Homogeneous Immunoassays: Historical Perspective and Future

Waters Symposium: Immunoassay

Homogeneous Immunoassays: Historical Perspective and Future Promise Edwin F. Ullman Scientific Consultant, 135 Selby Lane, Atherton, CA 94027

A Commercial Experiment In 1966, when technology-based start-up companies were still rare, Syntex Corporation and Varian Associates decided to launch an experiment. They would set up a new company, Synvar Research Institute, based on the proposition that if you assemble a group of highly talented people and point them in a general direction they will achieve commercial success. I recall being somewhat hesitant when I was approached to lead this new group. During my interview trip I was hosted by Carl Djerassi, a Director of Syntex Corporation and chemistry professor at Stanford, who, together with Ed Ginzton, President of Varian Associates, had conceived the Synvar experiment. When I first met Djerassi, one of his legs had been injured in a skiing accident. Undaunted by this, he had recently taken up rock climbing and managed to escape with only a broken arm. I was a bit leery on finding that the chairman of this new venture seemed to think nothing of taking wild risks and hoped that he was a bit more conservative when it came to running a company. With Carl as my questionably reliable one-armed, stiff-legged chauffeur, I was gratified to arrive intact to meet Bill Little, a Stanford physics professor, who was consulting with Varian in the somewhat unlikely field of organic superconductors. Bill Little had just written a Scientific American article (1) that included a seemingly outrageous idea of building magnetically levitated trains. This was to be one of the lesser opportunities that would come out of his fundamental new theory, which would permit the construction of organic polymers that would be room-temperature superconductors. It soon became clear that this was to be the primary focus of the new company. If successful, the preparation of these polymers would revolutionize the world economy by permitting loss-free transmission of electricity across unlimited distances using uncooled power lines. Wondering vaguely whether the California cult culture had captured these two otherwise brilliant scientists (the highest recorded superconducting temperature was 18 K and no superconducting organic compound was known), I ventured the suggestion that Little’s structural concepts were at least 20 years ahead of the capabilities of existing synthetic chemistry, an assessment that sadly was far too optimistic. I further suggested that it would be foolhardy to build a company on the premise that such polymers could be synthesized. Needless to say, Little did not look kindly on these remarks (though we have since become good friends) and so it came as quite a surprise when Djerassi later told me that he agreed with my comments and offered me the position. The rationale for the new company gradually became clear. Room-temperature superconductivity had an indeterminate present value: immense profits multiplied by a vanishingly small probability of success. However, it would serve to bring together an excellent group of scientists who would quickly become aware of the limited patience of investors and the need to create a profitable product if the company was to

survive. Two other less dramatic projects offered some hope that this might be accomplished. One project was intended to capitalize on the recently developed spin-labeling concepts of Harden McConnell, also a professor of chemistry at Stanford and, like Little, a former consultant for Varian. The other project would be related to my own research interests in organic photochemistry. Even so, neither of these projects started with clear product objectives. Our goal was to find exciting new technology and discover applications for it. Toward this end we had the exceptionally able assistance of the founding Board of Governors consisting of Carl Djerassi, Alex Zaffaroni (Executive Vice President of Syntex), and Varian executives Ed Ginzton and Martin Packard; and a consultant group consisting of Bill Little, Harden McConnell, and Ronald Breslow, a leader in the new field of bioorganic chemistry and a former Harvard collaborator of mine. Discovery and Corporate Goals During the next three years the Synvar group made a number of important scientific discoveries. Under the guidance of Fred Gamble, a former McConnell graduate student, Synvar developed the first composite organic/inorganic superconductors and demonstrated the potential of superconductivity in two-dimensional systems. However, these materials, layered tantalum sulfide crystals intercalated with aromatic amines, were superconducting only at the conventional temperatures below 18 K and had no apparent practical value (2). A more useful development was a light-responsive sunscreen, which progressively protected the skin from solar UV light after a brief period of unprotected exposure intended to stimulate natural tanning. Unfortunately, the combined wisdom of our group failed to recognize that at the time sunscreens were sold primarily by advertising, not by their efficacy, a point harshly brought home when we approached Revlon with the idea. As an outgrowth of our initial studies relating to McConnell’s spin-labeling concepts, we designed a new class of stable radicals, α-nitronylnitroxides. These compounds could be easily derivatized at a position that affected their electron paramagnetic resonance (EPR) spectrum and thus they appeared to have sensing attributes that might be useful (Fig. 1). These compounds and some conventional nitroxides were assembled into a spin-labeling kit and became the company’s first products, although the income was minimal. In a search for some kind of profitable product, Prithipal Singh, then a postdoctoral fellow, prepared dozens of derivatives for pharmaceutical activity screening. Nearly all of them were completely inert pharmacologically, though the possibility of taking advantage of their excellent radical-trapping properties to protect against radical-induced tissue damage was never evaluated. Nevertheless, it soon became apparent that these compounds were useful for a variety of analytical applications. Postdoctoral fellow David Boocock demonstrated that one of these compounds could signal the presence of as little as 0.1% deuterium in

