Analytical Luminescence: Its Potential in The Clinical Laboratory

-
CLIN. CHEM. 25/9, 1531-1546 (1979)
Analytical Luminescence: Its Potential in the Clinical Laboratory
Thomas P. Whitehead, Larry J. Kricka, Timothy J. N. Carter, and Gary H. G. Thorpe
The various types of chemiluminescent and bioluminescent to reduce the quantities of specimen and reagent required in
reactions are described. Applications of luminescence in the initial enzymatic step, thus reducing the cost of the assay.
the analysis of substances of clinical interest are surveyed. The second major application of luminescence is the utiliza-
The advantages, disadvantages, and prospects for lumi- tion of luminescent molecules as replacements for radioactive
nescent assays are discussed. labels in immunoassay.
An important feature of luminescence as an analytical
Additional Keyphrases: chemiluminescence biolumi- -
technique is that its usefulness is not confined to clinical
nescence analytical applications in the clinical labora-
-
chemistry. During the next few years we believe it will find an
tory luminescent immunoassays immobilized re-
-
increasing role in other pathology disciplines, e.g., hematology,
agents -luminescent conjugates luminometer (lumi-
#{149}
immunology, bacteriology, and pharmacology.
nescence measuring device)
Terminology and Definitions
Chemiluminescence may be simply defined as the chemical
introduction production of light. In the literature it is often confused with
fluorescence. The difference between these two processes is
The Place of Luminescence Assays in Clinical the source of the energy that is producing molecules in an
Chemistry excited state. In chemiluminescence this is the energy of a
The clinical chemist is involved in measuring a variety of chemical reaction, and the decay from the excited state back
substances by many different analytical techniques. Although to the ground state is accompanied by emission of light (lu-
different, these techniques share the common principle of an minescence). In contrast, incident radiation is the source of
interface between chemistry and physics. The most commonly the energy that, in fluorescence, promotes molecules to an
used such interface in clinical chemistry is absorptiometry, excited state. Analytically, the types of luminescence that have
both at visible and ultraviolet wavelengths, but emission flame engendered the most interest are chemiluminescence and
photometry and radioactivity are also commonly used. A novel bioluminescence. The latter is the name given to a special form
interface technique, so far relatively unexplored in clinical of chemiluminescence found in biological systems, in which
chemistry, is luminescence. a catalytic protein increases the efficiency of the luminescent
Analyses based on the measurement of emitted light offer reaction. Indeed, in certain cases the reaction is impossible
several advantages over conventional techniques: high sen- without a protein component.
sitivity, wide linear range, low cost per test, and relatively
simple and inexpensive equipment. Historical Aspects
Luminescence is expected to make an impact in two main Luminescence of various types has almost certainly been
areas of clinical analysis. Firstly, it may have an important role known to man since ancient times. For example, Aristotle
as a replacement for conventional colorimetric or spectro- (384-322 B.C.) described the BL1 of dead fish (bacteria) and
photometric indicator reactions in assays for substrates of fungi in his De Anima. After Robert Boyle’s discovery that
oxidases and dehydrogenases. In this type of assay the sensi- oxygen was intimately involved, little further progress was
tivity of the luminescence indicator reaction may be used ei- made in studies of BL until the nineteenth century with the
ther to quantitate substrates not easily measured by con- pioneering work of Dubois (1) on the BL of firefly and clam
ventional techniques (e.g., prostaglandins and vitamins) or extracts. In contrast to BL, the history of CL, especially that
in the aqueous phase, is remarkably short. The fortuitous
discovery of lophine (2) and pyrogallol (3) were the first ex-
Department of Clinical Chemistry, Wolfson Research Laboratories, amples in solution. The important CL substances, luminol and
Queen Elizabeth Medical Centre, Birmingham B15 2TH, U.K.
lucigenin, were discovered in 1928 (4) and 1935 (5), respec-
It has not escaped our notice that we printed a review on this topic
in a recent issue [Clin. (‘hem. 25,512-520 (1979)1; however, we believe tively.
this review article is complementary to the first, in a research area that
is receiving increasing attention, as evidenced by the other papers ofl
this subject in this same issue.-Ed. Nonstandard abbreviations used: BL, bioluminescent or biolu-
Received Mar. 7, 1979; accepted Apr. 4, 1979. minescence; CL, chemiluminescent or chemiluminescence.
CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1531

For a more complete discussion of luminescence, or indeed Types of Luminescence
of any facet of its history, the reader is referred to the re-
markable book by E. N. Harvey (6). Chemiluminescence
Advantages and Disadvantages of Luminescence The CL reactions described in this section all have the
and Luminescent Assays common feature of being hydrogen peroxide detectors. This
makes them of particular interest to the clinical chemist, be-
Sensitivity. Of the several advantages of luminescent
cause they offer an alternative to the colorimetric peroxide
methods over their conventional counterparts, their extreme
detection methods now used in many clinical assays. The
sensitivity is the most important. For example, as compared quantum efficiency of CL is often very low; e.g., for luminol
with spectrophotometry, the BL assay of NADH is estimated it ranges from 0.01 to 0.05 (13). However, the development of
to be some 25 000-fold more sensitive (7), and BL assays for sensitive photomultipliers has made it possible to follow very
glucose and alcohol are, respectively, 55- and 10-fold more weak luminescence, even when the quantum efficiency is as
sensitive than conventional assays (8). The minimal detect- low as 1015 (13).
able concentration for an assay ultimately depends on how Today many CL systems are known, but despite intensive
sensitively light can be detected, and on the quantum effi- studies the detailed mechanisms involved are often obscure.3
ciency2 () of the reaction. Generally BL reactions are much CL, albeit very weak, is stated to be a virtually universal
more efficient than CL reactions; typical quantum efficiencies property of oxidizable organic substances (14).
are in the ranges 0.1-0.8 and 0.01-0.05, respectively. Detection CL can occur in the gas, solid, or liquid phase; however, only
limits for the luminescent assay of ATP, NADH, and H202 liquid-phase CL, which appears to be the most useful for ap-
are reported to be 10b0 mol (7), 1016 mol (7), and i0 mol/L plication to clinical chemistry, will be considered here. The
(9-11). mechanism of organic CL in solution involves three stages
Range of linear response. Since CL is an emission process (19): (a) preliminary reactions to provide the key intermedi-
(as opposed to absorption), response is usually linearly pro- ate, (b) an excitation step in which the chemical energy of the
portional to concentration from the minimal detectable con- key intermediate is converted into electronic excitation en-
centration up to the point where it is no longer possible to ergy, and (c) emission of light from the excited product formed
maintain an excess of other reactants relative to the analyte. in the chemical reaction. In reactions in which a fluorescent
In the case of ATP assay by the firefly reaction, response is compound is added to enhance the CL emission, an efficient
linear over six orders of magnitude (12). energy transfer occurs and the resulting CL is known as
Speed of analysis. This largely depends on the type of lu- “sensitized CL.”
minescent reaction. In some instances a rapid flash is obtained Diacylhydrazides. The CL of a cyclic diacylhydrazide, lu-
(<1 s), the peak height of which may be related to analyte minol (5-amino-2,3-dihydro-1,4-phthalazinedione), was first
concentration; in other cases a more protracted glow occurs described by Albrecht in 1928 (4). Since then, many acylhy-
with a time course lasting several minutes. In the latter case drazides have been checked for their ability to luminesce, but
the integral or partial integral of the light-time curve has been strong CL is obtained only from cyclic diacylhydrazides of the
used as a measure of analyte concentration because it is much same type as luminol. A shift in the position of the amino
less sensitive to mixing efficiency, but this drastically reduces group reduces efficiency; e.g., isoluminol (6-amino-2,3-dihy-
the through-put and speed of analysis. drophthalazine-7,4-dione) is 10% as efficient as luminol (20).
Cost. The major cost benefit of luminescent assays arises Substitution in the ring structure markedly influences the
from their extreme sensitivity, which allows assays to be luminescence. Electron-withdrawing (electrophilic) substit-
performed with much less reagents, hence reducing the cost uents in the benzene ring decrease luminescence, but elec-
per test. For example, luminescence offers a means of reducing tron-donating (electrophobic) substitutents increase light
the cost of cholesterol assays involving cholesterol oxidase (EC yield, substitution at positions 5 and 8 being more effective
1.1.3.6), because the sensitivity of CL detection of peroxide than at 6 and 7. A complete loss of light occurs if the hetero-
allows the initial peroxide-producing reaction involving cyclic ring is substituted. Annelated analogs of luminol have
cholesterol oxidase to be scaled down. been produced that are 300% more efficient than luminol (15,
Specificity. Specificity is conferred on BL and CL by using 20) (see Figure 1). The efficiency, wavelength, and pH opti-
the luminescence as an indicator reaction coupled to inter- mum of light emission in luminol depend greatly on reaction
mediates (such as ATP, NADH, and H202) produced enzy- conditions. In general, hydrogen peroxide is the most com-
matically. Generally, BL reactions are specific because they monly used oxidant; catalysts include Fe(CN6) and Cu2
are enzymic processes, but CL reactions are nonspecific. Lu- (10). Other oxidants used include hypochlorite, iodine, per-
minol, for example, will undergo a CL reaction with various manganate, and oxygen in the presence of a suitable catalyst.
oxidants (oxygen, peroxide, superoxide, iodine) and its reac- Perhaps the most efficient catalyst in this reaction is heme,
tions are subject to interferences by reducing agents such as and the best medium would appear to be carbonate buffer
uric acid. (21). The optimum pH for CL varies somewhat with the cat-
Mechanisms. A disadvantage of both CL arid BL systems alyst and oxidant, but that for most oxidizing systems is near
is that in many instances the reaction mechanism is incom- pH 11. The luminescence of luminol follows the stoichiometry
pletely understood. Thus, it is particularly difficult to control in equation 1 (22), and it has been shown that the amino-
such reactions when they are used in analysis and to predict phthalate ion, which is formed in the singlet excited state, is
or explain analytical interferences. the light emitter in the reaction (23).
Toxicity and stability. CL and ilL reagents present no
known hazard to the environment. CL reagents (e.g., luminol) Equation
are stable and have been used as non-toxic, stable alternatives
Mo_
to radio and enzyme labels in immunoassay. BL reagents such 1uinoI + 5202 - 1L..._:::ico;
light+ (425 n)
as luciferase suffer from the same stability problems as many $5.
other enzymes. NH2
2 Quantum efficiency, = the number (or rate) of molecules lu- The reader is referred to several reviews (1.5-18) for further in-
minescing/the number (or rate) of molecules reacting. formation on studies of CL mechanisms.
1532 CLINICAL CHEMISTRY, Vol. 25. No. 9, 1979

