Biomedical Engineering Reference
In-Depth Information
as a signal-to-noise ratio for data analysis. Relative fold differences
between the treated and control groups are calculated by dividing the
signal-to-noise ratio of the treated group with that of the control group
(Sharrow, 1991).
4.3.3 Colorimetric assays
Colorimetric assays use a colorimeter (spectrophotometer) to determine
concentration of a chemical compound in a solution by measuring spectral
absorbance of the compound at a particular wavelength. A color is formed
during the reaction of a detection chemical with the target substance. The
intensity of the color, determined via spectrophotometer, is an indicator
of the amount of the target substance. Colorimetric assays are often used
for protein analysis. For example, the BradFord assay is used to measure
protein concentration (Simonian and Smith, 2006), and the p-nitrophenol
phosphate assay is used to measure the alkaline phosphatase (ALP) activity
(Gallagher, 2008b). Though simple, fast and economical, colorimetric assays
are not highly sensitive and are easily affected by pH and temperature of a
solution.
4.3.4 Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive immu-
noassay that can be used to quantitatively measure the concentration of a
protein of interest in a sample solution. The detection is based on two dif-
ferent mechanisms categorized as indirect and direct ELISA; the indirect
approach attracts the antibodies of interest from a sample solution onto an
antigen-coated microtiter plate whereas the direct, also called 'sandwich',
approach pre-coats antibodies that can recognize and capture the immuno-
specifi c antigens in a sample (Fig. 4.4) (Smith, 1993; Sumanas, 2002). ELISA
plates with selectively bound antibodies or antigens are detected by specifi c
reporter-linked antibodies. The reporters are chemical or fl uorescent dyes
that can be detected by colorimetric, chemiluminescent or fl uorescent meth-
ods. ELISA is a highly sensitive assay that is extremely useful for determin-
ing trace amounts of proteins or peptides in a sample, for example, secretory
proteins in culture media or soluble proteins in sera.
4.3.5 Cell viability and proliferation assays
Cell viability and cytotoxicity assays can be divided according to mecha-
nism, with proliferation assays relying on DNA presence/damage indicative
