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Antibody Validation

 

Some general considerations on antibodies and their validation

Antibodies can be powerful diagnostic and research tools to detect, isolate, or visualize their corresponding antigens in many different experimental setups such as western blotting, immunocytochemistry, immunohistochemistry, immunoprecipitation, ELISA, and more. In many cases, the antigen is a protein, but it can also be a chemical modification or almost any other small structure or compound. 

 

Antibodies long had the reputation of a magic bullet, and thousands of researchers still use them every day to identify and isolate proteins in a large variety of applications. But in the last few years, the fame of antibodies has become tarnished: cross-reactivity and variability of the antibodies are the main culprits. The "Reproducibility Crisis" or "Antibody Crisis" are two names for this phenomenon.
We at Synaptic Systems aim to characterize and validate our products as best as possible to provide antibodies of highest quality and reliability. All our antibodies are produced in-house and therefore we have full control over batch testing and quality control. To date nearly one fifth of our antibodies are Knock-out or Knock-down validated. We strive to raise this number, so if you have a Knock-out model for one of our antibodies which has not been tested yet, please contact us at marketing1@sysy.com.

The quality of an antibody-based reagent mainly depends on two major properties: affinity and specificity.
Affinity measures the strength of an interaction between an epitope and an antibody's antigen binding site. The higher the affinity of an antibody, the higher the sensitivity of an antibody-based assay. Specificity measures the degree to which an antibody differentiates between different antigens. This implies that even the best antibodies have a detection limit or may show false positive results in extreme situations. If, for example, the target antigen of a highly specific and sensitive antibody is very rare in the sample to be analyzed, the sample amount is often increased and the antibody is used at higher concentration to amplify the signal. If another target is present in the sample that is weakly bound by this antibody, the unfavorably high concentrations of this off-target and the antibody itself can then result in unspecific binding and false positive results. 

Different experimental approaches require different antibody properties since the antigen is presented in different ways. The antigen may be a rather linear stretch of amino-acids in western blot detection after denaturing SDS-PAGE, or a more or less native structure in immunoisolation of cells, immunoprecipitation, or ELISA-based approaches. In immunostainings, cross-linked, chemically modified structures in fixed cells or tissues have to be detected. This means that one antibody may show excellent performance for one application but completely fail in another. This also means that specificity in one assay does not guarantee specificity in a different application.
Validation has to be carried out for all applications independently.

 

What validation methods are available and what do they tell us?

Omitting the primary antibody

This is a useful and important control to test for potential background caused by the secondary reagent. However, this experiment gives no information about the quality of the primary antibody.

Signal of the antibody is in accordance with literature data

The observed molecular weight in WB, the tissue distribution, and the staining pattern are consistent with the literature. This validation method is easy to perform but has also some restrictions; e.g. the right molecular weight of a band in WB does not necessarily tell you that the band is the desired target protein. There are easily hundreds of proteins running at the same velocity in a WB that can lead to misinterpretation of the result. The data in the literature can also be incorrect or incomplete.

Blocking/pre-adsorption of the antibody with the immunogen

Especially for polyclonal serum, this is a useful experiment to determine if an observed signal is related to the immunized antigen. If a signal disappears after pre-adsorption, the signal has a high probability of being specific. However, the possibility of cross-reactivity to other proteins sharing a similar antibody binding epitope cannot be excluded (suggested protocol for pre-adsorption can be found here).                   

 

Fig. 1: Detection of Chil3/YM1 with polyclonal Guinea pig antibody cat. no. 443 004 in mouse spleen homogenate. Lane 1: Chil3/YM1 antibody (dilution, 1:1000). Lane 2: Chil3/YM1 antibody (dilution, 1:1000) blocked with 5 µg immunogen.

Fig. 1: Detection of Chil3/YM1 with polyclonal Guinea pig antibody cat. no. 443 004 in mouse spleen homogenate. Lane 1: Chil3/YM1 antibody (dilution, 1:1000). Lane 2: Chil3/YM1 antibody (dilution, 1:1000) blocked with 5 µg immunogen.

Transfection of cells to overexpress the target antigen

Overexpressing the target protein in a cell-line that exhibits only weak or no endogenous expression can give a basic idea of the specificity and also sensitivity of an antibody.
Reactivity with different isoforms of the same protein can easily be assessed by this approach. However, artificially high levels of the target protein generally do not reflect the endogenous expression level. A clean signal of expected size or localization in tissue or cell lysate that is known to contain the protein of interest in combination with a positive result in an overexpressing system is a good indicator for specificity.

