Antibody Validation

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The reproducibility of scientific studies has become a major issue, leading to a lack of trust in scientific results from the academic and pharmaceutical research communities.  The ongoing dialogue on reproducibility of results includes the role of improperly validated research reagents, such as antibodies. Antibodies are invaluable tools in the life sciences and have become workhorses of basic research. Their high specificity and selectivity for unique protein targets make them indispensable research reagents. However, although antibodies are designed to recognize a target protein, they may not be able to do so in all applications (e.g. those that alter the target protein’s structure).

The number of antibody suppliers contributing commercial antibodies to the Research Use Only (RUO) space has steadily increased over the past several years, with inconsistent validation data to confirm sensitivity, specificity, reactivity and lot-to-lot consistency. 

For this reason a number of initiatives brought forth by a committee of international scientists and leaders from academic and industry labs, journal editors and commercial antibody vendors, including Adipogen Life Sciences, have suggested broad guidelines for antibody validation as well as detailed context-dependent measures that should be followed by antibody suppliers (see Ref 1 for the meeting overview, Ref 2 and 3 for validation proposals and section below "Experiments performed for Antibody Validation").

In addition, the researchers approach has to be adapted as well. One step that researchers can take is to review the data sheet carefully with respect to the applications for which the antibody in question has been validated. It is also recommended that users of antibodies carry out at least one of the validation strategies described below in their own particular application or sample context.

The last point is a proper citing of the individual antibodies used in publications. A simple indication of the brand name is not enough. Not only can the correct batch information be useful, but also the correct reference of the product identification number (PID#). Often antibody suppliers provide several antibodies to the same target and the correct identification number is necessary for the reproducibility of the results.

For both antibody suppliers and researchers, the ultimate solution to the antibody validation problem will be to rely on the fundamental principles of research, “doing good science”. Developing an antibody and validating it is not different from doing an experiment correctly and doing the right experimental design with the proper positive and negative controls.

Ref 1: Antibody validation: a view from the mountains: M.J. Taussig, et al.; New Biotechnol. 45, 1 (2018)
Ref 2: A proposal for validation of antibodies: M. Uhlen, et al.; Nat. Methods 13, 823 (2016)
Ref 3: Ten Basic Rules of Antibody Validation: M.G: Weller, et al.; Anal. Chem. Insights 13, 1177390118757462 (2018)



Experiments performed for Antibody Validation


Validation Check Boxes

Validation criteria for antibodies include specificity ("off target" binding properties), sensitivity ("on target" binding properties) and reproducibility (lot-to-lot consistency) experiments. In addition, it is important that antibody validation approaches must be carried out in an application- and context-specific manner (to detect a protein in a native or a denatured conformation). At Adipogen Life Sciences we follow for each antibody two or more of the different below-indicated criteria with recombinant and endogenous targets that are performed in each validated application.


- Detection of properly folded recombinant target protein, protein tags and closely related proteins.

- Analysis of cell lines with known target expression levels (positive controls).

- Analysis of cell lines known not to express the target protein (negative controls).

- Genetic approach: Target-specific signal verified in transfected cells, knockout cells, or knock-down (CRISPR/Cas9 or siRNA-treated) cells.

- Comparison of results with antibody and isotype control to ensure acceptable signal-to-background ratio.

- Blocking with antigen peptide to confirm elimination of specific signal.

- Side-by-side comparison of a new lot with previous lots to ensure lot-to-lot consistency.

- Treatment of cells with appropriate triggers or physiological induction to detect an increase or a decrease of the target protein expression.

- Analysis of secreted targets in biological fluids (cell culture supernatant, plasma, serum, urine, others) using naturally occurring variable expression (e.g. healthy versus pathological conditions).

 

Functional/Application Validation


AdipoGen Life Sciences focuses on experimental applications that are of highest interest to the research community. We follow the ongoing research together with academic experts in their field of research to continuously improve experimental setups and provide optimized, detailed protocols. In addition, our technical support team provides a quick and reliable service to address your special inquiries and experimental setup. Below is an overview of validated application types performed at AdipoGen Life Sciences depending on the antibody and target.


- ELISA Antibody-Protein Binding assays performed in-house.

- Western blot, Immunoprecipitation and Flow Cytometry applications performed in-house or in collaboration with academic experts.

- Immunofluorescence application performed in collaboration with academic experts or industry partners.

