Chemiluminescence:
Applications for the Clinical Laboratory
ROGER L. BOECKX,PhD*
The chemical features and applications of chemiluminescence
are reviewed, with special attention to bacterial and firefly bioluminescence and to uses of chemiluminescence in direct substrate assays, enzyme assays, solid-phase reactions, and immu.
noassays. HUM PATIIOL15:104--111, 1984.
In 1667, Robert Boyle, the English chemist and
physicist, carried out the first systematic investigation
of bioluminescence, demonstrating the oxygen dependence o f the luminescence of a luminous fungus
growing on wood.
Since ancient times, this "cold light" has fascinated us. Whether it comes from Boyle's "glowing
wood," tile e n c o d e d flashes of a firefly on a late
spring evening, or the "flashlights" of deep-sea fishes,
our attention is always drawn to this phenomenon.
Bioluminescence is actually quite common in nature and is displayed by many diverse organisms
ranging from bacteria and other microscopic organisms to vertebrates. As a reldt of advances in the fields
of spectroscopy and photochemistry, tile chemical
mechanisms that lead to chemiluminescence are now
beginning to be tmderstood. With this understanding
has come increasing exploitation of chemiluminescence in analytic chemistry. T h e clinical laboratory,
with its need for accurate and precise measurements
of a wide variety of compounds in biologic fluids, is
beginning to look to chemiluminescence as an analytic tool.
THE CHEMICAL FEATURES
OF CHEMILUMINESCENCE
Chemiluminescence refers to tile emission of
light by exergonic chemical reactions, usually oxidative in nature but without the input or ouptput of any
significant heat or the input of energy in the form of
electromagnetic radiation. Emission of light in the
visible region of the spectrum requires energies in tile
range of 40 to 70 kcal/mol. Usually, light emission
occurs from an excited singlet state, 1 and it occurs
with a wide range of efficiencies. T h e overall efficiency of light production, the quantum yield (the
ratio of the number of molecules luminescing to the
n u m b e r o f molecules reacting), can range from a
value close to 1, the theoretical maximum (as is ob* Director of Clinical Chemistry, Children's ttospital National
Medical Center, and Associate Professor, Child Heahh and Development, The George Washington University School of Medicine and Heahh Sciences, Washington, DC
Address correspondence and reprint requests to Dr. Boeckx:
Department of Clinical Chemistry, Children's Hospital National
,Medical Center, 111 Michigan Ave NW, Washington, DC 20010.
served in the firefly luciferase reaction) to values as
low as 10 -15 (as seen in the neutralization o f hyd r o n i u m ion). Typical q u a n t u m yield vahtes for
"bright" chemiluminescent reactions are in the range
of 0.01 to 0.25. 2
In this century, a number of chemiluminescent
compounds have been synthesized. These substances
have allowed us to study the details of the reaction
leading up to the production of the emitting molecule. Perhaps the best known of these is hmlinol (5amino-2,3-dihydro- 1,4-phthalazinedione). Luminol
and a number of similar cyclic diacylhydrazides will
emit light following oxidation. The reaction of luminol with hydrogen peroxide is shown in figure 1.
The reaction requires a catalyst, which can be either
a metal such as ferricyanide or copper or an enzyme
such as a peroxidase. T h e reaction is now known to
invoh'e the production of a diazoquinone intermediate, which, following nucleophillic attack by hydrogen peroxide, is converted, through a series of
other intermediates, to an excited aminophttlalate dianion, the emitter molectde. 4 This mechanism, referred to as chemically initiated electron exchange
luminescence (CIEEL) is now thought to be the basic
mechanism of light emission in several types of luminescent reactions.5, 6
Bioluminescence is a special case of chemiluminescence that involves the action of a specific enzyme
system, generically referred to as a luciferase, and a
specific substrate, usually referred to as a luciferin.
Several different types o f bioluminescent systems are
known, but for the purposes of this review, it would
seem appropriate to limit our attention to only two.
