Q&A Part 2: Industry Leaders Discuss the Importance of Antibody Testing and Validation

First, we don’t obtain antibodies from other sources. We manufacture all our antibodies and sell only our antibodies. We don’t partake in rebranding or allow other companies to sell our product without our brand. You’ll always know it’s a Proteintech product. To make it to the catalog, our antibodies need to have a successful ELISA and Western blot and then we continue validation with IHC, IF, IP, FC, CHIP and knockdown in primary tissues and unmodified cell lines. We were the first to implement a gold standard in validation using the knockdown technique. New lots are tested and older lots are retested frequently to insure quality and reproducibility. Our data is organized and original in that we can pull the original film of a Western blot, for example, to show researchers upon request. Manufacturing, validating and managing the catalog of our own antibodies allows us to maintain a high level of quality control with traceability. A higher level of quality control means more reliable and consistent results for the scientist.

Every lot of every antibody is tested in a consistent manner before it is sold. We balance the rigor of antibody validation with the value the antibody has to the researchers. For instance, a newly developed antibody may be initially validated by Western blotting using recombinant proteins or lysates that overexpress the target protein. But as the antibody is used more by researchers we increase the rigor of validation to include native cells or tissues that may have been treated by genetic manipulation or drug studies or presented as multiple lysate panels with expression levels known based on proteomic studies of the target protein. This information is then correlated with the observed band intensity after antibody staining as part of our validation strategy.

Antibody validation is both lot specific and immunoassay specific, meaning each newly manufactured lot or batch or antibody is validated independently, and that the validation strategies developed, say, for Western blotting may not necessarily apply to immunohistochemistry or ELISA. Furthermore, we perform every validation experiment with the appropriate negative and positive controls and we strive to ensure that this information, in its entirety, is communicated to the researcher to foster transparency so that results can be repeated when protocols are precisely followed by users.

The proposal and community discussion are the start of a long process that will require continuous cultivation and collaboration. Thermo Fisher Scientific has already adopted the validation standards for our antibody portfolio in line with the recommendations of the International Working Group for Antibody Validation (IWGAV). Using these new Thermo Fisher Scientific specificity validation standards, Invitrogen antibodies are being tested for both target specificity and robust application validation aligned to a variety of applications such as imaging, flow cytometry and Western blotting.

To ensure that an antibody is binding to the correct target, we are testing our antibodies using one or more of the following four Thermo Fisher Scientific specificity pillars:

• Genetic modification where antibodies are tested using cell lines following knockdown or knockout technologies
• Independent antibody verification in which measurement of target expression is performed using two differentially raised antibodies recognizing the same protein target
• Functional or orthogonal testing in which functional activity or changes of target levels are measured in different cells or following cell treatments
• IP-mass spectrometry, where antibody targets are identified by mass spec following isolation by immunoprecipitation

The specificity validation data will be combined with application validation in a relevant biological model system. As we expand specificity and application testing of our extensive portfolio of antibodies, data showing specificity verification will be increasingly seen on our product detail pages and information sheets.

Atlas Antibodies is an original manufacturer, and thereby we have full control of both the validation and reproducibility of all our products. Most of our research antibody products are polyclonal antibodies, and for polyclonal antibodies the main issue is to ensure that an antibody purified from a new animal serum performs equal to the same antibody purified from another animal serum.

All new sera go through a rigorous material control where purified antibodies from both the new sera and antibodies from previous sera, the reference lot, are tested in parallel on consecutive samples in all assays. The parallel testing and comparison on consecutive samples is crucial since the sample material may differ from time to time. The main application for our products is immunohistochemistry (IHC) and the standard test set up for quality control of an antibody for the IHC application consists of a tissue microarray of more than 20 different samples of both normal and cancerous tissues.

The same rigorous testing is also performed for other applications such as immunocytochemistry- immunofluorescence and Western blot.

At Atlas antibodies, each new production lot as well as new purifications from the same serum, is given a unique lot number to secure traceability. Each product lot has a letter prefix, and lot numbers with the same letter prefix are purified from the same serum. We believe that transparency is very important, and with the letter prefix the end users will always know if different lots are purified from the same sera or not.

