Immunoassay labels based on chemiluminescence and bioluminescence

Immunoassay

Labels Based on Chemiluminescence and Bioluminescence W. RUDOLF

Department

of Chemistry,

University

Reagents required for reactions that produce chemiluminescence (CL) or bioluminescence (BL) may be coupled to antibodies or antigens and used as labels for immunoassay. Because methods based on CL and BL have very low detection limits, they have the potential to replace assays that currently employ radioisotopes as labels. The feasibility of several BL and CL labels has been demonstrated. To date, isoluminol derivatives have been most widely studied. Several steroid assays involving isoluminol labels have been reported, and labelled compound has been detected at levels approaching 10 I’ moles. Acridinium ester-labelled compounds have also been detected at this level. In addition to systems in which the label is a reactant required for light, CL and BL can be used to analyze the amount of product generated by enzyme labels. This approach has also yielded very low detection limits. Systems have been developed using enzyme labels that catalyze formation of ATP which is then assayed by the firefly reaction or that catalyze formation of peroxide which is determined by either luminol or peroxyoxalate CL.

KEY WORDS: luminescence, immuno enzyme techniques hemiluminescence (CL) is light accompanying a chemical reaction. Bioluminescence (BL) is light C accompanying a chemical reaction derived from nature (e.g. light from a firefly). Since CL and BL are essentially the same phenomenon, the two collectively are widely referred to as “luminescence” in the clinical literature. Instruments for measuring CL and BL are correspondingly known as “luminometers”. Because the term luminescence properly refers to all forms of light emission from electronically excited states including photoluminescence, triboluminescence etc., it will be avoided here. For a reaction to be accompanied by light, it must lead to an electronically excited product which in turn must emit light upon returning to the ground state. The CL efficiency, i.e. the fraction of reacting molecules that actually give rise to light emission, is a product of the excitation efficiency (the fraction of reacting molecules forming excited state product) and the emission efficiency (the fraction of excited state product molecules emitting light). Except for some BL processes, notably the firefly reaction, efficiencies are usually low. Even a relatively bright reaction such as the wellknown luminol reaction has an efficiency of only a few percent under optimum conditions. To use CL and BL for analysis, the reaction is performed under conditions such that the magnitude of the light output is a function of the concentration of species to be determined. Experimentally, the reactants reThis paper is based on presentation by the author at the Joint Congress on Clinical Chemistry, Quebec City, June 26-30, 120

1983.

SEITZ

of New Hampshire,

Durham,

NH 03824

quired for light are brought together under controlled conditions, and intensity is measured as a function of time as reactants are consumed. Usually, concentration is related to intensity integrated over some time interval after mixing, although it is possible simply to use maximum intensity as a measure of concentration. CL and BL assays are capable of extremely low detection limits with very simple instrumentation. Several factors contribute to this. One factor is that it is possible to measure extremely low levels of light using photomultiplier tubes. Another is that, since the light is generated chemically, the only serious instrumental source of background is dark counts from the photomultiplier. By contrast, fluorescence methods are subject to background from scattered excitation radiation. The apparatus for CL and BL measurements must provide for reproducibly bringing the reactants together to initiate light production as well as for measuring the light output. In a few situations it may be useful to have a filter to resolve the CL of interest from unwanted background emission. The reaction is generally initiated by forcefully injecting one of the necessary reactants for light production into a small tube containing the remaining reactants. The tube is usually already positioned in front of the photomultiplier detector, so that light can be measured immediately. Modern instruments provide for programmed sequential addition of two or more reactants as required for certain assay protocols. While CL and BL may be used to advantage for various enzyme and substrate assays, the greatest clinical interest has been directed toward the possibility of exploiting the sensitivity of CL and BL in conjunction with immunoassays. An antigen or antibody is labelled with one of the reactants required for light emission. After the appropriate chemical steps, the amount of free or bound label is then determined by adding the remaining reactants and measuring the resulting light emission. Often, bound and free label differ sufficiently in reactivity and/or CL efficiency that they can be distinguished without a separation. However, lower detection limits are generally achievable if a separation step is included in the procedure. The primary goal is to develop labels that can be used in assays requiring very low detection limits which presently can only be achieved with radioisotope labels. The use of CL labels would avoid the hazard and disposal problems associated with radioisotopes and would provide reagents with longer shelf-lives. Since the first reports of CL and BL labels in 1976 (l-31, considerable effort has been devoted to developing practical labelling systems with detection limits CLINICAL

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CHEMILUMINESCENCE & BIOLUMINESCENCE H2N NH oxidizing

