With the progress of science and medicine, antibodies have become potent tools to recognize, detect, isolate, or visualize their corresponding antigens in basic science research and clinical assays. Whether or not antibodies work in experiments and are stable and reliable is crucial for researchers, clinical users, antibody manufacturers, and journal publishers. It has been reported that ample antibody-based research failed to meet expectations. Therefore, it is necessary to carry out functional verification of antibodies to ensure their quality.
This article mainly focuses on antibody validation, which will be elaborated on several aspects, including the definition of antibody validation, the significance of antibody validation, as well as methods of antibody validation, and their comparison.
Antibody validation is the process of demonstrating if specific antibody is suitable for an intended application or purpose by using a specific laboratory investigation. It should be demonstrated to be specific, selective, and reproducible in the context where the antibody is to be used.
Antibody functionality is affected by sample immobilization, sample denaturation degree, sample preparation environment, culture conditions, and other factors. In addition, the target proteins of the same primary sequence can also show small specific differences such as post-translational modifications (e.g. glycosylation, phosphorylation), which can change their conformation and epitopes. Consequently, the antibodies acting on them will also affect their binding properties with the change of the conformation of the target protein. Therefore, antibody validation should be carried out in the context of specific application and use. All applications must be validated independently.
An non-validated antibody may bind to proteins other than their target protein thus producing false positives or may fail to bind to the target protein thus generating false negatives. These problems could lead to invalid experiment data. It is estimated that the life sciences sector loses $800 million a year due to poor antibody quality.
Antibody validation is essential for the reliability and reliability of the experimental data, that is, to confirm that the antibody specifically recognizes the target antigen and to ensure that all users can obtain the same results according to the correct experimental protocol.
The assessment of antibody validation is based on several factors, including the characterization of antibody and antigen, binding specificity and selectivity, concentrations of antibodies and additives, documentation, affinity constant, influence of non-target substances, stabilization and storage, application protocols, and user feedback [1].
Antibody validation should minimally include four main criteria: binding specificity, affinity, selectivity, and reproducibility [2].
Specificity: It can be regarded as a measure of the goodness of fit between paratope and epitope, or represents the ability of the antibody to discriminate similar or even dissimilar antigens in the intended application. An antibody with low specificity binds to several different epitopes.
Figure 1: The specific binding between an antibody and an antigen
Affinity: It measures the intensity of the interaction between an antibody and an antigen (epitope). A high-affinity antibody firmly binds to a specific antigen, while a low-affinity antibody weakly binds to the antigen. The higher the affinity of an antibody, the higher the sensitivity of an antibody-based assay. The equilibrium association constant (Ka) is the basic parameter to evaluate the binding affinity. The Ka is the ratio of antibody association rate (Kon) to the antibody dissociation rate (Koff). Compared with a low-affinity antibody with a low Ka, a high-affinity antibody with a high Ka will bind more antigens in a shorter period.
Selectivity: It describes how well an antibody binds to its intended target antigen within a complex mixture. An antibody selectivity for a certain antigen means that it shows little cross-reactivity for other antigens.
Reproducibility: It means that the validation data can be reproduced in any lab. However, antibodies vary from batch to batch. The batch-to-batch variability can produce significantly differing results. Compared with polyclonal antibodies and monoclonal antibodies, recombinant antibodies exhibit great superiority in reproducibility.
Generally speaking, what we usually refer to as antibody validation actually refers in most cases to the validation of antibody specificity.
Validation of antibodies needs to meet the applicability of an antibody in a specific application. Frequently used validation methods include Western blotting (WB), ELISA, immunofluorescence (IF), immunohistochemistry (IHC), immunocytochemistry (ICC), immunoprecipitation (IP)-mass spectroscopy (MS), flow cytometry (FC), knockout cell line, and knockdown cell line. Here is the list of the comparison among different methods.
Table 1. Methods of antibody validation
Validation types | Detection mechanism | Advantages | Disadvantages |
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WB | Proteins in a sample are detected through initial molecular weight separation after SDS-PAGE and subsequently blotted on a membrane, finally visualized through a proper antibody (e.g. colorimetric, chemiluminescent, fluorescent, and radioactive detection) |
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ELISA | Proteins in a sample are detected via specific antibodies and secondary enzyme-conjugated antibody |
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IP-MS | Protein complexes are first immunoprecipitated from a cell lysate and then separated via SDS-PAGE, followed by excision of protein bands of interest, and finally analyzed with mass spectrometry |
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IHC | Proteins in tissue sections are detected via specific antibodies and the antigen-antibody reaction is visualized by color-labeled secondary antibody |
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ICC | Proteins in single layers of cells grown in culture or from a patient sample are detected via specific antibodies and the antigen-antibody reaction is visualized by fluorophore-labeled secondary antibody |
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FC | When the target antigen is recognized by the fluorescence-labeled special antibody, the fluorescence signal will be acquired by Flow cytometry |
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IF | The target protein is detected by a specific fluorescence-labeled antibody, and the fluorescence signal is observed under a fluorescence microscope |
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Knockout (KO) cell line | Cell lines where the protein-encoding gene of interest is deleted with genetic tools such as CRISPR |
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Knockdown (KD) cell line | Protein-encoding gene expression is lowered using post-transcriptional gene regulation tools, such as siRNA |
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Additionally, the International Working Group for Antibody Validation (IWGAV) suggests five pillars to guide antibody validation in specific applications [2]:
(1) Genetic strategy
Knockout or knockdown target genes in control cells or tissues using technologies such as CRISPR/Cas or RNAi, and then detect relevant signals in these control cells or tissues.