KO-cells or tissue

The gold standard to test the specificity of an antibody. A loss of signal in the KO compared to the wildtype is the most reliable proof for specificity. Please keep in mind that a successful KO validation in a western blot does not guarantee specificity in other applications such as immunostaining. Unfortunately KO-cells or tissue are not always available. We try to KO-validate as many of our antibodies as possible. If you have access to a KO system for a target we have not validated yet, please contact us.    

KD-cells or tissue

The knock down (KD) of a target can also tell you a lot about specificity of an antibody. Downregulation of a protein via RNAi is often faster and easier to accomplish compared to a KO. In accordance with the reduced mRNA levels, the signal or staining should be reduced in the KD model compared to the control system. Sometimes the KD is not very efficient and interpretation of the results can be difficult.

Fig. 2: Indirect immunostaining of PFA fixed wild-type (WT) and knock-out (KO) mouse brain sections with Guinea pig polyclonal anti-LAMP5 (cat. no.: 412 005, dilution 1:500; red). Nuclei have been visualized by DAPI staining (blue).

Courtesy: WT and KO brains were kindly provided by M.-C. Tiveron and H. Cremer, Developmental Biology Institute of Marseille, Marseille, France.

Fig. 2: Indirect immunostaining of PFA fixed wild-type (WT) and knock-out (KO) mouse brain sections with Guinea pig polyclonal anti-LAMP5 (cat. no.: 412 005, dilution 1:500; red). Nuclei have been visualized by DAPI staining (blue).

Courtesy: WT and KO brains were kindly provided by M.-C. Tiveron and H. Cremer, Developmental Biology Institute of Marseille, Marseille, France.

Fig 3: Detection of HSP90 alpha and beta with rabbit polyclonal anti-HSP90 alpha (cat. no.: 380 003) and polyclonal rabbit anti-HSP90 beta (cat. no.: 380 103) in wt and KD HeLaT-Rex cells.

Courtesy: Silvia Dalba and Prof. Stephen High, Faculty of Life Science, University of Manchester

Fig 3: Detection of HSP90 alpha and beta with rabbit polyclonal anti-HSP90 alpha (cat. no.: 380 003) and polyclonal rabbit anti-HSP90 beta (cat. no.: 380 103) in wt and KD HeLaT-Rex cells.

Courtesy: Silvia Dalba and Prof. Stephen High, Faculty of Life Science, University of Manchester

IP/MS validation for IP or ELISA applications

Immunoprecipitation (IP) with subsequent protein identification via Mass Spectrometry (MS) is a powerful tool to analyze the antibody specificity for IP or ELISA applications. However, this approach does not tell you a lot about the antibody specificity in other applications like westernblot or immunostaining.

Antibodies against different epitopes of the same target

This validation method can contribute valuable data regarding the specificity of an antibody, especially in immunostaining or western blot applications. A matching staining pattern that is obtained with antibodies against the same target but with different epitopes gives good evidence for its specificity. It is unlikely that different antibodies that bind different parts of the target protein will have the same unspecific binding partners.
 

Know your antibody better - What do the different applications tell us about antibodies

Western Blot (WB)

In standard western blot approaches, denatured protein samples are separated by their molecular weight with SDS-PAGE and transferred to a membrane. Western Blot gives very valuable molecular weight information that can be compared to theoretical calculations and published data.
The analysis of different organs, cell-types, and subcellular fractions like membranes, versus cytosol or different organelles may also provide useful information about antibody specificity. However, this necessary background information about differential expression patterns is not always available.

Immunocytochemistry (ICC)

This method provides no information about the molecular weight of a protein. It gives insight into the sub-cellular localization of the target protein which can be compared to the literature or verified by co-staining with established antibodies positive for the compartment of interest. The use of different cell systems that have different expression levels or patterns of the target protein can be very helpful in analyzing specificity.

Immunohistochemistry (IHC)

Immunohistochemistry or Immunohistochemistry on paraffin-embedded samples (IHC-P) gives insight into the tissue specificity and cellular and subcellular expression patterns of the target protein. Again no molecular weight information is provided. Comparison to the literature, in-situ hybridization data or co-staining with an established antibody positive for cell-type or tissue compartment is mandatory. 

IP/ELISA applications

In these applications, the antibody must be able to detect the target molecule in its native conformation with high specificity.
Protein complexes may still be intact in the analyte leading to a masking of the antibody epitopes. Since these approaches are very different from denaturing SDS-PAGE, the resulting specificity data are difficult to compare.
However, the analysis of an immunoprecipitate with an antibody established for western blotting can provide additional information about the specificity of the IP.

Conclusion

If you have a KO model, the validation of an antibody is very straightforward and easy. If this gold standard is not available, as much information and validation data as possible has to be gathered about the target and the antibody itself to generate the highest probability of specificity.