Introduction to selectivity and integrity

In our previous blog we introduced the Aeonian Rating® and the RRID as annotations to our released antibodies. We now like to focus a little on the quality data presented on the product sheet.

All vendors and manufacturers claim their products fit for certain applications and they often call this validation. Too often we see no such evidence. At best, we see some Western blots (WB) and/or Immunohistochemistry (IHC) on one or a few tissues or cell lines, without any indication how meaningful the staining pattern really is. For example, we can find antibodies to brain or kidney proteins “validated” in WB on HeLa and in IHC on breast cancer. Likewise, claims of antibodies fit for flow cytometry (FC) are in many cases demonstrated as a peak away from background on a single cell line. Extra efforts are appreciated when an isotype control is included. But the most amusing evidence can be found in Immunocytochemistry (ICC), aka Immunofluorescence (IF), when data show the antibody lighting up entire cells of a cell line. Logically, a strong peak away from background can be expected on the same cell line in FC in such case as well. This confirms that the antibody binds strongly to this cell line. How specific is that? Well, we wouldn’t know until compared to other cell lines or cell types from the same species with much lower levels of the protein of interest. The right controls are part of a proper validation effort. But the choice of tissue or cell line the antibody is tested on is equally important, as such experiments should confirm the staining in the right cell type or cell compartment in physiologically relevant tissues. We aim as much as we can to test antibodies in relevant cell types and tissues.

Apart from poor quality data, meant to show fitness for an assay type, in most catalogs we miss any evidence of selectivity and integrity. Selectivity means that the antibody will not bind (as strongly) to other related proteins. Selectivity can be addressed in any type of immune assay, as long as it involves the presence of the closely related other proteins. We should realize though that such comparisons are ideally applied to the assay for which the antibody is required. For example, an antibody required for native assays, such as ELISA, lateral flow and multiplex assays, may show different cross-reactivity in assays on denatured proteins, like in WB and IHC. In the latter cases, epitopes that would be masked under native conditions may become available for binding after partly or complete denaturation. Hence, cross-reactivity tested under native conditions may validate the antibodies for assays under native conditions, but we must be cautious when the antibody is used on denatured proteins. And also the other way around.

The Aeonian Antibodies show, apart from specific signals in assay types like WB and IHC, data to demonstrate molecular integrity by SDS-PAGE and Coomassie staining of the antibody under non-reducing and reducing conditions. Finally, majority of the antibodies come with protein array data to demonstrate selectivity. Thus, we demonstrate that these antibodies are fit for assays both under native and denatured conditions. Those antibodies still lacking protein array data are waiting for them to arrive shortly. Our catalogue is still very small as it takes at least an hour per antibody to generate the datasheet and the new product's web page. This is because we don’t relentlessly copy all data over from our supplier. We vet all data carefully and remove the data we are not happy with. Consequently, some products do not make it to our catalogue. Data generated by Aeonian antibodies are all manually checked and evaluated on their scientific merit. Captions are rewritten, and protein sequences blasted to identify possible related proteins antibodies might cross-react with. Such efforts make these antibodies slightly more expensive compared to their competing products, but consequently they also become more reliable for the customers. Those products with a stiff competition will be delivered to USA and Europe without delivery charges so to accommodate the scientists’ ever tighter budgets.

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