The interested reader is referred to Hasting's excellent review of this topic. 6
Bacterial Bioluminescence
T h e first of these two bioluminescent systems is
that of the marine bacteria. Photobacte~qum phosphoreum, Photobacterium leiognathi, and Photobacterium
fisheri all occur as symbionts in the specialized "light
organs" of fish. These species can also be found free
in sea water and are particularly plentiful in areas
where their specific hosts are commonly found. A
fourth species, Beneckea harveyi, is not usually folmd
as a symbiont and is especial)' common in coastal surface waters.
Light emission in these bacteria occurs by what
is essentially a shunt o f the electron t r a n s p o r t
pathway. The reaction involves the oxidation of reduced flavin mononucleotide (FMNH2) by molecular
oxygen in tile presence of a long-chain aliphatic aldehyde (dodecanal). During the reaction, the alde104
CHEMILUMINESCENCE[Boeckx)
hyde is converted to the corresponding acid (fig. 2).
The exact structure of the emitting species is not yet
known, but it is most likely an enzyme-bound flavin
in the excited state. The bacterial luciferase is a dimer
with a molecular weight of 79,000, consisting of two
non-identical subunits. 4 Bacterial luciferases, when
isolated, invariably have oxidoreductase activity and
can couple the oxidation of NADH or NADPH to the
reduction o f FMN. Therefore, the concentration of
N A D H or N A D P H can be determined by this reaction.
o
!
Ch'F"
,"-rcoo,.
HO~).~'.-.S" "$~
Luciferin
--.
y
HO~$"
"$~
HO.J~sN~syO 4, C02 + AMP 4 h,
FIGURE3, The reaction sequence of the firefly luciferase reaction.
Reprinted with permission from Mayer and Boecloc3
intracellular ATP in bacterial cultures, allowing an
estimate of bacterial growth, and has been exploited
as a method of determining bacteriuria. 9-12 By measuring all the intracellular ATP present in a bacterial
culture, an estimate of bacterial growth can be made.
Nillson et al. 13 have used this assay for biomass to
measure the concentration of antibiotics in a serum
specimen. They described a simple assay of this t y p e
for gentamicin, ampicillin, and doxycyclin. These au-:
thors used Enterobacter aerogenes LU6 as a test organism. Aliquots of the drug-containing serum were
added to a growth medium containing the test organism. Following an appropriate incubation period,
extracellular ATP was hydrolyzed and intracellular
ATP was liberated and assayed using the firefly luciferase reaction. With this approach, the authors
were able to determine the concentrations of antibiotics in samples of 10 ~L of serum. Similar procedures have been developed for the assay of cephaloridine and tobramycin. 14
By using appropriate coupling reactions, it is
possible to assay other compounds. For example, by
exploiting phosphodiesterase (A), myokinase (B), and
pyruvate kinase (C), it is possible to use the firefly
ludferase (L) reaction in an assay for cyclic AMpIS-16:
Firefly Bioluminescence
In contrast with bacterial bioluminescence, light
emision by fireflies is not dependent on pyridine nucleotides, but it is dependent on the concentration of
ATP. In addition, a specific luciferin, 4,5-dihydro-2(6- hydroxy- 2-benz~176176176
acid, is required. In the firefly reaction, an enzymebound luciferin-adenylate complex is formed (fig. 3)
which, in the presence of oxygen, reacts to form a
dioxetanone intermediate. 7 This compound is converted to an excited carbonyl compound by way of a
CIEEL mechanism which involves decarboxylation.
After emission of light, the final product, oxyluciferin, is released from the enzyme. Firefly luciferase
was purified and studied by McElroy and DeLuca and
their coworkers. 8 Tim enzyme is a hydrophobic dimeric protein consisting of two nonidentical subunits,
each having a molecular weight of 50,000.
APPLICATIONS OF CHEMILUMINESCENCE
Direct Substrate Assays
T h e simplest application of chemiluminescent
reactions is in the assay of a compound that is a substrate for the light-emitting reaction itself. The earliest applications of chemiluminescent systems were
m a d e using the A T P - d e p e n d e n t firefly system.