NH

reagents

> b

0

base

coo

-*

coo-

H2N

0 b Figure low enough to replace radioisotopes. Many labels have been demonstrated in principle. Some of these have been successfully applied in assays. The field has developed to the point where it is reasonable to expect immunoassay systems based on CL and BL soon to become generally available and to take their place as one of the routine tools of the clinical chemist. The intent of this manuscript is to examine the current status of CL labels. Individual labelling systems will be considered with particular emphasis on systems that seem to have the necessary characteristics to be useful in practical clinical analysis. Before considering individual systems, it is useful to divide the reported approaches to labelling into categories. For more complete general information on CL and BL anaysis as well as more specific information on clinical applications, the reader may consult available books and reviews on this subject (4- 12). CL and BL labels CATEGORIES

It is possible to distinguish different types of labelling systems. This distinction is of value because the different categories tend to have common advantages and disadvantages. The first category involves labels that are consumed in the analytical reaction. For example, luminol derivatives used as labels are oxidized to produce light. These labels generally contain the structurally unique features that promote CL. In this case there is generally little if any background CL. Detection limits for the label depend on the ability to measure low level CL with adequate precision. To achieve the lowest possible detection limits, it is necessary to use a high sensitivity detection system and to couple the label in a manner that does not interfere with its ability to chemiluminesce efficiently. In addition, the CL reaction conditions should be chosen to promote maximum light output. This type of label is very similar to a hypothetical radioisotope label in which one could control both the half-life and the initiation of decay. However, the counting efficiency, i.e. counts registered per molecule reacted, is much lower than with radioactive labels, both because the CL efficiency is less than one and because instrumentally it is CLINICAL BIOCHEMISTRY, VOLUME

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APRIL 1984

coo+

blue light

coo-

1

not possible to measure every single photon. Factors establishing theoretical detection limits have been considered (13 I. A second category of CL label is not consumed in the light producing process. This category mainly involves labels that act to catalyze light production but also includes labels that emit after accepting energy from an electronically excited molecule produced chemically. In these systems the amount of label does not limit total light output. Greater light production can be achieved by adding larger amounts of the reactants that are consumed. This advantage does not necessarily lead to lower detection limits, however, because these systems are often subject to background CL. Variations in this background establish the detection limit. Thus the sensitivity of the detection system is less critical for this type of label. The third category involves the use of CL and BL to analyze the amount of product generated by an enzyme label. This is a form of enzyme immunoassay rather than a true CL immunoassay. However, by using CL and BL for product analysis, detection limits are lowered because less product is required for a detectable signal. LABELS

THAT

ARE CONSUMED

Luminol

Luminol (3-aminophthalhydrazide) is oxidized in base to yield CL corresponding to the fluorescence of the aminophthalate product (Figure 1). The reaction is general for aromatic hydrazides but luminol is the best known and one of the most efficient. In early work, luminol was coupled to ligands via reactions involving the amino group (14- 161 and by addition at the 6-position (17). The resulting conjugates, however, had lower CL efficiencies than the parent compounds. Labels derived from isoluminol have been more successful. For example, the isoluminol derivative 4-13-amino-2-hydroxypropylaminol-phthalhydrazide (Figure 2) can be coupled via reactions involving the primary amino group (3). Considerable effort has been devoted to evaluating the effect of structure on the CL efficiency of isoluminol derivatives suitable for coupling as well as on determining the oxidizing reagents that provide for 121

SEITZ TABLE

Immunoassays

Employing

1

Isoluminol

Derivatives

Sample

Type of Assay

Thyroxine Progesterone Progesterone Cortisol

Serum Plasma Plasma Plasma

Heterogeneous Homogeneous Heterogeneous Homogeneous

Cortisol

Serum

Heterogeneous

lo- 1400 nmol/L

Estriol-16a-glucuronide Pregnanediol-3a-glucuronide

Urine Urine

Homogeneous Heterogeneous

20-2ooopg

Hepatitis B surface antigen Estrone-3-glucuronide Estradiol-17P

Serum Urine

Two-site Heterogeneous Heterogeneous

25- 170 nmol/L 2-500 pmol/L

Analyte

0

OH

YH NH

Working

as Labels

Range

15-150

pg/L pg 15- 1000 pg 20-400 p.g/L 25-400

10-100

pg

-

Comments

Ref.