We routinely apply rigorous testing and validation of research antibodies we manufacture in-house or supply, using stringent validation workflows. When we bring in a hybridoma from an external source, we apply the same criteria as to our own cell lines. Validation strategies for antibodies can be complex, and we develop appropriate strategies for each target, sample and desired application(s) of interest. Our highly qualified antibody development and validation team is composed of experienced scientists who focus on a specific technique such as immunofluorescence, IHC, flow cytometry, Western blot, ELISA or functional assays. All data for each antibody is reviewed by a panel of scientists in a central review process to ensure only the highest performing clones are commercialized.

One important thing to note is that our antibody development and validation teams are distinct from our production teams. Any antibody transferred from development to the production team needs to meet the exact same validation and performance characteristics as specified by the antibody validation team during development. In short, if any clones or methods do not meet our rigorous specifications, we do not release them. This way, we are building reproducibility and robustness of methods into each quality clone.

At Bio-Techne, our aim is to provide only the highest quality affinity binders with the highest specificity, sensitivity and consistency. We have industry-leading validation standards, and access to the latest tools and technologies. For example, we are applying specificity testing methods using CRISPR knockout. We are probably one of the few companies that tests both the raw material lot as well as lots after the bottling process, even though this is resource intensive. We follow internationally accredited quality-management systems such as ISO 9001 to ensure our production and QC/QA processes are working well, and this ultimately helps with reproducibility.

CST is not just an antibody product company. Since our foundation, we have also done important basic and applied cancer research. We invest heavily in new technology development and our internal research programs. Like our customers, we also publish our research, so we understand the cost of failed experiments and the importance of researchers’ time. For these reasons, we only commercialize antibodies that were made and tested by us and for which we can provide the protocols and technical support to make sure they work in the hands of our customers the first time and every time. We place very strong emphasis on application-specific validation, quality control and technical support. For CST, product quality is simply a matter of straight business ethics. Our antibody development process has a high attrition rate. We would never sell an antibody that doesn’t work or is of poor quality. Thus we don’t have a very large catalog, but what we offer is likely the best quality in the market.

When we develop an antibody ourselves, or through a collaboration or licensed source, we ensure appropriate process controls and testing are in place to support performance consistency and proper product identification, handling and storage practices before we release them for sale.

When we source antibodies from other companies we follow similar steps to make sure that the products work as we describe them. This may involve direct testing, auditing and in some cases removing the product from the market when it does not meet our standards.

We also have a vigorous monitoring process once a new product is released to identify performance issues so we can address them directly.

The validation approaches vary from antibody to antibody and are highly dependent on the marker and the expression pattern of the marker. To test the specificity of an antibody, we use various approaches like siRNA knockdown, overexpression of the target protein and related proteins in cell lines, or cross-blocking or staining with other antibodies that recognize nonrelated epitopes. In addition, all antibodies are tested on cells—wherever possible on primary cells—to evaluate the sensitivity in detecting endogenous levels of the antigen.

The majority of the mouse antibodies are also validated with several primary tissue samples such as organs or tumor tissues. This gives us an idea of whether the sensitivity might be compromised when the tissue undergoes a harsh dissociation process. Another test, which is integral to our production and QC processes, is lot-to-lot testing of the antibodies. During production, the performance of the new lot is set to match the performance of the last lot. At the quality control department the lot-to-lot performance is tested again, but after the product has gone through one round of storage at our inventory site. This mimics the conditions at the customer site.

At Bio-Rad, we have been focusing on setting standards for validating antibodies for Western blotting. We decided to focus on Western blotting because it is one of the most widely used immunoassays in the life sciences. All of our antibodies are tested in Western blotting on cell or tissue lysates that express the native target at endogenous levels. In order for an antibody to pass our validation, it must detect its target protein with minimal non-specific binding. An antibody will not pass our validation if it strongly detects a protein in samples not expected to contain the target protein. Finally, the antibody must be sensitive enough that it doesn’t require overexpression or purification of the target protein in order for detection. Only 10–20% of the antibodies that we test pass these criteria, ensuring high specificity and sensitivity. Once we have validated an antibody for a particular assay, we validate every new batch of the antibody using the identical assay to ensure lot to lot consistency. For more information from Bio-Rad refer to this article: http://www.bioradiations.com/how-does-an-antibody-qualify-to-become-a-precisionab-antibody/

BioLegend manufactures antibody products at our main facility in San Diego, where we are certified to operate under ISO 9001:2008 and ISO 13485:2003 standards. Our Quality Management System ensures traceability, reproducibility and continuous improvements in manufacturing.