NM.2 to interferences are claimed to be superior to other CL re-
0 agents.
5(4 Diaryl oxalates. Diaryl oxalates such as bis(trichloro-
11
phenyl) oxalate undergo a CL oxidation reaction with hy-
11 0
drogen peroxide (11, 31-33), via a peroxyoxalate intermediate
(equation 3). Efficient CL occurs in ester solvents, and the
Equation 3 cs
1_H. 11.11112 Loo1 An ..1. of .rn.1..d Loci.ni.
1,Ao.o1 Anriv.ti*
1NH2. 1.H I.o-1Lno1
TCFO + 5202 - ClOC0.C0.00H + Cs5l0H
(I) (II)
x_Hco g_W 5011105
0-0
(I) - c’.o + (II)
An accidinic. phenyl Lophin. 1_H pyrogallot

carboxylot.
(III) Fluorescer (F) - F 2C02
-o H. 1_2H o11ic odd
+ +
\ ,.Sjl
H_f’
s
I.o_ [ciI$oco_}. F’ - F + light
( o’” ? intensity is increased by organic bases and inhibited by or-

ganic acids (34). A problem with diaryl oxalates is their sus-
ceptibility to hydrolysis and the subsequent effect on light-
Silool.. TCPO emission characteristics. Bis(trichlorophenyl) oxalate is one
bi. (2,4 ,6-trich1o,oph.ny1)oo1ot of the more stable diaryl oxalates and no hydrolysis-related
Fig. 1. Structures of some chemiluminescent compounds problems have been encountered in its analytical applications.
It has been used, in a ethyl acetate/methanol/aqueous buffer
The question of how excited states are produced in the CL (pH range 4-10) system containing triethylamine, for the
of the cyclic hydrazides remains in doubt, but electron- analysis of peroxide. A fluorescent molecule, perylene, has
transfer mechanisms (21, 24), mechanisms involving the in- been used as the light emitter in this reaction (sensitized CL)
tercession of diazoquinones and dioxetanes (21), and (more because of its stability and its favorable efficiency and wave-
recently) chemically initiated electron-exchange luminescence length range of emission (11). The detection limit is 7 X 108
mechanisms have been proposed (25). mol/L and the linear response range extends up to 10 mol
Comparison of some different oxidation systems for de- of hydrogen peroxide per liter. Although the bis(tricloro-
tecting luminol reveals that sensitivity is greatest with hy- phenyl) oxalate system is not as sensitive as luminol (see
drogen peroxide and catalysts containing heme. above) for the detection of peroxide it does have advantages
An interesting older observation (26) is that plant perox- of a lower background CL and less sensitivity to interferences
idases and luminol in the presence of H202 will react to pro- such as uric acid.
duce light at near-neutral pH. The detection limit for luminol Other CL systems. Many other liquid-phase CL systems
with the H202-microperoxidase system is 1 pmolfL (20). other than luminol and lucigenin have been described. For
Acridinium salts. Lucigenin (bis-N-methylacridinium example, lophine is oxidized in basic conditions to give a yel-
low CL (2), and siloxine (35) is oxidized in acidic media to
nitrate) is similar to luminol in that it luminesces on oxidation
by peroxide in basic solution in the presence of metal ion produce a yellow-red CL, depending upon the oxidant and
catalysts, the quantum efficiency of the process being about degree of oxidation. Polyhydric phenols such as pyrogallol (37)
0.01 to 0.02 (equation 2). The reaction is catalyzed by some or gallic acid (37) also undergo luminescent oxidations. Many
metal ions, such as Pb2+, that do not catalyze the1uminol re- other CL reactions are known in solution but their usefulness
action and thus provides the basis for analytical applications in analysis has not been explored.
not possible with luminol. Considerably less research effort
Bioluminescence
has been expended on the mechanism of light production in
Numerous assays for substances of clinical interest are
lucigenin than in luminol. What work has been done (27,28)
is largely in relation to its enzymic reactions, especially with based on reactions involving cofactors such as NAD+/NADH
xanthine oxidase (EC 1.2.3.2). and ATP. The BL reactions described in the following sections
offer sensitive alternatives to the conventional spectropho-
tometric or colorimetric assays for such cofactors.
Although terrestrial luminescent organisms such as the
Equation 2
0 firefly and glow-worm are the best-known BL organisms, most
HO
M.N + light (470 n) of the other examples are sea-living organisms, ranging in
lucigenin + 5202
55+ complexity from microscopic bacteria and plankton to many
species of fish. So widespread a phenomenon is BL that two-
\/ thirds of the organisms in the upper 2000 m of oceanic waters
are BL (38). To date, fewer than 1% of the known luminous
biological species have been studied in great detail (39), but
McCapra et al. (29, 30) describe novel acridinium phenyl
sufficient data have been accumulated to allow the various
carboxylates, which undergo CL oxidation with hydrogen types of BL to be classified mechanistically (40), as follows:
peroxide. The optimum pH of oxidation of these substances
depends on the nature of the substituent in the phenyl group (a) Pyridine-nucleotide linked systems
(see 30), thus allowing an acridinium salt to be tailored to fit (b) Adenine-nucleotide linked systems
particular pH requirements. Their shelf life and susceptibility (c) Enzyme-substrate systems

