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Department of Microbiology and Immunology

Antibody Validation and Reproducibility resources

First – be sure to search by lot number in addition to catalog number!!  Just because one lot was validated, it does not validate another lot!!

Antibody Registry   

The Antibody Registry includes commercial antibodies from hundreds of commercial vendors and thousands of individual labs. However, many of the links don’t go anywhere useful. Give it a try:   http://antibodyregistry.org/

Or use BenchSci  https://landing.benchsci.com/academic    ‘We use AI to screen the literature for published antibodies so you don’t have to.’

Companies:  practices vary immensely – do your homework before buying!!

BD Biosciences: [awaiting response]

BioLegend page on Antibody Validation Challenges: Reproducibility and Validation Webpage, including steps to improve reproducibility:  https://www.biolegend.com/reproducibility

Capricobio:  ‘We are an ISO certified cGMP facility and validate every lot of antibody that is produced. We validate our antibodies through cell binding specificity COA and other validation information can be obtained at customer request for their lot of interest.’

Cell Signaling Technology Antibody Validation:  https://www.cellsignal.com/contents/our-approach/cst-antibody-validation-principles/ourapproach-validation-principles

Jackson Immuno Research  (mostly secondary antibodies):  ‘Most of JIR’s products are polyclonal secondary antibodies, and because each preparation can recognize many epitopes on the primary antibody targets, the secondaries will react with the primaries wherever they are bound. If the primary has bound its target antigen (under any fixation condition), the secondary antibody will recognize it. Our Quality Assurance testing is designed to confirm that the antibodies behave as we describe them. The ability of the secondary antibody to bind to its intended target is confirmed by immunoelectrophoresis and/or a solid-phase immunoaffinity assay (target protein coupled to agarose beads). Each antibody purification event is also tested by a sensitive ELISA assay to confirm that products we describe as having “minimal cross-reactivity” to other species meet our standard (<1% reactivity as compared with the intended target). We have similar standards for products that are Fc or F(ab’)2 fragment specific, and for our anti-mouse subclass-specific reagents. All the products are tested in-process, and retested after vialing / lyophilization to confirm their performance.’  https://www.jacksonimmuno.com/

Milllipore/Sigma-Aldrich  Antibody Enhanced Validation – Additional Validation Data to Help Ensure Specificity and Performance

MiltenyiBiotec – Miltenyi Biotec has engineered new recombinant antibodies designed specifically for flow cytometry: REAfinity™ Recombinant Antibodies. These antibodies eliminate binding to Fc receptors and their recombinant production improves lot-to-lot consistency and reproducibility across multiple experiments. All antibodies are validated to work in flow cytometry. Additionally, enhanced validation data for antibody-specificity, sensitivity, and reproducibility are available on our website under each specific antibody clone. Please visit www.miltenyibiotec.com/reafinity for more information.

Santa Cruz  ‘We have validated all of our antibodies but only in Western Blotting. We have not done any validations for Immunofluorescence, Immuno Histochemistry, ELISA, or any other applications.’   – DO NOT USE SANTA CRUZ ANTIBODIES FOR FLOW CYTOMETRY UNTIL YOU HAVE VALIDATED THEM!!

ThermoFisher

https://www.thermofisher.com/us/en/home/life-science/antibodies/invitrogen-antibody-validation.html

 

UNDER CONSTRUCTION:

AbCam, BD

Other tips and resources: 

Information from the experts on antibody validation from BenchSci  https://blog.benchsci.com/reproducible-research

All antibodies should be validated prior to reporting of results. Recommendations for research using antibodies (page 7-8) ‘Although vendor-supplied technical information may help investigators select reagents such as antibodies, this information is insufficient for validation’.
Antibody Validation: Standards, Policies and Practices Workshop Report 2016
Best practices for using antibodies for flow cytometry:
  1. Always Titrate
  2. Validate Specificity (If manufacturer has not done this, then you need to!!)
  3. Integrate critical controls like Fluorescence Minus One controls (FMO)
  4. See: Uhlen et al., A Proposal for Validation of Antibodies, Nature Methods (2016)
  5. More at: https://expertcytometry.com/4-steps-to-validate-flow-cytometry-antibodies-and-improve-reproducibility/
  6. Also on the EuroMab network:
    Guidelines Website: https://www.euromabnet.com/guidelines/index.php   Guidelines Manuscript: https://www.ncbi.nlm.nih.gov/pubmed/26418356   Pos/Neg Controls Website: https://www.euromabnet.com/guidelines/positive-negative-controls.php   Additional Articles about Ab Validation: https://www.euromabnet.com/guidelines/articles-about-antibody-validation.php

Attn: other antibody manufacturers who wish to be added to this list, please provide links your antibody validation process and where investigators can access the validation data for each lot. I will be happy to include you in this list!  ncfisher@med.unc.edu.

Investigators: if a company you work with is not fully linked on this list, be sure to research your antibody source and validation before purchase!!