- In vitro Functional applications performed in-house and in vivo experiments performed in collaboration with academic experts.



Antibody Validation Program


Evaluation Program

AdipoGen Life Sciences' goal is to continuously improve the product data description of our manufactured antibodies. Since our in-house validation focuses on experimental applications that are of highest interest, we might not have comprehensive application or cross-reactivity data for all antibodies.  To address this fact, we have implemented an Antibody Validation Program, which allows researchers to test a defined group of antibodies in their experimental setups. These are provided free of charge in exchange for test results.


If you are interested in participating in our Antibody Validation Program please contact us at info@adipogen.com with the following information a) what experiments you plan to do and, b) which antibody you would like to test (see below list). You can also download our Antibody Evaluation Agreement (right side), fill in the form and return it by email.







Below is a list of antibodies that are included in our Antibody Validation Program and ordered by Product Name:

PID Product Name   PID Product Name   PID Product Name
AG-25A-0042 ACE2 (human), pAb AG-25A-0097 CTRP7 (globular domain) (human), pAb AG-25A-0033 Nampt (Visfatin/PBEF) (rat), pAb
AG-20A-0056 Adiponectin (human), mAb (ADI 943) AG-25A-0117 CTRP7 (human), pAb AG-20B-0039 Netrin-4, mAb (Nely-1)
AG-25B-0010 Adiponectin Receptor 1 (human), pAb (AL238) AG-25A-0109 CTRP9 (human), pAb AG-20A-0087 NMNAT2 (human), mAb (Nady-1)
AG-25B-0012 Adiponectin Receptor 2 (mouse), pAb (AL241) AG-25A-0091 DLK1 (human), pAb AG-25A-0113 NMNAT2 (human), pAb
AG-25A-0068 ANGPTL2 (human), pAb AG-25A-0092 DLK1 (human), pAb AG-25B-0013 Nod1 (human), pAb (AL184)
AG-25A-0060 ANGPTL3 (CCD) (human), pAb AG-20A-0058Y DLK1 (mouse), mAb (PF183E) AG-20A-0086 NQO1 (human), mAb (Skiny-1)
AG-25A-0064 ANGPTL3 (FLD) (human), pAb AG-25A-0062 DLL1 (human), pAb AG-25A-0105 NQO1 (human), pAb
AG-20A-0039 ANGPTL3 (human), mAb (Kairos-37) AG-20A-0085 DLL1 (mouse), mAb (D1L357-1-4) AG-25A-0043 Obestatin (human), pAb
AG-25A-0052 ANGPTL3 (human), pAb AG-20A-0080 DLL4 (human), mAb (DL86-3AG) AG-20B-0031 Omentin (human), mAb (Lecty-1)
AG-20A-0046 ANGPTL4 (CCD) (human), mAb (Kairos4-153AD) AG-25A-0102 DNER (human), pAb AG-25A-0051 Omentin (human), pAb