Scheme 1. Mechanism Mg2 of firefly luminescence
Table 1. Classification of BL Reactions
(a) Pyridine-nucleotide linked LH2 + E + ATP .- E:LH2:AMP + PP
e.g., Vibrio fischeri, Beneckea l-sarveyi(marine bacteria)
E:LH2:AMP+02 #{149}
E+L=O+C02+AMP+light
reductase
NADH + FMN 5’ FMNH2 + NAD hi’ max., 562 nm
luciferase
BL - 1
FMNH2 + RCHO + 02 FMN + RCO2H + light
where:
(b) Adenine-nucleotide linked
e.g., Photinius (firefly) 502H
luciferase
Luciferin + ATP + 02 2+ ADP + CO2 + light
HOSS
(c) Enzyme-substrate systems LH2, luciferin

e.g., Pholas (clam)
lucif erase {O
Luciferin + 02 5’ light
(e) Activation of precharged” systems L = 0, oxyluciferin

e.g., Aequorea (a coelenterate) PP, pyrophosphate
Ca2+
Precharged protein -e blue-fluorescent protein + light of the most hydrophobic enzymes known (51), and this may
explain its poor solubility. The activity of firefly luciferase
preparations depends greatly on the mode of preparation.
Type d (peroxidation systems) have not as yet found application in clinical Procedures involving precipitation with (NH4)2S04 of fire-
chemistry. fly-lantern extracts give the best preparations, which may be
safely stored at -20 #{176}C (52-54). Enzyme activity has also been
(d) Peroxidation systems stabilized by bovine serum albumin, sucrose, ascorbic acid,
(e) “Precharged” systems or thioethanol (55, 56).
Table 1 gives examples of the most important reaction types Specificity. The reaction is not specific for ATP; other
in terms of applications in clinical chemistry. nucleotides such as cytidine-5’-triphosphate, inosine-5’-tri-
phosphate, and iso-ATP (57) can stimulate light emission.
Firefly. ADP, uridine triphosphate, and guanine triphosphate have
Firefly luminescence undoubtedly has been the most ex- also been shown by some workers to stimulate light emission,
tensively studied BL system. The light-producing reaction but these results are controversial (58-60). Manganese cations
requires the enzyme luciferase, luciferin, Mg2+, ATP, and may replace Mg2 in the reaction (61).
molecular oxygen.4 Numerous reviews deal with the reaction Inhibitors. Certain anions inhibit the reaction: SCN
(18, 40,42-45) and the mechanism now proposed is shown in <1<N03<Br<C1 (62). Anesthetics such as procaine and
Scheme 1 (41, 46-48). The initial reaction is the rapid con- lidocaine are also inhibitors, and this fact has formed the basis
version, in the presence of Mg2+ and ATP, of luciferin to lu- of assays (63).
ciferyl adenylate, which, in the presence of luciferase, com- Marine bacteria.
bines with molecular oxygen to give an oxyluciferyl adenyl- Most studies of luminescent marine bacteria have centered
ate-enzyme complex in the excited state. After emission, the on two types,5 Beneckea harveyi (Photobacterium fischeri
ground-state complex disassociates to form enzyme, AMP, strain MAV) and Vibrio fischeri. Characteristics of the re-
oxyluciferin, and carbon dioxide, the last being derived from action such as kinetics, absolute quantum yields, and emission
the carboxyl group of luciferin. The reaction is best carried spectra vary with the type of bacterium from which the lu-
out at 25#{176}C in glycine buffer pH 7.8 (46). The color of the light ciferase is obtained. The BL quantum yield per enzyme turned
emitted differs for different species of firefly, and this is over, or product formed, is in the range 0.1 to 0.2 (64). Gen-
thought to be due to inter-species differences in the structure erally the emission spectra are characterized by a broad
of luciferase, because the structure of luciferin is identical for emission, with a maximum near 478-505 nm. Recently, how-
all species (49). Intensity and wavelength of maximal emission ever, a yellow-emitting strain of P. fischeri has been described
are also altered by changes in pH, ionic strength, temperature, (65). In vitro, the components required for luminescence are
and the presence of chlorides of Zn2+ or Cd2+ (50). FMNH2 generated from FMN by the oxidation of NADH or
Luciferase. Firefly luciferase is a dimer, each sub-unit NADPH with the aid of FMN reductase, a long-chain ali-
having a relative tholecular mass of 50000. Only one of the
sub-units exhibits enzyme activity in the BL reaction. Studies
of its amino acid composition have shown luciferase to be one The nomenclature of the various luminescent marine bacteria has
not been fully settled (see ref. 64 for fuller details). Three general
groups have been recognized, based on mode of flagellation, guanine
Lucife,-ase--a generic term referring to an enzyme that catalyzes + cytosine content of the DNA, and DNA/DNA and DNA/RNA hy-
the oxidation of a substrate, such as luciferin, with light emission. bridization studies. Group A: Vibrio fischeri or Photobacterium [is-
Luciferin-a generic term referring to a reduced compound that can then or Achnomobacter fischeri. Group B: Photobactenium phos-
be oxidized in an appropriate environment to produce an electroni- phoreum and Photobacterium leiognathi. Group C: (a) Beneckea
cally excited singlet state; light is produced on its return to the ground harveyi or Lucibactenium harveyi or P. fisheri MAy, (b) Beneckea
state. - ,splendida, and (c) Vibrio cholerae.
1534 CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979

