Titration Direct ELISA Protocol

Posted on Dec 8, 2014

The following is AxioMx’s recommended ELISA protocol. This protocol is applicable when primary antibodies are either FLAG®-labeled scFvs or IgGs.

A. Buffers

Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:

10X PBS

P80 g NaCl
2 g KCl
14.4 g NaHPO4
2.4 g KHPO4 Bring volume to 1L and pH 7.4.

Blocking solution

Commonly used blocking agents are 1-3% BSA, serum, non-fat dry milk, casein, gelatin in PBS. AxioMx recommends 3% BSA/PBS. Dilute the 3% bovine serum albumin (BSA) in 1X PBS (3 g of BSA per 100 ml of 1X PBS; mix with stir bar). Blocking solution should be made fresh daily.

Wash solution

PBST- 1X PBS with 0.1% Tween 20
Primary and secondary antibodies should be diluted in 1x blocking solution to reduce non-specific binding.

B. Protocol

Day 1: Coating NeutrAvidin Plates (For use with Biotinylated Antigen)

  1. Coat Maxisorp® 96-well plates (Nunc, #430341) with 100 µl/well of NeutrAvidin at a concentration of 10 µg/ml. Incubate all plates overnight at 4°C.

Day 2: Titration Protein ELISA

  1. Remove TMB solution from 4°C and allow it to warm to room temperature.
  2. Wash NeutrAvidin-coated plates three times with 250 µl/well of 1X PBS using a handheld electronic multichannel pipette or on a plate washer.
  3. Discard the last wash and gently tap the plates on a dry paper towel to remove residual PBS wash.
  4. Add 250 µl/well of 3% BSA/PBS block to each plate using a handheld multichannel electronic pipette.
  5. Incubate the plates at room temperature for 1 hour.
  6. While the plates are in block, prepare the antigen dilutions in an untreated 96-well plate:
    1. In tubes, dilute each target antigen to 200 nM.
    2. Serially dilute 1:2 serial dilutions for 7 points, 8th point should be 0 antigen (PBS only). Be sure to use fresh pipette tips for each serial dilution.
  7. Wash the NeutrAvidin-coated plates three times with 250 µl/well of 1X PBS by hand or on a plate washer.
  8. Using a hand-held multichannel pipette, transfer 100 µl of the serially diluted antigens from the untreated 96-well plate to the NeutrAvidin-coated 96-well plate.
  9. Incubate the plates at room temperature for 1 hour.
  10. Wash the plates three times with 250 µl/well of 1X PBS by hand or on a plate washer.
  11. Using a hand-held multichannel electronic pipette, add 250 µl/well of 3% BSA/PBSblock solution to the washed wells of the plates.
  12. Incubate the plates at room temperature for 1 hour.
  13. While the plates are in block, prepare the scFv dilutions in 1.5-ml microcentrifuge tubes: add 1 µg of scFv protein to 1 ml of 3% BSA/PBS block solution for a final concentration of 1 µg/ml. Mix well.
  14. Wash the antigen-coated plates three times with 250 µl/well of 1X PBS by hand or on a plate washer.
  15. Using an electronic multichannel pipette, add 100 µL of scFv (or IgG) antibody (at 1 µg/ml) to the corresponding positions on the plate.
  16. Incubate the plates at room temperature for 1 hour.
  17. Wash the plates four times with 250 µl/well of PBS/0.1% Tween 20 by hand or on a plate washer.
  18. Discard the last wash and gently tap the plates on a dry paper towel to remove residual PBS/T wash.
  19. Add 100 µl/well of secondary antibody-HRP conjugate (i.e. anti-FLAG®-HRP) diluted 1:5000 in 3% BSA/PBS per well to all ELISA plates.
  20. Incubate the plates at room temperature for 1 hour.
  21. Wash the plates three times with 250 µl/well of PBS/0.1% Tween 20 by hand or on a plate washer.
  22. Add 100 µl/well of Ultra TMB developing reagent that has come to room temperature. Develop at room temperature for 2-3 minutes.
  23. To stop the developing reaction, add 50 µl/well of 2 M H2SO4 stop solution.
  24. Place plates on plate reader and measure the absorbance at 450 nm.
MaxiSorp® is a registered trademark of Thermo Fisher Scientific Inc.
FLAG® is a registered trademark of Sigma-Aldridge Co. LLC