AG-25A-0066 ANGPTL4 (CCD) (human), pAb AG-25A-0040 FABP3 (human), pAb AG-25A-0112 Progranulin (human), pAb
AG-25A-0038 ANGPTL4 (human), pAb AG-25A-0041 FABP4 (human), pAb AG-25A-0021 RANK (ectodomain) (human), pAb
AG-25A-0055 ANGPTL4 (human), pAb AG-20A-0066 FGF-19 (human), mAb (FG369-1) AG-25A-0016 RANKL (human), pAb
AG-25A-0069 ANGPTL5 (CCD) (human), pAb AG-20A-0065 FGF-19 (human), mAb (FG98-6) AG-20A-0060 RELM-alpha (mouse), mAb (MREL 127)
AG-25A-0037 ANGPTL6 (human), pAb AG-25A-0076 FGF-21 (mouse), pAb AG-25A-0010 RELM-alpha (mouse), pAb 
AG-25A-0095 ANGPTL7 (CCD) (human), pAb  AG-20A-0051 FGF-21, mAb (FG224-7) AG-20A-0061 RELM-alpha (rat), mAb (RREL 803)
AG-20A-0053 ANGPTL7 (human), mAb (Kairos 108-4) AG-20A-0073 FGF-23 (human), mAb (FG322-3) AG-25A-0011 RELM-alpha (rat), pAb 
AG-20A-0055 ANGPTL7 (human), mAb (Kairos 397-7) AG-25A-0035 FOXP3 (mouse), pAb AG-20A-0012 RELM-beta (human), mAb (HRB 149)
AG-25A-0050 ANGPTL7 (human), pAb AG-25A-0084 FTO (human), pAb AG-25A-0012 RELM-beta (human), pAb 
AG-25A-0006 beta-Pix (mouse), pAb AG-25A-0089 FTO (mouse), pAb AG-25A-0063 RELM-beta (mouse), pAb
AG-25B-0033 Betatrophin (human), pAb (IN108) AG-20A-0007 GITR (mouse), mAb (MGIT 02) AG-25A-0015 Resistin (rat), pAb 
AG-25B-0035 CD112R (mouse), pAb (IN109) AG-25A-0073 GPX3, pAb AG-25A-0078 SENP2 (mouse), pAb
AG-25A-0049 Clusterin (human), pAb AG-25A-0090 Granulin C (human), pAb AG-25A-0083 Sirtuin 2 (human), pAb
AG-25A-0054 Clusterin (mouse), pAb AG-25B-0026 IGFLR1 (human), pAb (IN101) AG-25A-0100 Sirtuin 5 (human), pAb
AG-25A-0114 CTRP1 (human), pAb AG-20A-0027 IL-23p19 (human), mAb (I 178G) AG-20A-0091 Sirtuin 6 (human), mAb (S6R82-2)
AG-25A-0115 CTRP2 (human), pAb AG-25A-0045 IL-33 (human), pAb AG-25A-0101 Sirtuin 6 (human), pAb
AG-25A-0110 CTRP3 (globular domain) (human), pAb AG-20A-0042 IL-33, mAb (IL33068A) AG-25A-0106 TDO (human), pAb
AG-25A-0107 CTRP3 (human), pAb AG-25B-0032 IL-36beta (human), pAb (IN107) AG-20A-0029 Tim-3 (mouse), mAb (TI 339H)
AG-25A-0096 CTRP5 (globular domain) (human), pAb AG-25A-0111 IL-37 (human), pAb AG-25B-0011 TRAM (human), pAb (AL239)
AG-25A-0103 CTRP5 (human), pAb AG-25A-0087 Lipocalin-2 (human), pAb AG-25A-0059 TRB-3 (human), pAb
AG-25A-0116 CTRP5 (human), pAb AG-25A-0088 Lipocalin-2 (rat), pAb AG-25A-0075 Vaspin (mouse), pAb
AG-20A-0093 CTRP6 (human), mAb (256-E) AG-25A-0061 MFAP4 (human), pAb
AG-25A-0108 CTRP6 (human), pAb AG-25A-0028 Nampt (Visfatin/PBEF) (mouse), pAb