Scheme 2. The reaction sequence and intermediates postulated for the luciferase-mediated bioluminescent oxidation of NADH2 (including
nonenzymatic and dark pathways
E-FMNH
0H
(intermediate II) dar1c” reaction
RCHO
E-FMNH2 E + FMN + H202
(intermediate I)
E-FMNH
7citerase)
FMNH2 RCHO
/\ OOH
(Intermediate IIA)
NAD (02 (auto-oxidation) Ir
FMNreductase\ H0 RCOOH
NADH2
FMN E-FMN . HOH*
-* light
E-FMN . HOH
I
E + FMN + H20
(modified from 64 and 66)
phatic aldehyde, oxygen, and bacterial luciferase. The pro- Luciferase contains between 12 and 15 sulfhydryl groups,
posed sequence, intermediates, and alternative pathways are and luciferase from Beneckea harveyi has a reactive sulfhy-
presented in Scheme 2. The total light produced in the reac- dryl group on the a-subunit, at or near the active center (74).
tion is proportional to the amount of each of the substrates Treatment of bacterial luciferase with 8 mol/L urea or 5 mol/L
(02, FMNH2, and RCHO) when they are present in limiting guanidine hydrochloride leads to separation of the subunits
quantities. The same can be said of the luciferase because and rapid loss of activity. The subunits can be recombined and
excess FMNH2 is auto-oxidized so rapidly as compared with the activity recovered, but this recovery is absolutely depen-
the rate of light emission that FMNH2 has been reconverted dent on keeping the sulfhydryl groups reduced (75).
to FMN by the time the luciferase finishes its catalytic cycle. pH profile. The two luciferases show different pH profiles.
Thus, like the other reactants, the luciferase only acts once Vibrio fischeri luciferase shows a broad profile with nearly
in the in vitro reaction. It is, however, capable of turnover on optimal activity over the pH range 6.4-7.2. Beneckea harveyi
repeated addition of FMNH2. Although the role of the aIde- luciferase shows a quite different response, having its opti-
hyde was not understood for a long time, it is now established mum (measured by use of the initial maximal light intensity)
that it is oxidized to the corresponding carboxylic acid (64). in the region pH 5.6-6.8, declining sharply at higher values
Various induced mutants of Beneckea harueyi are known (64). (70). In both these cases the luciferase was assayed directly
These show altered BL properties, such as color of light by use of FMNH2. The pH profile obtained by an indirect
emission, temperature sensitivity, and luciferase turnover rate. assay involving the use of NADH (FMNH2 being provided via
Recently, a dim mutant requiring myristic acid (67, 68) and FMN reductase) exhibits a sharp optimum at pH 6.8 that
a nonluminous mutant that only produces light in the pres- reflects the response of the two enzymes (70).
ence of cAMP have been described (69), but their analytical Stability. Thermal-stability studies in which the enzyme
potential has not yet been fully explored. is incubated at various temperatures for 5 mm and the residual
Luciferase. Although luciferases from different strains have enzymic activity determined have shown that luciferase ac-
similar requirements and appear to involve similar reaction tivity begins to lessen between 25 and 35 #{176}C (76), presumably
pathways, the occurrence of two distinctly different luciferases because of heat-denaturation of the enzyme. At 40 #{176}C this
have been reported (70). The luciferases from Vibrio fischeri process takes just a few minutes. Heat-denatured luciferase
and Beneckea harveyi have been shown to be heteropolymers may be reactivated by dissolving it in 8 mol/L urea and di-
(Mr 79 000) composed of two non-identical subunits, and luting (75). Luciferase is inactivated by proteases (77), but this
contain no metals or other cofactors (71). The a and i9 sub- is reduced dramatically on binding of FMN, phosphate, or
units of luciferase from these bacteria have relative molecular sulfate anions (78). Phosphate anions appear to have a sta-
masses of 41 000 and 38 000, and 42 000 and 37 000, respec- bilizing effect on luciferase.
tively. No active hybrids are formed between subunits of the Specificity. Luciferase activity is highly specific for
two luciferases, and tryptic peptides of the two luci- FMNH2, but the enzyme also shows weak activity towards
ferases also differ (72). The role of each subunit in the BL other flavins and flavin analogs (79, 80). Only aliphatic al-
reaction appears to be quite different and it has been shown dehydes with a chain length of eight or more carbon atoms are
that the active site is located specifically on the a-subunit. The effective in the luminescent reaction.
role of the fl-subunit still remains to be elucidated, although Inhibitors. Luciferase is particularly sensitive to thiol re-
it is essential for BL activity (73). agents such as p-chloromercuribenzoic acid and to reagents

that react with lysyl, cysteinyl, and histidinyl residues (64). the protein in the presence of Ca2+ to oxyluciferin, with pro-
Volatile anesthetics (82), riboflavin, and cyanide, and copper, duction of light. The spent photoprotein is termed “blue-
iron, and other heavy metals also inhibit the enzyme (81). fluorescent protein” (BFP) (87, 88).
FMN reductase. Specificity. Metal ions other than Ca2 trigger the BL of
Flavin mononucleotide reductase (flavin reductase, the Aequorea system, notably Sr2+, Ba2+, and all of the lan-
NAD(P)H: dehydrogenase, or NAD(P)H-FMN oxidoreduc- thanides (88).
tase) appears to associate in vitro with bacterial luciferase (83). Other BL systems. Many other BL organisms are known-
The reductase is also postulated to supply reduced FMN in e.g., fungi (Collybia velutipes, Armillaria mellea), sea pansy
vivo as a substrate for the BL reaction. The enzyme is present (Renilla reniformis), marine worm (Chaetopterus), earth-
not only in luminescent bacteria but also in aerobic and an- worm (Otochaetus, Diplocardia), protozoa (Gonyaulax
aerobic nonluminescent bacteria such as Escherichia coli polyedra), fish (Apogon, Photoblepheron), shrimp (Heter-
(84). ocarpus), beetle (Pyrophorus)-but generally their re-
There is still some controversy over the types of FMN requirements for BL and mechanisms are poorly characterized
ductase present in the cell. Separation of two reductases, a free (12). We anticipate that analytically useful BL reactions may
form and one associated with luciferase, has been reported. be discovered among such organisms.
Other workers maintain, however, that only a single free re-
ductase exists (83).
Applications in Clinical Chemistry
Distinct FMN reduct.ases specific for NADH and NADPH The extreme sensitivity of luminescence assays and their
have been isolated from extracts of Beneckea harveyi by af- applicability to the important analytical intermediates ATP,
finity chromatography (85). The NADH- and NADPH-spe- NAD(H), and peroxide have led to the exploitation of both
cific FMN reductases have relative molecular masses of 30 000 CL and BL as analytical tools in clinical chemistry. The range
and 40 000, Km’s of 4.75 X i0 mol of NADH per liter and 4.0 of analytes quantitated by CL or BL assays is assembled in
x i0 mol of NADPH per liter, and specific activities of 31 Tables 2-7.
pmol of NADH and 51 tmol of NADPH oxidized per minute
Direct and Coupled Reactions
per milligram of protein, respectively. Vibrio fischeri appears
to have only one nonspecific reductase of Mr 45 000. Measurement of direct reactants. Perhaps the simplest
pH profile. FMN reductases isolated from Vibrio fischeri assays in which luminescent techniques are used involve
and Beneckea harveyi both show appreciable activity in measurements of substances that take part directly in various
NADH oxidation over a realtively broad pH range, from about luminescent reactions. Measurement of ATP with firefly ex-
pH 5 to pH 10. With the Beneckea harueyi enzyme, a bimodal tracts; of NADH, FMN, aldehydes, or oxygen with extracts
relationship is evident; with that of Vibrio fischeri the second from luminescent bacteria; and of hydrogen peroxide with
peak appears as a shoulder (pH optimum 6-6.4) (76, 83). compounds such as luminol are important examples. The only
Stability. One of the problems encountered in the purifi- requirements for the measurement of direct reactants are
cation of FMN reductase is loss of activity. But this has been appropriate reaction conditions and that the concentration
shown to depend on dithiothreitol, and at a dithiothreitol of the substrate to be measured is rate-limiting in the lumi-
concentration of 0.1 mmol/L, activity is slowly but reversibly nescent reaction.
lost. Maximal activity can be assured by incubation (24 h, 0 Coupled reactions: assays with luminescence as an indi-
#{176}C)
with higher concentrations of dithiothreitol (1 mmol/L) cator for other enzymatic reactions. With this system the
(85, 86). The oxidoreductase may be briefly frozen and then range of luminescent analytical applications is considerably
thawed with no loss of activity, but long freezing adversely extended. It relies on the fact that many substrates or enzymes
affects enzyme stability, loss of activity occurring several days can be coupled to measurable species in luminescent reactions.
after thawing. A 50% recovery of activity after rapid thawing Appropriate coupling enzymes and reaction conditions that
and dialysis may be obtained after storage in liquid nitrogen. make the substance to be measured rate-limiting are the only
FMN reductase is stable to lyophilization (86). requirements. Thus any reaction that produces or utilizes
Purified FMN reductases have only recently become ATP, NAD(H), NADP(H), FMN(H2), or H2O2 could be
available and the following data have been obtained with coupled to a luminescent reaction.
relatively impure reductase preparations. The maximal rate Continuous monitoring assays. The complexity of ana-
of NADH oxidation (followed by changes in absorption at 340 lytical procedures based on BL has encouraged several groups
nm) is obtained at 30 #{176}C, followed by a rapid decrease in en- to study methods of simplifying such procedures (89, 91, 116).
zyme activity (84). Studies on the initial rate of NADH oxi- Reaction conditions have been defined for ATP concentra-
dation at different temperatures and FMN concentrations tions of less than 10 mol/L, in which firefly BL is neither
have indicated that the reductase in the absence of FMN is significantly affected by consumption of ATP nor is it affected
denatured above 20 to 25 CC. With added FMN the reductase by inactivation or product inhibition of luciferase (89). A
retains its catalytic activity at higher temperatures, but in a constant ATP concentration leads to constant light output
modified form (83). The NADH-and NADPH-specific re- and ATP-converting reactions may be monitored simply by
ductases differ in their thermostability; for example, after 5 measuring light intensity. The continuous monitoring of ATP
mm of incubation at 55 #{176}Cthere is still 40% residual NADH- concentration by firefly luciferase may thus be used for the
spec ific activity, whereas the NADPH-specific reductase ac- kinetic determination of ATP-producing and -consuming
tivity is completely abolished (76). Both enzymes appear more enzymes and their substrates, and for end-point analysis of
heat stable than luciferase. substrates. This procedure is ideal for routine applications
Specificity. Riboflavin, FAD, and isomers and analogs of where ultimate sensitivity is not required (92, 93) and suitable
FMN may act as substrates for FMN reductase (80). reagents are now available (94). A similar continuous-moni-
Aequorea. toring approach has been applied to simplify the assay of
The BL system of the jellyfish Aequorea consists of pro- NAD(P)H-producing and -consuming reactions by the bac-
tein-chromophore complexes (termed “photoproteins”), terial luminescence system (95-97).
which react with Ca2+ to produce a bluish luminescence (Xmsx Met hocLs based on ATP. Reactions involving the production
469 nm) independent of dissolved oxygen. The photoprotein or utilization of ATP have extended the use of the firefly
consists of a species-specific protein in close association with system to the analysis of numerous substances of clinical in-
a chromophore component (luciferin), which is oxidized by terest (Table 2). Creatine kinase (EC 2.7.3.2) in serum can be