U n d e r proper conditions, as little as 10 -15 mols of
A T P can be measured using the firefly luciferase
reaction. This approach has been used to measure
A
c-AMP
~ AMP
B
AMP + A T P ~
2 ADP
C
2 ADP + 2 phosphoenolpyruvate
2 pyruvate + 2 ATP
~O- 'HH- L u m+i n2H202
ol
+
> ~ !
L
ATP + luciferin + O 2 - - ~
AMP + PPi + oxyluciferin + CO z + light
~ + N2 + 3H2~ + h="
FIGURE 1. Luminal chemiluminescence resulting from the oxidation of luminal by hydrogen peroxide. Reprinted with permission
from Mayer and Boeck)c3
H+ + NAD(P)H . . ~
f
O
- id(
xoreductase
vi
NAD(P}+
FMN
y
Lucifer~e
FMNH2 ~ ~ ' ~ -
H20 * RCOOH
. , f , o , x . ~ h~,
o2* RCHO
FIGURE 2, Bacterial bioluminescence as exhibited by Beneckea
harveyi. Reprinted with permission from Mayer and Boecloc3
105
A coupled enzyme system for the assay of chloramphenicol has been described by Boeckx and
Brett, 17 in which the reaction o f a bacterial chloramphenicol acetyl transferase is coupled to the
firefly luciferase reaction by means of acetyl CoA
synthetase (fig. 4). This assay is linear over the therapeutic range of chloramphenicol and can be completed in about one hour using only 20 tzL of serum.
A similar assay for gentamicin has also been developed. 18
F.irefly-luciferase-based assays for adenine nucleotides,15,19-27 creatine, 19 glucose, 19 triglycer-
HUMAN PATHOLOGY
CM + AcCoA
X
AMP + PPi
CO2 § Ox),lucife
I
Volume 15, No. 2 (February 1984)
~ CoA + AcCM
ATP + kcetQle
ciferin + 0 2
hv
The reaction sequence in a bioluminescent assay for
chIoramphenicol. CM indicates chloramphenicol; AcCoA, acetyl
coenzyme A; AcCm, ace~,l chloramphenicol. Reaction I is catalyzed by chloramphenicol acetyl transferase isolated from a chloramphenicol-resistant strain of Escherichia coll. Reactions 2 and 3
are catalyzed by aceiyl coenzyme A synthetase a n d firefly luciferase, respectively. Reprinted with permission from M o y e r a n d
Boeckx.3
FIGURE 4.
ides,28, 29 glycerol, 29 and a number of other metabolic
intermediates have been describedA 6
T h e bacterial system has also seen wide application. Assays for NAD +, NADH, and NADPH by direct reaction with the luciferase system are obvious
developments.19, 25,30-3a In addition, assays for
malate, 31,35 oxaloacetate,~9, 36 p y r u v a t e y ethanol, 3s,39
glucose, 31,38 g l u c o s e - 6 - p h o s p l m t e y 3-hydroxybutyrate, 35 and ammonia 40 have been described.
Kather et al 4~ have recently described a very sensitive assay for glycerol utilizing bacterial luciferase
and have exploited this method for the measurement
of lipolysis in small numbers of fat cells collected by
needle biopsy.
