ELISA plate map
Figure 1: ELISA plate map for setting up an assay. Purple circles = Standards, Grey circles = Samples, White circles = Blanks.
ELISA plate map example
When carrying out an ELISA assay it is important to layout where you place your standards, blanks, controls and samples on the ELISA plate for analysis of your results. Each 96 well ELISA plate allows researchers to analyze up to 40 samples at once on a ELISA plate reader.
As most Sandwich & Competitive ELISA kits have a 7-point dilution of their standard, 14 wells are taken up by standards on the ELISA plate. In addition to standards, it is also necessary to have a blank control which is just sample/standard dilution buffer. Researchers may also use additional wells for positive, negative or spike recovery controls on the plate.
ELISA plate standards
Figure 2: 7-point serial dilution of stock standard using standard dilution buffer.
ELISA standards allows researchers to determine the amount of analyte they have in their sample by extrapolating absorbance readings of samples to known concentrations of standards, thus allowing researchers to quantitatively determine analyte amounts. In an ELISA kit, researchers are provided with a stock solution of standard which will be diluted by the sample/standard diltution buffer.
Standard dilution guidelines
Note: Refer to datasheet provided with the kit for comprehensive sample dilution guidelines.
Dilute each standard vial provided with 1ml (Volume will depend on kit) sample standard dilution buffer to create the Standard Stock Solution. Keep tube at room temperature for 10 min and mix thoroughly.
To create the standard series, label 6 microcentrifuge tubes and aliquot 300µl of the Sample/Standard dilution buffer into each tube. Add 300µl of the standard stock solution into 1st tube and mix thoroughly. Transfer 300µl from 1st tube to 2nd tube and mix thoroughly. Repeat this dilution process until the standard series is complete (see Figure 1 above for details).
As mentioned above, a blank well is used on an ELISA plate to set an absorbance threshold for samples. This also allows for users to account for high background levels that may affect absorbance read out. Furthermore, using a blank will also allow users to determine if capture and detection antibody have any reactivity which may result in a false positive read out.