Scheme 3. Luminescent measurement of creatine kinase (CK)
Table 2. Assays Based on Firefly BL (ATP subunit B activity

Measurement)
Analyte Ref. 1. CK(MM,MB,BB) + AbM subunit -* CK(MB,BB) +
Substrates
CKMM:AbM + CKMB:AbM
ADP 54, 103, 110
Adenosine phosphosulfate 116
2. ADP + creatine phosphate + CK(MB,BB) -. ATP +
AMP 54
cAMP 55 creatine
ATP 89, 91, 103, 104, lucifera,e
105, 110 3. ATP + luciferin + 02 AMP + PP + oxyluciferin
Adenosine tetraphosphate 106
+ CO2 + light
Coenzyme A 109
Creatine 54
demonstrate decreased ATP concentrations in erythrocytes
Creatine phosphate 54, 105 from patients with sickle cell anemia, alcoholic cirrhosis, viral
Cytidine triphosphate 54 hepatitis, or chronic renal disease (44).
Glucose 54 Methods based on NAD(P)H. It has been appreciated for
Glycerol 107 some time that, as a result of the involvement of NAD(P)H
Guanosine triphosphate 54 (via FMN reductase) in the bacterial BL system, it should be
Phosphoenoyruvic acid 54 possible to measure NAD(H)-, NADP(H)-, NAD(P)-depen-
dent enzymes and their substrates by using BL assays. The
Pyrophosphate 117
use of the bacterial BL system opens the way to a wide range
Triglycerides 107 of analytical applications at the micro level, because a great
Uridine triphosphate 54 variety of biochemical reactions can be coupled to the con-
Enzymes version of pyridine nucleotides, thus yielding a measurable
Adenosine 3’,5’-monophosphate 55 species. Table 3 lists clinical analytes determined by methods
phosphodiesterase based on NAD(P)H. In addition to NADH the 3-acetylpyri-
Apyrase 54 dine analog of NADH (AP-NADH) can be measured directly
ATPase 54 with the bacterial BL system. The high redox potential of the
ATP sulfurylase 116 couple NAD/NADH has tended to limit the application of
dehydrogenases in coupled assays, as equilibrium does not
Creatine kinase 54, 92, 98, 99, 100
favor NADH formation. The way to obviate this difficulty is
Creatine kinase (B subunit) 92 to use 3acetylNAD+ or an auxiliary enzyme to alter the
Guanosine 3’,5’-monophosphate 108 equilibrium in favor of reduced-nucleotide formation (111).
phosphodiesterase A further analytical possibility for the bacterial BL system
Hexokinase 54 is the measurement of ATP by the reactions shown in Scheme
Myokinase 54 5. This potentially useful series of reactions would in theory
Nucleotide phosphokinases 54 enable assays now quantitated by use of firefly BL to instead
be performed with the bacterial system.
Pyruvate kinase 54
An exciting possibility for the utilization of BL is in the
determination of weak enzyme activities in small tissue
samples, biopsy materials, tissue cultures, and cell suspensions
determined in 10 iL of sample and, because the BL method (101). The bacterial system has been used to study isocitrate
measures one of the reactants directly, it does not require up dehydrogenase (EC 1.1.1.42) in normal human skin, some
to three coupled indicator reactions as is the case in some maculosquamous disease of the skin, and in skin biopsies
colorimetric and fluorometric assays for this enzyme. This during treatment with Dithranol (112). D-3-Hydroxybutyrate
enzyme has been determined in a drop of dried blood from a dehydrogenase (EC 1.1.1.30) has also been determined in
heel-prick by BL and the method has been proposed as a mouse pancreatic islets (112). It is envisaged that the tech-
screening procedure in newborns for possible Duchenne nique may eventually be used in testing for genetic enzyme
muscular dystrophy (98, 99). deletions in cells obtained by amniocentesis.
A method for the determination of creatine kinase isoen- Methods based on H202. It is well known that oxidases or
zymes, based on differences in their Km, has been devised by their substrates may be assayed by measuring the peroxide
Witeveen eta!. (100); however, Lundin and Styrelius (92) have formed during the reaction of the substrate with the oxidase.
described a novel assay for total creatine kinase and its B The peroxide is usually quantitated by using a chromogenic
subunit activity, by using specific immunoinhibition of en- reagent such as 4-aminophenazone (126). CL offers a highly
zyme activity and a BL ATP assay. B subunit activity was
obtained by use of an antibody inhibiting M subunit, which
Scheme 4. Luminescent measurement of c-AMP
gave a 99.5 and 50% inhibition of the MM and MB isoenzymes,
phosphodiesterase
respectively (Scheme 3). Adenosine 3’,5’-cyclic monophos- c-AMP AMP
phate (cAMP) has also been measured luminescently (Scheme
4). The sensitivity (picomoles), specificity, and linear range myokinase
(three orders of magnitude) of the cAMP assay cannot be AMP+ATP. ‘2ADP
matched by other techniques, and the application of the
method for cAMP measurements in urine has been demon-
2 ADP + 2-phosphoenolpyruvate ± 2 pyruvate + 2 ATP
strated (101). The sensitivity suggests its wider application
to small tissue samples, biopsy materials, tissue cultures, and Iuciferase/Iuciferin/02
cell suspensions. BL assays have already been utilized to ATP light

