Analysing ELISA Data

When running an ELISA it is always advised to run standards and samples in duplicates or triplicates, this enables statistical analysis to be ran on the results collected. To begin analysis the mean OD value should be calculated for each set of values, to provide an average. This is easily done by adding the values up and dividing by the number of values there. From this the standard deviation may also be calculated which expresses how much the members of each group differ from the mean value. A low SD indicates data points are all relatively close to the mean value, whilst a high SD indicates data points that are widely spread out.

Coefficient of Variation

Also known as the relative standard deviation, this measures the amount of variability relative to the mean. This can be used instead of the standard deviation if the data collected has different units or means. It is often expressed an a percentage and is defined as the ration of the standard deviation to the mean. It is often used with analytical research to express the precision and repeatability of an assay.

CV= Standard deviation/ Mean

A high CV% indicates inconsistencies and inaccuracy in the results collected. This may be caused by a number of factors such as:

  • Cross contamination between wells
  • Temperature variations during incubation
  • Wells left to dry out, influencing non specific binding
  • Inaccurate pipetting, differences in volumes of reagents added

Intra-assay precision

Describes the variation between a set of results collected from the same assay. It indicates the level of variation from the one experiment if each sample is measured several times.

The CV% is be calculated for each set of samples by dividing the standard deviation by the set mean, this value is then multiplied by 100 to produce a percentage. The CV% for each of the set samples is then averaged to produce the intra assay CV.

Inter-assay precision

Describes the variation between a set of results collected from running an assay repeated times. It indicates the reproducibility and precision from running the results in a different assay on different days.  The CV% is calculated using data collected for the same sample across different assays. The mean from each assay is calculated and from this the standard deviation of those means.

When calculating the CV% the following points are advised:

  • For calculating the CV% the concentration values should be used rather than the OD values
  • The intra-assay CV% should be calculated at less than 10
  • The inter-assay CV% should be calculated at less than 15
A low CV%, particularly intra-assay precision often indicates poor pipetting technique or problems with the sample preparation and handling. It would be advised to pay particular attention to these to improve the accuracy of the results collected. As with any statistical analysis the more data collected the better the validity of the statistical results.