Reflections on the problem of antibody reagents by Chinese researchers
June 15, 2015 Source: Bio Valley
Window._bd_share_config={ "common":{ "bdSnsKey":{ },"bdText":"","bdMini":"2","bdMiniList":false,"bdPic":"","bdStyle":" 0","bdSize":"16"},"share":{ }};with(document)0[(getElementsByTagName('head')[0]||body).appendChild(createElement('script')) .src='http://bdimg.share.baidu.com/static/api/js/share.js?v=89860593.js?cdnversion='+~(-new Date()/36e5)];On May 19th, 2015, Nature magazine experienced the current status and problems of current antibody reagents from the experience of several senior scientists in the United States and Canada, and sounded the alarm for researchers all over the world. As a Chinese scientific researcher, the problem of antibody reagents we encounter is only more than that of our foreign counterparts. In addition to complaints, we should further reflect on how to obtain high-quality, suitable antibody reagents.
Obtaining high quality and suitable antibody reagents is not difficult. We need to identify the following three aspects: 1. Excellent antibody supplier; 2. Excellent cold chain logistics system; 3. Regular sales (direct sales/distribution) channel .
1. Excellent antibody supplier
Excellent antibody suppliers should have the following information or can provide customers with the following information: antigen sequence selection, antibody purification methods, antibody validation data, application guidelines.
i. antigen sequence selection
The selection of antigenic sequences is critical, directly determining antibody specificity/low cross-reactivity and antibody production. Therefore, antigen sequence selection not only ensures the lowest homologous sequence in the whole genome, but also excludes signal peptide and protein transmembrane region sequences.
Ii. Antibody purification
The three main methods of antibody purification are: salting-out crude, Protein A/G universal purification and specific antigen purification column. The purification effect and antibody titer of the three methods are sequentially improved. Among them, Protein A/G general-purpose purification has moderate effects and cost, and most antibody reagent companies use this method for large-scale antibody production. In order to obtain higher quality antibodies, it is necessary to pass a specific antigen purification column, but the cost is correspondingly highest due to operations involving immobilization of specific antigens. However, the antibodies purified by this method have the highest purity and specificity.
Iii. Antibody verification data
Many vendors currently have antibody validation data, but we don't know the specific source of these validation data. Many antibodies have WB validation data, but there are very few trademarks in which cell lines are verified. If it is an overexpressed cell line, WB verification is of little significance. Similarly, immunohistochemistry or immunofluorescence has similar problems, and the data to be validated specifically from which tissue and which pathological sample is rarely mentioned. The results will vary greatly depending on the source. Suppliers often experiment with samples that are the easiest to get laboratory data, but when such antibodies are used in our own experiments, there are often multiple problems.
Iv. Application guide
Antibody vendors generally recommend relevant applications based on validation data, so validation data is decisive for the application. It should be noted that the same antibody can be used as WB, but not necessarily for immunofluorescence or immunohistochemistry, and vice versa. Therefore, the supplier's antibody validation data is very important.
The above lists the four hard requirements of excellent antibody suppliers. In fact, which ones can do the above well? The following is an example of an HPA gold standard antibody supplied by Sigma-Aldrich. The HPA gold standard antibody was designed and validated by Human Protein Atlas (HPA) Project.
1) HPA gold standard antibody antigen selection
The immune antigen of the HPA gold standard antibody is a recombinant human protein epitope recognition tag (PrESTs), and the epitope recognition tag generally contains 50-150 amino acids. Epitope recognition tags are designed and screened such that they contain unique epitopes and are suitable for eliciting highly specific antibody production. By means of a human genome-wide scan, it is ensured that the protein epitope recognition tag used to produce the antibody has the lowest homology to other proteins. At the same time, antigenic epitopes also exclude signal peptides and protein transmembrane regions.
2) HPA gold standard antibody affinity chromatography purification
HPA gold standard antibody was purified by affinity chromatography (PrESTs antigen as a ligand, specific antigen purification column), using three-step immunochromatography purification process: HisABP specific label removal, PrESTs specific capture, and finally passed The exclusion of the buffer by the exclusion chromatography not only ensures the purity of the antibody, but also facilitates the long-term preservation of the antibody.
3) HPA gold standard antibody verification
Each HPA gold standard antibody has been verified by protein chip, immunohistochemistry, WB and immunofluorescence. The amount of data verified is not only huge - more than 700 experimental verification images per antibody (pathologist certified and annotated) online The query, and, however, validates data from different tissue and cell line samples, and combines references and bioinformatics predictions to ensure the reliability of each experimental data.
4) HPA Gold Label Antibody Application Guide
HPA gold-labeled antibodies are mainly immunohistochemical/immunofluorescence, and most antibodies support WB experiments at the same time. Based on the verification results of various organizations or cells, it provides valuable data reference for researchers.
In summary, HPA gold-labeled antibodies can be regarded as a model of high-quality antibodies, and Sigma-Aldrich is one of the excellent antibody suppliers.
2. Excellent cold chain logistics system
At present, the cold chain logistics system is very mature, and various suppliers generally can guarantee the low temperature transportation of antibodies. For suppliers, the main reason is to invest, PDA-Aldrich is more prominent. After the antibody is received, the average ice box can still last for 2-3 days, which is enough for users to feel at ease.
3. Formal sales (direct sales / distribution) channels
In China, the sales channel is the key link of the problem, especially the fake antibody reagents. Many products can be discerned on the packaging (scientists pay little attention, often price-oriented). Driven by the interests, some dealers often make their own antibody reagents and put them on counterfeit labels to refill them. Another common phenomenon is that antibody reagents are purchased from regular channels and then sold through small workshops. To researchers. Whether it is shoddy or diluted, it will bring a lot of experimental risks to researchers. Finally, the majority of Chinese researchers are always paying money and manpower. It is more difficult to make up for the precious time cost. .
Therefore, in the purchase channel, it is recommended that you try to buy antibody reagents from the official designated dealers to suppress the circulation of fakes. At the same time, with the maturity of the e-commerce platform, you can order reagents directly on major official websites, which is the safest and most convenient way to purchase. In this regard, it is inevitable to mention Sigma-Aldrich again. Sigma-Aldrich is the earliest provider of life science products for e-commerce in China. It is dominated by direct sales, avoiding the problems of intermediate circulation and its powerful logistics system. It greatly reduces the experimental risks that may arise from the sales process.
In the face of the complicated antibody market, it is recommended that you choose carefully when purchasing antibody reagents. Do not seek small profits, purchase antibodies from excellent suppliers through regular sales channels, and minimize the losses caused by antibody quality problems.
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