Table 3. Assays Based on Bacterial BL INAD(P)HI Measurement
Sensitivity
Analyla or range Ref.
Substrates
Acetylpyridine-NADH 0.1-1.4 pmol 113
Aldehydes 54
Ammonia 114
Ethyl alcohol 0.0004-0.015% 8, 115
FAD 132
Fatty acids 68
FMN 7, 54, 104, 132
FMNH2 7, 54
Glucose 150-1500 pmol 8, 113
Glucose 6-phosphate 112
Glucose 1-phosphate 112
L-GIyCeroI 1-phosphate 96
Glycogen 112
3-Hydroxybutyrate 96
Malate 96, 113
NAD 7,54, 104, 113, 118
NADH 1014_107 mol 104, 113
NADP 104, 119
NADPH 10 pmol-200 nmol 104, 119, 120
Oxaloacetate 7, 117
Oxygen 54
Pyruvate 112
Enzymes
Alcohol dehydrogenase 0.0 15-3 pmol 8
ATP:NMN adenylyltransferase 118
Glucose-6-phosphate dehydrogenase 0.0015-0.1 pmol 8
Hexokinase 0.1-2.0 pmol 8
D-3-Hydroxybutyrate dehydrogenase 112
Isocitrate dehydrogenase 121
Lactate dehydrogenase 0.003-0.7 pmol 8
Malate dehydrogenase 0.007-0.7 pmol 8
Proteolytic enzymes 77
Trypsin 20 ng 77
sensitive alternative to the colorimetric determination of method for vitamin B12, but this has only been successfully
peroxide. For example, the enzymic generation of peroxide applied to pharmaceutical preparations (124). The use of CL
from glucose by glucose oxidase (EC 1.1.3.4) has been coupled in specific binding reactions is discussed below.
to the CL of luminol (9, 122) and the method has been auto- The major disadvantage of the determination of hydrogen
mated by using immobilized enzymes (130). Table 4 lists other peroxide produced by oxidase enzymes with use of luminol is
oxidases and substrates of oxidases determined by this that a high pH is required for light production. A two-stage
method. reaction with a pH change before luminol addition is therefore
A CL technique is also available to determine the reduced required. An alternative analytical manoeuvre, first reported
form of NADH (33), in which NADH reduces oxygen to hy- by Harvey (26) and subsequently by Cormier and Pritchard
drogen peroxide in the presence of methylene blue. The hy- (125) as a model system for the investigation of the BL system
drogen peroxide is then determined by measuring the CL in Balanoglossus, is the use of a peroxidase-like enzyme to
produced from its reaction with excess bis(2,4,6-trichloro- intercede between the H202-producing oxidase and the lu-
phenyl) oxalate in the presence of perylene. This method has minol indicator reaction. This is analogous to the intercession
been used to measure lactate dehydrogenase (EC 1.1.1.27) and of peroxidase in the glucose oxidase method for glucose
is potentially applicable to other assay systems involving
NAD(H).
Certain metal ions catalyze the CL oxidation of luminol and Scheme 5. Measurement of ATP by use of bacterial lumines-
lucigenin (12, 123) under alkaline conditions, and this has cence
formed the basis of CL analyses for trace metals. An important
limitation is that several metal ions may enhance the same CL ATP + NMN-*NAD + pp
reaction, so that separation or masking is required when such
assays are done. The technique has, however, been applied
NAD + malate oxaloacetate + NADH
successfully to the measurement of some metal ions, e.g.,
chromium in biological samples. The catalytic effect of cobalt
on the luminol reaction has been utilized as a sensitive assay NADH + bacterial luciferase system -k light
1538 CLINICALCHEMISTRY,Vol. 25, No. 9, 1979

Table 4. Methods Based on CL (H202 Detection) Scheme 6. Luminescent immunoassays
LIA
Sensitivity
Analyte or range Ref.
Agstaad or unkn,,wn + Ag-L + Ab =± Ab:Ag + Ab:Ag-L
B12 2 X iO mol/L 124
Catalase 104tg 129
Chromium(Ill) - 123, 128 Ag-Lh0d or free + luminogenic co-reactants -* light
Cytochrome C 101kg 129
Ferritin 129 LEJA
Glucose 2X 10-8 mol/L 9, 11, 122, 130,
131 Ag + Ag-E + Ab ± Ab:Ag + Ab:Ag-E
Hematin io tg 129
Hemoglobin io tg 129 Ag-Eh00d or free + luminogenic co-reactants -* light
Hydrogen peroxide iO mol/L 10
Hypoxanthine 133, 134 LCIA
Lactate 33
dehydrogenase Ag + Ag-CF + Ab ± Ab:Ag + Ab:Ag-CF
Myoglobin 104tg 129
NADH 2 X 1O-1O mol/L33 or free + luminogenic co-reactants - light
Uric acid iO-1O mol/L 135
LEMIT
analysis now commonly used in clinical laboratories (126). In Drug-E + Drug + Ab z± Ab:Drug + Ab:Drug-E
this way, the pH optimum of the light-producing step may be
decreased to a value nearer that of biological systems. The Drug-E
obvious advantages for the quantitation of hydrogen peroxide,
and hence oxido-reductases, seems to have been overlooked Substrate + NAD NADH + product
until recently (127), although this procedure has been used NADH + luminogenic co-reactants -* light
for the sensitive quantitation of enzyme immunoassays (see
below).
Methods based on precharged systems. The specificity of Legend
Aequorea BL for calcium ions provides the basis for a quan- LIA = luminescent immunoassay; LEIA = luminescent en-
titive micromethod that has a detection limit of 100 nmol of zyme immunoassay; LCIA = luminescent cofactor immu-
Ca2+ per liter. The method has been used to study ionized noassay; LEMIT = luminescent enzyme-multiplied immu-
calcium in stimulated muscle fibres, mitochondria, and nerves noassay technique; L = chemi-or bioluminescent substance;
(87, 88, 136-1 38), but when applied to the measurement of E = enzyme; CF = cofactor; luminogenic co-reactants = e.g.,
ionized calcium in serum (139) it appears to overestimate the H2O2 for an LIA where L = luminol; luminol/I-1202 for an
true concentration (140). LEIA where E = peroxidase; firefly luciferase/Mg2 for an
LCIA where CF = ATP; bacterial luciferase/FMN reduc-
Luminescent Immunoassays tase/FMN for an LEMIT where E = a dehydrogenase.
Luminescent substances have been used in immunoassays
and specific protein binding assays (a) directly as labels and assays for enzymes, such as peroxidase (EC 1.11.1.7) are
indirectly for the luminescent quantitation of (b) enzyme and considerably more sensitive than are conventional colorimetric
(c) cofactor-labeled ligands (Scheme 6). Four types of lumi- assays, a factor that has been exploited in the quantitation of
nescent immunoassay have been developed: enzyme conjugates in enzyme immunoassay (Table 6). A lu-
(a) Luminescent immunoassays-immunoassays in which minescent enzyme immunoassay for cortisol involving lumi-
are used luminescent labels such as luminol, isoluminol, lu- nescent quantitation of a peroxidase-cortisol conjugate (145)
ciferase, or the luminol derivative (141) cited as a universal has a sensitivity comparable with that of the radioimmuno-
labeling agent for antigens have been reported by several assay.
groups of workers and are the subject of some patents. Interest (c) Luminescent cof act or immunoassay. Several workers
in such labels is mainly due to their possible role as replace- have recently investigated potentially very useful enzymic
ments for radioactive labels. Compared with radioimmuno- methods for monitoring specific binding reactions, using Ii-
assay, luminescent immunoassay (LIA) has many of the ad- gand-cofactor conjugates quantitated by use of a BL reaction
vantages associated with enzyme immunoassay: the reagents (146-148) (Table 7). The ligand has, for example, been cova-
are cheap and stable, assays are rapid and can be automated, lently coupled to an enzymically active derivative of NAD.
a separation step may not be required, radiation hazards are After reduction with alcohol dehydrogenase (EC 1.1.1.1) and
obviated, and, in addition, these methods may offer the added ethanol, these conjugates can be measured quantitatively by
advantage of very sensitive detection of the label (142). means of light production by Jsing the BL bacterial luciferase
Heterogeneous and homogeneous luminescent immu- system. The light production by ligand-NADH can be in-
noassays have been described (see Table 5). The luminescent hibited by a protein that specifically binds to the ligand; thus
activity of a luminol-IgG conjugate reportedly is unaffected specific binding reactions can be assayed without separating
when bound to an antibody (143). In contrast, the luminescent free and bound labeled ligands. As ATP can be measured in
activity of an isoluminol-biotin conjugate increases 10-fold very low concentrations with firefly luciferase, the possibility
when bound to avidin, a binding protein specific for biotin. of labeling ligands with this cofactor has also been investigated
This enhancement in light output has been ascribed to in- (146).
creased CL efficiency mediated by the protein (144). (d) Luminescent enzyme-multiplied immunoassay
(b) Luminescent enzyme immunoassay-luminescent technique. It has been suggested (149) that considerable cost
CLINICAL CHEMISTRY. Vol. 25, No. 9, 1979 1539

