To decide whether an antibody is suited for its intended use, it has to be validated, the confirmation of which usually comes through experiments. It is important to understand that antibody validation is application and context specific. Atlas Antibodies provides antibodies that are strengthened by enhanced validation and are, thus, highly distinguished. The antibodies by Atlas Antibodies are also recommended by leading researchers. Their description is provided below.
Triple A Polyclonals
Triple A Polyclonals are originally developed within the academic Human Protein Atlas (HPA) project. HPA uses the antibodies to characterize all human proteins in the majority of normal and cancerous tissues, as well as on a subcellular level. What makes the antibodies so unique and specific are the comprehensive selection of antigen regions, affinity purification of the polyclonal antibodies, a stringent selection of approved antibodies and validation using different methods.
Atlas Antibodies also provides a selected number of mouse monoclonal antibodies, under the brand name PrecisA Monoclonals. PrecisA Monoclonals are developed with meticulous antigen design and are isotyped for multiple possibilities.
Transparency of Data
- Human Protein Atlas portal provides free access to data about the characterization of all antibodies achieved using different application
- For immunohistochemistry (IHC), every antibody is supplied with 500 images, from 44 normal tissues and 20 cancers
- The antigen sequences are provided on the product pages for all antibodies
- Where available, precise epitope information is supplied for PrecisA Monoclonals
- The scientific support team can provide all relevant information about production and quality control of each antibody, if required
Enhanced Validation of Antibodies
Based on the recommendations by the International Working Group for Antibody Validation (IWGAV), and as published in Nature Methods, Uhlén et al1, enhanced validation of antibodies is applied. This strengthens the reliability of the antibodies. The group consists of researchers from institutions in USA and Canada, such as Stanford University, Yale University, MIT, UCSD, University of Toronto, NIH, EMBL, Niigata University in Japan and the Science for Life laboratory in Sweden.
Five conceptual pillars for antibody validation are presented in the article. Those are suggested to be used in an application specific manner. Based on these pillars, HPA and Atlas Antibodies have developed an Enhanced Validation Strategy. There are five main methods to the strategy:
Five Conceptual Methods
1. Genetic validation:
Target confirmed by siRNA knock-down.
2. Orthogonal validation:
Specificity confirmed by a non-antibody based method.
3. Independent antibody validation:
Specificity confirmed by another antibody targeting a different epitope of the protein.
4. Recombinant expression validation:
Target confirmed by an overexpressed version of the protein.
5. Capture MS validation:
Presence of target verified by Mass Spectrometry.
At least one of the methods must be used for an antibody to receive Enhanced Validation Status in a specific application.
Figure 1. Example of genetic validation by siRNA knock down in Western blot using the Anti-PPIB antibody. U-251 cells have been transfected with control siRNA and two target specific siRNA probes. Downregulation of antibody signal confirms target specificity. The remaining intensity relative control lane is indicated as a percentage.
Figure 2. Examples of orthogonal validation in WB and IHC. Left: WB analysis in human cell lines SK-MEL-30 and Caco-2 using Anti-RAB27A antibody (HPA001333). Corresponding RAB27A RNA-seq data (TPM values) is presented for the same cell lines. Right: IHC staining of liver and colon tissues using the Anti-SLC2A2 antibody (HPA028997). The corresponding RNA-seq data (TPM values) for the same tissues are presented below. In both examples, samples with known high and low RNA expression are chosen and correlation to antibody signal is shown by both WB and IHC.
1. Genetic Validation
The target protein can be down-regulated on a genetic level using siRNA or CRISPR-Cas9. A possible way to determine if the antibody is specific for its purpose is to check if the knock-down (by siRNA) or knock-out ( by CRIS- PR) of the corresponding gene correlates with absence or decrease of antibody signal. If yes, then it is specific. This is exemplified in Figure 1 where the Anti-PPIB antibody AMAb91245 is used in Western Blot (WB) analysis in U-251 cells. The antibody signal is down regulated in the cells silenced by PPIB siRNA.
2. Orthogonal Validation
When the results from the different methods across several samples are compared, it can be decided whether the antibody can be validated. This is done by using a method that is non-antibody based for target quantification.
One approach is to compare the antibody staining intensities over multiple samples with varying expression of the target gene, with RNA-seq data in the same samples. This is illustrated in Figure 2, using WB analysis in human cell lines SK-MEL-30 and Caco-2 using Anti-RA- B27A antibody HPA001333. Corresponding RAB27A RNA-seq data (TPM values) are presented for the same cell lines.
3. Validation by Independent Antibodies
It is also possible for two antibodies to validate each other. When they are directed against different regions of the same target protein, a validation is possible when compared in a set of relevant tissues. This is exemplified in Figure 3 with two antibodies directed against different regions of the A2ML1 protein used in the immunohistochemistry application. Both antibodies show positive staining in liver, but are negative in tonsil, colon and kidney.
4. Recombinant Expression Validation
An over-expressed or tagged version of the target protein can also validate an antibody signal. When over-expressing the target protein in a cell line, the antibody is validated by comparing the signal from the over-expressed version with the unmodified endogenous target protein. This approach is exemplified in Figure 4a.