Chemiluminescent reactions based on the oxidation of luminol or luminol-like compounds have
also been developed. Hydrogen peroxide can be directly m e a s u r e d using this reaction. For example,
since hydrogen peroxide is a product of the glucose
oxidase reaction, the luminol system can easily be
used to measure glucose concentration. 42-a6
Another exciting application of luminol chemiluminescence involves its use in the evaluation of phagocytosis by leukocytes. It is well known that phagocytosis and subsequent bactericidal activity involve a respiratory burst with the production of singlet oxygen,
superoxide, and hydrogen peroxide. 47 The production of these active forms of oxygen results in a native
chemiluminescence. This native luminescence can be
amplified by the addition o f luminol, 4s t h e r e b y
making the post-phagocytic production of excited oxygen detectable by m e a s u r i n g the light emitted
during the process. This approach can be used to
measure phagocyte oxidative activity, as Other compounds, in addition to luminol, have been used as
luminogenic probes in such assays. For example, lucigenin, 10,10'-dimethyl-9,9'-biacridinium dinitrate,
has been successfully used in a sensitive assay of polym o r p h o n u c l e a r leukocyte r e d o x activity. 49 T h e
chemiluminescent evaluation of polymorphonuclear
leukocyte function has been used by DeChatelet and
Shirley 5~ in the evaluation of patients with chronic
granulomatous disease. These authors used luminol
as a luminescence amplifier and stimulated the respiratory burst o f p o l y m o r p h o n u c l e a r leukocytes
with phorbol myristate acetate. By this approach they
were able to quantify the luminescent response in microliter quantities of whole blood and to demonstrate
that cells from patients with chronic granulomatous
disease were not responsive to stimulation.
The ability to detect phagocytosis in whole blood
has been adapted for use as an assay for the opsonic
activity of serum.Sl By adding an appropriate bacterium and luminol to diluted whole blood (a source
of p o l y m o r p h o n u c l e a r leukocytes), it is possible to
evaluate the opsonic activity o f an a d d e d s e r u m
sample by measuring the light emitted as the phagocytosis process proceeds.
Enzyme Assays Using Chemiluminescence
In addition to assays of substrates, a number of
enzymes have been assayed using chemiluminescent
methods. O f most interest to the clinical chemist is
the assay of creatine kinase using the firefly luciferase
reaction as a detector for ATPA 9,52-55 Lundin and
Styrelius 52 have described a highly sensitive and specific assay for creatine kinase (CK) B subunit activity
involving the use of an antibody that inhibits M subunit activity (Ab):
CK(MM,MB,BB) + Ab--*
CK(MB,BB) + CK(MM):Ab + CK(MB):Ab
ADP + creatine phosphate + CK(MB,BB)
ATP + creatine
ATP + luciferin + 0 2
AMP + PPi + oxyluciferin +
CO 2 + light
Assays for cyclic A M P p h o s p h o d i e s t e r a s e , 15
ATPase, t9 hexokinase, 19 myokinase, 19 and pyruvate
kinase 19 have also been described.
Considering the number of reaction systems that
involve NADH, either as a substrate or as a product it is not surprising that a n u m b e r o f enzyme
assays using the bacterial luciferase system have
been developed. Alcohol dehydrogenase, 38 glucose6-phosphate dehydrogenase,3S hexokinase, 3s betah y d r o x y - b u t y r a t e d e h y d r o g e n a s e , 37 and lactate
dehydrogenase 3s have all been assayed using the bacterial system.
Solid-phase Luminescent Reactions
In 1977, Lee et al. 56 reported the immobilization
of firefly luciferase on glass rods and demonstrated
that, although enzyme activity was reduced, sufficient
activity was maintained to allow the immobilized enzyme to be used in analytical applications. In their
method, crystalline firefly luciferase was attached to
alkylamine glass beads ( 1 2 5 - 1 7 7 Ixm in diameter)
that had been cemented to a glass rod (1.7 • 40 mm).
The authors used these rods to assay creatine kinase
activity, which they found they could assay to as low
as 1.27 IU. The rods were stable enough to allow 30
assays to be completed in sequence.
Jablonski and DeLuca 59 immobilized bacterial lu106
CHEMILUMINESCENCE(Boeckx]
ciferase and FMN reductase on glass rods and were
able to use these rods in assays for N A D H and
NADPH, and Watanabe and Hastings 57 have co-immobilized N A D P H - F M N reductase, B harveyi luciferase, and alcohol dehydrogenase (ADH) onto sepharose 6B. They used this unique coupled system to
measure the activity of a number of dehydrogenases.
