528
Chemiluminescence and Bioluminescence, Analysis by
Tab. 3
Detection limits for labels used in immunoassay and nucleic
acid hybridization assays
Detection limits
(zeptomoles: 10–21 mol)
a
Label
CL
BL
Acridinium ester
500
Alkaline phosphatase
1
10
β
-D-galactosidase
30
0.2
Glucose oxidase
6 200
Glucose 6-phosphate dehydrogenase
3
1 000
Horseradish peroxidase
1
400
Isoluminol
50 000
Xanthine oxidase
3 000
CL, chemiluminescent assay; BL, bioluminescent assay.
effective alternatives to radioactive labels
(
32
P,
125
I), and generally, CL and BL
reactions are superior to colorimetric
and fluorescent reactions used to assay
conventional enzyme labels.
4.2.1
Labels
Luminol, isoluminol, acridinium ester,
acridinium sulfonyl carboxamide, Fre-
fly luciferase, marine bacterial luciferase,
Renilla luciferase, Vargula luciferase, ae-
quorin, and ruthenium(II) tris(bipyridyl)
have all been tested as labels. Acri-
dinium esters are one of the principal
types of CL label in current use in
immunoassay and hybridization assays.
A rapid flash of light is produced by
reacting the label with a mixture of
H
2
O
2
and NaOH. Nonseparation (ho-
mogeneous) nucleic acid assays can be
formatted with an acridinium ester la-
bel, as in the ‘‘hybridization protection
assay.’’ In this three-step assay, Frst the
sample and an acridinium ester–labeled
probe are incubated together. Next, the la-
belonanunhybridizedprobeishydrolyzed
to a nonchemiluminescent product (probe
bound to target nucleic acid is protected
from hydrolysis), and Fnally a chemilu-
minescent signal is obtained from the
acridinium ester label on the bound probe
by treatment with oxidant in basic con-
ditions (±ig. 4). The recombinant photo-
protein aequorin is the most promising
BL label. Light emission (rapid flash) is
triggered by reacting the aequorin with
calcium ions.
4.2.2
Detection Reactions
The two most popular enzyme labels,
alkaline phosphatase and horseradish per-
oxidase, can be quantitated by a range
of CL and BL reactions. These enzymes
are used in several ways: as direct labels
for nucleic acid, indirectly as avidin or
streptavidin conjugates with biotin-labeled
nucleic acids, or via antigen–antibody
coupling, such as anti-digoxigenin con-
jugates with digoxigenin-labeled nucleic
acids.
±or alkaline phosphatase, the CL as-
say using adamantyl 1,2-dioxetane aryl
phosphate
substrates
(e.g.
AMPPD
,
CSPD
) (±ig. 5) is convenient and very
sensitive (detection limit,
1 zmol).
In
membrane-based assays
(e.g.
Western
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