: Can you share your perspective on the reproducibility issue associated specifically with the use of antibodies?
JZ: Reproducibility is the key factor in scientific research. It will not only affect the robustness of a researcher’s results, but it also impacts the reliability and development of biological science. Unfortunately, more than 70% of researchers have tried but failed to reproduce another scientist's experiments, and more than half have failed to reproduce their own experiments. One of the major factors contributing to the lack of reproducibility is the reagents used. In biological research, immunology-based approaches, such as ELISA, Western blot, IP, IF, IHC, and FACS, are the fundamental techniques. As a result, the antibody reagents used have an important role in the repeatability of publication data.
We recognize that as a qualified antibody reagents provider, we are participants in researchers’ studies and are as responsible for the data as the authors are.
Antibodies are produced by host animals. Precise antigens are a prerequisite for screening reliable antibodies. We have the professional teams and various expression systems—from prokaryote to eukaryote cells and also cell-free expression systems—for protein/peptide reconstruction. Sino Biological owns one of the largest protein libraries in the world. We assure the quality of various protein antigens, which includes not only the sequence, but also the correct configuration and post-translation modifications. Using our professional staff and protein production expertise, we can choose the best antigen design for a certain target. For example, to obtain an antibody against a membrane protein, we would try the whole natural protein, the exocellular structure, and/or the DNA or whole cell immunization technique. To ensure reproducibility we screen the best antibody clone by comparing as many candidates as possible.
: Can you tell us what your company is doing on a routine basis to make sure you are testing and validating the antibodies you manufacture or obtain from other sources?
JZ: 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.
: Do you have any advice, recommendations, and/or best practices scientists should follow when selecting and validating an antibody?
JZ: Selecting the right antibody is a complex process and we recommend careful review of the following considerations based on your experiment:
- The species of the sample: Human, mouse, rat, virus or other;
- The nature of the sample: Protein/peptide, whole organisms: cell or bacteria. You need to know as much as possible about the target protein expression and cell type, distribution, and quantity;
- The species of primary antibody host: Normally, the antibodies are raised from mouse or rabbit. Then a mono- or poly-clonal antibody corresponding with the experiment requirements is chosen;
- The applications verified by the supplier: Because of the complex nature of biological samples, one antibody can not be used for every technique. We can only guarantee the applications approved for each product.
- The secondary antibody: Antimouse or antirabbit secondary antibodies are raised from different species than the primary antibody. Note that these anti-IgGs should not cross-react with the sample;
- The proper fluorochrome and chromogen labels: Scientists should take into consideration the mechanism and settings of equipment detection.
There are many tips for antibody-based experiments. Choosing the right antibody, following good protocols, and paying close attention to detail can make antibodies a reliable tool for biological research.
: What are your thoughts on the current efforts that are underway to tackle the reproducibility problem? Do you think they are realistic and implementable?
JZ: We are aware that procedures and standards for validation differ among suppliers. Sometimes, a biochemical condition of the storage buffer can influence the performance of an antibody. Therefore, establishing universal criteria or an authorized third-party validation might be the best approach. At Sino Biological, we compare our new products with the most popular antibodies utilized in publications for similar or enhanced performance.
However, because of the complicated nature of biological samples, the reproducibility issue might not merely be a problem of ensuring the consistent quality of antibodies by producers. Enhancing communication between users and vendors would be helpful for both sides. Scientists at antibody companies should participate more deeply in customers’ research efforts to help improve “precise design” for each individual study.
We look forward to new techniques that are cost-effective, high-throughput, and acceptable to the public. All of which is essential to improve the efficiency and precision of antibody validation. It will eventually benefit all antibody users.
: What else is your company doing to address reproducibility?
JZ: As mentioned above, the reproducibility crisis is in part derived by different methods and reagents from various sources. Sino Biological strives to make sure our antibody reagents meet the highest criteria in the field. A Certificate of Analysis is issued for each lot of product, which includes:
- Product information: Product name, catalog number, lot number, immunogen, clone ID, Ig type, formulation, preparation process;
- Antibody usage guide: Specificity, application, storage;
- QC-approved images;
- Lot-specific test results that are helpful for using the product
We guarantee our products for approved applications. Furthermore, our technical support department is available to address researchers’ special requests. We have put considerable effort into tackling the reproducibility problem, not just for our products but also for reagents from different sources.
Author Bio: Jie Zhang, Ph.D., Associate Researcher, Sino Biological Chief Scientific Officer, has been responsible for the antibody research and development department since 2009. Under his creative leadership, advanced antibody technology platforms have been successfully established, and over 9,000 antibodies & 500 ELISA kits have been independently developed.
Prior to joining Sino Biological, Dr. Zhang received his Ph.D. in Medicine from Peking University and completed his postdoctoral studies at Columbia University, during which he focused on the research of B cells and autoimmune diseases.