The antibody or protein labeling refers to covalently linking a target substance (enzyme, fluorescein, biotin) to an antibody or other protein, and specifically reacts with the generation test substance to form a multi-complex. With the help of precision instruments such as fluorescence microscope, ray measuring instrument, enzyme label detector, electron microscope, and luminescence immunoassay instrument, the test results can be directly observed by microscopy or automatically determined at the cellular, subcellular, ultrastructure, and molecular level. Perform qualitative and localization studies on antigen and antibody reactions or apply various liquid and solid phase immunoassay methods to qualitatively and quantitatively determine haptens and antigens in body fluids. At present, antibody labeling technology has been widely used in analytical research and technical determination in the fields of medical pathology, immunohistochemistry, molecular biology, and biopharmaceuticals. Common antibody labeling techniques include enzyme labeling, biotinylation labeling, and fluorescein labeling.
Commonly used antibody/protein labeling technology
- Enzyme labeling
Covalently bond the enzyme to the antibody or protein by a suitable method to make an enzyme-labeled antibody. Then, by the specific catalytic action of the enzyme on the substrate, a colored incompatible product or particles with a certain electron density are generated. These colored products can be observed with the naked eye, optical microscope, and electron microscope. It can also be measured with a spectrophotometer. The color reaction shows the presence of enzymes, which proves that the corresponding immune response has occurred.
Protein-protein conjugation is usually carried out with a bifunctional linker cross-linking agent. One end of the cross-linking agent (NHS ester) reacts with the amino acid lysine and the amine (-NH2) found in the N-terminus, and the other end (maleimide) Amine) reacts with the thiol group (-SH) found in amino acids. However, SMCC-modified proteins are extremely unstable and are usually self-reactive.
- Biotin labeling
Avidin and biotin can be combined with proteins (antigens, antibodies, enzymes), fluorescent agents, and other molecules without affecting the biological activity of the latter. It is an ideal labeling agent. An antibody molecule can be coupled with dozens of biotin or Avidin molecules, and avidin or biotin molecules can be combined with enzymes or fluorescein to form a bio-amplification system, which significantly improves the sensitivity of detection. Commonly used avidin-biotin labeling method, avidin -Biotin bridge method, and avidin-biotin-peroxidase complex method.
- Fluorescent labeling
The combination of dye and protein requires the formation of a stable covalent bond between the dye and protein, and this covalent bond cannot be broken under most conditions. Compared with the inherent fluorescence of proteins, small organic dyes have greater flexibility in selecting labeling sites and enhanced fluorescence and have been widely used in protein labeling. After labeling with appropriate dyes, proteins can be visualized in various biological processes, so that they can be quantified in biochemical assays or located in cell research centers. Dye-protein conjugates have become very important tools for studying enzyme activities, protein interactions, conformational changes, and regulating and observing specific biological processes. They have been widely used in ELISA, Western blotting, flow cytometry, and cell imaging.