Microarray-Based Immunoassays - ACS Symposium Series (ACS

Chapter 14

Microarray-Based Immunoassays 1

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F. W. Chu , P. R. Edwards , R. P. Ekins , H. Berger , P. Finckh , and F. Krause 2

Downloaded by STANFORD UNIV GREEN LIBR on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch014

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Division of Molecular Endocrinology, University College London Medical School, Mortimer Street, London W1N 8AA, United Kingdom Boehringer Mannheim GmbH, Bahnhofstrasse 9-15, D-8132 Tutzing, Germany 2

Recent worldwide interest in the development of miniaturized, array-based, multianalyte binding assay methods suggests that the ligand assay field is on the brink of a technological revolution. Our own collaborative studies in this area have centered largely (but not exclusively) on antibody spot "immunoarrays" localized on "microchips" which are potentially capable of determining the amounts of hundreds of different analytes in a small sample (such as a single drop of blood). Analogous technology for genetic testing using oligonucleotide arrays is under active development both in the US and Europe. Array-based immunoassay methods are clearly likely to prove of particular importance in areas such as environmental monitoring where the concentrations of many different analytes in test samples are required to be simultaneously determined. In this presentation we review the general principles underlying this emerging technology.

Immunoassay "Binding" or "ligand" assay methods have, in the past 35 years, been applied to the assay of a wide range of substances of biological importance. Because antibodies can be raised against many such analytes, antibody-based "immunoassay" techniques have achieved particular prominence, but the principles on which these techniques rest can be exploited using many other classes of binding agent. Of particular and increasing importance is the use - in methods relying on identical analytical concepts - of oligonucleotide probes, which bind to single chain fragments of D N A with affinities and specificities of the same order as, or greater than, those characterizing antibody-antigen and other binding reactions. Such "binding assay" techniques were originally developed to determine the minute concentrations of hormones in biological fluids, but were subsequently exploited in many other areas of medicine in which the estimation of small amounts of biologically-important substances is required. More recently still, they have been increasingly adopted in fields such as environmental monitoring in which similar needs arise. During the period 1960-80 the "competitive" or "saturation" assay approach relying on the use of radiolabeled analyte markers, and typified by radioimmunoassay (RIA) - dominated the field. Radiolabeled antibody methods, usually referred to as © 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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"immunoradiometric assays" (IRMAs), were developed in the late 1960's by Wide (7), followed shortly by Miles and Hales (2, 3), and owed much to the development of immunosorbant techniques for the preparation of pure labeled antibodies. These methods were originally claimed (3) to be inherently more sensitive than RIA; however this claim was neither supported by rigorous theoretical analysis, nor persuasive experimental evidence, and remained controversial. Particular doubts on its validity were cast in 1973 by Rodbard's and Weiss' (4) detailed theoretical studies purporting to demonstrate that both labeled analyte and labeled antibody methods possess essentially equal sensitivities, IRMA methods being supposedly somewhat more sensitive for the assay of small polypeptides in which radioiodine incorporation into the analyte molecule was restricted, but less sensitive for the assay of high molecular weight analytes. These erroneous conclusions stemmed in part from the confusion surrounding the concept of sensitivity prevalent during this period; in part from the common misconception (which persists to the present day) that a crucial distinction in regard to assay performance lies between labeled antibody and labeled analyte methods per se. (In other words, the belief that labeling the antibody used in the system of itself affects assay sensitivity.) In reality, labeled antibody, or "immunometric", methods can be sub-classified as "competitive" and "non competitive", immunometric assays of competitive design being essentially identical in sensitivity to competitive assays relying on labeled analyte. In short, higher assay sensitivity is not a necessary consequence of labeling the antibody used, but is a possible consequence of the adoption of a non-competitive design. Nevertheless, the low specific activity of the radionuclides conventionally used in this context implies that non-competitive IRMAs do not demonstrate significant improvements in sensitivity when compared with competitive RIAs and IRMAs and other non-isotopic competitive methods, especially when high affinity antibodies are used. However, the theoretical demonstration that "non-competitive" assays using antibodies labeled with non-isotopic markers of high specific activity are potentially capable of greater sensitivity and may require far shorter incubation times (5, 6), when coupled with the development of methods of monoclonal antibody synthesis by Kohler and Milstein in 1975 (7) - provided the basis for the development of a new generation of fast, "ultrasensitive", immunoassays. The first of this generation developed by Ekins et al in collaboration with the instrument manufacturer Wallac Oy (8, 9) - relied on high specific activity lanthanide chelate labels and timeresolution techniques for the measurement of the slowly-decaying fluorescent signals such chelates emit. Subsequently other manufacturers have also introduced ultrasensitive methods based on identical principles and the use of other high specific activity, non-isotopic, labels (e.g. chemiluminescent substances, enzymes, etc), as recently reviewed by Kricka (70). These second generation technologies have progressively replaced the earlier isotopically-based methods during the '80s, enabling, inter alia, the development of the automatic immunoanalysers now widely used in clinical laboratories. Despite the increases in sensitivity and reduction in assay performance times resulting from these developments, current immunoassay methodology remains essentially limited to single analyte measurements. However, the need for simultaneous, sensitive, determination of large numbers of analytes in small test samples is clearly apparent in many areas of clinical medicine; for example, allergy testing, the screening of transfusion blood for viral contamination, forensic investigation, etc. But the area in which such a need very clearly arises, and that has probably attracted the widest popular attention, is that of genetic analysis. Genetic testing techniques rely on the detection of specific polynucleotide sequences within a D N A strand by their binding to complementary oligonucleotide probes. Conscious of the impending completion of the Human Genome Project, US Government agencies established in 1992 a five-year collaborative study on the development of

