ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins

1. ELISA analysis of the interaction between recombinant PH and Vn

NOTE: Controls need to be included to exclude nonspecific binding. Human Factor H (FH) or C4b-binding protein (C4BP) are used as positive and negative controls, respectively.

1. Dilute each of the human proteins (Vn, FH, and C4BP) separately to 50 nM in Tris-HCl, pH 9.0 (coating buffer). Dispense 100 µL of protein solution into each well of a Polysorp microtiter plate. Close the plates with the lid and store them at 4 °C overnight (16 h) to facilitate the immobilization of protein onto microtiter plate wells.
2. Discard the solution from the microtiter plate by tilting it upside down over the sink and wash the wells three times with 300 µL of PBS/well. Block the coated wells for 1 h at RT with PBS containing 2.5% (w/v) BSA (PBS-BSA).
3. After removing the blocking solution, wash the wells three times with 300 µL of PBS containing 0.05% (v/v) Tween 20 (PBST) per well. Add 100 µL of 50 nM recombinant His-tagged PH to each sample well and incubate for 1 h at RT. In control wells, add only 100 µL of PBS-BSA.
   NOTE: The lph gene encoding PH from Hif was amplified by PCR and cloned into the pET26b expression vector that adds a 6× His tag at the C-terminus of the expressed protein. The recombinant vector was transformed into E. coli BL21(DE3) for expression. Ni-NTA resin was used to purify the recombinant protein.
4. Discard the protein solution and remove the unbound proteins by washing the wells three times with 300 µL of PBST per well. Add 100 µL of PBS-BSA containing horseradish peroxidase (HRP)-conjugated anti-His pAbs (1:10,000 dilution) and incubate for 1 h at RT.
5. Prepare 20 mM solution A by dissolving tetramethylbenzidine in a solution of 5% acetone and 45% methanol. To prepare solution B, dissolve 19.2 g of citric acid in 1,000 mL of H₂O, adjust the pH to 4.25 by adding KOH, then add 230 µL of 30% H₂O₂. Store both solutions in the dark at RT. Just before use, mix 500 µL of solution B with 9.5 mL of solution A to prepare the ELISA detection reagent.
6. Wash the wells three times with 300 µL of PBST per well and detect antigen-antibody complexes by adding 100 µL of ELISA detection reagent to each well.
7. Add 50 µL of 1 M H₂SO₄/well to stop the reaction. Measure the optical density of the wells at 450 nm using a microplate reader.