ELISA

Overview

You can coat the plate directly with your protein of interest or (for biotinylated antigens), coat with streptavidin/neutravidin followed by antigen to preserve the native structure. Without a plate reader, just do washes by hand with a multichannel pipette, flicking the waste solution into the sink. I also like to slam the plate on a paper towel on the bench to get all the liquid out.

Reagents needed

• NeutrAvidin protein (Fisher Scientific: PI31000)
• Nunc MaxiSorp 384-Well Plates (Nunc 464718)
• Enough Fc-antigen (2 µL x 1 uM stock per colony == (25 µL*2 +50 µL) x 20 nM solution per colony)
• HRP-conjugated antibody (GE Lifesciences: 27-9421-01)
• KPL TMB Microwell Peroxidase Substrate (Fisher Scientific: 50-76-03)
• A good pipet with many straight pipet tips.
• 1 M phosphoric acid solution
• Blocking bBuffer
  • PBS + 0.05% Tween + 0.2% BSA (PBSTB; freshly prepared)
• Wash buffer
  • PBS + 0.05% Tween

Protocol

Shake the plate for each step at a fixed speed.

1. Coat each well of 384-well Fischer MaxiSorp plate with 50 µL of 0.5 µg/mL NeutrAvidin in PBS.
2. Incubate plate overnight at 4 ˚C or 2 hours at RT.
3. Remove coating solution and block plate with 100 µL of blocking buffer for 1 hour at RT.
4. Add 25µL of 20 nM biotinylated antigen or Fc and allow capture for 30 minutes.
5. Remove antigen solution, and block plate with 1 µM biotin in blocking buffer for 10 minutes.
6. Prepare a 96 deep well plate of binder diluted 1/2.5 with PBSTB.
7. Prepare a 384 well plate using the regular clear ones, for each binder prepare 3 wells: top left direct ( 50 uL 1/5 binder); top right competition (50 uL 1/5 binder + 20 nM antigen); bottom left Fc (50 uL 1/5 binder)
8. Wash the MaxiSorp plate three times with 200 µL wash buffer.
9. Transfer 25 µL of each well from step 7 to the MaxiSorp plate. (Transfer the direct and Fc first, competition should be done at the end, and fast!) Incubate at RT for 15 min. (cannot go longer!!!)
10. Remove binder and wash plate three times with 200 µL wash buffer.
11. Add 25 µL anti M13 HRP-conjugated antibody diluted 1:5000 in blocking buffer and incubate for 30 minutes at RT.
12. Remove antibody solution and wash plate three times with 200 µL wash buffer.
13. Add 25 µL of HRP TMB substrate and incubate at RT until signal appears bright (this step is very fast, go one plate at a time, before the color reaches blue it should be stopped).
14. Quench reaction with 25 µL 1 M phosphoric acid.
15. Measure OD 450nm on the plate reader.

TMB development/quench

If things are working well the solution will turn blue starting around ~1-3 minutes after you add it. It takes a minute to add developer to the whole plate, and you want each well to develop for the same amount of time.

The way I do this is:

1. After the last wash, prepare the TMB reagent by mixing 1:1 in a multichannel reservoir.
2. Prepare quench solution in a separate reservoir.
3. Start a timer.
4. Add TMB to each row 5 seconds apart.
5. Place plate on a piece of paper or white background, watch for blue to appear in first wells.
6. When the blue starts to get dark, add quench solution to each row 5 seconds apart.
7. Read at 450 nm on plate reader.