Antibodies are immunoglobulins specifically introduced by immunity response of high animals, with the responsibility for recognizing and cleaning out specific antigens. Antibody is not only a powerful weapon against pathogen invasion in the organism, but also a tool for specific molecular recognition used in basic scientific research. The diversity of antibody molecules resulted in the concept of antibody library; each individual animal is a natural antibody library. In the post-genome era, in order to fit various “omics”, especially for proteomics requirement of high throughput technology, some gene engineering antibody libraries and antibody alternative libraries have been constructed based on phage display technology. Yet, more and more in vitro display systems such as ribosome display, mRNA display have been used for antibody library study, and that present more advantages than phage display.
In the 1980s, the research results of antibody gene structure and function were combined with recombinant DNA technology to produce genetically engineered antibody technology. The early antibody genes used to construct genetic engineering were derived from hybridoma cells. Since obtaining hybridoma cells must undergo a long-term, complex process of animal immunization, cell fusion and clonal screening. Moreover, it is difficult to prepare humanized antibodies and autoantigens or weak immunogenic antigen antibodies by hybridoma technology, so it restricts the popularization and application of genetic engineering antibody techniques. In the 1990s, combinatorial chemistry technology and genetic engineering antibody technology combined to produce antibody library technology. From then on, the antibody engineering technology has entered a new stage of development. The production of antibody library technology is based on the breakthroughs of two key technologies: (1) the emergence and development of PCR technology enables people to use a set of primers to amplify the whole set of immunoglobulin variable region genes; (2) the antibody molecules fragment with antigen binding function can be successfully expressed in Escherichia coli.
In a narrow sense, the so-called antibody library technology is to clone the whole set of antibody heavy chain and light chain variable region genes by gene cloning technology, then restructure it into a specific prokaryotic expression vector, and then transform the Escherichia coli to express the functional antibody fragment and obtain the specific antibody variable region genes by affinity screening. Antibody genes screened by antibody library technology will be used to construct and express genetic engineering antibodies.
With the continuous improvement and development of antibody library technology, the definition of antibody library technology has been constantly updated. Depending on the screening technique used, the antibody library technology has undergone three stages of development: a combination antibody library, a phage antibody library, and a ribosome display antibody library. In 1989, House of the American Scripts Institute first expounded the combined antibody library technology. They used RT-PCR technology to clone a full set of genes of the antibody light chain and a full set of gene of the heavy chain Fd segments from lymphocytes, and respectively constructed them into the λ phage expression vector to obtain the light chain gene library and the heavy chain gene library. and then the light chain genes and heavy chain genes are randomly recombined into the same expression vector to form a combination antibody library. The antibody library was packed in vitro and infected with Escherichia coli and cultured in a plate. The Escherichia coli infected with λ phage was cracked by the proliferation of phage, and the released phage re-infects the surrounding E. coli cells to form plaques. The expressed Fab fragments were also released into plaques. The plaques were transferred to a nitrocellulose membrane, the specific antibody fragment was cloned with the antigen labeled by isotope or horseradish peroxidase to obtain a gene of the Fab fragment. It is not difficult to see that the combined antibody library technology has obvious advantages over the hybridoma technology: (1) The step over the fusion of cells saves time and labor; (2) The screening capacity is expanded, which is beneficial to obtain high-affinity antibodies; (3) The antibody gene is directly obtained, which saves the step of cloning the gene from the hybridoma cells to facilitate further construction of various genetically engineered antibodies; (4) The screened antibodies are more likely to be expressed in Escherichia coli; (5) There is no need to be immunized in the body, it is possible to obtain autoantigens, weak immunogenic antigens, antibodies against toxic antigens, and human antibody.
Combined antibody library technology has been replaced by phage antibody library technology less than a year after its appearance. The phage antibody technology is based on phage surface display technology, which has been the most mature and widely used antibody library technology by far. Using this technology, the immunization antibody library, the natural antibody library, the semi synthetic antibody library and the total synthetic antibody library have been constructed successfully, and the genes of functional antibodies have been screened out from them. In recent years, many new antibody library technologies have been developed, such as selective phage display antibody library and ribosome display antibody library. The construction and screening of ribosome display antibody library is completely carried out in vitro, and it is not necessary to carry out the transformation of Escherichia coli. It is easier to construct high-capacity, high-quality antibody library, screening high affinity antibody and transform the antibody by in vitro evolution. Therefore, it represents the future development trend of antibody library technology.
At present, the use of antibody library technology can not only screen specific antibody fragments, but also be able to modify existing antibody molecules, such as reducing the mouse origin of antibody, improving its affinity and stability. The latter is also known as antibody directed evolution, and the antibody library used is called a directed evolution antibody library. Since the whole process of ribosome display technology is carried out in vitro, it is not necessary to transform E. coli for cloning and expression, it is easier to introduce mutations, construct a high-capacity mutation library, and it is convenient to carry out multiple rounds of repeated screening, which is a good tool for in vitro directed evolution of antibodies.