There are multiple ELISA methods and detection chemistries that allow the researcher to fit the proper assay to answer a specific experimental question. Utilizing different methods will affect the observed signal and dynamic range of measurable protein concentration.
At CST, we start with highly validated antibodies to define optimal antibody pairs for each sandwich ELISA kit. ELISA products undergo rigorous testing in biologically relevant models, ensuring specificity and optimal signal-to-noise.
Measuring the absolute concentration of an analyte requires a standard curve generated from a serial dilution of a known quantity of antigen. Quantifying the signal in each sample is performed by comparing the value to the standard curve to determine the concentration.
It is also possible to determine relative quantification by comparing samples to each other or to a reference sample. Fold-change may be determined by using a baseline sample of serum antibodies to compare a sample of serum antibodies in response to an infection.
Qualitative analysis of ELISA determines the presence or absence of the target analyte and is used when research calls for just a positive or negative signal. The presence of the analyte can be determined by observing any signal compared to a blank sample or a negative control.
In general, the readout from each ELISA well is measured using a spectrophotometer and is given as a numerical value and reported as relative light units (RLUs) or relative fluorescent units (RFUs) vs the log of analyte concentration. Each well will be compared to each other or a control to determine the relative amount of protein, or the exact concentration can be determined when comparing to a standard curve.
In contrast to sandwich ELISA, competition/inhibition ELISAs have an inverse correlation between the signal and the amount of analyte.
