Many ELISA kits come with concentrated standards and not ready to use standards. This means we, as user have to reconstitute them to use in the assay. This becomes a critical step as post the running the assay, the interpolation has to be done using the standards. Any shift in the accuracy of the standards in terms of concentration would lead to inaccuracy in the results. Hence it is essential that reconstitution of the standards is done correctly and accurately.
Reconstitution of the standard
Since the ELISA standard is often lyophilized, it must be reconstituted by adding a buffer/distilled water to make a stock solution. Because the procedure is batch specific, assemble your standard according to the product insert. The stock solution should be allowed to dissolve for at least 10-15 minutes before being further diluted. Pipette the Stock solution into aliquots and store at -20°C or below. After initial use, the aliquots should not be refrozen.
Preparation of the Standard
Here is an example of a serial dilution series from a stock solution to generate a standard curve of
20 – 640 ng/ml :
- The supplied standard (1ug) has been reconstituted with 1 ml diluent to yield a concentration of 1ug/ml.
- Add 320 ul of the reconstituted standard (with a new tip!) to a vial with 180 ul of Diluent. This will generate the first standard point, with a concentration of 640 ng/ml.
- To make the first 2X dilution, transfer 250 ul from the 640 ng/ml tube into the 320 ng/ml tube containing 250ul diluent. Repeat this process to generate the remaining points of the standard curve (160, 80, 40, and 20 ng/ml).
- Diluent is used for background control (0ng/ml).
- Please mix thoroughly between dilution steps.
# Tips and Tricks
- Use fresh pipette tips after each dilution
- Use clean tubes to prepare reconstituted standards and standard dilutions.
- Ensure that pipettes are calibrated regularly.
- Aim to use fresh standards for every assay and avoid freeze-thaw cycles.
- Repeat in duplicate or triplicate for accuracy and to ensure that you can still use your data should something go wrong with one measurement or an outlier.
- Construct a new standard curve for every plate and each experiment
