Immunoassays are powerful techniques for understanding the role of specific components in complex systems. They work on the basis of the recognition of a specific component (target X) by an antibody or equivalent (affibody, RNA aptamer, recombinant antibody, etc.), which results in the production of a detectable signal. In most cases immunoassays are QUALITATIVE, providing information in terms of signal intensity. What is really wanted however, is a QUANTITATIVE assay providing information in absolute chemical terms, namely the concentration of target X.. Quantitative ImmunoAssays would allow:
- Detection of the absolute concentration of components
- Reduce inter-assay variation in data
- Permit successful statistical analysis of smaller sample sets
- Permits direct comparison of data generated at independent sites or occasions.
Quantitative ImmunoAssays are simple to construct. They require the simultaneous analysis of experimental (or test) samples and calibration standards. The signal intensity generated by calibration standards of known concentration permits conversion of the signals generated by the test samples into absolute units of concentration.
A calibration curve (or standard curve) establishes the relationship between the amount of material present and the signal intensity measured. In the case of immunoassays, this would represent the relationship between the epitope concentration and the signal intensity obtained. This relationship is often non-linear , and in many applications displays a dynamic range (or response range) of approximately two orders of magnitude in the concentration of target X.
To perform a Quantitative ImmunoAssay, a set of "calibration standards" containing the epitope in various concentrations are deployed in the immunoassay alongside experimental "test samples". Densitometry is performed on all data from the assay, and curve fitting used to define the relationship between epitope concentration and signal intensity. This mathematical relationship is then used to convert the signals generated by experimental samples into concentration of target X, which in our experience is highly accurate.
Molecular identity of Calibration standards: For Western Blot applications, a calibration standard is a molecule which contains the epitope feature of an immunoassay covalently bonded to a protein of known molecular weight. Two configurations of this structure are possible (Figure 1), where the epitope structure is either linked to the amino acid backbone (Fig 1a) in the form of a fusion protein or linked to a side chain of a specific amino acid (Fig 1b). Figure 1a and 1b: schematic representation of calibration standard molecules
Figure 1: Schematic representation of calibration standard molecules A set of calibration standards to common epitope tags (His6, c-myc, HA, FLAG, AU1, AU5, glu-glu, ) was analysed by SDS-PAGE/Western blotting (detected via the His6 tag). A single band of 55kDa was detected, and the intensity of signal decreased with decreasing calibration standard loading as expected (Figure 2). Figure 2: immunodetection of a serial dilution of His6-calibration standard
Figure 2: Immunodetection of a serial dilution of His6-calibration standard Densitometry of the data was performed and the data plotted to define the relationship between epitope amount and signal intensity (Figure 3). Mathematical fitting of the data was performed, with the best fit achieved by "one site-specific binding" analysis (GraphPad Prism) as shown in Figure 3. An excellent fit of the data was achieved using 6 calibration standard concentrations each analysed in quadruplicate. Similar excellent fits could also be achieved by analysis of fewer standards, with indistinguishable results obtained from 3 calibration standard samples analysed in triplicate. (Figure 3)
Figure 3: Mathematical description of a calibration curve To determine the epitope concentration of an experimental sample, the mathematical description of the calibration curve is rearranged to calculate epitope concentration from raw signal intensity. Figure 4 displays the quantitative measurement of three "test" samples. Test samples of 2pmol and 0.5 pmol were analysed and the results obtained were 2.153± 0.127 pmol (mean ± standard error, n=4), 0.552± 0.045 pmol (mean ± standard error, n=4), confirming the accuracy of the measure (Figure 4). Samples should only be analysed which fall within the calibration range, as errors are higher for observations beyond the confines of the calibration curve e.g. 0.125 pmol in this example. Figure 4: accuracy of Quantitative ImmunoAssays
Figure 4: Accuracy of Quantitative ImmunoAssays In summary, Quantitative ImmunoAssays are easy to construct and offer several valuable benefits to the researcher. They permit calculation of the absolute concentration of the component of study with high accuracy (error <10%) and high reproducibility. This enhances the quality of research results and also the productivity of research programs by facilitating the direct comparison of data obtained on separate occasions.