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

Capture antibody ((5 Jluorophor)

Solid ili[>jiari

Add developing antibody (afluorophor)

Add developing antibody (afluorophor)


ca. 10 microns (a photons)

/

\^

(a photons)

reacts with empty sites

reacts withfilledsites (P photons)

(P photons)

^^^^^^^^ COMPETITIVE

NON-COMPETITIVE

Figure 1. Following exposure of (fluorescent-labeled) capture antibodies within the microspot area to analyte-containing medium, either occupied sites (non-competitive approach) or unoccupied sites (competitive approach) are determined using an appropriate "developing" antibody labeled with a second fluorescent label. The ratio of signals yielded by the two labels reveals capture antibody occupancy.

Capture antibody Add developing antibody

Add developing antibody

(P fluorophor)

(afluorophor)

ca. 10 microns a photons

Non-Competitive

P photons

Competitive

Figure 2. Combined non-competitive/competitive approach, relying on developing antibodies labeled with different fluorescent labels, and measurement of the signal ratio.


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oligonucleotide-array-based analytical techniques (the 'Genosensor Project'). Albeit formally restricted to D N A analysis, this project is closely similar in its aims and approach to our own studies, and is clearly relevant to other similar multianalyte binding assays. The current widespread interest both in the US and in Europe in the development of array-based microanalytical methods, and the related instrumentation and technology, for use in diagnostic medicine clearly has major implications with respect to other areas in which immunoassay techniques are frequently employed, such as environmental analysis. In this presentation, we describe the concepts underlying the development of miniaturized, multianalyte, microspot array technologies, which are likely to constitute the next major revolution in the binding assay field. Array-based Multianalyte Binding Assays: Basic Concepts The basic concepts developed by Ekins et al (11) that underlie the array technologies currently under development jointly at U C L and Boehringer Mannheim GmbH have been previously reviewed on several occasions (72, 13) and only a brief summary is therefore necessary here. These concepts include the following: i. The "Binding Site Occupancy" Principle of Immunoassay and Other Binding Assays. This embodies the proposition that all such assays implicitly rely on detennination of the fractional occupancy by analyte of a binding agent - typically an antibody or oligonucleotide. Non-competitive assays directly determine occupied sites; competitive assays require observation of unoccupied sites, from which binding-site fractional occupancy is inferred. ii. "Ambient Analyte" assay. This term describes assays in which the binding site concentration is so low as not to affect the analyte concentration in the medium to which the binding agent is exposed. In practice, this implies that no more than 5%, and ideally less than 1%, of the total analyte is bound. In these circumstances binding-site fractional occupancy is independent of both the amount of binding agent used in the system and the sample volume. iii. Microspot Assays. Such assays are characterised by the use of small amounts of a binding agent localised at high surface density on a solid support in the form of a "microspot". If the system fulfils ambient analyte assay conditions, the microspot acts as an analyte "sensor", its fractional occupancy by analyte being indicative of the analyte concentration in the surrounding medium. iv. Dual Label, Ambient Analyte "Ratiometric" Binding Assays. These assays rely on observation of the ratio of signals emitted by two labels to determine binding site fractional occupancy and hence the ambient analyte concentration. This approach reduces errors arising from variations in the amount, or surface density, of binding agent located within the microspot area. Figure 1 shows a microspot immunoassay using two distinguishable fluorescent labels. One is coupled to the solid-supported capture or "sensing" antibody, the other to a second "developing" antibody. A n alternative approach locates the labels on two developing antibodies directed respectively against occupied and unoccupied sites of the sensing antibody (Figure 2). A system operating in this manner can be described as both competitive and non competitive. These four concepts underlie the development of antibody microspot-array technologies in which each individual microspot in an array relates to a different analyte in the medium to which it is exposed. In our initial feasibility studies, a