Table 5. Luminescent Immunoassays a
Labeled Type Detection Sensitivity
substance Label of assay Analyte system (range) Ref.
T4-isoluminol isoluminol CBA/He T4 H202/MP 20-150 g/L 150
lgG luminol CBA/He lgG H202/hemin 5-100 sg/tube 143
Insulin luminol CBA/He insulin 5-25 ng/mL 151
Digoxin luminol CBA/He digoxin 5-25 ng/mL 151
Biotin isoluminol CBA/Ho biotin superoxide or 50 nmol/L 144
H202/LP
Anti-lgG bacterial Iuciferase immunocytochemical cell surface lgG FMNH2/Iaurylaldehyde 34
Testosterone-ovalbumin luminol derivative CBA/He testosterone H202/cupric acetate 0.1-10 ng 141
Abbreviations CBA. competitive binding assay; He, heterogeneous; Ho. homogeneous; MP, microperoxidase; LP, lactoperoxidase; T4, thyroxine.
Table 6. Luminescent Enzyme lmmunoassays a
Labeled
Type of Detection
substance Label assay Analyte system Sensitivity Ref.
Cortisol peroxidase Double Ab and/ or insolubilized cortisol Iuminol/ 0.02-1 ng 145
Ab techniques/He H202
IgG (anti-SEB) peroxidase CBA/He staphylococcal pyrogallol/ 1 ng 152
fraction enterotoxin B H202
Anti-lgG peroxidase/ immunocytochemical cell surface IgG POL/ 34
glucose glucose! 34
oxidase Iuminol/
peroxidase
peroxidase CBA/He cortisol; insulin 10 pg; 1.2 mU/L 153
Goat anti- peroxidase solid phase anti-HSA; HSA luminol/ 7 X iO diln. of 212
rabbit lgG H202 antiserum; 10 ng
lgG antibody to peroxidase Sindbis virus pyrog 1101! 5 X i0 PFU/mL 213
the virus H202
a SEB, staphylococcal enterotoxinB; P01, Pholas dactylus luciferin; HSA, human serum albumin.
Table 7. Luminescent Cotactor-Immunoassays

Labeled Type Detection
substance Label of assay Analyte system Sensitivity Ref.
Biotin NAD CBA/Ho biotin EtOH/alc. - 148
derivative dehydrogenase
2,4DNFBa NAD CBA/Ho 2,4-DNFB bacterial - 148
derivative luciferase
2,4-DNFB ATP CBA/Ho M2,4-DNP)- luciferin/firefly luciferase 0.5 nmol/L 146
derivatives f3-alanine
a DNFB. dinitr otluorobenzene; EtOH , ethanol; other nonstd. abbrevs in footnote to Table 5.
benefit may accrue from the monitoring of EMIT6 by using the zotization reaction, although the enzyme is largely inactivated
NADH-dependent bacterial luminescence system, because during the coupling procedure (8, 120). The properties of
in many cases the enzyme label used is an oxidoreductase. immobilized luciferase with regards to pH-activity profile and
Most of the reports of luminescent immunoassays are pre- optimal substrate concentration are very similar to those of
liminary in nature, and as yet their sensitivity as compared the native enzyme. Moreover, the immobilized luciferase is
with radioimmunoassays has not been fully evaluated. reported to be stable indefinitely when stored at 0 #{176}C (8).
Jablonski and DeLuca (120) used luciferase/FMN reductase,
Immobilized Luminescent Reagents immobilized on glass beads cemented onto a glass rod, in
Immobilized enzymes have assumed an important role as analyses for NADH and NADPH. A single rod was used for
reagents in clinical analysis (154). The application of immo- over 100 consecutive analyses of NADH without apparent loss
bilized bacterial and firefly luciferases in the analysis of co- of activity.
factors, enzymes, and substrates has been explored, and pre- Firefly luciferase has also been immobilized chemically onto
liminary results have been encouraging (Table 8). Bacterial glass beads, but the bound enzyme retained only 0.07-0.16%
luciferase has been linked chemically to glass by using a dia- of its original activity. The pH optimum of the immobilized
enzyme is lower than that’ of the native enzyme and it emits
6 EMIT” is a registered trade name of the Syva Corp., 3181 Porter light with a major peak at 615 nm, as compared with 562 nm
Drive, Palo Alto, CA 94304. for the native enzyme. An assay for ATP in which immobilized

