Sandwich ELISA Protocol

The below protocol is a sample protocol for a sandwich ELISA using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of analyte present in their sample. This can be very useful when looking for increases in protein concentration, phosphorylation of proteins or decreases on protein concentrations.

Steps in a sandwich ELISA protocol. 1. Capture antibody is bound to the microtitre plate. 2. Analyte is incubated with the capture antibody and binds. 3. A second antibody against the analyte of interest conjugated to a alkaline phosphatase or horseradish peroxidase binds to the analyte. In the presence of TMB/pNPP the conjugate enzyme catalyzes the reaction resulting in the production of colour.
  1. Dilute the capture antibody to the appropriate concentration, add 50-100ul per well.
  2. Incubate for 2 hrs at room temperature.
  3. Remove the solution and wash the plate three times for 5 minutes each with 200ul of wash buffer on a shaker.
  4. Add 300ul of blocking buffer.
  5. Cover the plate and incubate at room temperature for 1 hr or overnight at 4°C.
  6. Remove blocking buffer.
  7. Add samples or standards to the selected wells in duplicate or triplicate. The volume per well should be the same as the capture antibody used in step 1.
  8. Cover the plate and incubate at room temperature for 1 hr.
  9. Remove the solution and wash the plate three times for 5 minutes with 200ul of wash buffer on a shaker.
  10. Dilute and add the biotinylated detection antibody to each well.
  11. Cover the plate and incubate at room temperature for 1 hour.
  12. Remove the detection antibody solution.
  13. Wash the plate three times for 5 minutes each with 200ul of wash buffer on a shaker..
  14. Add the appropriate concentration of enzyme conjugate to each well. The volume used should be the same as the capture antibody used in step 1.
  15. Cover the plate and incubate at room temperature for 1 hr.
  16. Remove the solution and wash the plate six times for 5 minutes each with 200ul of wash buffer on a shaker.
  17. Add the substrate solution to each well.The volume used should be the same as the capture antibody used in step 1.
  18. Develop at room temperature for 30 minutes or until the desired colour intensity is reached.
  19. Stop the reaction by adding an equal amount of stop solution.
  20. Measure the absorbance using the appropriate equipment
  21. Analyze data and plot signal vs concentration of the antigen.

Reagents required

Coating buffer: 0.2M sodium carbonate/bicarbonate, pH 9.4

Capture antibody: Dilute in coating buffer

Wash buffer: 0.1M phosphate, 0.15M sodium chloride, pH 7.2 with 0.02% TWEEN 20

Blocking buffer: 2% (w/v) Bovine Serum Albumin (BSA) in wash buffer.

Standard diluent: 2% (w/v) BSA in wash buffer

Detection antibody (Biotinylated): Dilute 1 in 5 in standard diluent

Enzyme conjugate: Streptavidin-HRP diluted 1 in 5 in standard diluent

Substrate: TMB substrate

Stop solution: 2M sulfuric acid