Authors: Shakun Sinha, Ishtapran Sahoo, Priyanka Swamynathan and Aparna Chandrasekaran

Researchers widely use antibodies due to their capability of detecting proteins, monitoring their expression levels or tracking their dynamics in cells or tissues. The ability of the antibody to bind to the right target with minimal cross reactivity is crucial to any experimental outcome. Poor specificity and inconsistent performance can lead to unclear or incorrect results and scientific assumptions as well as loss of valuable research time and funds. It becomes important, therefore, for antibody suppliers like Thermo Fisher Scientific to provide quality antibodies that customers can rely on for consistency and reproducibility.

The Invitrogen antibody portfolio offers a broad range of options for nearly all areas of biology and with application coverage across workflows like Western blot (WB), immunocytochemistry (ICC), immunohistochemistry (IHC), flow cytometry (FC), Chromatin IP (ChIP) and ELISA. Alongside standard testing for use in such applications, many of the antibodies undergo further advanced verification tests that help ensure they bind specifically to the target protein. Given the breadth of the antibody portfolio, it is critical to have a comprehensive approach to validating antibodies, that relies on the target protein’s biology, function, and stability, rather than relying on a single testing method.

CiteAb, an independent reviewer of life science data, has showcased Thermo Fisher’s dedication to verifying antibody specificity. In CiteAb’s 2022 analysis of supplier antibody validation, Thermo Fisher emerged as an industry leader, having validated over 11,000 antibodies for enhanced specificity validation. This blog provides a high-level insight into the different strategies used for advanced verification of antibodies.

Strategies for Antibody Validation

With a complex antibody portfolio covering >90% of the proteome, there arises a need to employ multiple testing methods to verify antibody specificity as no single method will work for all antibodies. The method(s) chosen to verify specificity for a target protein can depend on its biological function, sub-cellular localization, gene essentiality, rate of turnover and other factors. Thermo Fisher uses one or more of the strategies listed below to advance verify their antibodies:

Antibody validation using CRISPR-Cas9 knockout

Genetic alterations are powerful techniques that provide a direct link between the gene, the target protein, and its detection by the antibody. This strategy establishes antibody specificity through CRISPR-Cas9 mediated knock-out models, that delete or permanently deactivate a gene of interest, thereby leading to a loss of expression of the target protein. This technique allows for clear, precise, and unambiguous assessment of antibody quality, making it one of the most preferred strategies.

Figure 1 demonstrates knock-out validated antibodies for use in applications like western blot, immunocytochemistry, and flow cytometry.

Figure 1 – Demonstration of antibody specificity using CRISPR-Cas9 mediated knockout of target protein.

(A) Transferrin knockout cell line was produced using LentiArray™ Lentiviral sgRNA (Cat. No. A32042, Assay ID CRISPR944258_LV). A Transferrin Receptor Monoclonal Antibody (10F11) (Cat. No. MA5-11441) was tested in Western blot for specificity using the Transferrin knockout cell line along with appropriate controls. The presence of bands in control lanes 1 and 2 and the absence of a band in lane 3 demonstrate antibody specificity.

(B) Knockout of EpCAM was obtained using LentiArray™ Lentiviral sgRNA (Cat. No. A32042, Assay ID CRISPR701274_LV) in A431 cells which were subsequently used to test a CD326 (EpCAM) Monoclonal Antibody (1B7), eBioscience™ (Cat. No. 14-9326-82), for specificity in immunocytochemistry.

(C) Loss of signal was observed in CD19 knockout cells (blue histogram) when compared to Cas9 control cells (pink histogram) in flow cytometry using CD19 Monoclonal Antibody (HIB19), PE (Cat. No. 12-0199-42), suggesting the antibody was specific to CD19.

Antibody suppliers also use alternate strategies alongside CRISPR-Cas9 knockout models to demonstrate antibody specificity, as certain limitations of this technique are widely accepted. For example, if the protein of interest has an essential function, knocking it out could compromise cell viability, leading to loss of test sample. In addition, other strategies must be considered in cases where knockout strategies cannot be used on human tissue samples or body fluids, as discussed below.

Antibody validation through siRNA-mediated knockdown

Another commonly used strategy for antibody validation is siRNA-mediated knockdown, which involves silencing mRNA to decrease protein production and establish specificity as a direct read-out of protein levels detected by the antibody. siRNA-mediated knockdown helps in examining antibody specificity for proteins that belong to multigene families.

Figure 2 demonstrates examples of antibodies verified using siRNA knockdown models. RBBP4 is an essential gene and knocking it out results in cell lethality (1). Therefore, siRNA knockdown becomes the preferred specificity verification strategy. An obvious reduction in lane 3 establishes antibody specificity to RBBP4 (panel A). In panel B, a SUMO1 knockdown in ICC exhibits how the antibody being tested is specific to only SUMO1 and not to SUMO2 or SUMO3.

