Antibody Validation – How do we confirm the specificity of an antibody?

May 12, 2018

One common question that scientists always ask when they get the signal from an antibody based assay is: "Does this signal truly represent the presence and quantity of my protein?"

Antibody specificity is always a concern for scientists. Even though there are a number of validation methods that have been used for the specificity testing, the data is often over-interpreted. Research data from various groups have shown some widely used monoclonal antibodies on the market are actually not mono-specific. They can cross-react with other proteins. And the cross-reactivity may mislead researchers with false positive results, and potentially cause unexpected side effects and false diagnostic reports for clinicians.

So what are the controls we should use for the validation of antibody specificity?

Cell lines and tissues that express the target proteins in high amounts can be used as positive controls for antibody validation. It can be endogenous protein or overexpressed protein encoded by a cDNA clone. However, positive control won’t be able to confirm the specificity of the antibody. You can see a band at the correct size or positive staining in the right subcellular location, but all of them could be false positive signals from the cross-reactivity of antibody. So normally for antibody validation, a negative control is required for the assessment of non-specific binding.

Knockout validation is so far the best negative control for the assessment of antibody specificity. In knockout validation, the antibody is tested on a knockout cell line which doesn't express the target protein. Starting from 2017, OriGene has teamed up with EdiGene, a CRISPR innovator, to produce double-knockout cells in a high-throughput manner. Unlike the knockout cell line from Horizon, OriGene knockout cell lines are from the commonly used cell lines, such as HEK293T or HeLa, making the product more relevant for researchers. In this double knockout cell line, the antibody target is not present as the gene encoding the protein is eliminated or "knocked out". Samples from knockout cells and parental cells (wild type) are tested side-by-side against the same antibody, and if the antibody is truly specific, it should only detect the specific signal in wild type cell but not in the knockout cell line. Using the lysates from these knockout cells, we have validated more than 100 monoclonal antibodies ( KO validated antibodies). Knockout validation is believed to offer a true negative control for antibody specificity testing.

But is knockout validation enough to confirm the mono-specificity of an antibody? I would hold this conclusion with caution. First of all, the off target effects of knockout cells are not well studied at this stage. So you could have a knockout cell not only eliminate the expression of your protein of interest, but also eliminate or reduce the expression of another protein, that may be a protein cross-reacting with the antibody. So the negative signal doesn’t always mean antibody won’t react with any other proteins except for the protein of interest. Secondly, testing with knockout cell/tissue is only possible for the non-essential proteins. For the essential proteins, knockout is impossible. So for these proteins, you will need another way to test the specificity of the antibody.

Several years ago, OriGene developed a protein array method for antibody specificity testing. With the world’s largest collection of overexpression lysates, we have made a unique protein chip that contains >10k over-expressed human proteins in duplicate on a single nitrocellulose coated glass slide. This protein microarray technology has been used to validate the specificity of many OriGene UltraMAB® monoclonal antibodies. By increasing the protein numbers on the chip, the cross-reactivity of an antibody can be further studied across the whole human proteome.

In summary, antibody specificity testing is rather complicated. It needs to be validated through multiple tools and directions. As technology continues to advance, we will have more valuable assets and standardized procedures for antibody validation. Scientists will not face the same question when they get positive signals from their antibody based assays.