You must know the IVD antibody selection experience!
With the development of life sciences, there are more and more researches on proteins, and the importance of antibody reagents in in vitro diagnostics is becoming more and more important. Facing the complex antibody reagent market, how to choose the antibody suitable for your own experiment becomes particularly important.
1. Regarding the choice of specificity:
The choice of specificity mainly needs to consider four aspects: protein specificity, species specificity, experimental method specificity, and marker specificity.
1. Protein specificity:
To find antibodies for the proteins that need to be detected, several details need to be distinguished. The requirements for antibodies are different for the detection of recombinantly expressed proteins and endogenous proteins. Please check the detection instructions in the antibody manual. If the recombinant protein is not expressed in full length, it is necessary to pay attention to whether the immunogenic region of the antibody is within the recombinant protein region. For endogenous proteins, it is best to know how they are cut and modified. For proteins with special phenotypes, sequence comparisons should be performed and combined with antibody immunogen sequences to check for cross-reactivity. The detection of phosphorylated protein needs to determine the specific site, and the phosphorylation of different sites means that there may be different mechanisms, so it is not appropriate to generalize.
2. Species specificity:
Different species of the same protein have large or small differences. At present, most commercialized antibodies use human protein sequences as templates to recombine proteins or design polypeptide antigens, and cross-react with other species according to the homology of proteins. It is necessary to refer to the reactive species information indicated in the instructions. It is difficult to find antibodies for some rare species. You can select antibodies for homologous sequence immunity through protein sequence comparison. However, in general, manufacturers will not accept their complaints about quality. If you can apply for free antibody samples, This is beneficial for antibody selection.
3. The specificity of the experimental method:
At present, there are many experimental methods for using antibodies. During the process of using different methods, because the protein samples are processed in different ways, the protein content is different, and the requirements for the recognition epitope and titer of the antibody are different. Specifically, choose according to the product application method indicated in the manual. However, manufacturers generally do not test each antibody in various experiments. At present, only WB, IHC, IF, etc. are more tested. If no suitable antibody is found for a specific experimental method, protein sequence analysis and The antigen information of the antibody, select the antibody that is as effective as possible. Also, it is a good way to apply for free antibody samples.
4. Specificity of markers
Generally, experimental methods based on experimental operations do not use labeled primary antibodies, such as WB, IHC, etc., and the purpose of displaying results is achieved through the labeling of secondary antibody reagents. However, some experiments based on instrumental analysis may use directly labeled primary antibodies, such as flow cytometry experiments. Then you need to know the range of fluorescence that can be detected by the instrument you are going to use, and choose the corresponding marker according to the different parameter requirements. In immunofluorescence double labeling experiments, different fluorescent markers need to be selected.
2. Selection of source of antibody species:
Some people think that monoclonal antibodies are better than polyclonal antibodies. In fact, this is not a rigorous point of view. It can only be said that the specificity of monoclonal antibodies is better in possibility, while the affinity of polyclonal antibodies will be better than monoclonal antibodies, and the specificity is not good. It must be inferior to monoclonal antibodies, mainly depending on the level of antigen design. At present, the monoclonal antibodies in the commercialized antibodies are mainly mouse monoclonal antibodies and rabbit monoclonal antibodies, and the polyclonal antibodies are mainly rabbit polyclonal antibodies, goat polyclonal antibodies, etc., and there are fewer antibodies from other species. It is not recommended to be entangled in monoclonal antibody or multi-antibody is better, the antibody that can make experiments is a good antibody.
In terms of selection, it is generally recommended that the farther the affinity between the experimental species and the antibody species is, the better, and it is not suitable to be homologous. Different species of antibodies will affect the matching of secondary antibodies. Especially in the immunofluorescence double-labeling experiment, it is necessary to distinguish the source of the antibody species of the two indicators on the basis of the difference from the experimental species, so as to facilitate the identification of the secondary antibody.
Three, about the choice of cost performance:
The quality of the antibody still needs to be presented through the experimental results, but we cannot accurately judge the effect of the antibody before selecting the antibody. No matter what brand, whether imported or domestic, there will be good products, and there will be products that cannot be made. Antibodies are heterogeneous products. There are obvious differences in the quality of antibodies between different brands, or even between different products of the same brand, and there is no unified measurement standard. This is the most difficult problem in antibody selection. Many people are used to making selections based on the laboratory’s traditional consciousness or the products used in the references, but because the antibody effects between different batches are also different, the correct choice cannot be completely guaranteed. In this case, the after-sales service of antibodies is particularly important. When encountering antibody quality problems, it can ensure that the problem can be effectively solved without affecting the progress of the experiment. Now some brands can provide free antibody samples, which is undoubtedly a good solution to this problem. First determine whether the antibody is suitable for your experiment, so as to avoid delaying the experiment arrangement due to antibody quality complaints.
In terms of price, you need to keep in mind that the most expensive is not necessarily the best, but the best for your experiment. It is not uncommon to spend a lot of money on expensive antibodies and take a long time to complain. Buy what is right, not what is expensive. When choosing the price, you should also pay attention to the size of the specification, antibody concentration, potency range, etc., and consider it comprehensively. In terms of specifications, it is more accurate to measure according to the amount of working fluid that can be diluted into finally. The antibody products provided by many old brands are basically in large packages. As far as experiments on a single subject are concerned, oversized packages sometimes cause a lot of waste.
At present, Proteintech, Bioworld, Cell Signal and other brands have successively launched antibodies in small packages, which to a certain extent give the selectors greater freedom of choice. They can calculate the amount of antibodies they actually need according to the potency of the products used, and make to precise and quantitative procurement.
Fourth, the choice of the brand:
Because of the heterogeneity of antibody quality, everyone pays attention to the choice of antibody brand, but no matter what the brand is, it is inevitable that you will encounter antibodies that are not suitable for your experiment. Therefore, it is generally recommended to consider brands that are generally recognized in the industry, easy to purchase, professional technical support, and convenient after-sales processing. Some don't even have official agents, they can only be optional. At the same time, when purchasing, be sure to find a regular agent who can provide product guidance, experimental analysis, and after-sales processing, so as to avoid affecting the experiment due to the purchase of fake parallel imports.
5. About other small details:
In the process of antibody selection and use, some unexpected things will be encountered. Some details still need more attention. For example, when choosing to do blocking or neutralizing experiments, it is sometimes necessary to add antibodies to the cell culture medium, and it is necessary to pay attention to whether the antibodies contain sodium azide. If it is a large package of antibodies, it is more economical to sub-package. If it is a high-titer antibody, it can be made into a mother solution and then sub-packaged. For short-term transportation of antibodies, keeping the temperature with ordinary ice cubes can meet the requirements.
Also don't get bogged down with molecular weight in the WB process. Many times, we see that the same antibody, different brands, or even different product numbers of the same brand, have different molecular weights. In fact, it may be that the manufacturer only used different species and different sample types for testing, or referred to different literature. For users, the same sample, no matter which brand is used, as long as the antibody is correct, the purpose The bands will only appear in the same position.
Regarding the molecular weight, it is generally not recommended to read the product description. The position of the band in WB is determined by the sample and will not vary with different antibodies. Therefore, before detecting the protein, we can use the database or references to get a more comprehensive understanding of the protein to be detected, including its possible cleavage and modification, phenotype, etc. When using a new antibody for the first time, do not cut the membrane horizontally according to the molecular weight, you can keep a few full lanes to see the position or distribution of the bands.
