Supplier antibody validation: how much progress has been made?
In this blog:
- How much enhanced antibody validation is being carried out by suppliers overall?
- Which antibody validation strategies are being used by suppliers?
- How much of the human proteome is covered by validated antibodies?
Are you interested in the progress made in supplier antibody validation?
It is standard practice to test antibodies for the applications recommended by the supplier, but toaday we are taking a moment to look at enhanced antibody validation that goes further and aims to assess the specificity of the antibody.
Our CEO, Dr Andrew Chalmers, created CiteAb to help researchers find reagents which work for their experiments. Antibody validation is a really important factor to consider when finding a reagent, helping to ensure an antibody will work as expected, be specific to your target and lead to reproducible results (see our guide here for other factors to consider when finding an antibody). At its core, validation helps to save that all important time and cost when the wrong reagent is selected.
To help researchers looking for validated antibodies, we began collecting antibody validations over three and a half years ago and adding them into our database and search engine. In the CiteAb database, we collect both cited validations and supplier validations; today’s focus lies with supplier validations.
In this blog, we take a look at how much antibody validation is being carried out, which suppliers are leading the way with antibody validation and reflect on which validation strategies are used the most.
It is important to note that this is a first look at the data and does not cover all suppliers. One prominent example is Cell Signaling Technology, whose data is not featured in our analysis given their alternative approach to validation which we return to later in the blog.
This blog is now on version two, with expanded details of supplier initiatives (First published: 19/10/22, last update: 16/11/22). Look out for updates and further blogs moving forward as we collect even more information from a greater range of suppliers.
An overview of the ‘five pillars of antibody validation’
Firstly, we want to outline the five pillars of validation from which most of the supplier validations, and our enhanced validation analysis, is based upon.
These pillars, coined by Uhlen et al are as follows… 
- Genetic strategies
- Orthogonal strategies
- Independent antibody strategies
- Expression of tagged proteins or recombinant expression
- Immunoprecipitation followed by mass spectrometry
Below we give a brief overview of these methods;
Supplier antibody validation: the numbers
By collecting enhanced supplier antibody validations for our database, we are able to provide a snapshot of the current state of the field.
In total, we found that over 45,000 antibodies had supplier-reported enhanced validations, and this number is growing everyday. Breaking this down into strategies, we uncovered over 13,000 KO validations, 10,000 KD validations, 14,000 orthogonal validations, and over 10,000 other validations. Impressively, we discovered that almost 40% of the human proteome had a validated antibody against it, showing validation is perhaps more advanced than we would expect.
To explore the field a bit further, we took a look at the following questions for individual suppliers:
- Which supplier has the most distinct validated antibodies?
- Which supplier’s catalogue is the most validated?
- Which supplier has validated antibodies against the highest percentage of the human proteome?
Which supplier has performed the most enhanced antibody validations?
Counting antibody validations as the number of distinct antibodies with a validation from the pillars performed, we found that Thermo Fisher Scientific, with their Invitrogen branded antibodies, led the pack with over >11,000 validations. We noted that Thermo Fisher uses a range of approaches to achieve this impressive number of validations, including KO/KD validation, IP-MS, independent antibodies and relative expression.
Novus Biologicals, part of Bio-Techne, comes in next with >7,500 validations, followed by Proteintech, GeneTex and Abcam each with over >4,000 validations.
Which suppliers have the highest percentage of their catalogue validated to a pillar?
We thought it interesting to take a different approach and look not just at the overall numbers of enhanced supplier validations, but at what % of supplier antibody catalogues have been validated to a validation pillar.
Of the suppliers with catalogues containing <1000 antibodies, GenuIN Biotech lead the pack with a significant 62.55% of their catalogue validated. These antibodies were knockdown validated, using their proprietary shRNA-mediated gene knockdown platform.
Taking suppliers with antibody catalogues exceeding 1,000 products as a separate category, we found that Active Motif are leading the way with over 20% of their antibodies validated.
For suppliers with catalogues containing over 10,000 antibodies, Proteintech had the highest catalogue coverage: 28%. For the next level up, catalogues with over 100,000 antibodies, Thermo Fisher had the highest percentage of antibodies validated.
Which suppliers have validations covering the largest percentage of the human proteome?
Given the extensive supplier validations we observed in our data, we were interested to see how much of the human proteome was covered by supplier validations. Having validated antibodies against as many of the proteins within the human proteome as possible is extremely useful to a researcher for the study of disease, development and more.
For this analysis, we used the 20,398 Human Swiss-Prot reviewed proteins available, and discovered that 38.19% of the human proteome had a validated antibody against it.
Delving into specific supplier validations, we noted that Novus Biologicals and Thermo Fisher Scientific had the highest percentage of the human proteome covered by their validated antibodies. Novus had validated antibodies against 25.2% of the human proteome, and Thermo Fisher Scientific 21.7%.
Antibody validation by strategy
We then took a look at which of the five pillars of validation was most used by suppliers, counting distinct antibodies validated per method. We found the most popular validation strategies from the pillars to be:
- KO Validation
- KD Validation
- Orthogonal Validations
These three validation strategies totalled over three quarters of all enhanced validations performed by suppliers. We have also collected validation data on independent antibody strategies, protein overexpression strategies (encompassing cell treatment, biological strategies, expression of tagged proteins or recombinant expression validations), and IP-MS.
