Denosumab Antibody | AbD26781_hIgG1

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Human Anti-Denosumab Antibody specificity ELISA

A microtiter plate was coated overnight with various antigens at a concentration of 5 µg/ml. After washing and blocking with PBST+5% BSA, detection was performed using Human Anti-Denosumab:HRP Antibody clone AbD26781_hIgG1 (HCA283P) at a concentration of 2 µg/ml in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.

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Human Anti-Denosumab Antibody specificity titration ELISA

A microtiter plate was coated overnight with denosumab, human RANKL, human IgG1/kappa, human IgG2/kappa or human IgG1/lambda at a concentration of 5 µg/ml. For complex formation, denosumab, at a concentration of 2µg/mL, was added to RANKL coated wells after blocking. After washing and blocking with PBST+5% BSA, detection was performed using Human Anti-Denosumab:HRP Antibody clone AbD26781_hIgG1 (HCA283P) titrated to the given concentrations in PBST, followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data for binding to denosumab and to the RANKL-denosumab complex is shown as the mean of three measurements.

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Human Anti-Denosumab Antibody inhibition ELISA

A microtiter plate was coated overnight with recombinant human RANKL at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, denosumab was added (0.3 µg/ml), spiked with increasing concentrations of each of four Human Anti-Denosumab Antibodies, clone AbD26295_hIgG1 (HCA280), clone AbD26862_hIgG1 (HCA282), clone AbD26781_hIgG1 (HCA283) all here in monovalent Fab format, and clone AbD26296 (HCA288). Detection of denosumab was performed using Mouse Anti-Human IgG (Fc) CH2 Domain:HRP Antibody (MCA647P) followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data is shown as the mean of three measurements. Note, although HCA283 is a non-inhibitory antibody, it shows a slight decrease in signal in this assay. This is most likely due to different binding modes of the denosumab-RANKL complex at higher denosumab concentrations (Arthur et al., 2012).

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Human Anti-Denosumab Antibody bridging ELISA for pharmacokinetic (PK) assay development

A microtiter plate was coated overnight with one of two Anti-Denosumab Antibodies, either clone AbD26295_hIgG1 (HCA280) or clone AbD26862_hIgG1 (HCA282) at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, 10% human serum was added spiked with increasing concentrations of denosumab. Detection was performed using Human Anti-Denosumab:HRP Antibody clone AbD26781_hIgG1 (HCA283P) followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data is shown as the mean of three measurements.

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Denosumab antigen capture ELISA for pharmacokinetic (PK) assay development

A microtiter plate was coated overnight with human RANKL at a concentration of 5 µg/ml. After washing and blocking with PBST+5% BSA, 10% human serum was added spiked with increasing concentrations of denosumab. Detection was performed using Human Anti-Denosumab:HRP Antibody clone AbD26781_hIgG1 (HCA283P) in HISPEC Assay Diluent (BUF049A) at a concentration of 2 µg/ml, followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data is presented as the mean of three measurements.

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  • Human anti Denosumab:HRP
  • Human anti Denosumab
(Rated 0.0 out of 5 based on 0 customer reviews)
  • Product Type
    Monoclonal Antibody
  • Clone
    AbD26781_hIgG1
  • Isotype
    IgG1
2 Formats Available
    Product CodeApplicationsDatasheetMSDSPack SizeList PriceQuantity
    HCA283PEdatasheet pdfdatasheet pdf0.1 mg
    HCA283P
    HCA283Edatasheet pdfdatasheet pdf0.1 mg
    HCA283
    Summary
    Secondary Antibodies
    Negative Isotype Controls
    Useful Reagents
    Positive Controls
    Histology Controls
    More Images
    References
    Reviews
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    • Human Anti-Denosumab Antibody, clone AbD26781_hIgG1 is a non-inhibitory anti-idiotypic antibody that specifically recognizes free human monoclonal antibody drug denosumab, and the drug in complex with its target, RANKL (receptor activator of nuclear factor kappa-B ligand). The antibody does not recognize free RANKL and does not inhibit binding of denosumab to RANKL.

      A pair of anti-denosumab antibodies can be used to develop a pharmacokinetic (PK) bridging assay. This antibody clone AbD26781_hIgG1 is recommended as the detection antibody, paired with either clone AbD26295_hIgG1 (HCA280) or clone AbD26862_hIgG1 (HCA282) as capture antibody. It can also be used as the detection antibody in an antigen capture format assay. This antibody is not suitable for the development of an anti-drug antibody (ADA) assay.

      Denosumab (Prolia, Xgeva) is a fully human monoclonal antibody (IgG2/kappa) for the treatment of osteoporosis, treatment-induced bone loss, bone metastases, multiple myeloma, and giant cell tumor of bone. The drug specifically binds to human RANKL a protein that acts as the primary signal to promote bone removal/resorption and prevents RANKL from activating its receptor, RANK, on the surface of osteoclasts and their precursors.

