ELISA standards for accurate sample measurement of the target protein should be carefully prepared to set up a standard curve. A standard curve is usually prepared by assaying a serial dilution of a standard preparation of the target analyte, of which the starting concentration is known
When preparing ELISA standards, we’ve included some guidelines to remember.
- What kind of standard should I choose?
The standard curve should be prepared with purified protein. Some companies provide purified proteins that are suitable with ELISA assays. A purchased ELISA kit would contain the standard(s) in the kit itself. If purified protein is not available, then a recombinant protein may be used. In case the protein is semi-purified, purifiy it in the lab with the concentration determined by HPLC.
Most of the ELISA standards are lyophilized and must be reconstituted. Since the instructions may be lot-specific, remember to follow the manufacturer’s reconstitution instructions.
- What should the standard curve range be?
The standard curve will typically be in pg/ml, ng/ml, ug/ml range. However this is dependent on the antibody-antigen interaction and the range set by the manufacturer of the kit or the relevant previously published research papers.
The standard curve range also depends on the target protein concentration to be measured in the sample matrix.
- How to reconstitute the standard?
If the standard is supplied lyophilized, then it must be reconstituted prior to first use. To reconstitute the standard following steps are to be followed:
- Spin the tube to collect all powder on the bottom.
- Add the appropriate volume of standard diluent (the same diluent should be used for dilution of any samples).
- 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.
- How should the standard be diluted?
The standard curve is obtained by diluting the standard with known concentrations in a series of dilutions that should span the standard curve range. The target protein concentrations in the unknown samples must fall within this range.
For example, a serial dilution series from a stock solution to construct a 20–640 ng/ml standard curve is below:
Some tips to keep in mind:
- Make two- or three-fold dilutions.
- Do not make dilutions that require pipetting a small amount of volume (2 ul or less).
- Avoid making large, single step dilutions. If the dilution is <1:1000, use two steps.
- Prepare enough of each dilution to run in duplicate or triplicate.
- Always include a background, negative control sample containing sample diluent.
- Make dilutions fresh just before use.
- Make all dilutions in tubes that do not absorb protein (polypropylene or glass; i.e. do not make dilutions in microplate).
- Always use a fresh pipet tip for each dilution.
- Mix each dilution well but avoid creating bubbles or foaming.
