In this new era in which research antibodies are under scrutiny for their quality and origin (1,2,3), the challenge of finding the right antibody, meeting the precise criteria required by the scientist, is not getting any simpler. In addition to the larger vendors who have stepped up to the plate and started culling poor quality antibodies, improved search facilities in their own websites and started introduction of extensive validation data for their best products, there has been a surge of emerging consultation websites to assist in the search for the right antibody. Most of these consultation websites do not show bias towards certain brands, and each have their own quality criteria of ranking. iSpyBio has taken the initiative to scrutinize 12 of such consultation websites and made an inventory of the benefits for their users. In addition, a case study focusses on the choice of three different websites, each with a different set of selection criteria.

The first observation to be made is that these consultation websites apply their own principles, thus making many unique from the others. For the scientists, this means that for a thorough search they may have to use several different websites to get a better idea of what antibodies of their interest are on the market. For example, CiteAb uniquely ranks the antibodies based on the number of publications the product has been described in, while Seekquence (and also BenchSci, which is not included in this study) look at the actual data inside these papers. However, OneWorldLab and AbYss concentrate exclusively to original manufacturers, thus limiting the search based on publications as many of their listed products would have been described in many scientific papers as different brands (4) and thus are less known for their original brand. Then, one may like to choose an antibody that has been successfully tested by other scientists before, but does not have the benefit (yet) of publications. Antibody review websites such like PabMabs and AntyBuddy would provide such services. However, they depend on scientists for their submission of antibody reviews, so their list may be limited to what has been submitted. Biocompare is by far the biggest consultation site, as it has most commercial antibodies listed (in contrast to other websites). Its attractive feature is the ability to directly compare selected products side-by- side so to judge price, formulation and performance. This practical feature is also available at Antibody Resource, Ab-Y- ss, iSpyBio and SeekQuence. Finally, OneWorldLab and Antibody Resource offer the option of smaller aliquots to find out at lower cost which one to buy at full size.

The second observation to be made is that none of the consultation websites give definitive answers as to which commercially available antibody out of 100s, if not 1000s of products all against the same protein, is guaranteed going to work according to the scientist's demands. Hence, a choice between a set of candidates narrowed down from multiple consultations, will have to be made by further experimenting in the lab. The efforts of going through this process of narrowing down the best candidates to a handful can be daunting though, despite the available consultation websites. Aeonian Biotech provides custom-tailored service to assist in that process, while focussing on antibodies from the original manufacturer for best price and for long-term consistency between purchases. Not only scientists in an academic environment, but also assay developers who often rely on different antibody sources may find the Aeonian services more cost-effective than going through the search process themselves.

The third observation to be made is regarding the extra features some of the websites offer: they are extremely attractive to scientists for various reasons. These features are worth discussing in more detail as they benefit both academic and commercial users

Website features

The most common and popular features are:

• a search box and search filters to assist narrowing down the candidates of request. Most allow narrowing down based on host species, application, and species reactivity.
• The list of sponsors/business partners help to decide how wide-spread the search is going to be.
• Sign-up option helps to communicate effectively between provider and customer

Slightly less common, but very often used features are:

• A clear submit button for feedback or upload
• Independent antibody reviews with rating
• Complete index
• Out-link to vendor's product sheet
• Product comparison side-by-side (QC data, product formulation and price)
• Blogs and videos
• A fixed feedback-form
• News and events

On the other scale of the spectrum are the unique features shown by only one of the scrutinized websites. Some of them are extremely interesting and might very well be incentive for scientists to come back for. Examples are:

• antibody listed together with recombinant protein when available (OneWorldLab)
• Trial size "superstar"ter kits (Antibody Resource)
• Direct online chat with the provider (OneWorldLab)
• Toolbox (dilution & mass molarity) / online computations (CiteAb)
• List of custom antibody services (CiteAb)
• Useful links to educational sites, suppliers, books, databases, software, etc. (Antibody Resource)
• Discuss/Disqus (Biocompare)

Providing discussion facilities became increasingly popular since both newspaper websites and multimedia like Facebook initiated them. Professional portals like LinkedIn and ResearchGate demonstrate the demand from the scientific world to exchange ideas and thoughts on science and their ethics. Considering the success of Biocompare, CiteAb and PabMabs, others are following to provide scientist's feedback either in the shape of data sharing or in the shape of discussions. Ideally the two are being combined. We would like to see discussions on unexpected data obtained by scientists who are not sure what to think about the obtained results. Usually such data are meaningful but never get published until someone else re-discovers it and gets the honour.

