dIN.
CHEM. 25/4,
512-519
(1979)
Applications of Blo- and Chemiluminescence in the Clinical
Laboratory
Frans Gorus1 and Eric Schram2
Many papers have been published in recent years describing
the analytical uses of bio- and chemiluminescent
reactions (for
reviews see 1-10). Although several of these methods, which
are often very sensitive and specific, can be applied in clinical
laboratories, they are still little known and have in general not
been used for routine assays. We propose to survey existing
methods and to discuss their applicability.
Extensive information on the mechanisms of bio- and chemiluininescence
can
be found in several recent reviews (11-19) and books (20,21)
and will not be dealt with in the present article.
Chemiluminescence
is said to occur whenever a molecule
emits a photon as the result of an exergonic chemical reaction
in which one of the intermediate
or end-products
is left in an
electronic
excited state. This phenomenon
occurs adiabatically and without absorption
of light. Since a rather high
amount of energy is necessary to produce a photon (around
200 kJ, according to the wavelength),
most chemiluminescent
reactions are of the oxidative type. In general only the lowest
singlet state is involved. Sensitized
luminescence
may result
when the excited end-product
transfers its energy to another
substance with suitable fluorescence
characteristics.
Bioluminescence
is a special case of chemiluminescence
to
be found in living organisms. Such reactions are mostly catalyzed by an enzyme, “luciferase,” which creates the favorable
environment
for the luminescent
oxidation of a specific substrate, “luciferin.”
The quantum yield of a chemiluminescent
reaction is the
number of emitted photons vs. the number of reacting molecules. For nonbiological
chemiluminescent
systems
this
quantum yield is seldom greater than 0.01. In bioluminescent
reactions,
however, much higher yields are attained, even
approaching
unity. The hydrophobicity
of the active site of
the enzyme is considered
responsible
for the fact that the
excited molecule is protected against nonradiative
deactivation processes,
e.g., chemical quenching,
energy transfer to
nonfluorescent
acceptor molecules, etc.
instrumentation
Assays
The measurement
Academic
Hospital,
Laboratory
emitted
of Clinical
in the course
Chemistry,
Free
University
of Brussels,
Laarbeeklaan,
101, B-1090 Brussels,
Belgium.
2 Biochemistry
Laboratory,
Faculty of Sciences,
Free University
of Brussels,
Paardenstraat,
65, B-1640 Sint-Genesius.Rode,
Bel-
gium.
Received
512
Aug. 4, 1978; accepted
Jan.
19, 1979.
CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979
reaction
reaction
is equivalent
to the measurement
of a
rate:
X
dc(t)
dt
ICL(t)
=
‘CL
ICL(t)
=
CL
=
number of photons emitted
quantum yield
reaction
dt
per second
rate
-
In most chemiluminescent
reactions
luminescence
first
increases rapidly upon mixing of the reagents, goes through
a maximum,
and then starts to decrease. According to the
system being considered
and the experimental
conditions,
measurements
will be made on the ascending or descending
part of the curve, at the maximum of the peak, or, in the case
of steady-state
measurements,
after reaching a plateau. Two
types of instruments
are used to measure this luminescence:
analog meters, which are the most popular, and single-photon
counters. The following instruments
are now commercially
available:
American equipment
DuPont Biometer (E.I. DuPont
de Nemours
& Co., In-
strument and Equipment
Division, Wilmington,
DE 19898)
integrates the photomultiplier
output over a 3-s period after
mixing. The results are displayed on a digital register.
Aminco Chem-Glow
Photometer
(American
Instrument
Co., Division of Travenol Laboratories, Inc., Silver Spring, MD
20910), equipped
with a microammeter
that allows instant
reading. The analog output may be fed into either a recorder
or an integrator-timer
(the time-span
is adjustable between
land
60
5).
SAl ATP meter (SAl Technology Company, San Diego, CA
92121) can be operated in several ways, to measure peak intensity
or to integrate over a 6-s period after mixing or over
60s
for Blo- and Chemiluminescent
of the luminescence
of a chemical
after
a 15-s lag period. All results appear on a digital dis-
play.
Pico-Lite
(Packard Instrument
Company, Inc., Downers
Grove, IL 60515), microprocessor
controlled
for measuring
total light emission, maximum peak intensity, or decay rate.
It has built-in digital display, thermal printer, background
subtraction,
and temperature
control.
