How Do You Validate Antibody Specificity?

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One of the common questions about antibody production is to validate and confirm the specificity of an antibody. This is because the usefulness of antibodies in different essays depends on the specificity of epitome binding. Only antibodies that pass the minimum criteria of standard antibody validation should be published. 

Recently, there have been concerns that antibodies are not specific enough for the intended use and can show cross-reactivity with off-target proteins. Antibodies specificity measures the goodness of fit between the antibody and the corresponding antigenic determinant. The specificity measures an antibody’s ability to differentiate between foreign antigens. 

The antibody validation comprises several components. These components include proving affinity, demonstrating specificity, and demonstrating reproducibility. Here are the different ways you can validate and detect antibody specificity during custom protein expression.

Genetic strategies

You can determine antibody specificity using the cells or tissues that do not express the target protein of the antibody. The number of techniques you can generate as part of the antibody validation process includes CRISPR-Cas9-9 and RNAi. The CRISPR-Cas9-9 is popular among researchers because it’s cheaper and more accurate. You can also use an alternative approach to remove the target gene through RNAi techniques.

You can assess antibody specificity by comparing binding signals in cells expressing the target protein. The knockout cell lines provide the most direct route to high antibody specificity. The target gene is knocked-out using genetic methods such as RNA interference. This method can reduce the expression of the target protein. However, this knockout method can alter the DNA to prevent the target protein from being expressed.

Orthogonal strategies

This strategy for antibody specificity validation involves cross-referencing antibody-based results with data obtained using non-antibody-based methods. It’s a vital method to verify existing antibody validation data and identify any effect directly related to the antibody in question. In most cases, the orthogonal strategy uses data available in the public domain.

In its simplest form, the strategy dictates that the results obtained in other hallmarks require collaboration by non-antibody-based detection methods. It involves assessing the target protein abundance using an antibody-independent essay. You then compare the results with those obtained using antibodies across a range of relevant samples. 

The non-antibody-based methods can include mining previously published results or studying expression analysis via omics techniques. You can also employ established antibody-independent methods such as in situ hybridization or RNA sequencing. It is a quick method because of high throughput analytical methods. 

Independent antibody strategies

Another strategy involves assessing the antibody binding of two independent antibodies to improve the specificity. The antibodies are validated using independent antibody strategies with individual antibodies scored based on the similarity of staining with its sibling antibodies.

Using two independent antibodies for the same protein target is valuable when testing antibody specificity. This is where you use antibodies that target non-overlapping epitopes of an antigen. You then obtain comparable results from antibodies that recognize independent regions of the same target protein. The result allows you increased confidence that the antibodies are specific and suitable for detecting the intended target. 

You can use this method to validate Invitrogen antibodies for research use. While this strategy is theoretically straightforward, it’s challenging to predict since results vary from sample to sample.

Expression of tagged proteins

This method involves using a tagged protein as a standard for immunocytochemistry or western blotting comparison. You can evaluate antibody specificity by comparing antibody labeling with an expression of a tagged version of the target protein from the endogenous locus. If the distribution of the tagged protein overlaps with the immunofluorescence signal, then you validate the antibody specificity. 

Use this strategy to determine antibody specificity by comparing the signal from the antibody to the tag-specific signal. You analyze the antibodies targeting a subset of genes in cells stably expressing a tagged protein. We use an antibody to enhance the signal and detect low abundant tagged target proteins.

You can calculate the expression level by measuring the transcribed mRNA, the expressed protein, or directly staining the protein when it’s still in the cell. However, it requires additional time and skill to express a tagged protein successfully. 

Immunoprecipitation-mass spectrometry

This method involves isolating and analyzing all proteins bound by an antibody using Immunoprecipitation. You then follow mass spectrometry to reveal the target protein and any off-target binding. It detects proteins that interact directly with the antibody and form a complex with the directly-bound target.

The mass spectrometric approaches for antibody target verification can identify the antibody targets and ISO forms. This method focuses on detecting proteins that bind the antibody being evaluated. 

It uses defined biological or chemical modulation of protein expression to validate the antibody specificity of the target protein. You then compare the data across multiple cell lines, including positive and negative expressing cells.

However, not all antibodies and targets are suitable for Immunoprecipitation. The results of antibody specificity will vary depending on the relative abundance and binding strength of the target protein. 

Bottom line

Antibodies are used in a broad range of research and diagnostic applications. However, you should accurately detect the antibody of interest even at low expression levels to achieve reproducible results. There are several ways you can use to determine antibody specificity. These strategies will verify the capture of antibody targets and estimate relative antibody performance for a specific target.

Validate antibody specificity in the particular technique used by the antibody. This is because an antibody validated by Western blot will perform differently in immunocytochemistry. The validation of an antibiotic happens across different applications. The proof of antibody specificity is a continuous process that uses different strategies.

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