Luminescent immunoassay in clinical analysis

352

trenas in analytical chemistry, vol. 1,

economic sciences. An interesting area of research could evolve from the recognition of the fact that economics does have a link with analytical chemistry. It might well be that some methods from economics could be used with advantage in analytical chemistry and it is worth noting that an allied science, operations research, has already been used 11-13.

References

1 Christie, O., Lecture at the University of Birmingham, U.K., 10 February 1982 2 Wangersky, P.J. (1982) Trends Anal. Chem. 1, 150 3 Sandler, G. (1979) British Medical Journal, 2, 21 4 Durbridge, T. C., Edwards, F., Edwards, R. G. and Atkinson, M. (1976) Clin. Chem., 22, 968 5 Gossen, H. H. (1854) Entwicklung der Gesetze des Menschlichen Verkehrsund die darausfliessenden Regelnfiir das MenschlichenHandeln

no.

1~ 1982

6 Van Meerhaege, M. ( 1979)Handboek van de Economie,8th Edition, H. E. Stenfert Kroese, Leiden, p. 133 7 Acland, J. D. and Lipton, S. (1971)J. Clin. Pathol. 24, 369 8 Lindberg, D. A. B. and Watson, F. R. (1974)Meth. Inf. Med. 13, 151 9 Heite, F. H., Dupuis, P. F., Van 't Klooster, H. A. and Dijkstra, A. (1978) Anal. Chim. Acta, 103, 313 10 Coomans, D., Broeckaert, I. and Massart, D. L. (1982) Anal. Chim. Acta, 134, 139 11 Massart, D. L. and Kaufman, L. (1975)Anal. Chem. 47, 1244A 12 Vandeginste, B. G. M. (1982) Trends Anal. Chem., 1,210 13 Goulden, R. (1974) Analyst, 99, 929 D. L. Massart is Professorof Analytical Chemistry in the Free University of Brussels, Laarbeeklaan 103, B-1090, Brussels, Belgium. His scientific interests are mainly in statistical and mathematical methods, but include separation science, ion-selective electrodesand trace metal analysis. He is an advisory editorfor TrA C.

Luminescent immunoassay in clinical analysis Luminescent immunoassay is a novel method for determining the concentrations of numerous organic compounds in complex body fluids. Its advantages over conventional assays are extreme sensitivity, broad range of linear response, speed of analysis and stability of reagents. Hartmut R. Schroeder Elkhart, IN, U.S.A. The numerous analytical applications of luminescence measurements include the detection of trace metals, microbial detection, phagocytosis and cell stimulation, indicator reactions to measure substrates of oxidases and dehydrogenases and the monitoring of immunoassays 1-4. The major impact of luminescence for clinical analysis is expected in the last two areas. Highly sensitive measurement of light generated in simple monitoring reactions permit detection of as little as 10 -16 mol ATP, 10 -14 mol N A D H , 10 -12 mol H , O , , 10 -15 mol heme catalyst (e.g. catalase) 4 and 10 -17 mol chemilumigenic c o m p o u n d (e.g. aminonaphthalhydrazide) 5. In most cases the peak light intensity is attained in less than a second and is related to concentration o f a n a l y t e over five orders of magnitude. Consequently, the above compounds are also attractive as labels for monitoring immunoassays.

The principle of luminescent immunoassay (LIA) The basis of L I A is similar to that of radioimmunoassay (RIA). A fixed a m o u n t of labeled antigen Labeled Antigen Ag-L

Unlabeled Antigen +

A

Specific Antibody +

Ab ,

(Free)

and varying quantities of unlabeled antigen (sample) are allowed to compete for a limited number of antibody binding sites (Fig. 1 ). However, the radiolabel is replaced by a reactant label which is detected in a light-generating reaction. After an incubation step, the antibody-bound labeled antigen is separated from the free form (heterogeneous assay). C o m m o n separation techniques include: chromatographic separation, adsorption of the free antigen to charcoal, cellulose or ion-exchange resins and precipitation of the antigen-antibody complex by a second antibody directed against the first. Then luminescence of the bound fraction or the free fraction is measured in the monitoring reaction. The concentration of unlabeled antigen is determined by comparison of the assay result to a standard curve. Homogeneous assays based on enzyme cofactor labels or chemilumigenic labels are similar to heterogeneous assays, except that the separation step is omitted. Because the light-producing activities of the reactant labeled antigens in the monitoring reactions are modified when bound to antibody, it is possible to measure the free and bound species in the presence of each other. Unlabeled AntigenAntibody Complex

Labeled AntigenAntibody Complex ' Ab" • "Ag-L

+

Ab" • "Ag

(Protein Bound)

Fig. 1. Generalized competitiveprotein binding reaction of LIA. 0 165-9936/82]0000-00001501.00

