Team:Queens Canada/elisa

ELISAs

Introduction

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Materials

• Typical ELISA kit: Antibody-coated 96-well microplate, Detection antibody (usually biotinylated), Standard, HRP conjugate (antibody or streptavidin), Diluent Buffers, Wash Buffer, Chromogenic substrate (usually TMB), Stop solution, Plate covers
• Additional material required:
  • Absorbance-based microplate reader
  • Distilled or deionized warer
  • Squirt wash bottle
  • Sample

Procedure

• General Protocol

1. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.

2. Add 50-100 µL of prepared standard and sample wells. Cover plate and incubate at room temperature for 2 hours.

3. Thoroughly aspirate or decant solution from wells and discard the liquid.

4. Wash wells 4 times using a squirt wash bottle or an automated 96-well plate washer.

5. Add 100 µL of diluted detection antibody to wells. Cover plate and incubate at room temperature for 1 hour.

6. Thoroughly aspirate or decant solution from wells and discard the liquid.

7. Wash wells 4 times

8. Add 100 µL of diluted HRP conjugate to each well. Cover plate and incubate at room temperature for 30 minutes.

9. Thoroughly aspirate or decent solution from wells and discard the liquid.

10. Wash wells 4 times.

11. Add 100 µL of chromogenic substrate to each well.

12. Develop plate at room temperature in the dark for 30 minutes. A

13. Add 100 µL of stop solution to each well. The solution in the wells should change from blue to yellow.

14. The plate must be evaluated within 30 minutes of stopping the reaction. Read the absorbance of each well at 450 nm and 550 nm. Substract 550 nm values from 450 nm values to correct for optical imperfections in the microplate.

15. Use curve-fitting statistical software to plot a four-parameter logistic curve fit to the standards and then calculate results for the test samples.