一．Principle of Liquid Phase Screening of Library

This method uses liquid phase medium. First, biotin-labeled antigen is used to bind to recombinant phages with functional antibodies on the surface in solution, and then avidin-coupled agarose or avidin-coupled magnetic beads are used to capture recombinant phages bound to biotin-labeled antigens, thereby achieving enrichment of functional antibodies in the antibody library; by changing the antigen concentration and binding-elution time, high-affinity antibodies can be screened; or a reasonable screening procedure can be selected, such as using competitive secondary antibodies to remove irrelevant phage antibodies and screen antibodies targeting specific epitopes.

二．Advantages of Liquid Phase Screening

(1) High screening efficiency, increasing the chance of phage particles contacting antigens, and can effectively separate low copy number or low affinity antibodies from the library;

(2) Solve the problem of antigen epitope destruction after coagulation;

(3) The screening system can be flexibly adjusted according to the library capacity, antigen relative molecular mass and purity;

(4) The required antibody affinity can be predetermined to a certain extent. The screening system can be flexibly adjusted according to the library capacity, relative molecular mass and purity of the antigen;

(5) The affinity of the required antibody can be predetermined to a certain extent.

三．Comparison of Advantages and Disadvantages of Liquid Phase Screening

四．Common Methods for Liquid Phase Screening

Magnetic bead screening: The principle of magnetic bead protein purification is to use the specific binding between the affinity groups on the surface of magnetic beads and the target protein to separate the target protein from the mixture. Magnetic beads are usually composed of magnetic materials and affinity groups. The affinity groups can be metal ions, antibodies, enzymes, nucleic acids, etc. When the target protein exists in the mixture, the affinity groups on the magnetic beads bind to the target protein to form a magnetic bead protein complex. Then, through the action of the magnetic field, the magnetic bead protein complex is concentrated together to achieve the separation and purification of the target protein.

五．Magnetic Bead Screening Steps

Wash the avidin-labeled magnetic beads with Tris-HCl-Tween buffer (TBST) and block with BSA for later use.

(1) Coating magnetic beads: Incubate 500μL of blocked magnetic beads with 1nmol biotinylated antigen and 1nmol free biotin at room temperature for 1 hour. Use a magnetic rack to adsorb the magnetic beads on it, remove the supernatant, wash with TBST, and resuspend in TBS containing 1% BSA.

(2) Negative screening: Add 1 mL of amplified phage antibody library to biotin-coated magnetic beads and incubate at room temperature for 1 hour. Then use a magnetic rack to adsorb the magnetic beads.

(3) Antigen screening: Transfer the supernatant obtained above to biotinylated antigen magnetic beads, incubate at room temperature for 1 hour, then use a magnetic rack to adsorb the magnetic beads and remove the supernatant. Wash with TBST 10 times and remove the supernatant.

(4) Elution: Add 250 μL 10 μg/mL trypsin and mix for 30 minutes.

(5) Take 120 μL of eluted phage and add it to 1.5 mL TG1. Incubate in a water bath at 37°C for 30 minutes. After centrifugation, add 200 μL TES buffer and evenly spread on TYE-A-G plate. The rest is stored in glycerol.

(6) Preparation of secondary library: The next day, wash the bacterial moss and inoculate it in fresh culture medium for culture. Add helper phage KM13 to it. After infection for 30 minutes, resuspend the precipitate, culture it at 37℃ for 1 hour, add kanamycin to a final concentration of 50μg/mL, and culture it at 30℃ overnight. After centrifugation the next day, pour the supernatant into cold PEG/NaCl to precipitate the phage and prepare the secondary library.

六．Protein Coupling Step

Calculate the protein to be coupled. Generally, 20-500ug of protein (antibody) can be coupled per mg of activated carboxyl magnetic beads. Some carrier proteins, such as BSA Fraction V, can be added in appropriate amounts to increase the total protein amount in the reaction system, which can play a certain blocking role. Ensure the correct direction of antibody coupling.

(1) Add the protein to 5 mL of MES buffer. (Pipette 50 μL of protein solution into 950 μL of MES buffer to make a 1:20 dilution and mark it as the protein solution before coupling reaction. Set aside for the subsequent calculation of coupling rate.

(2) Add the remaining protein solution to the centrifuge tube containing activated magnetic beads and shake vigorously to mix. Place the centrifuge tube on a rotary mixer at room temperature for coupling reaction for 16-24 hours. (Place the centrifuge tube on a magnetic separation rack until the supernatant becomes clear. Use a pipette to carefully collect the supernatant and mark it as the protein solution after coupling for calculation of coupling rate.

(3) Resuspend the magnetic beads in 5 mL of MES buffer and shake well. Place the centrifuge tube on a magnetic separation rack until the supernatant becomes clear. Use a pipette to carefully remove the supernatant.

(4) Repeat step (3), add 5 mL of quenching solution, shake well, and place the centrifuge tube on a rotary mixer at room temperature for 30 minutes. Place the centrifuge tube on a magnetic separation rack until the supernatant becomes clear, then carefully remove the supernatant with a pipette.

七．Optimize Liquid Phase Screening Scheme

Immobilized metal affinity chromatography (IMAC) technology can be introduced into the screening of phage antibody libraries. The purified antigen with His tag is mixed with the phage antibody library. After the phage antibody and antigen are fully combined, the affinity medium is added to allow the phage antibody antigen complex to pass through the His tag. The s tag is combined with the medium, and then the non-specific phage antibodies are removed by sufficient washing. Finally, the specific phage antibodies are dissociated and infected with TG1 for the next round of screening. This is an improved liquid phase screening method.

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