© 1982 Elsevier Scientific Publishing Company

trends in analytical chemist;ry, vol. I, no. 15, 1982

353 TABLE

I. Luminescence

of Assay

Detection System

Analyte

Sample

Type

Biotin Digoxin Insulin Thyroxine Testosterone Progesterone

P

Pl Pl Pl PI, u

CBA/Ho CBA/He CBA/He CBA/He CBA/He CBA/Ho CBA/He CBA/HeSP CBA/He

HzOa/Hemin HaOz/Hegb NaOCl HnOz/Mxp H20z/Cu HzOn/Mxp HzOz/Mxp H10z/Mxp HzOz/Mxp

U s S P S

CBA/HeSP CBA/He Two-site-SP Two-site-SP Two-site-SP

HzOz/Mxp HzOz/Hemin HzOz/NaOCl HzOz/Mxp HzOz/Mxp

Estradiol Cortisol Estrone-3glucuronide Human IgG a-Fetoprotein Anti-Hu IgG HBsAg

I:; S

P = pure; S = serum; Pl = plasma; U = urine; CBA = competitive phase,

Mxp

binding

Sensitivity Reference

(Range) 10-100 0.2-2.0 5-25 15-150 0.1-10 25-460 5-500 5-500 lo-200 15-l 000 5-50 10-100 1-1000 l-1000

/kg/L /.LglL FgIL pg/L ng/tube pgltube pgltube pgltube pgltube

9 4 4 4 4 2 2 2 2

pgltube pgltube @g/L /.kg/L /_&g/L

10 2 2

assay; Ho = homogeneous,

2,6 6

He = heterogeneous,

SP = solid

= microperoxidase.

Preparation of lumigenic conjugates Various methods have been used to covalently couple luciferase, NAD, ATP or isoluminol to antigens of interest through stable amide bonds4. Activation procedures with N-hydroxysuccinimide, mixed anhydride chemistry or diazotization and coupling through carbodiimide are preferred when linking small molecules. Coupling to proteins is generally through bifunctional reagents such as glutaraldehyde, bisimidates and carbodiimides. In a few cases, the light generating capacity of the label is diminished considerably when coupled to the antigen2*4,5*6.

Light measurement Typically, luminescent reactions (150 pL) are carried out in 6 X 50 mm tubes mounted in a luminometer such as the DuPont 760 Luminescence Biometer. Three detection systems are commonly used: (a) NADH, long-chain fatty acid and bacterial luciferase; (b) ATP, luciferin and firefly luciferase; (c) isoluminol derivative, microperoxidase (metal catalyst) and peroxide. Light production is initiated by rapid addition of the final component (10 pL) to the reaction. The concentration of label is linearly related to the peak light intensity, total light produced or a portion of the light integral.

Luminescent cofactor immunoassay (LCIA) Several novel enzymic methods have been demonstrated for monitoring specific binding reactions in a homogeneous format using ligand-cofactor conjugates and bioluminescent detection reactions. Biotin can be covalently coupled to enzymatically active NAD+ and after reduction with ethanol and alcohol dehydrogenase the biotinyl-NADH can be quantitated by measuring the light produced in the bacterial luciferase system7. Luminescence is inhibited by the specific binding protein avidin, presumably because enzymic interaction with the NADH moiety is prevented. In a competitive protein binding (CPB) assay biotin reverses the inhibition. In this assay a separation offree -

immunoassays

I

and bound conjugate is not necessary. The lower detection limit is about 25 nmol/L biotin in the CPB reaction. A similar assay was demonstrated with 2,4dinitrofluorobenzene-NAD+ (DNP-NAD+), antibody to DNP, and DNP-6-aminocaproate. Because ATP can be measured with greater sensitivity than NAD it was also investigated. DNP was coupled through a phosphate group to the terminal phosphate of ATP producing P’-DNP-adenosine 5’tetraphosphate*. The conjugate is degraded by enzyme(s) in firefly luciferase to release ATP which reacts to produce light. Pre-incubation of the conjugate with antibody to DNP prevents this cleavage and diminishes the peak light intensity. In CPB reactions DNP-@alanine reverses this effect and is detected at 25 nmol/L. These model systems demonstrate the feasibility of homogeneous LCIA. However, endogenous NAD or ATP and enzymes in the sample that degrade the cofactor conjugate present serious impediments to practical application.

Luminescent enzyme immunoassay (LEIA) The high sensitivity ofenzyme immunoassays can be improved considerably by using a luminescent readout. A peroxidase-cortisol conjugate has been prepared which produces light in the presence of luminol and hydrogen peroxide4. In the LEIA free and bound conjugate are separated by using anti-cortisol antibody immobilized on Sepharose. The lower detection limit of 10 pg cortisol per assay is comparable to that of the RIA. The heterogeneous assay format eliminates interference from other components in the sample in the luminescent detection reaction and makes this approach feasible. Recently, antigens have been labeled with catalytically active bacterial or firefly luciferase (Wannlund et al. lo). These conjugates can be used in a similar LEIA with the appropriate solid-phase antibodies and bioluminescence readout reagents. As little as 10 pmol DNP-leucine, 2.5 pmol methotrexate and 50 fmole trinitrotoluene can be determined.