Table 8. Assays with Immobilized Luminescent Reagents
Sensitivity or range
Enzyme (source) Solid support Mode of attachment Analysis of optimal sensitivity Ref.
Luciterase/ porous glass diazo coupling NADH 1 pmol-50 nmol 120, 156
FMN reductase NADPH 10 pmol-200 nmol
(Beneckea NAD
harvey,) NADP
FMN
MDH 0.007-0.70 pmol
LDH 0.003-0.70 pmol
ADH 0.0 15-3.0 pmol
G-6-PDH 0.0015-0.10 pmol
Hexokinase 0.1-2.0 pmol
Glucose 150-15000 pmol
Ethanol 4-150 mg/L
Luciferase polyacrylic diazo coupling FMNH2 157
(Photobacterium hydrazide
Fischeri or P.
!eiognathi)
Luciferase aminoalkylated glutaraldehyde ATP 1 X 108 to 1 X iO 155
(firefly) glass beads mol!L
firefly luciferase is used has been developed and the peak light by oxidation of carbohydrate moieties with periodate and the
intensity shown to be linearly related to concentration in the activated IgG was allowed to react with luminol (143, 151).
range 1 X 108 to 1 X 10 mol/L (155). Also of note is the
immobilized analytical system of Freeman and Seitz (127), Applications in Other Pathology Disciplines
described below in greater detail. The analytical applications of BL and CL are not confined
to clinical chemistry.
Preparation of Luminescent Conjugates The need for a rapid, accurate means of counting microbial
Various chemical methods have been used to attach lumi- populations has led to the introduction of BL techniques in
nescent molecules such as luminol and luciferase to proteins microbiology. Although in the most widely applied methods
or solid supports. Bacterial luciferase and FMN reductase the determination of ATP by use of firefly luminescence is
have been coupled chemically to a solid support by using a used (160), it has also been suggested that an alternative ap-
diazo reaction (120, 157) and to anti-IgG by reaction with proach could be the determination of FMN with bacterial BL
glutaraldehyde (34). Similarly, firefly luciferase has been at- (32). Bacteria may also be detected luminescently by heme
tached chemically to a solid support by means of glutaral- protein catalysis of luminol oxidation, but the limit of sensi-
dehyde (155). Both enzymes appear to be very sensitive to tivity of this method is only 10” to i0 viable bacteria per
chemical coupling procedures, especially those involving ly- milliliter (161, 162). BL assays have been used to rapidly
sine, cysteine, and histidine residues, and considerable loss quantitate rapidly bacteriuria (163-167), and automated
of enzyme activity has been encountered (64, 158). techniques are available that require no pre-processing of
Labeling procedures with use of luminol and isoluminol are specimens. This method gives better sensitivity than do
well documented. The luminescent properties of these mol- conventional techniques. A specific advantage of the BL
ecules are not greatly impaired by reaction at the aromatic technique is that it enables a rapid screening of urine samples,
amino group. Indeed, N-alkylation of isoluminol increases so that only positive samples need be examined further for
light yield (20). Luminol has been coupled to insulin by means bacteria! identification and antibiotic-sensitivity testing.
of the bifunctional coupling reagent glutaraldehyde and it has Antibiotic sensitivity can be quickly determined with the
been claimed that other bifuncional coupling reagents (e.g., firefly luciferase system (101, 168-172) by inoculating growth
Woodward’s Reagent K, carbodiimides, cyanogen bromide, media containing various antibiotics with equal aliquots of
and cyanuric chloride) are also effective (151). Alternatively, urine, and incubating at 37 #{176}C.The change in ATP concen-
luminol may be activated by converting the aromatic amino tration with time, relative to a control, is then measured.
group into an isothiocyanate group. Sensitivity patterns can be defined in 30 to 120 mm, de-
More complex procedures have been used to prepare pending on the growth rate of the organisms, and such pat-
biotin-isoluminol (20, 144), glutathione-luminol (159), thy- terns yield fewer false-positive results than with the antibiotic
roxine-isoluminol (150), and thyroxine-aminonaphthylhy- disc-diffusion technique. BL ATP assays can quickly estimate
drazide conjugates (20). bacterial growth, and this technique seems feasible for assay
An important observation in these studies was that covalent of amino acids, carbohydrates, chemotherapeutic agents,
attachment of CL labels to thyroxine leads to a 20- to 50-fold hormones, vitamins, and other nutritional factors by moni-
reduction in their light yields. Increasing the size of the spacer toring the growth of microorganisms that require these com-
group between the label and the thyroxine molecule does not pounds. This approach appears superior to previous bioassay
improve the light yield. The reduction in light yields has been techniques based on turbidimetry, because more nearly ac-
attributed to the presence of iodine atoms, which are known curate measurements are obtained more quickly. A BL ATP
to be efficient fluorescence quenchers (20). The reactive na- assay is available for gentamicin, and an accurate result for
ture of the aromatic amino group of luminol towards al- only 150 tL of serum is available within 4 h (173, 174). The
dehydes forms the basis of a procedure for coupling this technique is potentially applicable to any group of antimi-
molecule to IgG. Free aldehyde groups were generated on IgG crobial agents, and with appropriate modifications it should

also be suitable for rapidly measuring vitamins such as B12 and reagents such as luminol and lucigenin are widely available,
folate in serum. Metabolites leak from cells when they are but often of doubtful purity. Commercial preparations of
shocked by temperature, starvation, or chemicals, or when marine bacterial luciferase/FMN reductase are not readily
they are incubated with lytic enzymes. Intracellular ATP available in large amounts ad often exhibit considerable
disappears particularly rapidly after cells are subjected to any batch-to-batch variation in their activity. A particular
one or a combination of these treatments (175). Mammalian drawback is that the preparations often contain very low ac-
cells frequently lose intracellular ATP with aging, and ATP tivities of FMN reductase. A major problem with firefly lu-
measurements with ilL techniques have been used to measure ciferase preparations is the presence of adenylate kinase (EC
the viability of blood (32, 176-181), of spermatozoa after 2.7.4.3), transphosphorylases, and ATP. Only in the case of
storage (182), and of vaccine (183). ATP measurements have firefly luciferase is a reagent of acceptable quality available
also been utilized to study the thrombin-induced release of (94). In our opinion, a major factor in the success of the future
ATP by platelets (184), and the shape and lysis of erythrocytes exploitation of luminescence in clinical chemistry will be the
(185, 186). The phagocytic uptake of particles or of bacteria quality of available CL and BL reagents.
activates the metabolism of polymorphonuclear leukocytes
and results in the production of CL (187-200). Although the
References
mechanism is not yet precisely defined, the CL is believed to 1. Dubois, R., Note sur Ia physiologie des pyrophores. C. R. Soc. Biol.
result from the relaxation to the ground state of singlet oxygen (Ser. 8) 2,559 (1885).
(‘02) or excited carbonyl groups produced by ‘02-mediated 2. Radzizewski, B., Untersuchung uber Hydrobenzamid, Amarin und
Lophin. Chem. Ber. 10,70 (1877).
oxidations. The CL produced can be quantitated for the
measurement of metabolic activation after phagocytosis, and 3. Eder. J. M., Phosphorescenzerscheinungen beim Hervorufen von
Gelatineplatten. Photogr. Mitth. 24, 74 (1887).
indirectly to assess nonspecific opsonic activity in human
4. Albrecht, H. 0., Uber die Chemiluminescenz des Aminophthal-
serum. It has also proved possible to detect specific opsonins saurehydrazids. Z. Phys. Chem. 136, 321 (1928).
and classical complement pathway activation. Such CL 5. Gleu, K., and Petsch, W., Die Chemiluminescenz der Dimethyl-
techniques may prove to be a valuable tool in the evaluation diaridyliumsalze. Angew. Chem. 48,57 (1935).
of factors important in the host-defense mechanism. 6. Harvey, E. N., A History of Luminescence from the Earliest Times
until 1900. The American Philosophical Society, Philadelphia, PA
Instrumentation 1957.
Until recently, most luminescence measurements have been 7. Stanley, P. E., Analytical bioluminescent assays using the liquid
made by using either “home-designed” apparatus (89, 103, scintillation spectrometer: A review. In Liquid Scintillation Counting.
113, 148, 201) or commercial equipment modified to meet the M. A. Crook and P. Johnson, Eds., Heyden, London, 1974, pp 253-
271.
peculiar requirements of luminescence. Instruments so
modified include photometers (63, 103, 122), fluorometers 8. Haggerty, C., Jablonski, E., Stay, L., and DeLuca, M., Continuous
monitoring of reactions that produce NADH and NADPH using
(202,203), stopped-flow apparatus (125,204), continuous-flow
immobilized luciferase and oxidoreductases from Beneckea harveyi.
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Analytical Luminescence: Its Potential in The Clinical Laboratory

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CLIN. CHEM. 25/9, 1531-1546 (1979)

Analytical Luminescence: Its Potential in the Clinical Laboratory

Thomas P. Whitehead, Larry J. Kricka, Timothy J. N. Carter, and Gary H. G. Thorpe

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1531

other enzymes. NH2

1532 CLINICAL CHEMISTRY, Vol. 25. No. 9, 1979

x_Hco g_W 5011105

(I) - c’.o + (II)

An accidinic. phenyl Lophin. 1_H pyrogallot

( o’” ? intensity is increased by organic bases and inhibited by or-

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1533

(c) Enzyme-substrate systems LH2, luciferin

(e) Activation of precharged” systems L = 0, oxyluciferin

1534 CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979

NAD (02 (auto-oxidation) Ir

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1535

1536 CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979

Table 2. Assays Based on Firefly BL (ATP subunit B activity

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1537

1538 CLINICALCHEMISTRY,Vol. 25, No. 9, 1979

CLINICAL CHEMISTRY. Vol. 25, No. 9, 1979 1539

Table 6. Luminescent Enzyme lmmunoassays a

Table 7. Luminescent Cotactor-Immunoassays

1540 CLINICALCHEMISTRY,Vol. 25, No. 9, 1979

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1541

1542 CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1543

CLINICAL CHEMISTRY, Vol. 25, No. 9, 1979 1545

1546 CLINICALCHEMISTRY,Vol. 25, No. 9, 1979

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