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

Protocol

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.

Materials

Name	Company	Catalog Number	Comments
AR2G sensors	Pall Life Science	18-5095	Sensor to immobilized protein by amino coupling
Acetone	VWR	97064-786	Analysis grade
Bovine Serum Albumins (BSA)	Sigma-Aldrich	A2058	Suitable for cell culture
C4BP (C4b-binding protein)	Complement Technology, Inc.	A109	Bought as Frozen liquid form
Citric acid	Sigma-Aldrich	251275-500G	American Chemical Society (ACS) grade
HRP-conjugated anti-His tag antibodies	Abcam	ab1269	Polyclonal
C4BP	Complement Technology, Inc.	A109	Frozen solution
Histidine affinity resin column (HisTrap HP)	GE Health Care Life Science	17-5247-01	Columns prepacked with Ni Sepharose
His-tagged PH			Recombinantly expressed and purified in our lab
Methano	VWR	BDH1135-1LP	Analysis grade
Plasmid containing C-terminal 6x His-tag on the backbone (pET26(b))	Novagen	69862-3	DNA vector
Polysorb microtitre plates	Sigma-Aldrich	M9410	For ELISA
Tetramethylbenzidine	Sigma-Aldrich	860336-100MG	ELISA grade
Vitronectin (Vn) from human plasma	Sigma-Aldrich	V8379-50UG	Cell culture grade