Antibody Validation & Quality Policy

At Hello Bio we are committed to providing antibodies of the highest possibly quality and carry out hundreds of validation experiments at our purpose-built laboratory facilities in Bristol, UK. Validation of antibodies is key to ensure that they bind only to their target in the applications as indicated on the antibody datasheet. This ensures repeatable and accurate results for researchers.

The importance of using highly validated antibodies is clear – many researchers are aware of cautionary tales from research groups such as those who spent years validating estrogen receptor beta (ERß) as a breast cancer biomarker only to subsequently discover that all the antibodies used also cross-reacted with ERα. This resulted in the discovery that ERß isn’t even expressed in breast cancer (Andersson et al., 2017)! For more information and a much deeper analysis please see our white paper on antibody validation. Antibody validation is a complex process with many factors that need to be considered and can influence the performance of an antibody.

Our Validation Principles

At Hello Bio we use the following principles to guide us when validating an antibody:

1. Validation starts from a robust understanding of the target

We analyse every target for similar proteins that may provide cross-reactivity as well as analysing every antibody’s epitope (where data available) to assess for potential cross-reactivities.
Every target is assessed for expression and matched up to samples with and without expression in our extensive tissue library.

2. Validation is done in as close as possible conditions, tissue and cell type to that intended for the experimental use of the antibody.

We assess every target for its applications and design experiments to mimic these as closely as possible.
We test all our antibodies in complex tissue samples prepared identically to those used in experiments as opposed to using purified proteins.

3. Validation is application and species specific

We test in every species that we approve an antibody for. Where we predict immunoreactivity with different species this is based upon epitope or immunogen homology.

4. Validation is integrated with the 5 pillars proposed by Ulhen et al., 2016 but we are mindful of the practical constraints between laboratories.

We design our experiments following these principles however sometimes do other experiments that we feel provide valuable information about an antibody.

5. Validation is replicable to ensure the antibody has utility

We test each antibody multiple times in the same experiment to ensure reproducibility of results.
We assess every new batch using a risk-based approach to ensure consistency between batches of antibodies.

6. Validation data is shared between user and suppliers using open science approaches

All of our validation data is available through the Open Science Framework.
We test all antibodies using our published protocols (see our WB, IHC and ICC protocols) and publish extensive details with each piece of data to enable easy replication.

7. Validation is a shared enterprise between Hello Bio and our customers

Whilst we test our antibodies rigorously in a range of applications it isn’t possible to check for every experimental configuration. Sometimes it will be necessary for researchers to perform further validation experiments in their own lab.

Western Blot

We design bespoke experiments for every target when validating an antibody for western blot. This ensures the best chance that an antibody is binding to the correct target and that any cross-reactivities can be identified. Our experiments can often be fitted into the following categories:

Differential expression: The antibody is tested against tissues known to contain the target alongside others that are known to show no expression. Only if the antibody reveals a band of the correct molecular weight in the positive tissues with no bands in the negative tissues can it pass.
Transfected proteins: The antibody is tested against cells or tissues that have been genetically modified to express the target. Only if the antibody reveals a band of the correct molecular weight in the transfected but not wild type cells/tissue can it pass.
Cell treatment: The antibody is tested against cells that have been treated pharmacologically to manipulate the expression of the target. Only if the antibody reveals a band of the correct molecular weight that responds to the treatment can it pass.
Assessing the concentration response relationship between the antibody and signal intensity using tissue known to express the target.

Immunohistochemistry

We design bespoke experiments for every target when validating an antibody for immunohistochemistry. This ensures the best chance that an antibody is binding to the correct target and that any cross-reactivities can be identified. Our experiments can often be fitted into the following categories:

Localisation: The antibody is tested in tissues known to express the target in a characteristic manner. Only if the pattern of staining is consistent with the expected pattern then the antibody can pass.
Assessing the concentration response relationship between the antibody and signal intensity using tissue known to express the target.

Immunocytochemistry

We design bespoke experiments for every target when validating an antibody for immunocytochemistry. This ensures the best chance that an antibody is binding to the correct target and that any cross-reactivities can be identified. Our experiments can often be fitted into the following categories:

Localisation: The antibody is tested in cells known to express the target in a characteristic manner. Only if the pattern of staining is consistent with the expected pattern then the antibody can pass.
Transfected proteins: The antibody is tested against cells that have been genetically modified to express the target. Only if the antibody reveals staining in a pattern consistent with the known expression of the transfected protein in the transfected but not wild type cells can the antibody pass.
Cell treatment: The antibody is tested against cells that have been treated pharmacologically to manipulate the expression of the target. Only if the antibody reveals changes in staining that responds to the treatment can it pass.
Assessing the concentration response relationship between the antibody and signal intensity using cells known to express the target.

For any further queries on our validation, please read our white paper on antibody validation or contact our technical support team at technicalhelp@hellobio.com