一．Introduction to Phage Antibody Library Technology

Phage display antibody library technology (phage display antibody library techniques) refers to the use of polymerase chain reaction (PCR) to amplify the full set of variable region genes of antibodies, and through phage surface display technology, express Fab fragments or single-chain antibodies (ScFv) on the surface of phages, and screen and enrich specific antibodies through the "adsorption-elution-amplification" process.

二．Basic Principles of Phage Antibody Library Technology

The principle of phage antibody library technology is to randomly recombine the antibody heavy chain variable region (VH) and light chain variable region (VL) genes with the phage coat protein III (PIII) or coat protein VII (PVIlI) genes, and then infect Escherichia coli, proliferate and express them on the phage surface in the form of antibody fragments Fab or ScFv-coat protein fusion proteins. This type of phage particle can specifically recognize antigens and infect host bacteria for re-amplification. After the "adsorption-elution-amplification" process, specific antibodies can be screened and enriched. The constructed antibody library is called a full set of antibody libraries, and the antibodies screened from it are called phage antibodies. Its biggest feature is that it realizes the direct connection between genotype and phenotype, and can quickly and efficiently screen expression-specific antibodies from a large number of clones.

三．Construction of Phage Antibody Library

First, extract the total RNA of the cells, amplify the variable region gene by RT-PCR, use two different restriction endonucleases to digest the purified light chain PCR product and the expression vector respectively, and then separate and purify the cleavage products, connect the vector and the light chain in a certain proportion, transform the competent cells and expand the culture, and the extracted plasmid is the light chain library. Then double-digest the heavy chain PCR product and the light chain library, purify them, connect them in a certain proportion, transform the competent cells and expand the culture, and the extracted plasmid is the light chain library. Then, the heavy chain PCR product and the light chain library are double-digested, purified, connected and transformed into competent cells in a certain ratio, added with helper phage culture, centrifuged to obtain the supernatant, added with polyethylene glycol (PEG) for precipitation and collected phage particles, thus obtaining the phage total antibody library.

四．Expression Vector of Phage Antibody Library

The expression vector of phage antibody library can be divided into three systems: λ phage (phageλ), single-chain filamentous phage (filamentous phage) and phagemid (phagemid), each of which has its own advantages and disadvantages. The phage expression system can be cloned by plaque lift, but the exogenous fragment cannot be too large, and its library capacity is small (10^6). Filamentous phage is mainly used by Tek Biotech. This vector includes two subsystems, monovalent and polyvalent, which can screen high-affinity antibodies. Phagemid is the most widely used expression vector and is also a simple and efficient prokaryotic expression system for simulating B cell production of antibodies. The purpose of building an antibody library is to screen out various specific antibody molecules, and phage antibody libraries have incomparable advantages in this regard.

五．Screening of Phage Antibody Libraries

There are two classic screening methods: one is to coat pure antibodies on solid phase media, such as ELISA plates, immunotubes or affinity chromatography columns, then add phages to be screened, wash off non-affinity phages, and recover high-affinity phages; the other is to connect antibodies to biotin groups, and then fix them on paramagnetic beads coated with streptavidin antibiotics to screen phages. During the screening process, the type of phage, the antigen density on the surface of the solid phase medium or the concentration of the antigen in the solution, and the washing time are three factors that have a greater impact on the efficiency of the screening. At the same time, each round of screening needs to be tested to confirm the effectiveness of the screening.

六．Advantages of Phage Antibody Library

1. The method is simple and easy, saving time, and can be prepared in large quantities through fermentation production;

2. The screening capacity is increased, and 1 million to 100 million clones can be screened within a few weeks to obtain high-affinity humanized antibodies;

3. The antibody gene is directly obtained from the lymphocytes of unimmunized humans or mice, so fully humanized antibodies can be obtained, overcoming the shortcomings of hybridoma cell instability and avoiding artificial immunization and hybridoma technology;

4. Simulate the in vivo immune process and the natural full set of antibody libraries, and freely "manufacture and clone antibodies against any antigen."

Tek Biotech has M13, T4 and T7 phage display systems. According to the customer's project requirements, different phages will be used for protein, antibody and cDNA display. In the completed cases, the M13 phage pIII gene display system is more commonly used, and the constructed library capacity can reach 10^11, of which the effective library capacity is 10^9.