trends in a&y&al

354 Luminescent immunoassay (LIA) Chemilumigenic labels (e.g. aminophthalhydrazides, aminonaphthalhydrazides, lucigenin) are used extensively to monitor both homogeneous and heterogeneous immunoassays (Table I). An interesting homogeneous CPB assay has been developed with a biotin-isoluminol conjugate and avidin (the binding protein) as modelg. On oxidation with lactoperoxidase and peroxide the conjugate produces ten-fold greater luminescence when bound to avidin. The enhancement is due to increased chemiluminescence efficiency. Competing‘biotin prevents this enhancement and can be determined without separation over a range of 50400 nmol/L. However, the sensitivity is limited by the background luminescence of the free fraction. Furthermore, with clinical samples, proteins and other components interfere with the luminescence reaction. Nonetheless, similar assays determine progesterone and estriol-16a-glucuronide in plasma at comparable levels to RIAs, but an extraction step is required in either case (Kohen at al. ‘*lo). Heterogeneous LIA formats are preferred because they take full advantage of the sensitivity inherent in chemilumigenic labels and eliminate interference problems. A number of assays have been developed for hormones and steroids (Table I). The two-site solid phase format gives the greatest sensitivity for bi- or multi-valent protein antigens. Recent applications in an LIA for hepatitis B surface antigen (HBsAg) suggest excellent commercial possibilitie?. A 100 PL serum specimen is incubated in an anti-HBsAg coated well of a microtitration plate. The antigen binds to the antibody and after an intermediate wash step the sandwich is completed by incubation with anti-HBsAg labeled with isoluminol. Following a final wash step, an experimental luminometer adds microperoxidase and peroxide to initiate luminescence, and provides automated readout. When this assay is compared to an RIA, it has about the same sensitivity (1 ng/mL) and specificity. Use of a chemilumigenic label eliminates the inconvenience and stability problems incurred with radiolabels. Although samples must be added individually, wash steps and reagent additions are done simultaneously on multiple wells using commercial instrumentation. The automated readout at 10 s intervals is much faster than the 1 min counting time of

RIAs. The convenience and speed of this LIA merit consideration of this technique for other large-scale screening applications.

Conclusion The feasibility, validity and advantages of luminescent immunoassays have been amply demonstrated. However, for these methods to be accepted in the clinical laboratory greater automation and precision is required. Considerable progress has been made in automated readout with an industrial prototype luminometer (LB9520, Laboratorium Fritz Berthold, Freiburg, F.R.G.) recently exhibited at Analytica 1982 in Munich. Hepatitis testing, thyroid evaluation and the group of steroid LIA’s already developed represent some possibilities that could bring luminescence-based techniques into the routine clinical laboratory.

References

Isacsson, A. T. and Wettermark, G. (1974) Anal. Chim. Acta. 68, 339

DeLuca, M. D. and McElroy, W. D. (eds) (1981) Proceedings of the Symposium on Bioluminescence and Chemiluminescence: Basic Chemistry and Analytical Applications, LaJolla, CA, August 26-28, 1980, Academic Press Inc., New York Gorus, F. and Schram, E. (1979) Clin. Chem. 25, 512 Whitehead, T. P., Kricka, L. J., Carter, T. J. N. and Thorpe, G. H. G. (1979) Clin. Chem. 25, 1531 Schroeder, H. R. andyeager, F. M. (1978)Anal. Chem. 50,1114 Schroeder, H. R., Hines, C. M., Osborn, D. D., Moore, R. P., Hurtle, R. L., Wogoman, F. W., Rogers, R. W. and Vogelhut, P. 0. (1981) Clin. Gem. 27, 1378 Schroeder, H. R., Carrico, R. J., Boguslaski, R. C. and Christner, J. E. (1976) Anal. Biochem. 72, 283 Carrico, R. J., Yeung, K.-K., Schroeder, H. R., Boguslaski, R. C., Buckler, R. T. and Christner, J. E. (1976) Anal. Biochem. 76, 95 9 Schroeder, H. R., Vogelhut, P. O., Carrico, R. J., Boguslaski, R. C. and Buckler, R. T. (1976) Anal. Chem. 48, 1933 10 Sergio, M. and Pazzagli, M. ( 198 1) Luminescent Assays: Perspectiues in Endocrinology and Clinical Chemistry, University of Florence, Florence, Italy, July 6-7

Hartmut Schroeder received his B.S. degree in Chemistry from Youngstown State UniversiQ (Ohio) in 19%. He then underwent post graduate training in Biochemistry under Dr M. F. Mallette at the Pennsylvania State University, receiving the M.S. degree in 1970 and his Ph.D. in 1972. Dr Schroeder is currently employed as a Senior Research Scientist at the Ames Research and Development Laboratories, a Division of Miles Laboratories, Inc., 1127 Myrtle Street, Elkhart, IN 46515, U.S.A., which he joined in 1974.

In Forthcoming Issues.. “XPS

in polymer

“Matrix “Solvation

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analysis”,

effects in the spectroscopic effects on spectroscopic

I

recognition

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of petroleum”,

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using liquid by Ante M. Krstulovic by Gabor

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techniques”,

“Microcomputer-interfaced interactive graphics”,

. by H. R. Thomas

“Clinical investigations of catecholaminergic systems chromatography with electrochemical detection”, “Pattern

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and Henri Colin

E. Veress

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