Compared with solid phase screening, the biggest advantage of using intact cells as screening substrates is that the receptors on the cell membrane surface maintain their natural conformation, and the receptor proteins do not need to be purified, which is more conducive to screening functional antibodies. Phage antibody libraries can be screened not only by binding to cells, but also by internalization. Since the ligand-receptor complex can be engulfed into the cell through cell internalization after ligand-receptor binding, this method can be used to screen phage antibodies that can be internalized through cell internalization. Antibody-antigen binding can simulate this process, so this method can be used to screen antibodies that can be internalized by cells.

Ⅰ. Principle of Cell Screening

There are a large number of signal molecules distributed on the cell surface, which reflect the characteristics of the cell and its functional state. Although molecules can be directly used as screening targets, for molecules such as receptors that are difficult to purify or expressed in low amounts, they need intact cell membranes or form complexes with other subunits on the cell membrane to function.

Ⅱ. Advantages and Disadvantages of Whole-cell Screening

Ⅲ. Whole-cell Screening Strategy

IV. Experimental Process of Cell Screening of Antibody Library

(1) Cell treatment: Add EDTA to the screening cells and centrifuge at 4℃, 1000rpm for 10min;

(2) Washing: Centrifuge the treated cells with serum-free DMEM medium at 4℃, 2000rpm for 1min, and wash 1-2 times;

(3) Incubate phage: Take the prepared phage solution and add it to the screening cells, add DMEM medium, and incubate at 4℃ for 2h;

(4) Washing: Centrifuge the incubated cells with serum-free DMEM medium at 4℃, 3000rpm for 1min, wash 8-10 times, and then wash 3 times with PBST;

(5) Elution: Centrifuge at 4℃, 3000rpm for 5min, discard the supernatant, add Tris-HCl (pH=3.0), resuspend the cells, centrifuge at 4℃, 4000r for 5min, and quickly neutralize with Tris-HCl (pH=8.0) to about pH=7.0. (6) Activation of 2738: Take two centrifuge tubes, pour LB liquid culture medium, add tetracycline, add a single colony from the 2738 plate to one of them, and use the other as a blank control. Incubate in a shaker at 37°C and 250r for 4h.

(7) Dilution of eluted product: Take three sterile 1.5ml centrifuge tubes, add sterile LB culture medium to each tube, add the eluted product to the first tube, mix well, take out the eluted product from the first tube and add it to the second tube, mix well, take out the eluted product from the second tube and add it to the third tube, mix well.

(8) Infection: Take three sterile centrifuge tubes, add the activated 2738 bacterial solution and the diluted eluted product (one tube for each gradient) to each tube, and place them at 37°C for 1.5h.

(9) Pour the plate: Add 3-4 ml of the top gel to which IPTG and X-gal have been added to the infected bacterial solution, mix well, and pour it on the IPTG+X-gal plate (the plate must be preheated in a 37°C incubator for at least 1 hour), place it in a 37°C incubator, and culture it overnight.

V. The Currently More Successful Cell Screening Methods are Mainly:

(1) Subtractive screening: This method is used to screen antibodies targeting melanoma cells from the phage antibody library. For example, the specific operation is to first perform several rounds of positive screening with melanoma cells, and then perform negative screening with normal melanoma to remove antibodies that do not specifically bind to positive cells. Due to the low concentration of cell surface antigens and the inherent defects of deductive screening, this screening method is very difficult.

(2) Cell internalization screening: Some antibodies bind to cell surface antigens, and the antigen-antibody complex can be engulfed into the cell. Therefore, the cell internalization screening method can be used to screen such antibodies. Studies have shown that this screening method is feasible and it is easier to obtain specific phage antibodies than cell surface screening.

VI. Application of Whole Cell Screening Method

Whole cell screening method can be applied to the search for new receptors, ligands and antigen epitope analysis, the development of new peptide vaccines, the screening of gene therapy targeting vectors, the screening of tumor targeted therapy vectors, the development of enzyme receptor agonists and antagonists, disease detection and treatment, etc.

Tek Biotech has complete upstream and downstream docking services, including antigen, antibody expression purification, antibody humanization, recombinant antibody expression and other services. It has mature and stable technology in phage library construction. Phage display technology provides a library capacity of 10^8-10^9 for the construction of level 1 immune library, and the insertion rate meets >95%. The antibody affinity obtained by screening is generally at the nM-pM level. It has a variety of screening systems, including direct screening method, competition method, negative screening method, cell screening method, etc., which can provide VHH screening, scFv sequence screening, Fab antibody screening, etc. to meet the different needs of different customers. High product delivery standards: for immune libraries, delivery of pre- and post-immunization sera, antibody display libraries, screening of eluted products, nanoantibody sequences with complementary CDR regions, QC quality control standards (including RNA extraction, cDNA preparation, etc.).

If you are interested in phage display antibody projects, or other related services, such as antibody sequencing, scFv, Fab and Fc fusion proteins, please contact us!