European
equipment
Celltester
Mod. 1030 and 1060 (Lumac Systems AG,
Reichensteinerstrasse,
14, CH 4000 Basel 2, Switzerland),
photodiode detector with digital display; integrates over 5, 10,
or 40 s, or reads luminescence
every 2.4 a.
luciferase
ATP + luciferin
Lumacounter
Mod. 2080 (Lumac Systems AG, Reichensteinerstrasse,
14, CH 4000 Basel 2, Switzerland),
photomultiplier instrument with digital display and analog output,
interfaced with microcomputer
HP97S for data processing
and programmed
operation.
Measuring modes-integral,
peak, and rate; preset counting times-lO,
30, and 60 s; temperature contol-25,
30, and 37 #{176}C
(available in the U.S.A.
from Lumac, Inc., Westlake Village, CA 91361).
Biolumat LB 9500 (Laboratorium
Prof. Dr. Berthold, D7547 Wildbad, G.F.R.); similar to Lumacounter.
Luminometer
1250 (LKB-Produkten
AB, S-16125 Bromma, Sweden), modular system including digital display,
printer, and potentiometric recorder; has temperature control
and mixing device. Integration time is 1 to lOs.
Pico-ATP (Jobin Yvon, Division D’Instruments S.A., 16-18,
Rue du Canal, 91160 Longjumeau, France), photomultiplier
instrument for measuring peak intensity. It has digital display
and an automatic injector.
Bioluminescence Analyzer XP-2000-2 (Skan AG, CH-4009
Bagel, Allschwil, Switzerland), equipped with automatic syringe and optional chart recorder or digital integrator. Integration time ranges up to 99 s.
Several firms are currently also supplying purified reagents
suitable for clinical analysis (Lumac, LKB).
Many people are still using scintillation counters (with the
coincidence circuit disabled), although such instruments are
more sophisticated
than necessary for the present purpose.
In the “repeat” mode, luminescence can be followed over the
course of time (22-25).
Where applicable, integration at the plateau or on the descending part of the luminescence curve is the most reliable
method because this region is less dependent on the efficiency
and rapidity of mixing. However, when the substrate responsible for luminescence is produced in situ, the slope of the
ascending part of the luminescence curve may be small enough
to allow for easy measurement.
This situation occurs, for instance, when dehydrogenases
are being assayed by means of
bacterial luciferase (26). Although in their case the slope was
much steeper, Lundin and Thore (27) were able to show that
luminescence on the ascending curve was proportional to ATP
concentration
in the case of firefly luciferase. Because of the
availability of purified firefly luciferase, they were further able
to reach a plateau for light production. This implies that other
ATP-converting enzymes are absent and that analysis is being
done at a low concentration
of reactants, which thus avoids
significant substrate consumption and end-product inhibition.
Such a system has already proved particularly useful for “ATP
monitoring” purposes, as in the assay of creatine kinase (EC
2.7.3.2) (28-30).
Compared with other analytical techniques, luminescence
measurements
offer many advantages. They have very high
sensitivity (1015 mole for ATP and 10’s mole for NADH),
great dynamic range, high specificity and rapidity, and they
require no sophisticated or expensive apparatus (light sources,
monochromators,
special optics, etc.).
Luminescence
measurements
have been automated
in
various cases. Flow-cells in particular are advantageous
because their use eliminates the phosphorescence
problems
encountered
with glass vials or plastic caps (or both). Optimization of flow-cell configurations
is discussed in recent
papers (31, 32). A few publications
refer to luminescence
measurements in connection with use of a centrifugal analyzer
(33-35).
Firefly Bioluminescence
The overall reaction
lows:
for firefly bioluminescence
is as fol-
Adenyl luciferin
+ 02
adenyl luciferin
-
adenyl oxyluciforin
+ light
The active components of the firefly light organ have now been
thoroughly studied and the mechanism of the reaction is fairly
well understood, although its control in vivo is still open to
much discussion. Commercial preparations
contain dried
firefly lanterns, crude lantern extracts, or purified luciferase.
Until recently, assays were performed with the crude extract,
which is more economical but has a higher “noise” background
(probably because of residual ATP) and is likely to react with
substances related to ATP because of the presence of adenylate kinase (EC 2.7.4.3) and transphosphorylases.
Several
commercial purified luciferase preparations now available are
very well suited for use in the routine clinical laboratory.