It is very important to us that every product that leaves our facility performs exactly as promised. This starts with validation of all new antibodies against endogenous targets in the applications that will be most relevant to customers. The confirmation of specificity and lack of off-target binding is confirmed with appropriate cellular controls, comparison with any pre-existing antibody clones and appropriate performance in multiple relevant assays. Subsequently, each production lot is QC tested by a variety of biochemical assays, and in the most relevant functional assay for that antibody’s molecular target.

As an antibody manufacturer with our own production facility and testing laboratory, GeneTex has over 20 years of experience validating antibodies. Our antigen affinity-purified polyclonal antibodies are routinely evaluated using Western blot, immunocytochemistry and immunohistochemistry. As with all polyclonal antibodies, there is the concern of batch-to-batch differences due to variations in the immune response of the host. Therefore, each subsequent lot must be retested in all applications and species. This is a time-consuming, costly and labor-intensive process, but is necessary to guarantee consistency in GeneTex’s polyclonal antibody products. A similarly rigorous screening process is applied to the selection of our mouse monoclonal antibody reagents.

In addition, given the reality that antibodies often perform best in an application-dependent fashion, obtaining convincing data for multiple applications and species with one antibody is often impossible. As a result, multiple antibodies to the same target are produced and validated for specific needs. At this time, RNAi-mediated knockdown validation is also utilized on an increasing scale.

As a complementary approach to validate our products, GeneTex also encourages researchers to submit “Star Reviews” where they evaluate our antibodies and provide independent user reviews. GeneTex also cites published papers where an antibody was used successfully.

GeneTex has more recently implemented a systematic analysis of our antibody reagents using more definitive means to confirm specificity. These efforts are encapsulated in our “Knockout/Knockdown Validation” program. Beginning with our most popular products, GeneTex is evaluating its antibodies through Western blot using cell lysates generated by either shRNA-mediated knockdown or CRISPR-mediated knockout of the target protein. In particular, GeneTex’s leadership has made a substantial commitment to incorporate CRISPR-mediated knockout validation as a central pillar of the QA protocol.

LI-COR’s near-infrared (NIR) fluorescent secondary antibody products are affinity purified and highly cross-adsorbed to ensure high specificity and minimal cross-reactivity. All IRDye® antibody conjugates are specifically tested and qualified for fluorescent Western blotting. Secondary antibodies labeled with spectrally distinct IRDye fluorophores can be combined for multiplex Western blot detection.

When purchasing antibodies from a vendor, we choose primary antibodies that are specifically validated for Western blotting. For multiplex Westerns, careful selection of primary and secondary antibodies is critical to prevent cross-reactivity. We choose primary antibodies raised in two different host species, which can be discriminated by the secondary antibodies. Secondary antibodies must be highly cross-adsorbed, and should be derived from the same host species if possible. To validate antibodies for multiplex Western blotting, we run three control blots—a separate blot with each individual primary antibody, and a third blot with the primary antibodies combined. These controls will characterize the banding patterns and verify that the primary antibodies can be multiplexed.

Manufacturing the best antibodies is a complex process. At Sino Biological, there are at least 12 steps. We can do it all, including designing antigens, choosing the host animals, optimizing immunizing procedures, comparing hybridoma and phage screening techniques, detecting serum and screen sub-clone, validating antibodies, expanding production and purification, re-verifing the batches, accelerating the life test, re-checking the stock within routine periods, and providing feedback.

We use an independent QC department to validate antibodies. Every antibody manufactured is verified for purification and isotype. The specificity validation is done with immunogens and natural proteins. Testing with available cell types, tissues, and species is also required. All the results are consolidated and corroborated. Our QC department is required to validate antibodies from batch to batch. We follow the strictest standards for antibody validation. The majority of our antibodies have similar or better performance than antibodies from other sources.