Compared to RIA (t-=0.98) Compared to RIA (r=0.974) Compared to RIA I r=0.98) Compared to RIA I r=O.98) Applied to methylene chloride extract. Compared to RIA (r=O.98) 0.1 pmol detection limit Compared to RIA I r=O.98) Compared to gas chromatography (,=0.96) Compared to RIA (r=0.94) Compared to RIA I r=0.97)

23, 24 25. 26 27.28 29

30 31.32 33 34 35 36

volve antibody-coated tubes. The amount of bound label is assayed after a hydrolysis step that gets the label back in solution prior to addition of the reagents required for CL. The hepatitis B surface antigen is determined by a two-site assay involving Table 1 indicates how CL methods

labelled antibody. (23 - 36 1have been

found to correlate with standard methods, usually RIA. Correlation Figure

2

o* ,OAr

are typically

Amounts as low as lo-” moles have Recently an elegant new approach +

&IQ

coefficients

H202

-&*+

C02+ArOH

r CH;

light

Figure

around

0.97

to

0.98. The coefficient of variation for intra-assay replicates is typically in the range of5- 10%. As the working ranges indicate, sensitivity is indeed excellent.

3

the lowest detection limit (18, 191. A more recently reported isothiocyanate derivative of isoluminol also allows for easy coupling while still yielding superior detection limits (201. A number of assays for steroids, hormones and other species have been developed using isoluminol as a label (21, 22). These are summarized in Table 1. In these assays a derivative of isoluminol with a pri-

mary amino group is coupled using N-hydroxysuccinimide as the coupling reagent. All assays except that for hepatitis B surface antigen involve labelling of the analyte and competitive binding. The homogeneous assays are based on the observation that binding to the antibody enhanced CL efficiency by roughly a factor of four. Except for thyroxine the heterogeneous assays in-

been detected. to homogeneous

CL immunoassay has been reported (37). It is based on the observation that an isoluminol-labelled antigen bound to a fluorescein-labelled antibody yields green emission when reacted with oxidizing reagents in base. The electronically excited aminophthalate derivative generated in the oxidation (Figure 11 transfers its energy to fluorescein. Since the free label is not close to fluorescein, it yields the usual blue light upon oxidation. Thus this approach offers the possibility of simultaneously measuring amounts of free and bound label by monitoring two wavelengths. Acridinium

esters

Aromatic acridinium esters chemiluminesce when oxidized by hydrogen peroxide as shown in Figure 3. Light is emitted as excited N-methyl acridone product relaxes to the ground state. Antigens with phenolic-OH groups may be derivatized by reaction with acridine-9carboxyl chloride followed by methylation of the nitrogen (38). Conjugates have been detected at a level of 2 x 10-l’ moles. Thus the performance of this label is similar to that of isoluminol although, so far, fewer assays have been developed using it.

ATP The best known BL reaction is the firefly which is widely used to measure ATP (3). ATP + luciferin

122

reaction

+ Mg*+ + 02 oxyluciferin* firefly 5 luciferase light

+ other products

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Amounts as low as 10 Ifi moles of ATP can be detected by measuring the intensity of BL from the firefly reaction. The feasibility of using ATP derivatives as labels has been demonstrated in an assay for dinitrophenol(2, 39). However, this label has not been pursued because of potential problems associated with endogenous ATP and/or ATP-degrading enzymes that would act on the label. In principle, firefly luciferin would be an attractive candidate for a label since it would not occur endogenously like ATP or participate in other enzymecatalyzed processes. However, the firefly reaction is so specific for the luciferin structure that it is not possible to prepare a conjugate with a high BL efficiency. NADH Small amounts of NADH may be determined by measuring light from the bioluminscent reaction derived from luminous marine bacteria. An NADH analog has been demonstrated as a BL label (1). However, this system is subject to the same kinds of problems as ATP, and has not been developed further. Fluorescein

THAT

AKE

NOT

ci

-A

Figure

dl

4

any function other than providing functional groups for covalent attachment of another species. Luminol is not the only substrate that chemiluminesces upon peroxidase-catalyzed oxidation by hydrogen peroxide. A procedure has also been reported using pyrogallol as the substrate (44). Femtogram levels of peroxidase have been detected using Pholad luciferin as the substrate in the presence of oxygen (43 1.Unfortunately Pholad luciferin is a complex protein from a luminescent clam which is either extinct or in very low abundance and thus is not a practical substrate. The low detection limits achieved here are probably due to the absence of peroxide.

Firefly luciferase is the enzyme that catalyzes the firefly reaction. It has been employed as a label in immunoassays for methotrexate (45) and TNT (46). In the analytical step ATP and luciferin are added in the appropriate medium and the resulting light emission is measured. The detection limit in the TNT assay, 10 fmol, is low but not as low as with luminol and acridinium ester labels. In view of this and the fact that firefly luciferase is relatively expensive, it probably will not be the preferred label in practical applications.