In their reaction system, the aldehyde (dodecanal)
required for the luciferase reaction is supplied as dodecanol and is converted to dodecanal by the alcohol
dehydrogenase, which is co-immobilized with the luciferase. T h e activity of A D H is dependent on the
provision of NAD + by the activity of the dehydrogenase to be measured:
TABLE 1. Applications of Luminescent Immunoassay
Anal)te
Alpha-fetoprotein
Anti-IgG
Biotin
Cortisol
Digoxin
Estradiol
Estriol
Estrone
Hepatitis B
surface antigen
IgG
Insulin
Pregnanediol
Progesterone
Testosterone
Thyroxine
DH
X(oxidized) + N A D H + H +-~-------~
X(reduced) + NAD +
C12-RCOH + NAD +~
ADH
~ CI2-RCHO + NADH
References
66-69
62, 94, 101
65, 70-72
63, 73-74, 76-78
80
63, 81-83
64, 74, 84-88
74, 81, 85
62, 89
67-69, 71, 90, 93-94
80
74, 81, 95
61, 63-64, 74, 78, 96-98
63, 99
60, 62, 65, 72, 100, 136
with antigen in tile sample for sites on a limited
amount of antibody. By attaching the antibody to a
solid support, such as a plastic tube or sepharose,
antibody-bound luminol can be detected by the addition of hydrogen peroxide and a catalyst. A variety
of oxidation systems are available, including hydrogen p e r o x i d e - m i c r o p e r o x i d a s e , hydrogen peroxide-hematin, hydrogen peroxide-lactoperoxidase, and persulfate. 61 Kohen et al. 64 have shown that
when a steroid-luminescent label conjugate is bound
to an antibody, the resulting luminescence is significantly enhanced. This antibody-enhanced chemiluminescence can be used in a homogeneous LIA. Boguslaski and Schroeder 65 have reviewed both homogeneous and heterogeneous approaches to LIA. In
recent years, a large number of assays using either
heterogeneous or homogeneous LIA have been reported (table 1).
In LEIA, the antigen is labeled with an enzyme
that can either catalyze a luminescent reaction (e.g.,
peroxidase or luciferase) or produce a substrate for
a luminescent reaction. This latter approach, referred to as amplified bioluminescent immunoassay,
has allowed a significant increase in sensitivity. Wannlund et al.lOZ have reported such an amplified assay
that can detect as little as i0 attomoles of trinitrotoluene. A number of LEIA assays have been described
(table 2).
In LCIA, an antigen-cofactor conjugate is used.
The cofactor (CF) can be either NADH or ATP, and
the amount of antibody-bound cofactor can be assayed by using either the bacterial or firefly luciferase
reactions.
R'ase
N A D P H + FMN -*-----~ NADP + FMNH 2
L'ase
F M N H 2 + C12_RCHO + 0 2
1.
C~2-RCOOH + H 2 0 + FMN + light
In this scheme, X is the substrate for the NADHspecific dehydrogenase (DH) to be assayed. R'ase is
the N A D P H - d e p e n d e n t FMN reductase, and L'ase is
the bacterial luciferase. The authors have used their
system in an assay for lactate dehydrogenase.
Other applications of the solid-phase approach
have been described. Lippman 5s has used mercaptoacetylisoluminol attached to sepharose 6B in an
assay o f cholinesterase activity, and Jablonski and
DeLuca59 have used glass-rod-immobilized bacterial
luciferase in a sensitive assay for glucose.
Chemiluminescent Immunoassays
Although the luminescent assay of many analytes
can be accomplished by the use of reactions that directly or indirectly alter the concentrations of the substrates of chemiluminescent reactions, it is obvious
that this approach is somewhat limited. However, the
coupling o f the sensitivity of chemiluminescence with
the specificity of immunologic reactions promises to
make luminescent reactions more widely applicable.
