Basic Rules and Regulations Involved in Antibody Validation

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By Kaleem Ullah

Antibodies are among the most reagents used in clinical tests, diagnostics, and life science research. Although they have such widespread importance and receive considerable investments in time and money, antibodies still lack standard guidelines to define their validation before use. 

As a result, most commercial antibodies either are not properly validated or lack sufficient experimental data. The inadequacies in antibody validation have led to unrepeated results and, in some cases, abandoned projects. Consequently, this causes the loss of large sums of money, valuable time, and effort. 

Hence, there exists a pressing need for a universal standard for validating antibodies. 

What is Antibody Validation?

Antibody validation is the process of proving specificity and an antibody’s ability to distinguish separate antigens. It also involves demonstrating specificity in the particular application it is intended for or establishing affinity, which means determining the strength of the bond an antibody can form with an epitope. Lastly, the process is also defined as providing the reproducibility of an antibody. 

But although these are practical definitions of antibody validation, its widespread implementation or application of a systemized process is still a major concern. 

Many antibody producers validate their products while others do not perform any validation whatsoever. These inconsistencies exist because, contrary to the drugs industries, there is no regulatory body in charge. 

Fortunately, some basic rules and regulations have emerged for validating antibodies, and scientists, clinicians, and concerned parties should be aware of them as outlined below. 

10 Basic Rules for Validating Antibodies

Antibody Definition

The binding molecule, usually an antibody, has to be identifiable in no uncertain ways. For this reason, an antibody ID, a clone number, commercial product number, or equivalent is appropriate. A definition that’s considered even more suitable is the antibody structure primarily defined according to the amino acid sequence. Also, it is necessary to categorically state whether the binder is a monoclonal or polyclonal antibody. The absence of this info means the work will be irreproducible. 

Defining The Target

Perhaps the most vital characteristic of antibodies is their ability to selectively connect with another molecule called an antigen (usually the analyte). The definition of the target antigen should be as precise as possible. When dealing with haptens, the immunogen structure must be designated as the chemical formula, including the hapten orientation and linker. Failure to define the target properly means failure to decide if the binder is adequately selective. 

Binding Selectivity/Cross-Reactivity

Cross-Reactivity (CR) is a reasonably sophisticated property and is commonly misinterpreted. Several researchers rely solely on the definition credited to Abraham GE, absent the full understanding of his research paper. Consequently, it’s a popular belief that monoclonal antibodies show fewer CRs when compared to polyclonals. However, this isn’t always true, so it’s vital to get the CR data right. That’s because, with more CR data, the probability of accurate evaluation of usability is higher in any given application. 

Concentrations of Additives And Antibodies

Determining antibody concentration is relatively tricky, even with the surface plasmon resonance (SPR). As an alternative, many companies decide either a protein concentration with a method that’s not selective, e.g., UV at 280 nm, bicinchoninic acid, Bradford assay for amino acid analysis. Ultimately, the conclusion can be drawn that antibody concentration determined on any product label is just a preliminary estimate for assay optimization. 


For every feature stated above, the derived information should be documented appropriately using a data sheet. Documentations that are almost empty and contain only the reagent’s name and the order numbers, should indicate the poor validation standards of the antibodies involved. These types of antibodies are often only helpful in contexts involving the task of screening a binder. Nevertheless, if there’s a need for a reliable reagent, it’s best to avoid such antibodies. 

Binding Potency of Antibody/Antigen Complex

Information about the binding strength of the complex formed between the antibody and antigen can be pretty insightful. This property of the antibody/antigen complex is commonly known as “binding constant” or “affinity constant.” Here, the law of mass action is used to calculate and define the binding potency and can be estimated using ELISA, SPR, equilibrium dialysis, or several other procedures. 

Influence of Nontarget Substances/Matrix Effect

Abraham’s definition, as referenced above, also proves beneficial for matrix components like humid acids, solvents, humic acids, salts, and other additives. The principal difference is the nonspecific mode of action and, therefore, the typically elevated concentrations that are applicable. Regrettably, this method is yet to get widespread application. However, it can be useful when examining an immunochemical reagent’s robustness. 

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Stabilizing And Storing

Despite their advantages, antibodies can be somewhat unstable reagents and require individual care. That is because of the high cost of most antibody reagents. Thus, antibodies can only be stored or stabilized using the applicable protocols. When using an antibody for a longer time, it’s quite helpful to use validation of stability but only under specified conditions. Hence, avoiding poor stability can save a lot of frustration and financial burden. 

Application Protocols

Application protocols serve as essential information sources for understanding a given antibody’s applicability under specific conditions. One most vital piece of information that can be deduced from application sheets is the possible application of the antibody in the intended sample matrix-like surface water, urine, or serum. This improves the likelihood of the successful execution of an immunoassay. Also, the number of accessible application protocols is valid documentation of the validation method’s practicality. 

User Feedback

Another essential addition to application protocols is user comments and ratings. Typically, these users must have already bought and used the antibody in specific applications. The unlimited and direct interchanging of user experience and data is an excellent example of the practice called “Open Science,” a highly effective approach to speed up and enhance scientific progress. Finally, antibodies that have many positive feedbacks and available good reports are generally of better quality. 


These ten basic rules can serve as the standard measures for assessing antibody validity in different cases. This makes the processes as hassle-free as possible, especially for those lacking any antibody experience. There are several references and supplementary resources to cross-check when evaluating any commercial antibody’s validation level. In case of failure to meet one or more of any five required rules, the use of the respective binding agent or antibody is not advisable for scientific works without extra validation effort. The various analytical methods covered in this article are simply examples. Therefore, validation depends purely on the particular application.