The main idea of antibody repertoire technology is to clone all the antibody variable region genes of an animal into plasmids or phage, and use different antigens to screen out clones carrying specific antibody genes to obtain corresponding specific antibodies.
A variety of important antibodies, such as membrane protein antigens, autoantigens, and antibodies to viral antigens, are screened from antibody libraries. These show the potential for application of antibody library technology.
Antibody library technology not only mimics the process by which the animal's immune system produces antibodies, but also has many unique advantages that make hybridoma technology difficult to compare. The antibody library technology does not require immunization. In theory, 10 to 10 storage capacity may contain all antibodies. By using the antigen, specific antibodies can be directly screened from the non-immune animal antibody library, and antibodies against the species' own antigen can be screened. A completely human-derived McAb can be obtained from a humanized monkey antibody library, overcoming the difficulty of obtaining a human McAb with hybridoma technology. In addition, due to the rapid proliferation of bacterial cells and low cost of cultivation, it is advantageous for large-scale preparation of high-purity antibodies for protein crystal structure research and application. Therefore, antibody library technology will play an important role in the development of biology and medicine.
The antibody library technology includes the following main processes: the complete set of variable region genes of the antibody is cloned, linked to the relevant vector, introduced into the recipient bacterial system, and the genes are expressed on the surface of bacteria, phage, etc. by using the conditions of synthesis and secretion of the receptor protein. Screening and amplification were performed to establish an antibody library.
The phage antibody library technology is a breakthrough in antibody library technology. It is not a soluble expression product of bacterial clones detected at the time of screening, but a Fab or scFV expressed on the surface of phage particles after phage vector transformation of bacteria. It is called a phage antibody. Through multiple rounds of antigen adsorption-elution-amplification, the desired antibody clones are finally screened, greatly simplifying the screening process. The selected phage antibody clones can be obtained by inserting amber mutants and recipient bacteria that cannot read amber mutations to obtain soluble Fab or scFV.
Burton et al. cloned human antibody-encoding gene into phage and expressed human antibody active fragment (Fab) with engineered bacteria, and its antigen neutralization ability
It is more than 1000 times higher than human natural antibodies. The surface-expressed phage antibody library utilizes a biphasic expression vector, ie, the vector appears in two forms during gene cloning, namely phage form and phage form. The phage system completely mimics the human B cell system, in which different phage are directed against different antigens, the genes encoding the antibodies are within them, and the expressed proteins are outside.
The phage antibody library (a full-featured antibody library) can realize the humanization, miniaturization and multi-functionalization of monoclonal antibodies, and theoretically, if a large-capacity antibody library is constructed, a monoclonal antibody technology against any antigen can be selected. The diversity of antibodies, and thus the effective epitope of the phage antibody library (referring to the selection of corresponding antibody-binding fragments from any antigen or hapten) directly affects its utility. A phage antibody is a single-stranded phage (or fibrillar phage, most commonly known as M13) that expresses an antibody molecule Fab or a single-chain antibody on the surface of a phage. This surface expression is through a Fab or single-chain antibody and a single-stranded phage coat. The protein (PIII or PVIII) is formed by the formation of a fusion protein.
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