Protein Protocols Protein Protocols

1090 Stott
Fig 1. Chemiluminescent oxidation of luminol by peroxidase.
rate. This reaction is catalyzed by metal ions at high pH, resulting in emission of blue
light (emission peak about 425 nm). At lower pH, the reaction is catalyzed by hemecontaining
enzymes, such as horseradish peroxidase, catalase, cytochrome C, and
hemoglobin (Fig. 1). However, the light output is low with a half-life of a few seconds.
The presence of any one of a series of “enhancer molecules” increases the light
emission from horseradish peroxidase by 1000-fold or more, and alters the kinetics so
that a steady glow is produced lasting several hours (6,7). Microparticles, plastic beads,
plastic tubes, microtiter plates, membranes, and plastic pins have all been used successfully
as solid supports in a wide range of enhanced chemiluminescence assays,
including competitive immunoassays, immunometric assays, and RNA and DNA binding
assays (6,8,9).
In common with all other sensitive detection systems, maintenance of the label
enzyme in its active state is important. The precautions detailed in Notes 1–3 should be
observed to maximize the sensitivity achieved. Reagents for enhanced chemiluminescence
can be prepared in the laboratory or are available commercially (see Note 4).
The purity of the substrate solution is important in achieving maximum sensitivity.
Therefore, the precautions detailed in Notes 5–7 should be followed if preparing
substrate solutions. The free base form of luminol undergoes rearrangement to a
mixture of luminol and a series of contaminants. Therefore, luminol should be purified
by recrystallization as the sodium salt before use (see Note 8).
1.1. Light Measurement Instruments (Luminometers)
Commercial luminometers range from low-cost manual single tube instruments to fully
automated high-capacity machines and have been reviewed previously (10). However,
application-specific requirements are rarely discussed, and the first-time user will require
some guidance in matching an instrument to the chemistry or chemistries to be used.
There is normally no requirement for wavelength selection, because very few reactions
produce significant light output. Therefore, a simple luminometer can consist
of a detector and some means of presenting a sample or samples in a light-tight compartment.
There may also be a system for adding reagents to the sample while in the
chamber. The detector is usually a photomultiplier tube for sensitive instruments, but
can be a photodiode in a portable instrument (7,11) or photographic film, if a
semiquantitative result is sufficient (2,12).