Synthetic luciferin is added after purificati#{224}n
of the luciferase
in concentrations
that will give maximum sensitivity. Proportionality of the luminescence with the ATP concentration
is observed for concentrations
extending over several orders
of magnitude.
Purified luciferase is particularly valuable for the continuous monitoring of ATP-conoerting
reactions,
as in the
clinical determination
of creatine kinase. Experimental
conditions are such that ATP consumption
by the luciferase is
negligible, and light emission therefore remains proportional
to the ATP concentration
at each moment (28-30). Kinetic
determination
of enzymes and metabolites
and end-point
determination
of metabolites can be performed in that way.
Many clinical applications deal with the measurement
of
bacteria. Firefly bioluminescence
can indeed be used as a
sensitive, reliable, and rapid screening method for testing the
presence of bacteria in biological fluids by measuring the
amount of the ATP of bacterial origin. For this purpose the
ATP of eukaryotic origin must have been previously removed
by selective lysis of the somatic cells and destruction of the
liberated ATP. Lundin and Thore (36) compared several
methods for extracting ATP from bacterial cells, and commercial reagents (from Lumac) are now available for the selective lysis of somatic and bacterial cells. In view of the
ubiquitous presence of ATP in living cells, it is also possible,
for instance, to investigate the growth and physiology of mycobacteria and filamental microorganisms, which are difficult
to study by classical techniques (6, 37, 38).
Firefly bioluminescence
has already been successfully applied to the detection of significant bacteriuria
(39-41).
Johnston
et al. (42) have elaborated
an automated
test
adapted to AutoAnalyzer equipment that provides results
within about 30 mm. Other automated procedures have been
described by Picciolo et al. (43) and Thore (44). The detection
limit lies around 108 cellsfL. False negatives, due to inhibition
of luciferase, are seldom observed. Rather, the bioluminescent
method is more likely to give false positives, because of incomplete removal of ATP from somatic cells. Detection of
germs in blood is now under study (45).
The susceptibility
of germs to various antibiotics such as
ampicillin, gentamycin, doxycycline, and nitrofurantoin
has
been related to the level of intracellular
ATP. Results are
obtained within hours and correlate well with the classical
techniques (45-48). Antibiograms
of Mycobacterium
bovis
could be obtained after only four days, instead of after three
to six weeks (49).
Determination of antibiotic levels in biological fluids is also
possible by following the depression of growth of a suitable
bacterial test strain (46, 50,51). Results are provided within
4 to 6 h and correlate well with plate-diffusion
tests. Other
compounds that influence bacterial growth, such as vitamins,
could be determined
the same way. Work is in progress to
automate the process (46).
Firefly bioluminescence
has further been used to evaluate
CLINICAL CHEMISTRY,
Vol. 25, No. 4, 1979
513
the viability and hence the biological potency of some vaccines
such as the BCG vaccine against tuberculosis (38). In our view,
further efforts in the field of rapid detection of life-threatening
situations such as septicemia and bacterial meningitis by
means of the firefly bioluminescence
system should be encouraged.
The determination
of ATP has also proved useful for the
study of the ATP-content
of erythrocytes in various pathological situations (52, 53); study of the critical effect of ATP
and ADP on platelet aggregation in vitro (54); monitoring of
the release of ATP by platelets under the influence of
thrombin (55); study of the shape and lysis of erythrocytes (56,
57); evaluation of the viability of stocked erythrocytes, leukocytes, and platelets (58); assessment of cell death (59);
evaluation of the viability of spermatozoa (60); and monitoring
of the leakage of ATP from erythrocytes during immunolysis
(44).
Firefly bioluminescence
provides a means of measuring
many substrates or enzymes that participate in a reaction
whereby ATP is formed or consumed, or in a reaction that can
be coupled to such a one. Although many other possibilities
exist, efforts of clinical chemists have concentrated up to now
on creatine kinase.
Because of its great sensitivity, firefly bioluminescence
proved suitable for elaborating a screening test for muscle
disorders, based on the determination of creatine kinase levels
in a drop of capillary whole blood (61, 62). As a result, early
detection of patients with Duchenne muscular dystrophy and
prevention of the so-called malignant hyperthermia,
which
can occur during anesthesia, have now become possible. When
collected and air-dried on an appropriate filter-paper disc, the
samples are stable for six weeks at room temperature and can
be mailed to a specialized laboratory for assay. Interference
from cellular ATP and adenylate kinase is eliminated by an
extensive preincubation period. Results are in good agreement
with serum creatine kinase values.
