Don't Make a Substandard ELISA Standard Curve

If you want to run a quantitative ELISA assay, then you will need to include a standard curve on your plate. While this task might seem a bit mundane, an accurate standard curve can be hard to achieve. It requires careful handling of the standard, accurate pipetting and the the right controls.

Follow these rules to make sure your ELISA standard curve isn’t substandard:

Start with a solid standard

Your ELISA standard curve is only as good as the protein you start with. Your standard should be protein that is as pure as possible and that has been accurately quantitated. Purchase a standard from a company or purify recombinant protein in the lab.

Reconstitute your standard carefully

Many standards purchased from a company will come as a lyophilized powder. Reconstitute and aliquot the standard with the recommended amount of water or buffer using the following steps:

Spin the tube to collect all powder on the bottom
Add the appropriate volume of standard diluent
Mix and let sit for 5 minutes at room temperature
Mix again and aliquot the stock sample into single-use vials
Freeze remaining standard solution at -20°C or below

Stick to one buffer

Different buffers can have different effects on color or light development. Therefore, always dilute your standards in the same buffer used to collect your unknown samples.

Choose your range carefully

The concentration of your unknown samples should lie within the linear range of your ELISA standard curve. Make sure you don’t miss this range by making an all-encompassing standard curve. A general standard range covers 0-1000 pg/ml. If you repeat the experiment several times and become comfortable with the protein concentration range, then you can narrow the standard curve on subsequent experiments.

Be diligent with your dilutions

Never has pipetting been so important as when making dilutions for an ELISA standard curve.

Take it slow : make two- or three-fold dilutions
Don’t try to pipette tiny volumes; stick with 2 µl or greater
Don’t make big leaps: if the dilution is <1:1000, use two steps
Plan to run samples in duplicate or triplicate and make enough the first time
Make your dilutions just before you are going to use them
Use tubes that do not absorb protein (polypropylene or glass; i.e. do not make dilutions in microplate)
Now is not the time to save on tips; use a fresh tip for each dilution
Mix each dilution well but avoid creating bubbles or foaming

Make a new standard curve for every experiment

It is tempting to use yesterday’s standard curve for today’s experiment, but it won’t be accurate. Minor day-to-day differences in things like temperature and equipment calibration can affect your results. Also consider running a standard curve on every plate to take variations in assay length in account.

Use your controls

Always include a background, negative control sample that contains sample diluent
When possible, use a positive control sample containing a known concentration of target protein
Consider spiking a sample with a known concentration of protein to control for matrix effects

Assess your ELISA standard curve

Once you have plotted your standard curve and found the best fit line, calculate the R² value. The R² value gives you a measure of the quality of your curve.

If you are extremely close to 1 (like 0.998), then give yourself a pat on the back and know that you are measuring your protein concentrations accurately.

Photo courtesy of Epic Fireworks.