Introduction to antibody library screening and screening methods

Posted by beauty33 on May 14th, 2019

Since the phage display technology was founded in 1985, the fields of cell biology, immunology, protein engineering, and the pharmaceutical industry have been deeply affected. It fundamentally changed the traditional monoclonal antibody preparation process (hybridoma indirect technique), and announced the specificity of antibody modification and affinity maturation in vitro. With the continuous development of this technology, various display technologies such as ribosome display, mRNA display, bacterial display and yeast display have emerged. This article mainly uses the phage display antibody library as an example to introduce the screening technology of the antibody library.

The screening of the antibody library refers to screening specific antibodies against a certain antigen from the antibody library, which is a key link in the process of obtaining high affinity antibodies. So what is an antibody library? The full-length antibody variable region gene in human or animal is cloned by PCR and DNA recombination technology, and expressed by display technology, and the complete antibody gene expression library obtained is an antibody library.

Due to the wide variety of monoclonal antibodies, screening of antibody libraries requires strict screening conditions and optimization of screening methods based on different monoclonal antibodies. Therefore, the screening technology of antibody libraries has been in a state of development and improvement, and is mainly divided according to the time of occurrence. Classic screening method and new screening method.

1. 1.classic screening method

Classical screening methods mainly include solid phase screening and liquid phase screening, which are suitable for antibody screening for antigens with clear and purifiable properties. The solid phase screening method enriches high-affinity phage by antigen coated on a solid phase medium such as an enzyme plate or an immunotube; the liquid phase screening method coats the biotinylated antigen with avidin On the magnetic beads or agarose, the phage antibody capable of specifically binding to the antigen is enriched by magnetic beads, and then washed, eluted, recovered, and the like. By repeating the screening several times in this way, a high affinity phage can be obtained. Both methods can reduce non-specific binding by adding skim milk or BSA.

2. 2.the new screening method

For cases where the antigen cannot be purified or the nature is unclear (such as cancer cell surface receptors), or the classical screening process may cause antigen inactivation, new screening methods need to be developed. The current novel screening methods mainly include cell screening method, tissue section or in vivo screening method, selective infection screening method and protein chip screening method.

Cell screening method:

Cell screening can maintain the natural conformation of antigens and antibodies, so it is widely used in screening tumor cells. This technique is also suitable for cell surface receptor screening and antigen identification. However, cell screening has certain difficulties. Due to the complex surface composition of the cell membrane, non-specific binding is increased, and the number of rounds of screening is too large to easily lose specific bound antibodies. In order to reduce non-specific binding, cell screening methods have developed methods such as subtractive screening, competitive screening, and internalization screening. Deduction screening is performed by reducing antigen-negative cells by binding to an antibody library before or after screening. The principle of internalization screening is that some antibodies that bind to cell surface antigens enter the cell, so antibody screening can be performed by internalization of cells. The specific operation is to first use the antigen-negative cells to screen the antibody library for subtraction screening, and then incubate the antibody library with the antigen-positive cells, wash away the antibody bound on the surface of the cell membrane, lyse the cells to obtain specific binding antibodies in the cells, and then perform amplification. With the next round of screening. Competition screening is to incubate excess negative and positive antigens with the antibody library, and the competition screening is divided into fluorescence activated cell separation method and immunomagnetic cell separation method for different recovery methods of positive cells. In the FACS method, fluorescein can be labeled with an antibody to be screened, washed, and sorted by flow cytometry.

Immunomagnetic cell separation is also known as immunomagnetic beads. The principle is that cell surface antigens can bind to an external magnetic field when bound to specific antibodies attached to magnetic beads, while cells that cannot be bound are separated.

Slice tissue or in vivo screening method:

Slice tissue and in vivo screening methods are closer to clinical applications. The slice tissue screening method uses fresh tissue sections for antibody screening, but due to the limited number of antigens, the recovered phage antibodies tend to be less. The in vivo screening method is to inject the antibody library to be injected into the mouse intravenously, and take out the tissue or the target organ several hours later, and recover the phage, so that after 3 to 4 rounds, the specific antibody can be enriched.

Select infection screening method:

The most commonly used phage display is the PIII display, which is the fusion of a foreign protein or polypeptide with the N-terminus of PIII. The PIII molecule contains three domains, the amino-terminal domain (NT) N1 and N2, and the carboxy-terminal domain ( CT), a link between NT and CT linked by glycine-rich linker peptides (G1 and G2). These three domains and linker peptides play an important role in the infection of bacteria. The principle of selection of infection screening (SIP) is to replace the NT end of PIII with a specific polypeptide or protein. When these polypeptides or proteins specifically bind to a ligand with an NT terminus, the phage has the ability to infect, thus It can be propagated in bacterial cells and then enriched.