W h i t e h e a d et al., 16 in a recent review o f this
topic, have classified luminescent immunoassays into
four categories: 1) luminescence immunoassay (LIA),
2) luminescent enzyme i m m u n o a s s a y (LEIA), 3)
luminescent cofactor immunoassay (LCIA), and 4)
luminescent enzyme-multiplied immunoassay technique (LEMIT). In LIA, luminescent labels essentially replace the radioisotopic label in the classic RIAtype assay. A variety of methods for the synthesis of
luminol or isoluminol conjugates have been reported. 6~ Such a luminol-labeled antigen competes
Ag + Ag-CF + Ab*---*Ab:Ag + Ab:Ag-CF
Ag-CF(bound or free) + luminogenic reactants --*
light
An assay for biotin in which NAD + is used as the
label has been described.122 In this case, the bound
Ag-NAD + is first converted to Ag-NADH by adding
ethanol and alcohol dehydrogenase, and then the
NADH is detected by adding the bacterial luciferase
107
HUMAN PATHOLOGY
YABU= 2.
Volume '15, No. 2 [Februan/t984)
and Chenfihmlinescence. Westlake Village, CA, State
Printing and Publishing, 1979, p 37
2. White EH, Rapaport E, Seliger HIt et al: The chemiluminescence and bioluminescence of firefly luciferin: an efficient chemical production of electronically excited states.
Bioorg Chem 1:92, 1971
3. Boeckx RL: Luminescence: a new analytical tool for therapeutic drug monitoring. In Moyer TP, Boeckx RL (eds):
Applied Therapeutic Drug Monitoring, vol 1. Washington,
DC, American Association for Clinical Chemistry, 1982, p
199
4. Hastings JW, Wilson T: Bioluminescence and chemiluminescence. Photochenl Photobiol 23:461, 1976
5. Koo J-Y, Schuster GB: Chemically initiated electron exchange luminescence: a new chemiluminescent reaction
path for organic peroxides.J Am Chem Soc 99:6107, 1977
6. Hastings JW: Biological diversity and chemical mechanisms
ill bioluminescence. In Schram E, Stanley P (eds): Proceedings of the International Symposium on Analytical Applications of Bioluminescence and Chemilmninescence. Westlake Village, CA, State Printing and Publishing, 1979, p 1
7. Koo J-Y, Schmidt SP, Schuster GB: Bioluminescence of the
9firefly: key steps in the formation of the electronically excited state for model systems. Proc Natl Acad Sci USA
75:30, 1978
8. DeLuca M: Firefly luciferase. In Schram E, Stanley P (eds):
Proceedings of the International Symposium on Analytical
Applications of Bioluminescence and Chemiluminescence.
Westlake Village, CA, State Printing and Publishing, 1979,
p 32
9. Johnston HH, Mitchell cJ, Curtis GDW: An automated test
for the detection of significant bacteriuria. Lancet 2:400,
1976
I0. Chappelle EW, Levine GV: Use of the firefly bioluminescent
reaction for rapid detection and counting of bacteriuria.
Biochem Med 2:41, 1968
11. Thore A, Ansehn S, Ltmdin A, et al: Detection of bacteriuria
by luciferase assay of adenosine triphosphate. Clin Microbiol 1:1, 1975
12. Alexander Dtt, Ederer GM, Matsen JM: Evaluation of an
adenosine 5'-triphosphate assay as a screening method to
detect significant bacteriuria. J Clin Microbiol 3:42, 1976
13. Nilsson L, Hojer H, Ansehn S, et al: A simplified luciferase
assay of antibiotics in clinical serum specimens. In Schram
E, Stanley P (eds): Proceedings of the International Symposium on Analytical Applications of Bioluminescence and
Chemiluminescence. Westlake Village, CA, State Printing
and Publishing, 1979, p 515
14. Harber, M.J. and Asscher, A. W.: A new method for antibiotic assay based on measurement of bacterial adenosine
triphosphate using the firefly bioluminescence system. J
Antimicrob Chemother 3:35, 1977
15. Johnson RA, Harman JG, Broadus AE, et al: Analysis o f
adenosine 3',5'-monophosphate with luciferase luminescence. Anal Biochem 35:91, 1970
