- Add 50-100 µL of a readymade standard and sample to wells.
- Cover plate and keep at room temperature for 2 hours.
- Separate the solution from wells and discard the liquid.
- Add 100 µL of detection antibody to wells.
- Cover plate and incubate at room temperature for 1 hour.
- Decant solution from wells and discard the liquid.
- Wash wells 3-4 times. (Gentaur Company sells an automatic washer for this.)
- Add 100 µL of diluted HRP conjugate to each well.
- Cover plate and again incubate at room temperature for 30 minutes.
- Decant the solution from wells and discard the liquid.
- Add 100 µL of peptide substrates that bind with proteolytic enzymes by color formation to each well.
- Incubate at room temperature for 30 minutes. By adding stop solution to each well, the solution in the wells will change from blue to yellow. The plate must be watched within 30 minutes of stopping the reaction. Read the absorbance of each well at 450 nm and 550 nm with an Elisa Reader.
- Subtract 550 nm values from 450 nm values to correct for optical imperfections in the microplate.
- Use curve-fitting statistical software to plot a four-parameter logistic curve fit to the standards and then calculate results for the test samples.
Generar ELISA Protocol