(2) Orthogonal strategy
Quantify multiple samples using antibody-independent methods, and then detect the correlation between antibody-based and antibody-independent quantification.
(3) Independent antibody strategy
Use two or more independent antibodies to identify different epitopes on the target protein, and then confirm the specificity by comparison and quantitative analysis.
(4) Expression of tag protein
Modify the endogenous target gene, that is, adding the affinity tag or fluorescent protein tag to the endogenous gene, and then correct the signal from the tagged protein by antibody detection.
(5) IP-MS
IP can be used to isolate the target protein by using antibodies to bind specifically to the target protein. Its combination with MS analysis (IP-MS) can identify target proteins that interact directly with purified antibodies or proteins that may form complexes with target proteins.
Proper controls hold the key to confirming specificity.
A positive control is a relevant cell line or tissue strongly expressing the protein of interest that can be used to confirm the selective binding of the antibody. Non-expressing cells that have been transfected with the protein of interest offer the finest positive controls [2].
A cell line or tissue known not to express the desired protein is an appropriate negative control [2]. Thus, knockout cells or animals offer the most effective negative controls. VG Magupalli et al. validated anti-NLRP3 and anti-ASC antibodies with respective knockout cells [3]. There are alternative methods that can be utilized to produce comparable control results because these reagents are frequently out of the price range of many labs. Frequently, it could find cell lines that have been biologically shown to not express a certain protein of interest. For instance, PTEN-null H1650 cells serve as an excellent negative control for PTEN antibodies. siRNA or shRNA knockdown controls are also used as negative controls. When a proper negative control is not available, a cell line or tissue expressing only a small amount of the target proteins can serve as an acceptable substitute.
Make sure that you have used an optimized protocol to maximally enable the antibody to pass the validation process. For instance, incubation can range greatly from a minimum of 60 minutes to overnight at about 4°C. This is carried out to establish the ideal incubation time for each antibody employed. You may experience sensitivity problems if the period is short in duration. But if the incubation goes on for a long time, background staining could happen. To undergo a unique optimization procedure, additional necessary parameters including blocking conditions, working dilutions, and denatured & native conditions are also taken into consideration.
It is crucial to be aware that TBS or PBS will be the most common forms of the buffer used in antibody experiments. However, you must choose the optimal size. Additionally, researchers must take into account details like pH.
Researchers can lower the hazards associated with doing an experiment without carefully considering the antibodies they obtain by following a number of minor actions. The data sheet for the product should explicitly specify the suggested usage of the antibody, including the applications for which it has been validated, the proper methods, and the required dilutions. Researchers should be aware of this information to get started on an experiment.
Of course, no matter what detection method is used, we need to know in advance the background investigation of the target, including the expression abundance of the target, the spatiotemporal specific expression of the target, the detection threshold of the target antibody, and the appropriate detection method. Only through the analysis of scientific background knowledge, the most suitable method can be selected and a scientific validation strategy can be formulated.
Finally, specific criteria and factors must be met in order to successfully validate and confirm the specificity of an antibody. The specificity of antibodies is dependent on background conditions. Antibodies can only be properly validated in the context of the applied technology and the conditions under which they are ultimately used.
Further reading about knowledge of antibodies:
How to Choose an Antibody for Scientific Research?
How to Choose? Polyclonal, Monoclonal, or Recombinant Antibody?
Cross-reactivity of Antibody: Beneficial or Harmful?
How to Choose the Right Secondary Antibody?
The Overview of Recombinant Antibody
How to Choose the Loading Control Antibodies?
References
[1] Michael G Weller. Ten Basic Rules of Antibody Validation [J]. Anal Chem Insights. 2018; 13: 1177390118757462.
[2] Jennifer Bordeaux, Allison W. Welsh, et al. Antibody validation [J]. Biotechniques. 2010 Mar; 48(3): 197–209.
[3] Magupalli V, Negro R, et al. HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation [J]. Science. 2020 Sep 18;369(6510):eaas8995.
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