Filters are essential for a properly focussed search. The three websites in the case study below all benefit from allowing the user to focus in to species reactivity, clonality, host species and applications. We have chosen to pick Biocompare, CiteAb and iSpyBio because each use complete different selection criteria: Biocompare on price, CiteAb on references and iSpyBio on the sum of four quality criteria: #QC images on product sheet, #application, #publications and #customer's reviews.

We will have a look at AIF1, also known as IBA1. This is a marker used to monitor activated microglia, although it is also expressed in macrophages and neutrophils. Because of its widespread use and high demand, we will have a look how the best unconjugated(!) antibodies can be found when using Biocompare, CiteAb, and iSpyBio. We will identify the highest-ranking products, and compare the QC data on their product sheets, their formulation, and prices. We may even be able to identify the original manufacturer when applicable.

At the time of writing Biocompare returned 450 different products across 34 suppliers when AIF1 was entered. Because this antibody is mainly used as a CNS marker, we will narrow down this search for IHC on rodents. This brings the list down to 176 different products across 22 suppliers.

Let's first have a look at monoclonal antibodies: This filter narrows our search further down to 21 mouse monoclonal antibodies (mabs), 3 rabbit mabs and 1 human mab. We prefer rabbit mabs over mouse mabs, because we use the antibody for rodent brain tissue. All three turn out Abcam's EPR clones (Epitomics Rabmabs, original manufacturer). However, one of them is only fit for human, so the filter did not work entirely correct. The two antibodies we have reasonably quickly identified are EPR16589 (40ul) and EPR6136(2) (100ul). Surprisingly, neither have been rated by Biocompare, although the latter is reported having one citation, while the former is reported having none. Going to the Abcam website, we find that EPR16589 (ab178847) is available as a 100ul unit (not as 40ul as reported by Biocompare) being 63.7ug protein A-purified IgG at £259. A trial size sample of 10ul being 53.8-63.7ug IgG at £105. The product shows nicely stained microglia on rat cerebellum with inserted negative control data of secondary only. It also shows staining of distinct cells in mouse endometrium being captioned as macrophages, again with negative control insert. Western blots show consistent clean 16kDa in human, mouse and rat spleen lysates. In contrast to Biocompare's indication, the datasheet indicated two citations for this antibody, both papers on human brain though. The product EPR6136(2) (ab178620) comes as a tissue culture supernatant (hybridoma-conditioned culture media). A vial of 100ul is priced £275 and a vial of 10ul costs £109. Although the product sheet shows many data (including many IHC pictures) the results are not as convincing as the former product, despite one citation. So, in a very easy way, Biocompare has identified us with one perfect rabbit mab for IHC work on mouse and rat brain from a potential pool of 450 different products. And it has been proven to work on human brain by citations. Should the scientist need the antibody on human samples but rabbit antibody is already used for another marker in a double labelling experiment, we can search for rodent unconjugated mabs with human reactivity. After clicking on the tab “product view”, we get the list of 32 products across 14 suppliers, but the ranking is unclear as none of them have any stars. The top 4 (one Cloud-Clone, one LifeSpan, and two Antibodies-online products) are listed in pink and separated from the rest of the list by a yellow bar. This suggests priority listing based on sponsorship. The first product underneath this short list is Biorbyt antibody orb89047. It is offered as a 50ug affinity purified IgG unit for £370 and it shows IHC data on human spleen. However, the clone code is not mentioned. The top short-listed Cloud-Clone MAC288Hu22 only shows a 23kDa band in a Western blot with just recombinant protein, although claimed fit for IHC. The price is £290 for 200ug antigen-affinity purified IgG. The second short-listed LifeSpan LS-C96253 (clone 1022-5) shows IHC data on human spleen and costs $870 for 200ug affinity purified IgG. This product might, or might not be identical to the Biorbyt product mentioned above. The third short-listed antibody is Antibodies-online ABIN1175627. One must login to have access to any details, or have an online chat with a sales person. This product is a rebranded Cloud-Clone product. Despite some complications around rebranding, the search for a proper monoclonal antibody has been relatively easy and quick with the identification of the recombinant rabbit antibody clone EPR16589, and mouse clone 1022-5 (the latter to be confirmed by other searches below).

