一．Buffer Selection

The selection of buffer should not only consider the stability of the target, but also the actual application of the combination with the microenvironment, and minimize nonspecific binding. The use of irrelevant proteins, non-ionic detergents and physiological salt concentrations is conducive to reducing background. At the same time, there are some other conditions, such as some targets may require the addition of divalent cations or other auxiliary factors in the buffer. Protein auxiliary factors can be co-fixed with the target, or added to the sorting buffer, or even used as a method to capture the target.

二．Consideration of Selection Pressure

Common selective pressure considerations include: concentration of target molecule, binding time and washing intensity. Other techniques to increase selection pressure include the use of denaturants (such as acids, urea and guanidine), organic solvents, increasing temperature or increasing the concentration of competing ligands or targets in the washing buffer. The first round of screening is particularly important, and its purpose is to capture as many positive clones as possible from the constructed library while removing non-specific clones. Multi-porous post-coating targets or a large amount of soluble targets should be used during screening to ensure that a large number of binding phages are captured, thereby maintaining the diversity of the library.

三．How to Reduce the impact of Antigen Tags and Carrier Materials on Screening Efficiency

During the screening process, conjugates targeting all substances on the antigen, such as antigen tags, fusion proteins, and carrier materials, may be screened out. To overcome this problem, negative screening against non-targets can usually be used to limit the enrichment of antibodies targeting other than the target antigen. For example, before using streptavidin magnetic beads to screen biotinylated proteins, the library can be pre-bound with streptavidin magnetic beads and biotinylated non-target proteins to reduce the proportion of such unwanted conjugates.

四．How to Remove Complex Antigen Mixtures

For unknown or complex target molecules, such as whole cells or impure proteins, strict negative screening can be performed. For example, negative screening of whole cells is performed when the target protein is not known in advance, but the non-target protein is known. During negative screening, the phage display library is first incubated with recombinant proteins corresponding to the non-target antigen. The unbound phages are then transferred to the target antigen for screening.

五．Choice of Solid Phase and Liquid Antigen Screening Methods

When the antibody library capacity is large, the expected copy number of the target antibody is high or the affinity is high, and the antigen source is rich, the solidified antigen screening method is reliable and easy to use, which is conducive to obtaining high-affinity and high-specificity antibodies. When the target antibody is expected to be difficult to obtain or the solidified antigen screening is unsuccessful, the biotinylated antigen liquid phase screening method can be used. When the purpose of the screening is to select high-affinity antibodies, the liquid phase screening method has obvious advantages, especially the dissociation rate screening is more efficient, rapid and repeatable, which is a more ideal means.

六．Selection of Library Screening Methods

In the first case, if the structural characteristics of the receptor are known and the linear neutralization region or polypeptide fragment of the receptor can be easily obtained, then the classical screening strategy or high-throughput screening can be used. This strategy is simple and easy to use, and can screen antibodies with high affinity, but it is possible that the antibody has no binding activity with the natural receptor. In the second case, if the structural characteristics of the receptor are known, but there is no purified protein, the whole cell optimization screening strategy or high-throughput screening can be used. The third case is when the receptor is unknown, and appropriate negative cells are used to screen the common antigen, and then the target cells are screened to obtain specific antibodies.

七．Antigen Coating Method

Schematic diagram of direct coating and indirect coating 

八．Use of Combined Screening Method

During the screening process of phage antibody library, there will be some non-specific binding. If a single screening method is always used, these non-specifically bound phages will also be dissociated and amplified, thus being retained. The combination of several panning methods, such as the solid phase and liquid phase combined alternating panning method, can effectively avoid some non-specific adsorption.

九．Optimization of Dissociation Conditions

After the bound phage antibody is dissociated, it only shows medium or low affinity, which affects the enrichment and screening of specific antibodies. The dissociation process needs to be optimized. For example, gradually increase the pH value of the dissociation solution in the last round of screening; use the target protein solution as the dissociation solution to compete for the dissociation of phage antibodies; alternate dissociation method, etc.

Teck Biotech has established a complete and mature phage antibody display technology platform. Based on the phage display technology platform, Tek Biotech can provide major experimental links including antigen design, alpaca immunization, library construction and screening (solid/liquid screening), and active function verification, and provide highly specific and high-affinity camelid VHH antibodies to scientists around the world.