一．Technical Difficulties in Cell Screening

The main challenge in screening phage display gene libraries on cells (or tissues) is that the cell surface contains a large number of receptors and proteins. Unlike screening fixed antigens, the high abundance of a large number of proteins and certain protein components on the cell surface can easily interfere with the screening of cells that overexpress antigens, and even make it difficult to separate the target antibodies.

二．How to Improve the Efficiency of Cell Screening?

1. Reduce the interference of background signals: Use the corresponding negative cell line (negative cell line) or tissue for background subtraction, which is a negative selection process. The main factors affecting the background elimination effect are: cell line selection and cell number for background elimination, cell/phage ratio, binding and washing conditions, phage and cell separation methods, and selection rounds. In actual operation, how to set positive and negative selection depends on the type and capacity of the phage antibody library used, the cell or tissue type, the level of antigen overexpression, and the characteristics of the target antigen.

2. Improve the sensitivity and specificity of selection: mainly achieved by optimizing the cell screening process. In addition, ultra-large capacity antibody libraries are also the key to successful cell screening.

3. Improve reactivity: Due to the expression amount and variability, tumor antigens and receptors on the cell surface are not ideal immunogens for preparing antibodies, so the selection efficiency must be improved in in situ screening to obtain relevant antibodies.

三．Cell Sorting Method

For adherent cells grown on cell culture plates, the binding, washing and elution of phage antibody libraries are similar to traditional solid phase screening. For the sorting of suspended cells, phages can be mixed and incubated with the cells to be screened, and the cells bound to the phages can be separated into organic phases for recovery by low speed or differential centrifugation. For labeled target cells, negative selection cells can also be added to the screening, and FACS (fluorescence activated cell sorting) or MACS (magnetic activated cell sorting) methods can be used to sort the labeled target cells.

四．Main Applications of Cell Screening

In the screening of membrane proteins such as tumor antigens and receptors, it is crucial to maintain the native conformation, which is difficult to achieve with solid phase screening. Complete cell screening can avoid the problem of conformational changes. In addition to intact cells, target materials for antigens on cell membrane preparations and tumor tissue sections. The research on cell-based antibody screening for tumor cell screening is becoming more and more extensive.

五．Selection of Cell Materials

Among the targets for cell screening, some are identified antigens with known gene sequences, some are tumor cell lines that have not yet been identified, and there is also a type of tumor tissue for which little information about antigens is known. For a few targets with clear sequences and structures, purified antigens or expressed recombinant proteins can be screened, but because of conformational changes, the ability of antibodies obtained in this way to recognize and bind to natural antigens varies greatly. Through cell transfection, antigen proteins can be located on the cell surface, and specific antibodies can be screened using intact cells or cell lysates as target materials.

六．Selection of Antibody Libraries

In antibody screening for cells, immune antibody libraries prepared from antibody genes amplified from immune animal B cells can be used, as well as natural antibody libraries, synthetic libraries, and semi-synthetic libraries without antigenic bias. The preparation of immune antibody library is simple, the antigenic tendency of its coding sequence is strong, and the screening background is simple, so it is expected to quickly obtain high-affinity antibodies under limited library capacity. The inconvenience is that each antigen needs to be re-immunized and the library is built, but some antigens are difficult to work due to immune tolerance or toxicity. At this time, a large-capacity non-immune library or synthetic library can be selected to complete the screening, especially for unknown antigens and autoantigens with low immunogenicity. It is crucial to increase the library capacity to increase the chance of successful screening. The sequence composition of natural library and fully synthetic large-capacity antibody library is non-biased, large in capacity, and can overcome the inhibition of autoantibodies during antibody recombination in vivo, so it has wide applicability. In the semi-synthetic antibody library, the sequence of one chain (generally light chain) of the antibody molecule is fixed, and diversity is introduced only in the heavy chain, so the constructed antibody library may have antigenic tendency.

Tek 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.