Below is the same list of antibodies in our Antibody Validation Program and ordered by Product Number:

PID Product Name   PID Product Name   PID Product Name
AG-20A-0007 GITR (mouse), mAb (MGIT 02) AG-25A-0033 Nampt (Visfatin/PBEF) (rat), pAb AG-25A-0090 Granulin C (human), pAb
AG-20A-0012 RELM-beta (human), mAb (HRB 149) AG-25A-0035 FOXP3 (mouse), pAb AG-25A-0091 DLK1 (human), pAb
AG-20A-0027 IL-23p19 (human), mAb (I 178G) AG-25A-0037 ANGPTL6 (human), pAb AG-25A-0092 DLK1 (human), pAb
AG-20A-0029 Tim-3 (mouse), mAb (TI 339H) AG-25A-0038 ANGPTL4 (human), pAb AG-25A-0095 ANGPTL7 (CCD) (human), pAb 
AG-20A-0039 ANGPTL3 (human), mAb (Kairos-37) AG-25A-0040 FABP3 (human), pAb AG-25A-0096 CTRP5 (GD) (human), pAb
AG-20A-0042 IL-33, mAb (IL33068A) AG-25A-0041 FABP4 (human), pAb AG-25A-0097 CTRP7 (GD) (human), pAb
AG-20A-0046 ANGPTL4 (human), mAb (Kairos4-153AD) AG-25A-0042 ACE2 (human), pAb AG-25A-0100 Sirtuin 5 (human), pAb
AG-20A-0051 FGF-21, mAb (FG224-7) AG-25A-0043 Obestatin (human), pAb AG-25A-0101 Sirtuin 6 (human), pAb
AG-20A-0053 ANGPTL7 (human), mAb (Kairos 108-4) AG-25A-0045 IL-33 (human), pAb AG-25A-0102 DNER (human), pAb
AG-20A-0055 ANGPTL7 (human), mAb (Kairos 397-7) AG-25A-0049 Clusterin (human), pAb AG-25A-0103 CTRP5 (human), pAb
AG-20A-0056 Adiponectin (human), mAb (ADI 943) AG-25A-0050 ANGPTL7 (human), pAb AG-25A-0105 NQO1 (human), pAb
AG-20A-0058Y DLK1 (mouse), mAb (PF183E) AG-25A-0051 Omentin (human), pAb AG-25A-0106 TDO (human), pAb
AG-20A-0060 RELM-alpha (mouse), mAb (MREL 127) AG-25A-0052 ANGPTL3 (human), pAb AG-25A-0107 CTRP3 (human), pAb
AG-20A-0061 RELM-alpha (rat), mAb (RREL 803) AG-25A-0054 Clusterin (mouse), pAb AG-25A-0108 CTRP6 (human), pAb
AG-20A-0065 FGF-19 (human), mAb (FG98-6) AG-25A-0055 ANGPTL4 (human), pAb AG-25A-0109 CTRP9 (human), pAb
AG-20A-0066 FGF-19 (human), mAb (FG369-1) AG-25A-0059 TRB-3 (human), pAb AG-25A-0110 CTRP3 (GD) (human), pAb
AG-20A-0073 FGF-23 (human), mAb (FG322-3) AG-25A-0060 ANGPTL3 (CCD) (human), pAb AG-25A-0111 IL-37 (human), pAb
AG-20A-0080 DLL4 (human), mAb (DL86-3AG) AG-25A-0061 MFAP4 (human), pAb AG-25A-0112 Progranulin (human), pAb
AG-20A-0085 DLL1 (mouse), mAb (D1L357-1-4) AG-25A-0062 DLL1 (human), pAb AG-25A-0113 NMNAT2 (human), pAb
AG-20A-0086 NQO1 (human), mAb (Skiny-1) AG-25A-0063 RELM-beta (mouse), pAb AG-25A-0114 CTRP1 (human), pAb
AG-20A-0087 NMNAT2 (human), mAb (Nady-1) AG-25A-0064 ANGPTL3 (FLD) (human), pAb AG-25A-0115 CTRP2 (human), pAb
AG-20A-0091 Sirtuin 6 (human), mAb (S6R82-2) AG-25A-0066 ANGPTL4 (CCD) (human), pAb AG-25A-0116 CTRP5 (human), pAb
AG-20A-0093 CTRP6 (human), mAb (256-E) AG-25A-0068 ANGPTL2 (human), pAb AG-25A-0117 CTRP7 (human), pAb
AG-20B-0031 Omentin (human), mAb (Lecty-1) AG-25A-0069 ANGPTL5 (CCD) (human), pAb AG-25B-0010 AdiponectinR1 (human), pAb (AL238)
AG-20B-0039 Netrin-4, mAb (Nely-1) AG-25A-0073 GPX3, pAb AG-25B-0011 TRAM (human), pAb (AL239)
AG-25A-0006 beta-Pix (mouse), pAb AG-25A-0075 Vaspin (mouse), pAb AG-25B-0012 AdiponectinR2 (mouse), pAb (AL241)
AG-25A-0010 RELM-alpha (mouse), pAb  AG-25A-0076 FGF-21 (mouse), pAb AG-25B-0013 Nod1 (human), pAb (AL184)
AG-25A-0011 RELM-alpha (rat), pAb  AG-25A-0078 SENP2 (mouse), pAb AG-25B-0026 IGFLR1 (human), pAb (IN101)
AG-25A-0012 RELM-beta (human), pAb  AG-25A-0083 Sirtuin 2 (human), pAb AG-25B-0032 IL-36beta (human), pAb (IN107)
AG-25A-0015 Resistin (rat), pAb  AG-25A-0084 FTO (human), pAb AG-25B-0033 Betatrophin (human), pAb (IN108)
AG-25A-0016 RANKL (human), pAb AG-25A-0087 Lipocalin-2 (human), pAb AG-25B-0035 CD112R (mouse), pAb (IN109)
AG-25A-0021 RANK (ectodomain) (human), pAb AG-25A-0088 Lipocalin-2 (rat), pAb
AG-25A-0028 Nampt (Visfatin/PBEF) (mouse), pAb AG-25A-0089 FTO (mouse), pAb







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