Let's have a look at the polyclonal antibodies (Pabs). We start again with a search for reactivity on rodents in IHC. Biocompare returned, under tab “product view”, 148 different products across 22 suppliers. There was no scope to further narrowing down in a meaningful way. So, Biocompare identified 102 rabbit pabs, 37 goat pabs, and some bird, rodent and sheep individual products. The short-listed top 4 (sponsored) were three Cloud-Clone products followed by one LifeSpan product. They are all rabbit antigen-affinity purified IgG. The first is PAC288Ra01, specific to rat at $380 for 100ug IgG, the second and third PAC288Mu01 and PAC288Mu02, specific to mouse at $360 for 100ug IgG, all with unconvincing IHC data. The LifeSpan rat-specific product is not that much better with odd IHC data on human (!) tonsil. The top two underneath the short-list are one Aviva BioSys and one Biomatik product, followed by six different Biorbyt products. The Aviva OAGA01616 is a rabbit antigen-affinity purified pab priced $289 for 100ug. It has IHC data on breast cancer tissue and 15-16kDa band in undefined tissue extracts. The Biomatik CAE02146 is an antigen-affinity purified goat pab, specific for isoform 3, priced £363 for 100ug. However, this product is manufactured by a different party (see below), and it is better known under larger brands (including Biorbyt) with QC data and literature citations and customer reviews. The Biorbyt antibodies are either rabbit or goat pabs, some of them sourced from different manufacturers. So, we see that Biocompare lets us down a bit when we cannot narrow down our search to under five real-different candidates and in addition to poor short-listed products, we hit identical products occurring under different brands with different prices and different data on their product sheets.

A search of IBA1 resulted in almost the same products, but with a higher variety of brands.

Biocompare is a good start for a search, especially when 100s products are on offer. However, its filter system is not always capable to narrow down further when extra stringency is required.

CiteAb lists 89 AIF1 antibodies. As discussed, CiteAb lists according to the number of literature citations. The winner is ProteinTech 10904-1-AP with 49 citations. It is a rabbit pab costing $279 for 47ug antigen-affinity purified IgG. Its datasheet shows proper IHC staining on human brain sections. The second place with 6 citations is for Synaptic Systems 234-003. This is another rabbit product, with on the product sheet 10 references, and no QC data. It costs 310 Euro for 50ug affinity purified IgG. Following products are two GeneTex rabbit pabs with 5 and 4 citations and a BIOSS rabbit pab with 3 citations. After that, goat (rebranded Everest products) and more rabbit products with one citation. Monoclonal antibodies are first found on the second page with zero citations.

We repeated the exercise, this time we entered IBA1. Now, CiteAb lists 374 IBA1 antibodies. This time, the top antibody with 155 citations is Abcam's goat pab ab5076 (121 citation on its product sheet, see above). The second with 49 citations is Proteintech's rabbit pab 10904-1-AP, also the winner in the AIF1 search. Third with 41 citations is Novus goat pab NB100-1028, which is identical to ab5076, but it was listed with 46 citations as an AIF1 antibody (see above). As number four with 36 citations, we find our first mab from Abcam: Ab15690 (clone 1022-5), followed with 22 citations by Santa Cruz SC-32735 with the same clone 1022-5. Abcam's Ab15690 costs £279 for 100ug IgG fraction with data from the LifeSpan LS-C96253 product sheet, and Santa Cruz's SC-32735 costs $279 for 200ug IgG with IHC on rat C6 glioblastoma. It is worth mentioning that the following two products in the list are Abcam's goat pab Ab107159 raised against the C-terminal 100aa of rat IBA1 with 21 citations, and Everest Biotech's EB05419 with 14 citations. The latter has been discussed above as rebranded Ab5076 and NB100-1028. This Everest product features in many catalogues as many brands.