      View a summary of all anti-denosumab antibodies.
    • Intended Use
    • Product Form
      Human IgG1 antibody (kappa light chain) selected from the HuCAL® phage display library and expressed in a human cell line. Conjugated to horseradish peroxidase (HRP) – liquid
      Human IgG1 antibody (kappa light chain) selected from the HuCAL® phage display library and expressed in a human cell line - liquid
    • Reconstitution
    • Preparation
      Purified IgG prepared by affinity chromatography on Protein A
      Purified IgG prepared by affinity chromatography on Protein A
    • Preservative Stabilisers
      0.01% Thiomersal
      0.01% Thiomersal
    • Immunogen
      Denosumab
    • Purity
    • Affinity
      The monovalent intrinsic affinity of this antibody was measured as KD= 19 nM by real time, label-free molecular interaction analysis on immobilized denosumab and as KD= 17 nM on the RANKL/drug complex.
    • Approx. Protein Concentrations
      IgG concentration 0.1 mg/ml
      IgG concentration 0.5 mg/ml
    • Reagents In The Kit
    • Preparing The Antibody
    • Test Principle
    • Buffer Solution
      Phosphate buffered saline
      Phosphate buffered saline
    • Storage
      Store at -70oC.
      Storage in frost-free freezers is not recommended.
      This product should be stored undiluted. This product is photosensitive and should be protected from light.
      Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
      Store at +4oC or at -20oC if preferred.
      Storage in frost-free freezers is not recommended.
      This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
    • Shelf Life
      12 months from date of despatch
      12 months from date of despatch
    • Acknowledgements
      Sold under license of U.S. Patents 6753136, 7785859 and 8273688 and corresponding patents. This antibody was developed by Bio-Rad, Zeppelinstr. 4, 82178 Puchheim, Germany. Prolia and Xgeva are registered trademarks of Amgen Inc.
      Sold under license of U.S. Patents 6753136, 7785859 and 8273688 and corresponding patents. This antibody was developed by Bio-Rad, Zeppelinstr. 4, 82178 Puchheim, Germany. Prolia and Xgeva are registered trademarks of Amgen Inc.
    • Licensed Use
      For in vitro research purposes and for commercial applications for the provision of in vitro testing services to support preclinical and clinical drug development. Any re-sale in any form or any other commercial application needs a written agreement with Bio-Rad
    • Regulatory
      For research purposes only
    • This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.

    • Application NameYesNoMin DilutionMax Dilution
      ELISA
    • Application NameYesNoMin DilutionMax Dilution
      ELISA

    • Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
    • Technical Advice
      Recommended protocols and further information about HuCAL recombinant antibody technology can be found in the HuCAL Antibodies Technical Manual
    • Technical Advice
      Recommended protocols and further information about HuCAL recombinant antibody technology can be found in the HuCAL Antibodies Technical Manual
    • Recommended Protocol
    • Recommended Protocol
    • ELISA
      This antibody can be used as a detection antibody in a bridging ELISA together with HCA280 (clone AbD26295_hIgG1) or HCA282 (clone AbD26862_hIgG1) as capture reagents. This antibody can be used in an antigen capture ELISA. Protocol: PK bridging ELISA to measure free drug.
    • ELISA
      This antibody can be used as a detection antibody in a bridging ELISA together with HCA280 or HCA282 as capture reagents. This antibody can be used in an antigen capture ELISA. Protocol: PK bridging ELISA to measure free drug.
    • Immunohistology
    • Immunohistology
    • Histology Positive Control Tissue
    • Histology Positive Control Tissue
    • Immunofluorescence
    • Immunofluorescence
    • Western Blotting
    • Western Blotting
    • Instructions For Use
    • Instructions For Use

    Additional Denosumab Antibody Formats

    Formats Clone Applications Sizes available
    Denosumab Antibody : HRP AbD26781_hIgG1 E 0.1 mg
    Denosumab Antibody : Purified AbD26781_hIgG1 E 0.1 mg
    • Copyright © 2017 Bio-Rad Antibodies (formerly AbD Serotec)

    Recommended Secondary Antibody

      Recommended Negative Isotype Control

        Useful Reagents

          DescriptionProduct CodePack SizeApplicationsList PriceQuantity
          Human anti DenosumabHCA2800.1 mgE
          HCA280
          Human anti DenosumabHCA2820.1 mgE
          HCA282
          Hispec Assay DiluentBUF049A50 mlIY
          BUF049A
          DescriptionProduct CodePack SizeApplicationsList PriceQuantity
          LYNX Rapid HRP Antibody Conjugation KitLNK001P1 Conjugation For 400µg AntibodyCJ
          LNK001P
          Human anti DenosumabHCA2800.1 mgE
          HCA280
          Human anti DenosumabHCA2820.1 mgE
          HCA282
          Hispec Assay DiluentBUF049A50 mlIY
          BUF049A

          Recommended Positive Controls

            Histology Controls

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