ELISA analysis

• Qualitative ELISA only determines whether the antigen is present or not in the sample. It requires a blank well containing no antigen or an unrelated control antigen.
• Semi-quantitative ELISA allows the relative comparison of the antigen levels between the samples.  
• Quantitative ELISA allows calculating the amount of antigen present in the sample. It requires comparison of the values measured for the samples with a standard curve prepared from a serial dilution of a purified antigen in a known concentration. This is the most commonly reported ELISA data. 

ELISA standard curve
​​The standard or calibration curve is the element of the quantitative ELISA that will allow calculating the concentration of antigen in the sample.
The standard curve is derived from plotting known concentrations of a reference antigen against the readout obtained for each concentration (usually optical density at 450 nm). 
Most ELISA plate readers will incorporate a software for curve fitting and data analysis. The concentration of the antigen in the sample is calculated by extrapolation of the linear portion of the standard curve.​ 

Figure 1: Example of a quantitative ELISA standard curve from Human ICAM1 SimpleStep ELISA^® Kit (ab174445).

Curve fitting software allows using different models to plot your data. 

• The linear plot presents the concentration of the antigen on one axis and the readout on the other. R2 values are normally used here to determine fitting, with values higher than 0.99 representing a very good fit. However, linear plots tend to compress data points on the lower end of the curve resulting in decreased resolution. 
• The semi-log plot helps to counteract the compression at the lower end caused by linear plots. Semi-log plots use the log of the concentration against the readout. This method commonly results in a sigmoidal curve that distributes more evenly the data points.
• Log/log plot provides good linearity for the low to medium range of the concentrations. The higher end of the range tends to lose linearity. 
• 4- or 5-parameter logistic (4PL or 5PL) curves are more sophisticated methods that take into account other parameters such as maximum and minimum and therefore require more complex calculations. 4PL assumes symmetry around the inflection point, while 5PL takes asymmetry into account, which normally is a better fit for immunoassays.