Witteveen et al. (63) devised a bioluminescent
method in
which the different isoenzymes of creatine kinase can be determined without a separation procedure; based on the difference in Km of the creatine kinase isoenzymes, this method
requires both bioluminescent
and spectrophotometric
measurements. A more promising bioluminescent method involves
use of a specific antibody to inactivate the M-subunits (30)
and use of purified luciferase reagent to continuously monitor
the ATP formed in the reaction. This bioluminescent method
gives results that correlate well with those obtained by spectrophotometry
but is more sensitive, thus allowing the assay
of B-subunit activities as low as 1 UIL. As a consequence, this
method could be of considerable clinical interest in the early
diagnosis of acute myocardial infarctions.
Examples of other substrates or enzymes that have been
determined by firefly bioluminescence
are listed in several
previous reviews (e.g., 3, 5). We shall therefore only mention
some recent work of particular clinical interest:
Nanomole quantities of serum glycerol (64) can be assayed
by bioluminescent
monitoring of ATP consumption
in the
following reaction:
glycerol
kinase
(EC
2.7.1.30)
Glycerol + ATP
Mg
glycerol 3-phosphate
+ ADP
Triglycerides have been determined in an analogous way after
saponification.
A bioluminescent
microdetermination
of creatine phosphate and ATP in plasma has been reported (65). These two
compounds are believed to be valuable metabolic indices for
prognosis in life-threatening
conditions.
Lee et al. (66) have linked firefly luciferase covalently to
514
CLINICAL CHEMISTRY, Vol. 25, No. 4, 1979
arylamine glass beads cemented to glass rods, to be used in the
same way as “dipsticks.” Considerable activity is lost upon
immobilization,
but, on the other hand, the enzyme displays
enhanced stability and can be reused many times or incorporated into automated systems.
Recently, the use of firefly bioluminescence
has been suggested for monitoring protein-ligand binding, by using a ligand
covalently coupled to ATP in such a way that the ATP can still
be recognized by the firefly luciferase as long as the ligand has
not been bound with the protein (67). However, attempts to
elaborate solid-phase
immunoassays
by using antibodies
coupled to firefly luciferase have been unsuccessful so far
(44).
Firefly bioluminescence has been used on several occasions
determine metabolites or enzymes in biopsy material or in
single cells (68-70). Additional information on the methodological problems associated with the use of the firefly luciferase has been presented (71-73).
to
Bacterial Bioluminescence
Bioluminescence
is displayed by a certain number of bacteria, the most studied of which are Photobacterium
fischeri
and Beneckea
harveyi.
Luminescent
bacteria are readily
cultured at room temperature
and represent an inexpensive
and easily accessible source of luciferase. To cause light
emission, purified bacterial
luciferase
requires oxygen,
FMNH2, and a linear, saturated long-chain aldehyde. Thus,
a specific bacterial luciferin, in the sense of a specific substrate
for the luciferase, does not exist. Crude luciferase extracts
contain an oxidoreductase activity, which is closely associated
with the luciferase activity. Under the conditions just mentioned, NADH (and to a lesser extent, NADPH) can also
generate luminescence in the presence of FMN:
oxidoreductase
NAD(P)H
+ FMN + H
NAD(P)
+ FMNH2
luciferase
FMNH2 +02+
RCHO
-
FMN
+ RCOOH + H20 + light
In this way FMN, NADH, or NADPH can be determined by
bacterial bioluminescence,
provided that the assayed substance is the limiting factor (2, 3, 5, 23, 74-79).
The use of coupled reactions makes it easy to determine
numerous substrates and enzymes involved in NAD(P)Hproducing or -consuming reactions (2, 3, 5, 23, 74-81). Bacterial bioluminescent
techniques are indeed applicable to
several clinically important dehydrogenases
such as lactate
dehydrogenase
(EC 1.1.1.27), alcohol dehydrogenase
(EC
1.1.1.1), malate dehydrogenase
(EC 1.1.1.37), and glucose6-phosphate dehydrogenase (EC 1.1.1.49) (3,5,80,81).
Other
clinically important enzymes such as creatine kinase, aspartate aminotransferase
(EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) might further be determined by coupling
the reaction they catalyze to a suitable dehydrogenase
reaction.