Figure 2 – Antibody specificity determined using siRNA mediated knockdown of target protein

(A) We transfected HeLa cells with RBBP4-specific siRNA (Silencer® select Cat. No. # s11838, s11837) and observed a reduction in signal intensity in lane 3 compared to lanes 1 and 2 when using the antibody product RbAp48 Monoclonal Antibody (11G10) (Product # MA1-23273) in Western blot, indicating its specificity to the target protein.

(B) We transfected HepG2 cells with siRNA targeting SUMO1, SUMO2, and SUMO3. We observed a decrease in signal intensity only in the SUMO1 siRNA transfected cells upon testing with a SUMO1 Monoclonal Antibody (T.243.0) (Product # MA5-14877), demonstrating the antibody’s specificity.

Although widely used, siRNA knockdown has limited utility when the target protein has a long half-life or residence time, making it challenging to draw definitive conclusions about antibody specificity. Additionally, as with knockouts, this technique cannot be used with human tissue samples or body fluids. These challenges associated with genetic alteration are key drivers to explore and adopt alternate strategies such as relative expression and cell treatment.

Antibody validation through Relative Expression

Proteins display varying expression levels depending on the cell type. For example, proteins with house-keeping functions are expressed ubiquitously, while others are expressed in a cell-type specific manner. Relative expression strategy exploits this natural variation in protein expression across different cell or tissue models and uses this as a hallmark of specificity. Limitations of protein half-life and essential functionality can easily be addressed using this technique provided the protein is expressed uniquely across cell or tissue models. The examples shown in Figure 3 demonstrate how target biology can clearly help establish antibody specificity across WB, ICC and IHC applications.

Figure 3 – Antibody specificity demonstrated by detection of differential basal expression of the target protein.

(A) Western blot and (B) ICC using CD19 Monoclonal Antibody (6OMP31) (Cat. No. 14-0194-82) shows differential expression of CD19 in B cells (Raji, Ramos and Daudi) in comparison to other cell types (SW480, Jurkat and MOLT-4) which are null expressors of CD19.

(C) Immunohistochemistry using Cytokeratin 5 Monoclonal Antibody (SP27) (Cat. No. MA5-16372) shows differential expression of Cytokeratin 5 in Mouse Skin compared to brain tissue section, exhibiting antibody specificity.

Antibody validation through Cell treatment

Under specific conditions, chemical agents or drug molecules can alter protein expression. Researchers can utilize the change in expression pattern upon treatment with chemical agents to demonstrate antibody specificity. You can apply this strategy in multiple scenarios such as targets that require treatment with specific inducing agents leading to enrichment due to their low endogenous basal levels, targets that undergo post-translational modifications (e.g., phosphorylation) because treatment agents activate the associated kinases, and targets that translocate between cellular compartments in response to altered signaling cascades. In Figure 4A, Western blot using a Phospho-EGFR antibody shows increased levels of protein phosphorylation at the tyrosine residue upon EGF treatment, whereas pre-treatment with EGFR-antagonists, Gefitinib and Afatinib, resulted in inhibition of EGFR phosphorylation, showing antibody specificity. Similarly, we demonstrate induction of IFIT3 protein upon treatment with interferon in ICC application (panel B).

Figure 4 – Altered expression of target protein upon cell treatment demonstrates antibody specificity.

(A) Western blot using Phospho-EGFR (Tyr 1068) antibody (Cat. No. PA5-17848) shows increased expression of phospho-EGFR with EGF treatment. Pre-treatment with EGFR antagonists (Gefitinib and Afatinib) resulted in inhibition of expression of phospho-EGFR.

(B) ICC using IFIT3 Monoclonal Antibody (OTI1G1) (Cat. No. MA5-25003) shows specific detection of IFIT3 in A549 cells upon treatment with IFN alpha (500U/ml for 24 hours). Untreated A549 cells did not show expression of IFIT3.

In summary, researchers must validate the specificity of antibodies using a combination of techniques due to the inherent variability of proteins in terms of expression, function, and stability, although they play a crucial role in protein research. Depending on target biology, it is important to choose a validation strategy that provides the most confidence in an antibody product. As mentioned above, if essential genes or long protein half-life are present, researchers cannot apply strategies like knockout or knockdown. In such cases, they must consider an alternative such as relative expression or cell treatment. Reproducibility in antibody performance is essential to the progress of science and implementing rigorous testing standards such as those explained above, increase confidence in the data. Thermo Fisher demonstrates commitment to providing high-quality antibodies by utilizing one or more specificity testing strategies across multiple applications.

Additional Antibody Blogs

References:

Miao X, Sun T, Barletta H, Mager J, Cui W. Loss of RBBP4 results in defective inner cell mass, severe apoptosis, hyperacetylated histones and preimplantation lethality in mice†. Biol Reprod. 2020 Jun 23;103(1):13-23. doi: 10.1093/biolre/ioaa046. PMID: 32285100; PMCID: PMC7313262.