Antibody validation and speedy peptide mapping expertise

First, can you give our readers a brief introduction to antibody validation – what is it, why is it used and what does it contain?

The intrinsic ability of antibodies to bind antigens is widely used in the life sciences, especially as detection tools for target proteins in research. To ensure the accuracy of the detection, antibodies must be validated to demonstrate that they are specific, selective and reproducible in the same context of their intended use.

In traditional validation, the interactions between the antibodies and their binding partners are tested through controlled experiments. This function-based validation ensures that the antibodies purchased or manufactured have the desired properties. However, it does not guarantee that the antibody tested is exactly the same as that received in a previous batch or that it has the same sequence as that to be produced.

What is the difference between antibody validation tests for specificity and antibody validation tests for reproducibility?

A specificity test ensures that the antibody only binds to the target it is intended to bind to. A reproducibility test usually involves testing the same antibody over an extended period of time on different lots to ensure that each antibody always gives the same result.

Why is protein sequence verification critical to antibody validation?

First, lot-to-lot variability is a major problem in the research antibody market. There have been cases where people trusted a kit and they saw a positive reaction only to unfortunately realize that the kit actually detected a different protein because the kit’s antibody lot had changed.

A certificate that proves that the protein sequence of an antibody remains unchanged could easily have avoided this mishap. Second, more and more antibodies are now being produced recombinantly from a known amino acid sequence. It must be ensured that the final product, the protein produced by the expression system, actually has the same exact sequence.

How has peptide mapping developed in recent years?

Peptide mapping involves the enzymatic digestion of the protein and the separation and detection or identification of any resulting peptide. The separation is usually carried out using liquid chromatography (LC). The detection or identification can be done either with a UV detector or by mass spectrometry (MS) or tandem mass spectrometry (MS / MS). More recent efforts have also begun using multiple enzymes to digest the protein to increase sequence coverage.

Can you tell us about your MATCHmAbTM rapid peptide mapping technology. tell??

Our MATCHmAbTM service is designed to check the antibody sequence. To use the service, a customer sends their antibody sample and the antibody’s amino acid sequence. We digest the antibody with one or two enzymes in our laboratory and analyze the resulting peptides using LC-MS / MS and our proprietary peptide identification software.

If the sequence of the antibody is correct, the analysis can usually result in 100% or almost 100% sequence coverage. This means that each amino acid of the sequence is covered by at least one peptide that was identified from the mass spectrometry data. Conversely, a significantly lower sequence coverage usually indicates a sequence error.

What was the main inspiration behind MATCHmAbTM?

The reproducibility crisis caused by lot-to-lot variation is a major problem in the antibody industry. Scientists often rely on our services to overcome bad experiences (e.g. significant loss of money and time) caused by reproducibility. Many of our own scientists also have a molecular biological and biochemical background where they have relied on antibodies, and they too are well aware of the inconsistencies in antibody batches.

Because an antibody is uniquely determined by its amino acid sequence, we believe the surest way to address this issue is to ensure that the antibody has the exact same sequence in each batch. Many of our customers already routinely ensure the sequence consistency of de novo Sequencing of the antibody with our REmAb® service and comparison of the sequence of each batch. However, we would like to offer a cheaper and faster solution if the customer already knows the sequence (either because the antibody is recombinantly produced from a known sequence or the antibody has already been sequenced de novo in another batch). For this reason we created MATCHmAb ™.

When developing MATCHmAbTM, what were the main obstacles you encountered and how were they overcome?

Fortunately, we previously had the basics to develop MATCHmAb ™ as a REmAb. placed® Platform has sequenced thousands of antibodies de novo. Our main effort in developing MATCHmAb ™ was to optimize costs and lead times. This involved many decisions, from determining the best combination of enzymes to fine-tuning our internal sample processing workflow. Automation was also the key to reducing costs and lead times. Our goal was to make MATCHmAb ™ an affordable and fast solution for any researcher to validate any batch of antibodies.

How is MATCHmAbTM different from other peptide mapping technologies?

When it comes to MS-based peptide mapping, most laboratories do similar experiments. The main difference from MATCHmAb ™ is that it uses the same laboratory and analysis software as our REmAb® de novo sequencing service. The performance of both the laboratory and the software has been tested in the past by de novo sequencing of several thousand antibodies and continuously optimized. This ensures that more peptides can be correctly assigned and that customers receive the greatest possible coverage of their antibodies.

If MATCHmAb ™ discovers that the antibody may differ from the specified sequence, a customer can optionally upgrade to the REmAb® service to receive the full sequence of the antibody. In this way, the customer is not only limited by knowing that the new antibody is different, but can also secure the exact sequence of the new antibody.

What are the advantages / advantages of fast peptide mapping?

With MATCHmAb ™, customers were able to reduce the risk of failure and at the same time save time and money due to possible downstream problems. The most attractive thing about our fast peptide mapping service is its excellent reproducibility testing while the customer is running specificity experiments.

In addition, unlike other peptide mapping services, it is an easy-to-use service to screen many routinely used antibodies at an affordable price. It ensures that internal processes and pipelines remain robust. It’s comforting to know that the antibody used is the same molecule that was intended to be used.

Which MATCHmAbTM applications are there – who can use them?

MATCHmAb ™ can be used by any scientist who routinely relies on immune reagents or is developing therapeutics for their work. Perhaps more relevant, MATCHmAbTM is a great complementary tool that antibody manufacturers can add to their validation toolbox: routine controls on established hybridoma or recombinant cell lines, checkpoints during the development of antibody-producing cell lines, or to protect intellectual property.

MATCHmAbTM could very well be the first check-in to resolve binding issues, save manufacturers time and money, and ensure the reproducibility of assays across experiments for their customers. In this way, MATCHmAbTM goes beyond checking a sequence: it ensures trust. But as mentioned earlier, this really is an invaluable weapon that scientists can add to their arsenal for validation. For example, antibody engineering researchers could quickly verify that their expressed protein matches the engineered sequence, while also gaining invaluable information about PTMs and insights that could warrant further fine-tuning. The latter would be particularly useful for developing biosimilars.

Where can readers find more information?

Readers can find more information in our antibody validation resources, why protein sequence is important for antibody validation, and on our MATCHmAbTM peptide mapping and analysis services page.

About Rapid Novo

Rapid Novor is the world leader in next generation protein sequencing (NGPS). Through breakthroughs in proteomics and bioinformatics, the Rapid Novor team routinely helps biotech and pharmaceutical companies unlock the full potential of the humoral response for immune reagents, diagnostics, and / or therapeutic development.

Thanks to Rapid Novor’s NGPS-based antibody discovery services, researchers have advanced thousands of projects, patented antibody therapeutics, and introduced the first recombinant polyclonal antibody diagnostics.

Our technology has empowered thousands of researchers with the potential to unlock a wide variety of discoveries to shape future research and advance global health. At Rapid Novor, we believe that the path to a world of more effective treatments that cost less and are easier to develop begins with deciphering immunity.

About Dr. Am Ma

Dr. Bin Ma has been doing bioinformatics research and technology marketing for more than 20 years. With his breakthroughs in MS-based protein sequencing technology, Dr. Ma to provide new solutions to help humanity fight disease.