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The key step flow of phage display technology

author:Pro-Health Biotech AtaGenix

After more than 40 years of development, phage display technology has become increasingly mature and has become one of the mainstream platforms for antibody discovery technology. With the rapid development of antibody engineering, the industry of antibody drugs from murine to fully human sources has become a trend, and researchers have more demand for the transformation of antibody functions, the research and development of bi/multi-specific antibodies, and the technical requirements for antibody screening and optimization have been further improved, and phage display technology has also played an increasingly important role in this process.

Phage display antibody library is currently the most mature and widely used genetic engineering antibody screening technology, through phage display technology to make genetic engineering antibodies in the form of fusion proteins in the form of phage coat protein gpIII or gpVIII. N-terminus, so as to connect the genotype and phenotype of the antibody, with solid phase or liquid phase antigen incubation with antibody library, through "adsorption-elution-amplification" several cycles to enrich specific clones, so it has the advantages of simplicity, speed and efficiency.

The key step flow of phage display technology

Advantages of bacteriophage display technology

1. The method is simple, fast and time-consuming. It takes only a few weeks from mRNA extraction to antibody library construction and screening for positive clones of specific antibodies.

2. Large screening capacity and high efficiency. The phage antibody library usually contains 106-109 antibody molecule clones with different antigen binding specificity, which can select millions or even billions of molecules, and each round of "adsorption-elution-amplification" can enrich clones with strong antigen specificity, and it is very easy to screen specific antibodies from the antibody library.

3. Easy to operate at the gene level, protein phenotype and genotype are unified. According to different purposes, the antibody structure can be modified "as you like", genetic mutations can be carried out, and various new antibodies can be prepared; In addition, bacteriophage antibodies can also be conjugated with some functional molecules to form immunotoxins, enzyme-labeled antibodies, etc., further broadening the application range of antibodies.

4. Easy to scale production.

Phage display technical process

The key step flow of phage display technology

In general, phage display technology has the following 5 steps:

Step 1: Build a phage display library

Recombinant DNA technology is used to integrate foreign cDNA into viral DNA. Different genomes are inserted into the genomes of multiple bacteriophages. Spliced into a gene for a coat protein that shows up on the outside of phage particles, these isolated phages exhibit only one protein, peptide, or antibody. These collections of phages are called libraries, such as antibody phage banks, protein phage banks, or random phage libraries.

Step 2: Combine

These libraries are exposed to selected targets, with only some phages interacting with them. These goals are to plan the recognition of specific ligands, such as fixed proteins, cell surface proteins, or tissue cell surface proteins.

Step 3: Wash

Unbound phages can be washed away by the wash buffer, leaving only those that have an affinity for the receptor.

Step 4: Elute

Elution buffer elution recovers phages that have an affinity for the target

Step 5: Zoom in

Demonstrated specificity of eluting phages are used to infect new host cells for expansion, or directly for bacterial infection and expansion of recovered phages.

The key step flow of phage display technology

Phage display technology has attracted much attention due to its wide application fields, which requires the majority of practitioners to be familiar with and master phage display technology at the same time, carry out extensive exchanges and learning, and constantly make new breakthroughs while applying phage display technology.