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water (3, 4); and postdoctoral fellow Jan Becker, with one of our permanent staff, Richard Schneider, showed that using EPR spectroscopy these compounds could be employed for other analytical applications (such as measuring pH and the clinically relevant enzymes alkaline phosphatase, glutamicpyruvic transaminase, amylase, and acetylcholinesterase) (5). Since EPR measurements were not subject to optical interferences the possibility that such assays could be carried out inside cells or in whole blood instead of serum seemed attractive. Thus by the end of 1968 it appeared that clinical chemistry might offer the most promising direction for the company. With this opportunity in mind, Avram Goldstein, then head of the Stanford Pharmacology department and an expert in addiction research, was persuaded to join our advisory group. At a seminal meeting at Lake Tahoe in the fall of 1969, Goldstein pointed out the growing need for urine testing for drugs of abuse and suggested that our spin-label technology could be applied toward this goal. Thus began a crash program leading to the development of a method for the rapid detection of drugs. The concept was simple. It was only necessary to combine a solution containing antibodies to morphine, a morphine–nitroxide spin label conjugate, and a urine sample. The presence of morphine in the sample would be signaled by a sharpening of the characteristic three-line EPR spectrum of the nitroxide radical. In the absence of morphine, most of the conjugate would be bound to the antibody and its relatively slow tumbling rate would be insufficient to average out the anisotropy of the electron spin coupling with the S = 1 nuclear spin of the nitroxide nitrogen atom. When morphine was present it would compete with the conjugate for a limited number of antibody binding sites, and the released conjugate would display the sharp averaged spectrum characteristic of freely tumbling nitroxides. The nitroxide line peak intensity could therefore be used as a measure of the morphine concentration (Fig. 2).

Figure 1. α- Nitronylnitroxides. A: Phenyl derivative showing a 5line EPR pattern caused by hyperfine coupling to the two identical nitrogen atoms. B: Additional coupling by the underlined hydrogen atoms in other derivatives causes each of the lines to appear as a 4-line pattern that changes upon enzymatic hydrolysis.

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Spin Labels and Vietnam Within a few months, a spin immunoassay for morphine (6, 7) was successfully demonstrated by Richard Leute, a postdoctoral fellow who had most recently been working in the field of organic photochemistry. Assays for other abused drugs followed rapidly. Although the assays were manual, the protocol was simple. The combined reagents and sample were mixed in a cup and drawn into a glass capillary, which was then plugged with clay at one end. The capillary was then dropped into a Varian E4 EPR spectrometer with most of the controls preconfigured and hidden to permit operation by an inexperienced technician. In 1970, when Nixon declared his war on drugs, this freeradical assay technique (FRAT ®) was already being clinically evaluated by a lab in Washington. Following an urgent request from the White House later that year, Syva (renamed because a synthetic varnish company had the name “Synvar”) hastily sent three of the modified Varian EPR behemoths, each weighing 450 pounds, to Vietnam along with three people and a few hand-labeled bottles of reagents. Thus was launched a highly controversial program to screen the urine of military personnel in Vietnam for abused drugs, the largest-scale field use of an immunoassay ever attempted. Rumors promptly proliferated on how to get around the tests—drink a quart of vinegar, use megadoses of vitamin C, eat asparagus, etc.— but only the vitamin C story made any chemical sense because ascorbic acid reduces nitroxides. However, this problem had been foreseen and circumvented by the inclusion of potassium dichromate in the assay mixtures. Amid banner headlines, complaints about abridgment of civil liberties, and stories of false accusations of drug use, the testing succeeded in identifying the vast majority of people in need of treatment. The success of the program clearly hinged on the simplicity of the method, which permitted relatively untrained military personnel to manually process up to 120 samples an hour. Although other immunochemical methods had been known for many years, they were either clumsy or analytically unsatisfactory. The detection of insoluble antigen–antibody

Figure 2. Spin immunoassay for morphine. Morphine present in the sample displaces nitroxide-labeled morphine that is bound to an anti-morphine antibody. When the nitroxide is bound to an antibody its tumbling rate is insufficient to average the anisotropically broadened hyperfine EPR coupling. When not bound, the nitroxide tumbles freely and a sharp averaged signal is observed.