Substrates that have already been determined are malate,
oxaloacetate, NAD, NADP, glucose 6-phosphate, glucose,
ammonia, and ethanol (3,5,80, 81). By taking advantage of
the specific conversion of urea into ammonia by urease (EC
3.5.1.5), it should also be possible to determine urea in biological fluids. Notwithstanding
the widespread use of dehydrogenase reactions in the clinical laboratory, classical spectrophotometric
methods have so far seldom been replaced by
the more sensitive bioluminescent
techniques. The recent
availability of purified enzyme preparations
and the use of
immobilized enzymes are likely to change this situation.
Jablonski and DeLuca (82) have attached highly purified
NADH: and NADPH:FMN
oxidoreductase
and luciferase
from Beneckea harueyi on arylamine glass beads cemented
to glass rods. Such reusable sticks show a great potential
usefulness in clinical analyses such as the determination
of
glucose, ethanol, and several important dehydrogenases
(81).
Using oxidoreductases
that react exclusively with either
NADH or NADPH confers a great specificity to the system
and obviates the need to destroy one of these two cofactors
before assaying the other one. This fact was early recognized
by E. Gerlo, who characterized
the two oxidoreductases
of
Be,teckea harueyi in our laboratory (80,83). Immobilization
of luciferase in particular increased the stability of the enzyme.
Bacterial bioluminescence
has been used on different cccasions to determine various substrates and enzymes in microgram quantities of tissue (76, 77,84-86). In a few instances
bacterial bioluminescence
was used to determine ATP by
means of coupled reactions (87), e.g.,
+ 2H2O2 + OH
Luminol
monoanion
ioxidant
+ N2 + 3H20 + he
catalyst
Aminophthalate
dianion
The luminol-ferricyanide-H2O2
system was used to determine
glucose in serum (99) according to the following reaction
scheme:
ATP:NMN adenyltransferase
ATP+NMN
‘NAD+PP
(EC 2.7.7.1)
glucose
3-D-Glucose
+02+
ozidase
H2O
(EC
1.1.3.4)
malate dehydrogenase
NAD
+ malate
D-gluconic
oxaloacetate
+ NADH + H
All applications of the firefly bioluminescence
based on the
ATP assay should therefore also be possible with the bacterial
bioluminescent system, highlighting once again the enormous
potential of this method.
In a very different way, bacterial bioluminescence has been
used to determine proteolytic enzymes (88) such as trypsin
(EC 3.4.21.4), chymotrypsin
(EC 3.4.24.1), and pepsin (EC
3.4.23.1) by following the inactivation
of the luminescent
enzyme as a function of time. And the most recent applications
of bacterial luminescence concern the assay of fatty acids by
means of a mutant strain of Beneckea harueyi that is dependent on these substances for luminescence,
but no clinical
applications have yet been quoted (89). Although NAD-ligand
conjugates have been used with bacterial bioluminescence
to
monitor protein-ligand
binding reactions (90), attempts to
obtain useful antibodies coupled to bacterial luciferase have
been unsuccessful so far (91).
Other Bioluminescent Systems
Amongst the multitude of other living organisms displaying
bioluminescence
is the Aequorea system, which has been
thoroughly studied and already been used as an analytical tool.
In particular,
it has been applied to the determination
of
ionized calcium in biological preparations. The specificity for
Ca2+ is not absolute, but the other cations that can also generate bioluminescence
are not present in biological samples
in appreciable quantities (92-94). For further information the
reader should refer to the reviews of Blinks (95-97).
Luminol Chemilumlnescence
To obtain chemiluminescence
from luminol (5-amino2,3-dihydrophthalazine-1,4-dione)
in an aqueous solution, the
analyst must include a base and a strong oxidant; for maximum efficiency a catalyst or a cooxidant
is required
as well.
Various metal ions such as Co(II), Cu(II), Fe(II), and Fe(III)
have been determined by means of their activating effect on
luminol chemiluminescence
(6, 7), and vitamin B12 has recently been assayed after reduction of the complexed Co(III)
to Co(II) (98), but the applicability of this method to physiological fluids is as yet undetermined. The chemiluminescence
of luminol is more valuable for the determination
of oxidants
such as H2O2, which is produced in a series of enzymatic reactions.
acid + H202
base
H202 + lummol + ferricyanide
light + product
-
Glucose is first oxidized enzymatically at neutral pH with simultaneous production of H202. A strongly buffered alkaline
reagent is then added to initiate the chemiluminescent
reaction, which is efficient only at high pH (10 to 11).
