Materials and reagents (1) Enzyme plate (2) Microplate reader (3) 0.5mol / L Na2CO3 (pH9.6) (4) 1% BSA (5) HRP-labeled anti-M13 phage polyclonal antibody (6) 2mol / L H2SO4 ã€Steps】 (1) Dilute the antigen to 10μg / ml with PBS or 0.5mol / L Na2CO3 (pH9.6); (2) For each clone to be tested, coat at least 2 wells and dilute the antigen with 200 μl per well. At the same time coated with negative control antigen. In addition, the positive control antigen is coated with anti-M13 antibody; (3) Coat at room temperature for 1 to 2 hours, preferably at 4 ° C overnight, shake off the liquid in the hole, and put it on a paper towel to dry. (4) Each well is blocked with at least 200μl 1% BSA, 37 ° C for 1h. Remove the liquid and let it dry. (5) The recombinant phage was blocked with an equal volume of blocking solution at room temperature for 15 min to 30 min in advance to block non-specific binding sites between various proteins. Using M13 phage as a positive control phage, also follow the above steps. (6) In each coated antigen well, add 200μl of diluted recombinant phage suspension at 37 ℃ for 1-2h. ? Add 200μl of positive control phage to the wells coated with positive control antigen. (7) Remove the liquid from the hole and put it upside down on a paper towel. Immerse the plate in PBS / 0.5% Tween20 (PBST), shake to remove air bubbles, and remove the plate. Repeat washing 5 times. (8) Put the board upside down on a paper towel to remove all liquid. (9) Dilute HRP enzyme-labeled Anti-M13 antibody with blocking buffer to an appropriate concentration. (10) Add 200 μl of HRP / Anti-M13 dilution solution to each well. (11) Incubate at 37 ° C for 1h. Wash 6 times as before. (12) Add 36 μl of 30% H2O2 to every 21 ml of 1 × ABTS substrate solution. Add 200 μl to each well. (13) Leave at room temperature for 20 to 60 minutes until a moderate color reaction occurs. Add 2mol / L H2SO4 to stop the reaction. (14) Read the light absorption value with a microplate reader at a wavelength of 415 nm. In good data, the absorption value of the phage antibody and the screening antigen should be 2 to 3 times higher than the absorption value of the negative control antigen.
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