Journal of Chemical Education • Vol. 76 No. 6 June 1999 • JChemEd.chem.wisc.edu


precipitates (precipitin) dates back to at least 1905 (8), and the first immunoassay to be widely used in clinical diagnosis, the Wasserman complement fixation test for syphilis (9), was described a year later; but the methods were insensitive and inaccurate. Not until 1941, when Coons (10) developed the concept of using fluorescent labels for visualizing antibody binding to tissue slices, did immunochemical methods become more broadly useful for clinical diagnostic applications. An important further step was made in 1959, when Yalow and Berson recognized that radiochemical labels, which were then coming into general use as tracers, could be advantageously employed to follow antigen binding and could provide a quantitative measure of antigen concentration in solution (11). (Radioimmunoassay, RIA, is separately discussed in this symposium by Dr. Yalow on pages 767–768.) Although quickly adopted for clinical use and available for a number of clinically significant analytes in the early 1970s, RIAs required labor-intensive separations and thorough washing of the bound phase. Three years earlier, Singer and Plotz (12) had demonstrated a method that avoided these troublesome separation and washing steps, but it provided only a qualitative result. They showed that the sensitivity of the precipitin method could be greatly enhanced by binding an antigen to latex particles and detecting the increased light scattering caused by antibodyinduced particle aggregation. Subsequently, Dandliker in 1961 (13) developed a more quantitative procedure that also avoided the separation steps of RIA. He found that the polarization of light emitted from a fluorophore-labeled antigen increased upon its binding to an antibody, and that the polarization was inhibited in the presence of a competing antigen. However this fluorescence polarization inhibition immunoassay (FPIA) was not generally useful because of the relatively primitive state of development of commercial fluorometers. Thus Syva’s spin immunoassay was the first quantitative homogeneous immunochemistry method that was suitable for clinical use. EMIT and Drug Testing Despite the overnight success of FRAT, I was concerned that its limited sensitivity (>100 nM) would be a serious impediment for more general clinical applications. It seemed that only the military was likely to have the wherewithal to handle the massive and expensive EPR instrumentation. Interestingly, our photochemistry project, which we had never abandoned, now provided the conceptual basis for an assay that would preserve the simplicity of spin immunoassay but have the needed higher sensitivity. We had been interested for some time in the possibility of developing a photographic film that was not based on silver halide. The main problem was to find a way to mimic the enormous amplification provided by silver halide, which can yield up to 106 silver atoms in response to the absorption of a single photon. The approach that we were considering was to photochemically turn on an enzyme, which would be able to catalyze the formation of many dye molecules during a development step. Though we did not have time to pursue this line of research, the idea led directly to the thought that the activity of an enzyme might also be modulated by an immunochemical binding event. The simplest approach was to attach an antigen to an enzyme that requires a bulky substrate. Antibody binding to the antigen