Bostick and Hercules (100) devised a chemiluminescent
method based on the same principle but making use of a flow
system and glucose oxidase immobilized on Sepharose. The
method gave good results for serum samples, but interferences
were observed in urine, which were ascribed to reduction of
H202 by uric acid in alkaline medium. Pretreatment
of the
urine samples to remove interfering reducing substances appeared necessary. A slightly modified flow system was used
to compare this method with the hexokinase method for the
determination of glucose in urine (101). The results correlated
well with those obtained with the hexokinase system, although
the latter gave consistently lower results at low glucose concentrations.
We ourselves elaborated a somewhat similar flow system
for the determination
of uric acid by means of uricase (EC
1.7.3.3) fixed on porous glass beads (102). Specific features of
our system include capillary tubing for the fluids lines and the
detection
flow-cell, and specially designed micro-mixers
containing a small ball actuated by an oscillating magnet. To
lower the blank value of the reagent, a delay-line of suitable
length was introduced between mixing the luminol with potassium ferricyanide and adding the mixture to the H2O2containing sample. Finally, under the conditions used, it was
not possible to demonstrate
any reduction of H202 by uric
acid.
Further applications have dealt with the determination
of
cholesterol (34), L-aminoacids (103, 104), hypoxanthine (105),
L-amino acid oxidase (EC 1.4.3.2) (104), and xanthine oxidase
(EC 1.2.3.2) (106).
In many cases the bacterial bioluminescent
system might
be replaced by luminol chemiluminescence
because of the
production
of H202 in the presence of methylene
blue
(MB):
H
+ NADH + MB05
MBred +02
H202 + luminol
NAD
+ MBred
MB05 + H202
-
The fact that iron-porphyrin
products
+ light
complexes
CLINICAL CHEMISTRY,
such as in hae-
Vol. 25, No. 4, 1979
515
moglobin or myoglobin intensify the chemiluminescence
of
luminol and H202 in alkaline solution (107) has been turned
to use by Ewetz and Strangert for the automated detection
of significant bacteriuria (108). The method does not require
any special treatment of the samples and might be of interest
as a mass screening technique, were it not for the possible
interference by blood traces. The experimental and theoretical
limits of the method were recently discussed by Miller and
Vogelhut
(109).
Freeman and Seitz (110) devised a chemiluminescent
fiber
optic probe for hydrogen peroxide, consisting of peroxidase
immobilized in a polyacrylamide
gel at the extremity of the
probe. By immersing the probe in a solution containing H202
and excess luminol, the chemiluminescence
generated
is
transmitted by the fiber optic to the detector. A steady-state
light output is reached within seconds when the rate at which
substrate diffuses into the enzyme phase equals the rate of the
enzymatic reaction. The reproducibility of the method should
still be improved, but the approach looks very promising.
Finally, hematin-labeled
antibodies that emit light upon
addition of alkaline luminol and sodium perborate have been
used in solid-phase immunoassays
(44); other investigators
have made use of horseradish peroxidase as a luminescent
label in immunological reactions (91).
Other Chemiluminescent Systems
Oxalate esters such as TCPO [bis(2,4,6-trichlorophenyl)
oxalate] will produce light when reacting with H202. To increase the efficiency of the reaction, a suitable fluorescerusually perilene-to
which energy can be transferred must be
added. Other oxalate esters such as bis(2,4-dinitrophenyl)
oxalate (DNPO) can be used, but TCPO is usually preferred
because of its greater stability and easier preparation.
The
overall reaction is as follows (111):
Cl
Cl
ciQ-o_c-c_o_-Oci
0
0
+ H20
Cl
TCPO
2Cl 0H
+
2C02
2,4,6-trichlorophenol
Because of the poor solubility of TCPO in water, ethyl
acetate is generally used as a solvent, and methanol is added
to enable mixing with aqueous H202 samples. Small quantities
of a base such as sodium salicylate or triethylamine accelerate
and intensify the chemiluminescence.