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.001

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Antibody concentration (x 1/K)

Figure 3. Increase in antibody concentration in the system (i.e. increase in microspot area) results in an increase in analyte binding, but a decrease in the surface density of captured analyte, and hence a decrease in the signal/background ratio.

Figure 4. As microspot area increases, the signal yielded by occupied sites fades into the background. Reproduced with permission from Ekins, R.P.; Chu, F.W. In Immunoanalysis of Agrochemicals, Emerging Technologies; Nelson, J.O.; Karu, A.E.; Wong, R.B., Ed.; ACS Symp. Ser. 586 American Chemical Society: Washington, D C , 1995, Ch. 11. Copyright 1995 American Chemical Society.


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commercially-available laser scanning confocal microscope was used to observe microspots in which were located sensor and developing antibodies labeled by a pair of conventional fluorophors - Texas Red and FITC (72). The latter have since been replaced by a polyfluorophor amplification system to increase assay sensitivity, but a detailed description of this modification is presently prevented by commercial constraints. This modified approach yields very high sensitivities (73). The use of fluorescent labels and a laser scanning confocal microscope has subsequently been adopted by several participants in the US Genosensor project, although other methods of observation of light-emitting arrays (relying, for example, on CCD-based devices) are also obviously feasible. Downloaded by STANFORD UNIV GREEN LIBR on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch014

Theoretical Considerations: Microspot Assay Sensitivity and Speed The proposition that microspot assays may be more sensitive and rapid than conventional systems challenges many accepted ideas in the binding assay field and often arouses skepticism. One widely-held belief is that - to determine a small amount of an analyte in a sample using a non-competitive assay strategy - it is necessary to 'capture' the majority of the analyte molecules present (14). Contradicting this precept, binding agent microspots sequester only an insignificant fraction of the total analyte in the sample. Another concept (deriving from the mass action laws) apparently contravened by the microspot approach is that the use of a large amount of binding agent maximizes the velocity of the binding reaction, thus yielding assays requiring shorter incubation times. These conflicts with generally accepted ideas should be briefly addressed. Sensitivity. As indicated above, all immunoassays essentially rely on measurement of antibody binding site occupancy, either directly (non-competitive assay) or indirectly (competitive assay). A non-competitive assay generally yields higher sensitivity since it is normally preferable, in practice, to measure a small quantity directly rather than indirectly, i.e. by subtracting one large quantity from another. In certain circumstances it may be advantageous - if high sensitivity is required - to maximize binding-site occupancy; however, increasing the number of binding sites to achieve this objective is also likely to cause a concomitant increase in the background signal. The proposition that, in a non-competitive microspot assay design, sensitivity is increased by reducing the microspot area (assuming the binding agent surface density remains constant) is illustrated in Figures 3 and 4. Figure 3 shows the fall in the signal/background (s/b) ratio (expressed as a percentage of the asymptotic (s/b) limit as the antibody-coated area tends to zero) as the amount of antibody increases. Also plotted is the percentage of the total analyte bound to the antibody. The calculations underlying these figures are based on the assumptions of equilibrium and the use of a relatively low total analyte concentration ( . .01

