Guidelines for Preparing ELISA Standards


ELISA (enzyme-linked immunosorbent assay) is a plate-based assay used to detect the concentration of a specific protein in a liquid sample. Three different types of data output can be obtained:

 Qualitative: ELISAs can be used to determine whether or not the protein of interest is present in the sample by comparing the sample to a blank well or an unrelated control antigen without the target protein.
 Semi-Quantitative: Since the signal intensity produced is directly related to the amount of target protein in the sample, ELISA allows samples to be compared by observing the relative levels of the target protein.
 Quantitative: For a precise calculation of the target protein concentration in assay samples, a standard curve with known target protein concentrations should be created using a purified antigen and compared with the ELISA data generated.
ELISA pipettes

To set up a standard curve, ELISA standards should be carefully prepared for accurate sample quantification of the target protein. We’ve provided some guidelines for you to consider when preparing ELISA standards.

1. What kind of standard should I choose?

A purified protein should be used to prepare the standard curve. Otherwise, use a recombinant protein which can be semi-purified in the lab and measure the concentration with HPLC. Some companies may provide purified antigens. Or, save time by purchasing Boster Bio’s PicoKine ELISA kits, which include the standard and other necessary ELISA reagents for your convenience.

Most ELISA standards are provided in lyophilized form and need to be reconstituted. Remember to follow the reconstitution instructions provided by the manufacturer since the instructions might be lot-specific.

2. What should the standard curve range be?

Usually, the standard curve will range from 0 to 1000 pg/ml. However, if the predicted target protein concentration is extremely high, the standard curve can reach up to 3000 pg/ml.

3. How should the standard be diluted?

The standard curve is prepared through serial dilutions of the standard with known concentrations that should span the standard curve range. This range is expected to be close to the target protein concentrations in the unknown samples.

Here is an example of a serial dilution series from a stock solution to generate a standard curve of 3.9 –2000 pg/ml:

ELISA Standards Protocol

4. Some tips to keep in mind:

 Use fresh pipette tips after each dilution
 Standard solutions should be used within 2 hours
 Repeat in duplicate or triplicate for accuracy
 Construct a new standard curve for every plate and each experiment

Now that you’ve read about ELISA assay standards, be sure to include them in your experiments and validate your results!

Access our Technical Resource Center for more ELISA tips and troubleshooting guidance.

Questions? Contact [email protected] and we will be happy to assist you.