16. Whitehead TP, Kricka LJ, Carter TJN, et al: Analytical luminescence: its potential in the clinical laboratory. Clin
Chem 25:1531, 1979
17. Boeckx RL, Brett EM: A bioluminescence micromethod for
measuring chloramphenicol in serum. Cliu Chem 27:819,
1981
18. Daigneault R, Larouche A, Thibauh G: Aminoglycoside antibiotic measurement by bioluminescence, with use of
plasmid-coded enzymes. Clin Chem 25:1639, 1979
19. Strehler BL: Bioluminescence assay: principles and practice.
Methods Biochem Anal 16:99, 1968
20. Holmsen H, Storm E, Day HJ: Determination of ATP and
ADP in blood platelets: a modification of the firefly assay
for plasma. Anal B~ochem 46:489, 1972
21. Hammar H: ATP and ADP levels in epidermal replacement
rate in the normal human skin and in some papulosquamous diseases of the skin. Acta Dermatovener 53:251, 1973
22. Balharry GJE, Nicholas DJD: A new assay for ATP-sulphurylase using the luciferin-luciferase method. Anal Biochem
40:1, 1971
Luminescent Enzyme Immunoassays
Analyte
Albumin
Alpha-fetoprotein
Antihuman serum
albumin
Bacterial cells
Chorionic
somatomammotropin
Cortisol
Cytomegalovirus
Dehydroepiandrosterone
and DHEA sulfate
Digoxin
Equine encephalitis
virus
Herpes virus
Methotrexate
Mumps virus
Pregnanediol
Staphylococcus
enterotoxin
Trinitrotoluene
References
113
92
103, 119
121
107-108
104-106
109-110
111
79
112
91-92
114-116
117
120
118
102
s y s t e m . C,'irrico et a1.123 h a v e d e s c r i b e d a n a s s a y f o r
N(2,4-dinitrophenyl)13-alanine that involves the use
of a 2,4-dinitrobenzene-ATP
c o n j u g a t e in h o m o g e n e o u s i m m u n o a s s a y . A d e n o s i n e t r i p h o s p h a t e is s u b sequently quantified using firefly luciferase.
T h e last f o r m o f l u m i n e s c e n c e i m m u n o a s s a y is
t h e L E M I T , in w h i c h a l u m i n e s c e n t r e a c t i o n is u s e d
to d e t e c t t h e c h a n g e in N A D H c o n c e n t r a t i o n in a
typical enzyme-multiplied immunoassay. This app r o a c h h a s b e e n u s e d b y S t a n l e y 124 in a n a s s a y f o r
phenytoin.
CONCLUSIONS
T h e s e n s i t i v i t y o f c h e m i l u m i n e s c e n t assays a n d
t h e p o t e n t i a l f o r c o u p l i n g t h e s e s y s t e m s to s p e c i f i c
i m m u n o l o g i c r e a c t i o n s p r o m i s e to p r o v i d e t h e clinical
laboratory with a novel analytic approach. A number
of commercial manufacturers are marketing highly
purified luciferases and luciferins, making the develo p m e n t o f m o r e a s s a y p r o c e d u r e s a n e a s i e r task.t33
High-quality luminometers are also available, although much luminescence work can be accomp l i s h e d b y a d a p t i n g p h o t o m e t e r s , 2~
fluorometers, 126-127 s t o p p e d - f l o w 128-129 a n d c o n t i n u o u s - f l o w
apparatuses,39,126,130 a n d l i q u i d s c i n t i l l a t i o n c o u n t e r s 25,32
I n t h e n e x t few y e a r s , c h e m i l u m i n e s c e n c e will
e m e r g e f r o m t h e r e s e a r c h l a b o r a t o r y a n d t a k e its
p l a c e in t h e clinical l a b o r a t o r y . F o r t h o s e r e a d e r s int e r e s t e d in a m o r e d e t a i l e d e x a m i n a t i o n o f this t e c h nique, a number of excellent reviews are available. 16,75,134-135
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1976
49. Allen RC: Lucigenin chemiluminescence: a new approach to
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In DeLuca M, McElroy WD (eds): Bioluminescence and
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