COVID-19 Spike-ACE2 Binding Assay Kit

1. COVID19 S-protein Microplate (Item A): 96 wells (12 strips x 8 wells) coated with recombinant COVID19 S-protein RBD domain.
2. Wash Buffer Concentrate (20x) (Item B): 25 ml of 20x concentrated solution.
3. Assay Diluent (Item E2): 15 ml of 5x concentrated buffer. For diluting testing reagent, ACE2 protein (Item F), detection antibody (Item C) and HRP-conjugated IgG Concentrate (Item D).
4. ACE2 protein (Item F): 2 vials of purified human recombinant ACE2 protein (1 vial is enough to assay half microplate)
5. Detection Antibody ACE2 (Item C-1): 2 vials of goat anti-ACE2 (1 vial is enough to assay half microplate).
6. HRP-conjugated Anti-goat IgG (Item D-1), 15 μl of 1000x concentrated HRP-conjugated anti-goat IgG.
7. TMB One-Step Substrate Reagent (Item H): 12 ml of 3,3',5,5'-tetramethylbenzidine (TMB) in buffered solution.
8. Stop Solution (Item I): 8 ml of 0.2 M sulfuric acid.

1. Microplate reader capable of measuring absorbance at 450 nm.
2. Shaker.
3. Precision pipettes to deliver 2 μL to 1 mL volumes.
4. Adjustable 1-25 mL pipettes for reagent preparation.
5. 100 mL and 1 liter graduated cylinders.
6. Distilled or deionized water.
7. Tubes to prepare sample dilutions.

• Evaluate inhibitor activity
• Screen inhibitors of the SARS-CoV-2 Spike-ACE2 complex
• Characterize antibody function
• Vaccine development
• Selection of patient serum for antibody therapy

Mix testing reagent (e.g., small molecule, antibody) with ACE2 protein concentrate, then dilute the mixture with 1x Assay Diluent dilute to make a 1x ACE2 protein working concentration. Each sample should contain the same 1x ACE2 protein concentration.

1. Bring all reagents to room temperature (18 - 25 °C) before use. It is recommended that all samples should be run in at least duplicate.
2. Add 100 μL of each sample into appropriate wells. Cover well with plate holder and incubate for 2.5 hours at room temperature or over night at 4 °C with shaking.
3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 μL) using a multi-channel pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
4. Add 100 μL of prepared 1x detection antibody, anti-ACE2 (Reagent Preparation step 5) to each well. Incubate for 1 hour at room temperature with shaking.
5. Discard the solution. Repeat the wash as described in Step 3.
6. Add 100 μL of prepared 1x HRP-conjugated anti-goat IgG (see Reagent Preparation Step 6) to each well. Incubate for 1 hour at room temperature with shaking.
7. Discard the solution. Repeat the wash as described in Step 3.
8. Add 100 μL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with shaking.
9. Add 50 μL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Determine the average absorbance across the replicate readings per sample.