KO Validation Supplier Initiatives
KO Validations are often felt by researchers to be the most reliable method for antibody validation. Commonly using CRISPR/Cas9 technology, this strategy compares the signal produced from the tested antibody with and without the target protein.
Collecting KO validation data from over 25 suppliers for over 13,000 antibodies, we found that the leading supplier for KO Validations was Abcam with over 4,000 validated antibodies, followed by Origene. How have they achieved this success?
For Origene, their KO-cell lysate library, created in 2017, has certainly contributed to the amount of KO validations they have been able to perform. For Abcam, their acquisition of EdiGene cell lines and lysate portfolio in 2019 has aided their capacity for KO cell line validations.
KD Validation Supplier Initiatives
As another genetic strategy for validation, knockdown is an effective way to validate an antibody. Although this method may not remove all the protein like the KO technique, KD validation does allow for a temporal angle in validation given that RNAi down regulation is not a permanent genetic alteration. This means that the target protein will return to its normal levels, enabling a comparison of antibody specificity at both low and normal levels of target expression.
For our KD validation analysis we looked at data from 10 suppliers and over 10,000 antibodies. We discovered that Proteintech led the way with KD validated antibodies with >4,500 KD validated antibodies. They were followed by Thermo Fisher Scientific having just under 3000 KD validated antibodies available.
Orthogonal Validation Supplier Initiatives
Orthogonal validation is another popular and robust method of antibody validation used in the life sciences. We have collected, to date, orthogonal validation data from 8 suppliers, contributing to a total of over 13,000 antibodies.
The suppliers with the most orthogonal validated antibodies were Thermo Fisher Scientific (>6,400 antibodies), Novus Biologicals (<4,300 antibodies) and Genetex (<2,300 antibodies).
Independent Antibody Strategies, Protein over-expression and IP-MS Supplier Initiatives
To date, we have collected data from five suppliers for over 4,000 antibodies using independent antibody strategies. Novus Biologicals and Genetex performed the most validations using this strategy. Both of these suppliers are also leading in orthogonal validation.
For protein-overexpression strategies, we have amassed data from eight suppliers and over 6,000 antibodies. We found that Thermo Fisher Scientific performed the most of these validations, followed by Novus Biologicals.
Finally, so far we have collected data on over 250 antibodies from two suppliers for IP-MS validations. Thermo Fisher Scientific have performed the majority of these validations, with Genetex also validating some of their catalogue with this method.
A key player with an alternative approach
Cell Signaling Technology, a key player in the antibody space, take a slightly different approach by focusing on validation for specificity in each application independently. They use an adapted version of the pillars of antibody validation outlined above, which includes additional strategies. They have coined their approach the six ‘Hallmarks of Antibody Validation’, and a significant portion of their antibodies have been validated using multiple, complementary combinations of strategies from this approach.
Their frequent use of multiple pillars or ‘Hallmarks’ for assay-specific validation of their antibodies means a majority of their products have been validated using the definitions of this blog. However they do not use the same terminology, and their data have not been included for this reason.
CST assert that specificity and functionality of an antibody can only be determined when the antibody is used in the context of its intended application. In addition, they do not assume that validation in one application suggests specificity and functionality in another, which is why they perform each application validation independently of the others. CST will only recommend an antibody for an application if it has been validated in that specific application, and are willing to share with their customers available data that indicate why an application is, or is not recommended for a given antibody.
We will return to the CST approach in a future piece looking at antibody validation strategies in more detail, so do watch for this.
Further Validation Initiatives not included in this analysis
Validation initiatives mentioned in this blog do not cover all supplier initiatives out there. However, we are constantly expanding and exploring our data on validations and we will be delving into more detail on aspects of validation in the coming months, with the aim of highlighting progress in the industry.
An example of a supplier with significant validation initiatives which combine many of the strategies outlined above, but have not been mentioned in our data analysis, is Millipore Sigma.
Millipore Sigma use techniques such as orthogonal and genetic strategies – reported in our data to be highly used in supplier validation initiatives. They also make use of the remaining ‘pillars of antibody validation’ to validate their catalogue.
Outside of supplier in-house enhanced validation, we are also excited about initiatives led by alternative companies. A prime example of the kind of innovative work being undertaken is from YCharos, a not-for-profit with the aim of characterising every commercially available antibody against the human proteome. Their initiative follows open science principles, meaning the benefits will be available to the life science community easily.
Know of any further initiatives of interest? Do get in touch and let us know! We will certainly be returning to this analysis in the future, and we’re excited to see the number of supplier antibody validations increase over time.
Finally, we are really thrilled to be bringing back the International Antibody Validation Meeting to Bath next year, from the 13th-15th September. If you are interested in validation, whether from an academic, pharmaceutical or supplier background, do look out for when registration opens in the new year!
- Rhys and the CiteAb team
- Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., Rimm, D.L., Rodriguez, H., Hiltke, T., Snyder, M. and Yamamoto, T. (2016). A proposal for validation of antibodies. Nature Methods, 13(10), pp.823–827. doi:10.1038/nmeth.3995.