TCPO luminescence can be used in the same way as luminol
chemiluminescence
each time a substrate or an enzyme is
involved in a reaction in which H202 is produced or in a reaction that can be coupled to such a reaction. For example,
Williams and Seitz (112) used TCPO instead of luminol to
assay for NADH and lactate dehydrogenase. Background light
emission and detection limits in flow systems are lower than
with luminol. Moreover, the determination of H202 is possible
over a broader pH range, roughly from pH 4 to 10, which allows, for instance, an assay for glucose in urine in conditions
where interferences by reducing substances (113) are minimal.
Up to now, however, pure TCPO has not been commercially
available, and flow systems are somewhat more complicated
than for luminol. The analytical
applications
of TCPO
chemiluminescence
have recently been reviewed (114).
Several chemiluminescent
systems have been used to
monitor immunological reactions. Small numbers of microorganisms can be rapidly identified and quantitated by using
homologous antibodies linked to peroxidase, which catalyzes
516
CLINICAL CHEMISTRY, Vol. 25. No. 4, 1979
light production upon addition of pyrogallol and H2O2 (115).
As few as 30 to 300 bacterial cells could be detected. Peroxidase-labeled
antibodies are furthermore
much more stable
than radioisotope-labeled
ones.
A number of derivatives of isoluminol (6-amino-2,3-dihydrophthalazine-1,4-dione)
and various aminonaphthalhydrazides were attached covalently to ligands and their yields
of light with different oxidation systems compared (116).
Similar conjugates of luminol (instead of isoluminol) were less
efficient because of steric hindrance.
Several of the chemiluminescent
conjugates were stable and
detectable in pico- to nanomolar quantities over a broad pH
range (8.6 to 13.0); they can be used as nonisotopic labels to
monitor competitive protein-binding
reactions, as already
demonstrated
in assays for biotin (117), thyroxine (118), and
testosterone
(119).
Low-Level
Biological Chemllumlnescence
Very weak luminescence, which is observed from rapidly
cells, in microsomal and mitochondrial
extracts, and
during phagocytosis of bacteria, has been ascribed to exergonic
oxidation reactions involving singlet molecular oxygen or
superoxide anion radicals (or both). Luminol can be added to
intensify this chemiluminescence.
The low-level chemiluminescence
accompanying
phagodividing
cytosis
can be used to measure
total
phagocytosis
capacity,
which provides us with a useful tool to study the mechanism
of phagocytosis and how various substances and pharmaceuticals influence it (120-129). Moreover, this method has
helped in the screening of pathological situations characterized by decreased bactericidal capacity, such as in chronic
granulomatous
disease (129-132).
Conclusion
Chemiluminescent
and bioluminescent
methods offer
considerable advantages for the clinical laboratory. Because
of the great sensivity of these methods, only a few microliters
of sample are needed for each determination; this makes them
suitable for screening tests and for analysis of neonatal blood,
cerebrospinal
fluid, and amniotic fluid. In addition, these
methods
are often highly specific and results are often obtained much more rapidly than would otherwise be possible.
Proportionality
of the luminescent
response is usually observed over several orders of magnitude. Bio- and chemiluminescence could be of great value in the elaboration of sensitive enzyme immunoassays, in specific binding tests, and in
the rapid detection of bacteria.
Finally, we must emphasize that chemiluminescent
or
bioluminescent
measurements
are easy to make and require
no complicated apparatus. Indeed, no monochromator,
light
source, or special optics are needed, and the instrumentation
can be used for a wide array of clinical analyses. Such instruments as well as ready-to-use reagents have now been commercialized. Moreover, luminescence assays lend themselves
easily to automation.
More work remains to be done on possible interferences in
clinical samples. Indeed, biological fluids can sometimes
contain extremely variable amounts of metabolites or drugs
that could either interfere with the production of light or absorb the photons once they have been produced. However, the
great sensitivity of luminescent
methods allows for ample
dilution of the samples, thus minimizing the probability of
such eventualities.
After this survey was written, a symposium was held in
Brussels on the analytical applications of bio- and chemiluminescence. Many of the papers presented at this meeting
dealt with various clinical applications: use of firefly luciferase
for the assay of creatine kinase; bacteriuria and antibiotic
susceptibility;
measurement
of dehydrogenases
with immobilized bacterial luciferase; and use of chemiluminescence
in
enzyme immunoassays,
measurement
of phagocytosis, and
assays of substrates with specific oxidases. Details of these
procedures will appear in the proceedings to be published
(133).
26. Gerlo,
ninescentie.
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