, 1

Ratiometric 10

100

1,000

TSH Concentration (uU/ml) Figure 9. The precision profiles obtained by separate analysis of the competitive and non-competitive data are compared with that obtained using the ratio of the signals emitted by both labeled developing antibodies (ie the combined competitive/non-competitive ratiometric method).



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labeled material is low). In other words, a Microspot format exploits to the full the sensitivity potential of competitive assay designs. In order to overcome the sensitivity limitations of competitive assays, certain workers (18, 19) have adopted a novel, albeit somewhat complicated, approach, i.e. that of exposing the initial incubation mixture (containing unlabeled antibody-analyte complex and residual anti-analyte antibody) to either an unlabeled anti-idiotypic antibody or an analyte-protein complex reacting with, and thus effectively blocking, residual, unoccupied, antibody binding sites. The incubation mixture is then further incubated with a second, labelled, anti-idiotypic antibody capable of binding to the analyte-antibody complex, but sterically prevented from reacting with blocked sensorantibody binding sites. The resulting "idiometric" assay system thus possesses a superficial resemblance to a two-site assay, and can legitimately be described as of non-competitive design. It is claimed to yield very high sensitivity; however the labelled anti-idiotypic antibody is blind to the nature of molecules occupying antianalyte-antibody binding sites, so that, in regard to specificity, the system relies solely on this antibody's characteristics, and thus functions essentially as a single-site assay. In principle, such an approach may be exploited using the Microspot format; however it requires additional incubation steps, and for this and other reasons we have not as yet examined it in detail. Another question occasionally asked in regard to the Microspot technology is its vulnerabilty to the presence of fluorescent substances in test samples. This constitutes a potential hazard in regard to samples encountered in both biomedical and environmental monitoring contexts. This problem is very largely obviated by the sequential nature of incubations carried out in our current procedures; thus the test sample itself is washed away following the initial incubation prior to exposure of the sample carrier to developing antibodies. Moreover since, in the final laser scanning of the sample holder, areas surrounding antibody spots (as well as the spots themselves) are examined, any fluorescent material non-specifically bound to the sample carrier is immediately apparent and its interfering effects - if not overwhelmingly large corrected for. Finally, of course, fluorescence of analyte molecules themselves may occur, but the resulting effects would inevitably be small in comparison with signals emitted by labeled antibodies, and would in any case implicitly corrected for by the standardisation procedure. To summarise, we have not experienced problems, in practice, due to the presence of fluorescent substances in biological samples, and would not expect them to arise in samples encountered in an environmental setting except in extremely exceptional circumstances. Other multianalyte immunoassay techniques have, of course, been known for many years. These have generally depended on the use of different labels to monitor the individual reactions, though some have relied on their spatial separation, for example on paper strips. The former are clearly restricted by the number of different labels that can be readily distinguished; the latter have been somewhat cumbersome, not readily automated, and have required relatively large sample volumes. The Microspot technology represents the ultimate miniaturized form of this approach, reflecting the realisation that the use of vanishingly small concentrations of antibody yields assays that are faster and more sensitive than any other. Immunosensor techniques and other similar sensing methods have frequently mentioned as candidate technologies for use in both the biomedical and environmental monitoring fields, and a great deal of time and money has been devoted by manufacturers on their development over many years. However the development of transducer-based sensing devices of adequate sensitivity and specificity in itself presents formidable difficulties (some of a fundamental nature), putting aside the obvious desirability of multiparameter testing in environmental monitoring and many other potential areas of application. For these reasons, we believe such sensors lie far in the future, and that the development of array


