Introduction and selection of commonly used blocking agents in immunoassay

In immunological experiments, blocking agents are often used to reduce non-specific binding. Commonly used blocking agents are skim milk powder, BSA , serum and Fab fragment single-strand secondary antibody. Select the appropriate sealer according to different platforms.

Skimmed milk powder
Function principle : Skim milk powder contains a variety of proteins, which can block the unadsorbed protein solid phase carrier (WB membrane, ELISA plate, etc.) sites, reduce the probability of antibody binding, and avoid the high background caused by non-specific binding.
Advantages: The price is cheap, because it contains a variety of different molecular weight proteins, so it is more comprehensively enclosed.
Disadvantages: complex composition and narrow application range. Not applicable to biotin and alkaline phosphatase labeled antibody systems. Moreover, the blocked vector is not easy to store for a long period of time and is therefore less used in the production of the kit.

Bovine serum albumin BSA
Principle of action: Same as skim milk powder, blocking the solid phase carrier site of unadsorbed protein.
Advantages: Single component, suitable for ELISA experiments, immunological detection experiments of biotin, alkaline phosphatase system or other experiments with higher specificity.
Disadvantages: A small amount of antibody residues in most BSAs can cause cross-reactivity between antigens/antibodies, resulting in a background.

Animal serum
Principle of action:
1. The sample to be tested will have some substances that are non-specifically bound to the protein, resulting in too high background, so this part of the non-specific binding can be blocked with serum. From this point of view, there is no significant difference between serum and BSA;
2. There may be an Fc receptor in the sample to be tested, which can bind to the constant region of the antibody, and the antibody is blocked in the serum, so that the binding of the primary antibody and the secondary antibody to the sample Fc receptor can be blocked by serum. BSA does not block the Fc receptor.
Advantages: Because serum can block Fc receptors and reduce non-specific binding between antibodies, it is widely used in immunohistochemistry and immunofluorescence experiments.
Disadvantages: Some blocked serum may contain sodium azide, so it is not suitable for the HRP enzyme labeling system.

Fab fragment single chain secondary antibody
Principle of action: Since bivalent antibodies (full IgG or F(ab')2 fragments) have two binding sites, they cannot be used for blocking. The Fab fragment single-stranded secondary antibody has only one binding site, thus completely blocking the immunoglobulin.
Application: In the IHC or IF double-label/multi-label experiment, the immunoglobulin on the surface of the sample is blocked, the endogenous immunoglobulin on the tissue section or cell surface is blocked, and the trouble of the two primary antibodies from the same genus can be solved.

※ No protein sealer --- New high-efficiency sealer <br> Guangxi Laidijia Biotechnology Co., Ltd. is proud to introduce Blocker protein-free sealer, which is a chemically synthesized compound with high hydrophilicity and no bio-blocking agent. Worried about biosafety, quality, poor batch and other issues.
main feature:
1) Coupling with the activated carboxyl group on the surface of the microsphere to form a stable covalent bond
2) Pure chemical synthesis, small difference between batches, high purity
3) The spatial structure is simple, and the surface of the microsphere is more fully enclosed, which reduces the non-specific adsorption of the microspheres and reduces the matrix interference.
4) Improve reagent sensitivity
5) Contains a large number of hydrophilic groups, which is beneficial to improve reagent stability
Application: Latex immunoturbine reagent / chemiluminescent reagent, instead of BSA as a blocking agent or combined with BSA.
Guangxi Ledijia Biotechnology Co., Ltd. Blocker sealant, this product is a very good protein-free immunoblocking agent. For more information, please contact QQ: 357497328 , the immune professional customer team is eager to serve you!
In addition, the products recommended by Ledijia Bio are: immune reagent stabilizer
Immune Reagent Stabilizer <br> Immune Reagent Stabilizer is mainly used for immunoassay detection reagent/antigen/antibody/protein preservation. This product is ready-to-use, no dilution, easy to use, and the substance to be preserved is dissolved in the stable solution. Put it in the refrigerator. The immunological reagent stabilizing solution is beneficial for maintaining the three-dimensional structure of the protein, maintaining the biological activity of the protein, prolonging the use period of the immunoassay reagent and protein, and ensuring the performance of the product.
Application areas :
1) Immunoassay reagent storage, such as immunoturbidimetric detection reagent / chemiluminescence detection reagent
2) Protein preservation, such as antigen/antibody/enzymes, etc.