Moreover, the target protein may be tagged by an affinity tag or a fluorescent protein. The pattern displayed by the tagged target protein is matched to the antibody signal.
If there is a match, then that means that the antibody recognizes its target protein. Validation by tagged proteins can be applied in immunocytochemistry-immunofluorescence (ICC-IF) and is shown in Figure 4b.
5. Migration Capture MS Validation
In this method, the staining pattern and the protein size detected by the antibody is compared with results obtained by a capture Mass Spectrometry (MS) method. In this method gel migration is crucial because the band generated by the antibody in WB should correlate with the target protein identified by MS in terms of gel migration.
Atlas Antibodies and Validation
The antibodies offered by Atlas Antibodies are extensively validated in collaboration with the Human Protein, which is what makes them highly characterized. The Enhanced Validation implementation ensures a further layer of security.
Enhanced validation offers increased security of antibody specificity in a defined context.
Figure 3. The two Anti-A2ML1 antibodies HPA038847 (Antibody 1) and HPA038848 (Antibody 2) target different regions of A2ML1. Antibody stainings across relevant positive and negative tissues are similar between the two, and the antibodies validate each others staining pattern in IHC. Here, the two antibodies are used for staining esophagus (positive for A2ML1 using both antibodies), tonsil, colon and kidney tissues (negative for A2ML1 using both antibodies).
Figure 4a. Example of recombinant expression validation in Western blot using the Anti-ACY3 antibody (HPA039219). Lane 1: marker, Lane 2: negative control (vector only transfected HEK293T lysate), Lane 3: ACY3 over-expression lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY408962).
Figure 4b. Immunofluorescent staining of transgenic HeLa cells using Anti-NES antibody HPA006286 shows positivity in intermediate filaments (in green). Antibody staining overlaps with GFP tagged nestin protein (in purple).
Selected References from the Human Protein Atlas Project
Uhlén M, Bandrowski A, Carr S, Edwards A, Ellenberg J, Lundberg E, Rimm DL, Rodriguez H, Hiltke T, Snyder M and Yamamoto T.
A Proposal for Validation of Antibodies.
Nature Methods 2016 13, 823–827.
Uhlen M, Zhang C, Lee S, Sjöstedt E, Fagerberg L, Bidkhori G, Benfeitas R, Arif M, Liu Z, Edfors F, Sanli K, von Feilitzen K, Oksvold P, Lundberg E, Hober S, Nilsson P, Mattsson J, Schwenk JM, Brunnström H, Glimelius B, Sjöblom T, Edqvist PH, Djureinovic D, Micke P, Lindskog C, Mardinoglu A, Ponten F.
A pathology atlas of the human cancer transcriptome.
Science. 2017 357(6352)
ftul PJ, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Ait Blal H, Alm T, Asplund A, Björk L, Breckels LM, Bäckström A, Danielsson F, Fagerberg L, Fall J, Gatto L, Gnann C, Hober S, Hjelmare M, Johansson F, Lee S, Lindskog C, Mulder J, Mulvey CM, Nilsson P, Oksvold P, Rockberg J, Schutten R, Schwenk JM, Sivertsson Å, Sjöstedt E, Skogs M, Stadler C, Sullivan DP, Tegel H, Winsnes C, Zhang C, Zwahlen M, Mardinoglu A, Pontén F, von Feilitzen K, Lilley KS, Uhlén M, Lundberg E.
A subcellular map of the human proteome.
Science. 2017 356(6340).
Edfors F, Danielsson F, Hallström BM, Käll L, Lundberg E, Pontén F, Forsström B and Uhlén M.
Gene-specific correlation of RNA and protein levels in human cells and tissues.
Mol Syst Biol. 2016 Oct; 12(10): 883.
Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, Olsson I, Edlund K, Lund- berg E, Navani S, Szigyarto CA, Odeberg J, Djureinovic D, Takanen JO, Hober S, Alm T, Edqvist PH, Berling H, Tegel H, Mulder J, Rockberg J, Nilsson P, Schwenk JM, Hamsten M, von Feilitzen K, Forsberg M, Persson L, Johansson F, Zwahlen M, von Heijne G, Nielsen J, Pontén F.
Tissue-based map of the human proteome.
Science. 2015 Jan;23;347(6220).
Atlas Antibodies provides the antibodies from the Human Protein Atlas. We offer a wide range of highly validated research antibodies – Triple A Polyclonals and PrecisA Monoclonals – targeting over 75% of the human proteome.
Triple A Polyclonals are primary antibodies developed and characterized within the Human Protein Altas project. The 21,000 rabbit polyclonal antibodies cover 75% of the human proteome and are validated in IHC, WB and ICC-IF. For each IHC antibody, you can explore 500 IHC images for each antibody.
PrecisA Monoclonals are primary mouse monoclonals with unique antigen design for optimal specificity. They are epitope mapped with defined specificity and isotyped for multiplexing.
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