ANTIBODY-BASED TECHNIQUES - A Guide to Methods in the Biomedical Sciences - page 77

transformation of chemical energy into light energy. For example, HRP
oxidizes the substrate luminal (a chemical reaction) with the concomi-
tant production of light, which is enhanced in the presence of a chemical
enhancer. The light emission can be detected on photographic film or
in chemiluminescent readers by digital cameras into a computer. While
the dynamic range of film is between one and two orders of magnitude,
chemiluminescent readers can detect and quantitated proteins over at
least three orders of magnitude, and therefore provide more quantitative
information.
Fluorescent antibodies
Antibodies can be covalently modified with a wide variety of fluo-
rescent compounds. Albert Coons was the first to directly conjugate a
fluorescent label to an antibody for visualization and demonstrate that
the antibody still retained antigen binding activity (31). From this humble
beginning, fluorescent antibodies have become one of the most useful
reagents for study of basic questions in immunology, cell biology and
neurobiology, and in clinical medicine. The power of techniques that uti-
lize fluorescent antibodies and the advances they have made possible in
biomedicine are underscored in subsequent sections devoted to these
methods, and in Chapter 5. Today, there are a large number of different
fluorescent molecules that have distinct excitation and emission spectra
and can be applied to fluorescence microscopy and/or fluorescence ac-
tivated cell sorting. These include fluorescein (FITC), Texas Red (TR),
phycoerythrin (PE), allophycocyanin (APC), various “Cy dyes” (includ-
ing Cy5, Cy7 etc.) and Alexa Fluor dyes (Alexa 488, Alexa 546, etc.),
to name but a few. The Cy dyes and Alexa fluor dyes were designed
to have higher intensities and reduced photobleaching properties than
conventional fluors.
Colloidal gold labeling of antibodies
Antibodies can be labeled with colloidal gold beads of defined size
to provide an electron dense tag which enables the use of antibodies
in immunogold electron microscopy (Chapter 5) Gold beads of 5 nm,
10 nm and 15 nm are most commonly used. The colloidal gold interacts
tightly with the antibody but is not covalently attached.
Methods to identify and quantitate antigens
Prior to the 1960s, the widespread use of antibodies was more qualita-
tive than quantitative. Assays to measure antibody-antigen interactions