Guidelines for Preparing ELISA Standards

Greetings Earthling,

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:

ELISA pipettes

Troubleshooting Guides

Download troubleshootingnhandbooks for IHC, Western blot and ELISA for FREE.

troubleshooting_box_image Troubleshooting guides

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.

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.

ELISA troubleshooting ebook guide download PDF

*Note to educators: you are encouraged to share BosterBio's resources and PDFs on your class and lab websites, please cite or link to origin

900+ ELISA kits, find yours: Shop now!

Buy Primary Antibody Get Secondary FREE

Buy Primary Antibody Get Secondary FREE

Boster Products for ELISA Kits

See more

Boster Guarantee

All Boster antibodies and ELISA kits are guaranteed to meet the specifications on the data sheet. We promise to thoroughly investigate any concerns about the quality of our products; if you encounter a problem with a Boster antibody or ELISA kit, our technical support team will respond with personalized advice within 24 hours. If we can’t make your experiment work, we will refund your purchase in full or provide a replacement free of charge.

100% Satisfaction Guaranteed

Product Review

Human IFN Gamma PicoKine™ ELISA Kit (EK0373)

Review : "Very Easy To Work With And Great Results With Least Standard Error. I used this assay kit for detection of the cytokines in the plasma samples. Only 100 uL of sample yielded absolutely flawless results. Plasma samples showed variable cytokine expression among independent groups which demonstrates the accuracy of this kit. Straightforward protocol. Very easy to perform. Highly recommended product for precise cytokine assay analysis." -Akshay Jain, University of Missouri, Pharmaceutical Sciences Research Assistant

Read More

About Boster Biological Technology

Here are some quick facts about



4.8 / 5
Rating on