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technologies of the kind described in this presentation offers a far greater prospect of success in the foreseeable future. Acknowledgments


We acknowledge the meticulous and dedicated technical work of Antony Burt of the Department of Molecular Endocrinology, University College London in the feasibility and other preliminary studies on which the project has been based. Later studies were possible as a result of the varied development work of the extensive Boehringer Mannheim Microspot project team. The generous financial assistance of the Wolfson Foundation to the Department is also acknowledged with gratitude.

1991,

References 1. 2. 3.

Wide, L.; Bennich, H.; Johansson, S.G.O. Lancet 1967, 2, 1105-1107. Miles, L.E.H.; Hales, C.N. Nature 1968, 219, 186-189. Miles, L.E.H.; Hales, C.N. In Protein and Polypeptide Hormones; Margoulies, M . , Ed.; Excerpta Medica: Amsterdam, Holland, 1968, Part 1; pp 61-70. 4. Rodbard, D.; Weiss, G.H. Anal. Biochem. 1973, 52, 10-44. 5. Ekins, R.P. In Radioimmunoassay and Related Procedures in Medicine; International Atomic Energy Agency Vienna: Vienna, Austria, 1977, Vol 1; pp 241-268. 6. Jackson, T . M . ; Marshall, N.J.; Ekins, R.P. In Immunoassays for Clinical Chemistry; Hunter, W . M . ; Corrie, J.E.T., Eds.; Churchill Livingstone: Edinburgh, UK, 1983, pp 557-575. 7. Köhler, G.; Milstein C. Nature 1975, 256, 495-497. 8. Marshall, N.J.; Dakubu, S.; Jackson, T.; Ekins, R.P. In Monoclonal Antibodies and Developments in Immunoassay; Albertini, A.; Ekins, R.P., Eds.; Elsevier: North Holland, Amsterdam, Holland, 1981, pp 101-108. 9. Soini, E.; Lövgren, T. CRC Critical Reviews in Analytical Chemistry 1987, 18, 105-154. 10. Kricka, L.J. J. Clin. Immunoassay 1993, 16, 267-271. 11. Ekins, R.P.; Chu, F.; Micallef, J. Journal of Bioluminescence and Chemiluminescence 1989, 4, 59-78. 12. Ekins, R.P.; Chu, F.; Biggart, E. Analytica Chimica Acta 1990, 227, 73-96. 13. Ekins, R.P; Chu, F. Tibtech. 1994, 12, 89-94. 14. Hay, I.D.; Bayer, M.F.; Kaplan, M . M . ; Klee, G.G.; Larsen, P.R.; Spencer, C.A. Clin. Chem. 1991, 37, 2002-2008. 15. Ekins, R.P.; Newman, B ; O'Riordan, J.L.H. Theoretical Aspects of 'Saturation' and Radioimmunoassay. In Radioisotopes in Medicine: In Vitro Studies, Hayes, R.L.; Goswitz, F.A.; Murphy, B.E.P., Eds.; Oak Ridge Symposia, USAEC: Oak Ridge, Tennessee, 1968, pp 59-100. 16. Crank, J. In The Mathematics of Diffusion, 2nd edition; Oxford U P: Oxford, U K , 1975. 17. Fodor, S.P.A.; Read, J.L.; Pirrung, M.C.; Stryer, L.; Lu, A.T.; Solas, D. Science 251, 767-773. 18. Barnard, G; Kohen, F. Clin. Chem. 1990, 36, 1945-1950. 19. Self, C. Determination Method, Use and Components. International patent application: PCT 01033.


Microarray-Based Immunoassays - ACS Symposium Series (ACS

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