological Inc.) was then applied to each well. The captured human or mouse MET-hIgG Fc were then detected with alkaline phosphatase conjugated goat anti-human IgG. Each combination was done in triplicate.

Li K., Tavaré R., Zettlitz K.A., Mumenthaler S.M., Mallick P., Zhou Y., Marks J.D, & Wu A.M. (2014). Anti-MET immunoPET for non-small cell lung cancer using novel fully human antibody fragments. Molecular cancer therapeutics, 13(11), 2607-2617.

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LASV GPC Trimer Binding Assay

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A 96-well plate (Corning) was coated with 50 μl/well of 5 μg/ml stabilized LASV GPC trimer in PBS buffer at 4 °C overnight. After blocking with 3% milk in 100% superblock buffer (Thermo Scientific) at room temperature for 2 h, 50 μl phage were added to the plate and incubated at room temperature for another hour. Binding of phage to the stabilized LASV GPC was detected by HRP-conjugated anti-M13 antibody (GE Healthcare). The cut-off value for positive binder was set as 3 × higher signal of antigen binding compared to background signal.

Gorman J., Cheung C.S., Duan Z., Ou L., Wang M., Chen X., Cheng C., Biju A., Sun Y., Wang P., Yang Y., Zhang B., Boyington J.C., Bylund T., Charaf S., Chen S.J., Du H., Henry A.R., Liu T., Sarfo E.K., Schramm C.A., Shen C.H., Stephens T., Teng I.T., Todd J.P., Tsybovsky Y., Verardi R., Wang D., Wang S., Wang Z., Zheng C.Y., Zhou T., Douek D.C., Mascola J.R., Ho D.D., Ho M, & Kwong P.D. (2024). Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability. Nature Communications, 15, 285.

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Phage Display Screening Optimization

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The plate was coated with 50 μg/ml mAbs. After washing and blocking, the amplified phages were added, and incubated for 1 hour at RT. After washing, diluted HRP-conjugated anti-M13 antibody (GE Healthcare) was added at RT for 1 hour. The plates were developed, and subsequently terminated by 3N HCl. The OD was measured at 490 nm.

Tang C.T., Li P.C., Liu I.J., Liao M.Y., Chiu C.Y., Chao D.Y, & Wu H.C. (2015). An Epitope-Substituted DNA Vaccine Improves Safety and Immunogenicity against Dengue Virus Type 2. PLoS Neglected Tropical Diseases, 9(7), e0003903.

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Screening Phages for hCEC Specificity

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To monitor efficient subtraction of fibroblast-specific and enrichment of hCEC-specific phages, polyclonal phage libraries before and after subtraction and/or panning round were collected and tested on fibroblasts and hCECs. Cells were grown to 90% confluency in FnC-coated 96 well plates, fixed with 10% formaldehyde and blocked with 400 μl 3% BSA per well. Hyperphage or KM13 helper phage were included as negative control. Bound phages were detected by HRP-conjugated anti-M13 antibody (GE Healthcare) and the assay was developed by addition of TMB (Promega). Reaction was stopped by the addition of H₂SO₄ and reading taken at OD_450nm.
To screen for hCEC-specific clones, soluble monoclonal scFv was expressed directly from the pIT2 vector. Well-isolated colonies were inoculated o/n at 37 °C in 2 × TY, ampicillin, 1% glucose. A vector only clone was used as negative control. The overnight cultures were mixed 1:6 with 2 × TY, ampicillin, 1% glucose and incubated for 4–5 h at 37 °C. Medium was exchanged with 2 × TY, ampicillin, 0.4 M sucrose, 1 mM IPTG, and cultures were incubated o/n at 30 °C and pelleted. 100 μl of the supernatant was tested in parallel on cultivated hCEC and fibroblast as described for the phage ELISA, except that the detection was done by a HRP-conjugated c-myc specific antibody (Pierce).

Dorfmueller S., Tan H.C., Ngoh Z.X., Toh K.Y., Peh G., Ang H.P., Seah X.Y., Chin A., Choo A., Mehta J.S, & Sun W. (2016). Isolation of a recombinant antibody specific for a surface marker of the corneal endothelium by phage display. Scientific Reports, 6, 21661.

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