KEACTEL)

Fluorophors

Peroxidase One of the the oxidizing systems that reacts with luminol to generate CL involves hydrogen peroxide as the oxidizing agent and peroxidase. The amount of a peroxidase-labelled compound may be assayed by adding excess peroxide and luminol and measuring steady state CL intensity. An assay for cortisol has been developed with a working range of 20-1000 pg (41). However, other researchers have not achieved low detection limits for peroxidase using luminol (42, 43). Problems have been encountered because other heme-containing species, e.g. hemoglobin, also catalyze the luminol reaction producing a background signal (42). Since peroxidase is an enzyme, systems employing it as a label might be considered forms of enzyme immunoassay. However, this type of assay differs from other forms of enzyme immunoassay in that it does not involve the accumulation of product. Instead, one observes a steady state intensity which is a direct measure of the catalytic activity of the label. Also, the oxidation is often carried out under highly basic conditions (42) where the enzymatic activity of peroxidase is very low. In this context the heme group is acting as the catalyst and the rest of the protein does not serve CLINICAL

L-

:I: fir fir * + zcoz c-c + b-b I 1 llqhl 1 tl

Firejly luciferase

Immunoassays based on CL from the reaction of hypochlorite with fluorescein have been described in the patent literature (40). The ease with which the isothiocyanate derivative of fluorescein can be coupled to many potential analytes is an attractive feature of this reaction. Interestingly, the detection limit for fluorecein coupled to a protein is much lower than for free fluorescein. This system has promise, but has yet to be described in the open literature. LAUELS

& BIOLUMINESCENCE

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excited by energy transfer

In several CL reactions, an excited intermediate produced chemically transfers its energy to a fluorophor which then emits. The fluorophor is not consumed in the reaction because its only function is to accept energy and emit. Because CL intensity is proportional to fluorophor concentration, this type of reaction may be used to excite fluorescent labels. The particular reaction that has been used involves the reaction of an aromatic oxalate ester with peroxide in the presence of a fluorophor. This is an example of a larger class of reactions involving various oxalate derivatives known collectively as peroxyoxalate reactions (47). The feasibility of using this reaction to excite fluorophor-labelled compounds has been demonstrated (48). Fluorscein, dansyl, fluorescamine and rhodamine labels are all excited. Detection limits as low as one fmol have been achieved. An attractive feature of this approach is that it involves fluorescent labels which are stable and easy to prepare. However, because this reaction can excite a variety of fluorophors, it is subject to error from background associated with fluorescent contaminants. It also requires a mixed solvent system because the oxalate derivatives that yield efficient light emission are not soluble in water (Figure 4). 123

SEITZ

Immunoassays based on the peroxyoxalate reaction have been patented (49). Their success depends on tinding conditions that minimize CL background problems. While the emission is actually CL because it is excited chemically, this approach is in many respects more similar to fluorescence than to other forms of CL analysis.

4. 5. 6.

ENZYME LABELS 7.

CL and BL may be used to analyze the amount of product generated by an enzyme label. Detection limits for this approach depend on the detection limit for the product as well as the amount of product generated.

8. 9.

Glucose oxidase Glucose oxidase catalyzes the oxidation of glucose and the formation of hydrogen peroxide. The hydrogen peroxide can then be analyzed by CL, either by adding luminol and an appropriate catalyst (Figure 1) or by adding an oxalate ester and fluorophor (Figure 4) (50-52). This system has achieved detection limits as low as lo-” moles (50). A method for 17a-hydroxyprogesterone has been developed using the oxalate ester system for peroxide detection (52).

10.

11. 12. 13.

Pyruuate kinase Pyruvate kinase catalyzes the formation of ATP which can be sensitively determined by the firefly reaction. The feasibility of pyruvate kinase as a label has been demonstrated (531. Glucose-B-phosphate

dehydrogenase

14. 15. 16.

Glucose-6-phosphate dehydrogenase catalyzes NADH formation. This NADH can then be determined by measuring BL using the reaction derived from luminous marine bacteria. This system has been applied in a BL procedure for TNT with a detection limit of 1 x lo-l7 moles (46).

17. 18. 19.

Summary The goal of developing low detection limit immunoassays with CL and BL labels has been achieved. Several assay procedures using luminol labels have been developed. More assays involving other labels may be expected. In the next few years CL and BL methods should find their way into routine clinical practice.

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F, Campbell AK. A new chemiluminescent label for use in immunoassay. Biochem Sot Trans 1982; 10: 224-5. 21. Schroeder HR. Luminescent immunoassays and binding assays monitored by chemilumigenic labels, Serono Symp Pub1 Raven Press, 1982, 129-46 through CA97: 51964c. 22. Kohen F, Kim JB, Lindner HR, Eshhar Z. Steroid immu-

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125