So, it turns out that when a certain protein has different names, CiteAb will return different results based on the name entered. For example, the AIF1 search did not find Abcam's goat pab ab5076 with 121 citations, nor its Novus (NB100-1028) version that was cited in 46 papers, both pulled up in the IBA1 search. This same rebranded antibody will come back with different citations dependent on which brand it carries. Price of this antibody can vary dramatically between the different brands. One product was not found at all: rabbit pab Wako #019-19741 is possibly the most used AIF1/IBA1 product in the world: a search of WAKO 019-19741 in Google Scholar returned 1930 results! Also the two Abcam mabs EPR16589 and EPR6136(2) identified by Biocompare, each having citations, could not be found by CiteAb.

When we ask for AIF1 unconjugated antibodies, we get 14 results. When we ask for IBA1, we get 15 results. Its ranking is based on the quality of the antibody measured by the numbers of images, applications, publications, and reviews.

Thus, searching for AIF1, the top product listed is ProteinTech's rabbit pab 10904-1-AP, which also came out as the top one in CiteAb when searched for AIF1. It became top item based on 4 images, 5 applications, 10 publications (CiteAb listed 49), and zero reviews. Runner up, with just 4 images and 3 applications, is Biorbyt's goat pab Orb19198, specific for isoform 3. It costs £250 for 100ug antigen-affinity purified IgG. The second runner up, with 4 images and 2 applications, is Millipore's mouse mab MABN92, costing £256 for 100ug purified IgG. All come with proper IHC data and WB data. Finally, it is worth noticing that the following three products in this list are all goat pabs coming from the same manufacturer (Everest Biotech): they are ProSci 45-220 at $350 for 100ug and Abgent's AF1039b at price to be enquired for 100ug, both isoform 3-specific and identical to Orb19198, and Bio-Rad's AHP2024 against the C-terminus costing £300 for 100ug. The latter is better known as rebranded Ab5076 and NB100-1028 (see above).

When searching for IBA1, the top product listed is Novus's NB100-1028, with 5 images, 6 applications, 26 publications (CiteAb IBA1 search: third position with 41 publications) and 4 reviews. The runner up is Proteintech's 10904-1-AP (winner in the AIF1 search), followed subsequently by Biorbyt Orb19198 (second in AIF1 search) and Millipore's MABN92 (third in AIF1 search).

When searching for the best IAF1/IBA1 antibody, there is some consistency across the last two tested consultation websites in finding the same strongest candidate antibodies. But there were also some differences. Apart from finding different products based on different quality criteria, the most important factor if complexity if the rebranding. Only when fully aware of this, and when the scientist is capable of recognizing the rebranded products as the same, will a search result in a good variety of candidates, regardless which one is used. However, certain products are identified by one search website and not by the others. And because many proteins have different names, one has to enter the different names in the same search in order to fish out your favourite candidates. Here follow the strongest products (three mabs and three pabs) we have identified making use of the three websites:

Literature

1. Uhlen et al. A proposal for validation of antibodies. Nat Methods. 2016 Oct;13(10):823-7.
2. Weller MG. Quality Issues of Research Antibodies. Anal Chem Insights. 2016 Mar 20;11:21-7.
3. Voskuil JL. The challenges with the validation of research antibodies. F1000Res. 2017 Feb 17;6:161
4. Voskuil J. Commercial antibodies and their validation. Version 2. F1000Res. 2014 Oct 2 [revised 2014 Oct 15];3:232