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02 January, 2024 by Anshul (neobio)
Antibody validation is the process of confirming that an antibody works specifically and consistently within a given experimental context. This involves demonstrating that an antibody not only binds to its target antigen but does so strongly and reproducibly. Antibodies are a critical tool in life sciences, playing key roles in research and clinical medicine applications such as Western blotting, Immunohistochemistry, ELISA, and more. Deciphering cellular mechanisms, diagnosing diseases, or unraveling the mysteries of human physiology all demand reliable antibodies. But without rigorous validation, these reagents may not function as expected, leading to unreliable results.The Role of Antibody Validation in Ensuring Reproducibility
The crux of scientific research is reproducibility — if an experiment’s results cannot be replicated, its conclusions are deemed unreliable. This is where antibody validation becomes absolutely essential. Properly validated antibodies help ensure experimental findings are not just a fluke but are solid, repeatable science that can be trusted and built upon.
Some of our antibodies that have undergone enhanced validation through HuProtTM Human Protein Arrays are noted below:
AKT1 (Prognostic Marker for Neuroendocrine Tumors) Antibody
Alpha-2-Macroglobulin / A2M Antibody
Bcl-6 (Follicular Lymphoma Marker) Antibody
Key Components of Antibody Validation
– Specificity: The ability of antibodies to distinguish between different antigens.
– Affinity: The intensity of an antibody binding to its epitope.
– Reproducibility: The ability to yield consistent results across different experiments and conditions.
Antibody validation is a complex process that requires multiple approaches to ensure the produced antibodies are specific, sensitive, and reliable. At NeoBiotechnologies, we follow the universally accepted five pillars of antibody validation. These pillars provide a comprehensive framework to ensure the highest level of specificity and efficiency for our monospecific monoclonal antibody portfolio.
The first pillar of antibody validation is the knockout/knockdown method. This technique involves creating cells or organisms in which the gene of interest is either completely (knockout) or partially (knockdown) inactivated. The absence or reduction of the target protein allows researchers to confirm the specificity of an antibody. If an antibody still shows a signal in knockout or knockdown experiments, it is likely that the antibody is not specific to the intended target.
The second pillar involves using multiple different antibodies that recognize the same target. If these antibodies, which are raised against different epitopes of the same protein, give a similar staining pattern, it increases confidence in the specificity of the antibodies. This approach is particularly useful when knockout/knockdown samples are not available.
The third pillar is a powerful technique that combines immunoprecipitation (IP) with mass spectrometry (MS). In this method, an antibody is used to pull down a protein from a complex mixture. The pulled-down proteins are then identified by mass spectrometry. This method provides direct evidence for the specificity of an antibody and can also reveal potential off-target proteins.
The fourth pillar involves using biological and orthogonal validation methods. Biological validation refers to using biological knowledge about the protein, such as its known location in a cell or response to certain treatments, to confirm the antibody’s specificity. Orthogonal validation involves using a different method, which does not involve antibodies, to measure the same target protein. Consistent results between the antibody-based method and the orthogonal method provide strong validation for the antibody.
The last pillar of antibody validation involves the use of recombinant protein expression. In this method, the gene encoding the protein of interest is cloned and expressed in a heterologous system. The recombinant protein is then used as a positive control in western blot analysis. The presence of a band at the expected molecular weight further confirms the specificity of the antibody.
In conclusion, antibody validation is a multifaceted process that requires a combination of several techniques to ensure the produced antibodies are specific, reliable, and suitable for their intended applications. By adhering to the five pillars of antibody validation, NeoBiotechnologies ensures the delivery of highly validated antibodies to support your research needs.
In this section, we will delve into some of the practical techniques utilized in antibody validation. These methods range from Western Blotting to Immunohistochemistry, each serving a unique purpose and contributing to the overall validation process.
Western blotting is a common method used in antibody validation. It is often the first step in determining an antibody’s specificity, as it can identify whether the antibody recognizes the denatured antigen. In this process, antibodies are validated using lysates from cells or tissues that express the protein of interest. A single band at the known molecular weight for the target is a strong indication of the antibody’s specificity.
Immunohistochemistry (IHC) and Immunocytochemistry (ICC) are powerful techniques used to validate antibodies. IHC is particularly useful for determining the localization of proteins in tissue sections, while ICC is used to study the distribution of proteins within individual cells. These techniques involve staining processes where the antibody of interest is used to target specific antigens within a tissue or cell sample. The staining is then observed under a microscope, providing visual evidence of the antibody’s specificity.
NeoBiotechnologies’ antibodies are rigorously tested for Immunohistochemistry using Formalin-fixed Paraffin Embedded Sections. View our extensive portfolio of these products for your research and diagnostic needs.
Protein arrays are another comprehensive validation technique that can quickly assess the specificity of an antibody against a large number of targets simultaneously. This high-throughput method can validate antibodies against thousands of proteins in a single experiment, making it highly efficient for large-scale antibody validation.
The use of both positive and negative controls is essential for successful antibody validation. Positive controls involve using samples known to express the target protein, while negative controls use samples known not to express the target protein. Comparing the antibody’s reactivity in these control samples can provide evidence of its specificity.
Each antibody may require a unique protocol for optimal results. Factors such as incubation times, working dilutions, blocking conditions, and the use of native vs denatured conditions may need to be optimized for each antibody. It’s essential to give the antibody the best chance of passing the validation process through the use of optimized protocols.
Finally, the choice of buffers can significantly impact the performance of your antibody. Most antibody assays use two buffer types: PBS or TBS. The optimal buffer for your experiment depends on various parameters that can influence buffer performance, such as pH.
Antibody validation is not merely an optional procedure in the research process; it’s a vital step to ensure reproducibility. The use of poorly validated antibodies can lead to irreproducible data, hampering the progress of scientific research. More than 70% of researchers have struggled to reproduce experiments conducted by other scientists, often due to issues with antibodies. Proper antibody validation methods can mitigate these issues and ensure that research data is reliable and reproducible.
Secondary verification of antibodies is a crucial step in antibody validation. It ensures that the antibody has the required specificity, affinity, and reproducibility for its intended use. The reliability of experimental data can only be guaranteed when a secondary verification is performed strictly according to the antibody detection standard. This step should never be overlooked in the validation process.
NeoBiotechnologies is committed to producing highly validated, monospecific monoclonal antibodies, both hybridoma and recombinant, for a variety of hosts such as mouse and rabbit. The company’s rigorous in-house testing guarantees that these antibodies meet their high internal benchmarks and perform as expected in their designated applications.
The future of antibody validation lies in collaboration and community efforts. The establishment of the International Working Group for Antibody Validation (IWGAV) and the publication of recommendations for antibody validation standards, known as the five pillars, is a testament to the research community’s commitment to raising the profile of antibody validation. Sharing knowledge and experiences can help other scientists find the best antibodies for their research and contribute to the overall improvement of antibody validation standards.
For further information, reach out to our dedicated technical support team.