might then sterically block access to the substrate (Fig. 3A). This was not an unreasonable supposition, in that others had shown that anti-enzyme antibodies could sometimes inhibit enzyme activity (14). As it happened, a newly hired scientist in our laboratory, Ken Rubenstein, had worked recently with lysozyme, a small single-subunit enzyme that acts on bacterial cell walls. Since a number of drug derivatives were available from our work on FRAT, Rubenstein needed only about a month to prepare a conjugate of lysozyme with a morphine derivative and demonstrate that anti-morphine antibodies could indeed inhibit its activity. A simple assay could then be constructed. Sample suspected of containing morphine was combined with anti-morphine antibody, a suspension of killed bacteria, and the lysozyme-drug conjugate. The rate of decrease in light scattering occasioned by cell wall hydrolysis signaled the presence or absence of the drug. The more drug that was present, the less antibody was available to bind to the enzyme conjugate and the faster the reaction (15). The door now appeared to be open to developing sensitive and simple mix-and-read assays for almost anything that would bind to a receptor. As so often happens in science, related work unknown to us was being carried out in other laboratories. An assay that worked just like RIA but with an enzyme substituting for the isotopic RIA label was demonstrated nearly simultaneously by Engvall and Perlman (16 ) and Van Weemen and Schuurs (17 ). Although this enzyme-linked immunosorbant assay (ELISA) proved to be a good alternative to RIA and is still widely used, it suffered from the same separation and washing problems as RIA. By contrast, our enzyme multiplied immunoassay technique (EMIT®) simply required mixing the reagents followed by a light-scattering measurement using an inexpensive photometer widely available in clinical laboratories. Since EMIT and the earlier nonseparation assays, FPIA and FRAT, were carried out solely in solution and did not involve the separation of the bound and free labeled reagent, we called these methods “homogeneous” immunoassays to distinguish them from the more conventional “heterogeneous” ELISA and RIA. The Vietnam war ended in 1972 and a field test for drugs of abuse was no longer needed. FRAT, with its clumsy lowsensitivity EPR instrument became history, and Syva’s modest one-year profits turned into large losses. Fortunately, by 1973,

Figure 3. Principle of EMIT using (A) antibody-induced steric hindrance to substrate binding and (B) antibody-induced conformational changes to modulate the activity of a drug–enzyme conjugate. When drug is present the antibody is unavailable to inhibit enzyme.


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an EMIT drugs-of-abuse panel was launched, which gradually replaced other forms of drug testing such as thin layer chromatography. However, it soon became clear that the EMIT assays had serious limitations. The killed bacterial lysozyme substrate worked well with urine samples but was practically useless for serum assays because serum agglutinated the bacteria. Fortunately, other enzymes were found, including malate dehydrogenase (MDH) and glucose-6-phosphate dehydrogenase (G6PDH), that overcame the problem. These enzymes had low-molecular-weight substrates, yet when conjugated to a drug they could be inhibited by anti-drug antibodies. Various lines of evidence suggested that the inhibition was an effect of the antibody on enzyme conformation (Fig. 3B) rather than steric exclusion of substrate (18). By using a bacterial G6PDH that utilized the coenzyme NAD, interference from the related NADP-utilizing enzyme found in serum could be avoided. With the identification of the utility of G6PDH, quantitative EMIT assays for monitoring drugs in serum could readily be constructed. This provided the first opportunity for widescale monitoring of therapeutic drug concentrations in serum to establish optimum dosage regimens. Unfortunately, the development of EMIT assays for proteins proved much more difficult because antibodies to the proteins could bind to protein–enzyme conjugates at sites well removed from the enzyme, where they had little effect on the enzyme activity. It proved possible to construct a commercial assay for C-reactive protein by using β-galactosidase as the enzyme and a synthetic polymeric substrate having exaggerated steric requirements (19), but the method required highly purified antigen and was not satisfactory for general use. One obvious alternative was to use the old precipitin method with modern instrumentation that would permit the use of rate nephelometry for quantitation. This approach was taken by Beckman, which launched its ICS® system for measuring serum proteins. Although this was a successful approach, the method was limited to higher-concentration proteins, at least in part because of variable light scattering caused by the serum samples themselves. Fluorescence Energy Transfer Immunoassay We decided to try to develop a homogeneous protein assay by taking advantage of dipole–dipole coupled (Förster) energy transfer (20). It had already been demonstrated that the distance between defined sites on a protein could be estimated when the sites were labeled respectively with a fluorescer and an energy acceptor (21). The efficiency of energy transfer is directly proportional to the overlap of the donor emission and the acceptor absorption and inversely proportional to the sixth power of the distance between the two groups. Since energy transfer up to 80Å was readily measured with available dyes, it should only be necessary to label a protein antigen with a donor and its corresponding antibody with a fluorescent acceptor. Light energy absorbed by the donor would be transferred to the acceptor and detected as acceptor fluorescence. Moshe Schwarzberg, who had recently joined Syva from the Weizman Institute, undertook this project. He labeled antibodies to albumin with a tetramethylrhodamine acceptor and labeled albumin with a fluorescein donor. On mixing these reagents, binding was expected to produce an induced rhodamine fluorescence. In the presence of unlabeled albumin the binding would be inhibited and the signal would be corre784

spondingly reduced. However, little increase in fluorescence could be observed over the strong emission produced by direct light absorption by rhodamine. Nevertheless, there was an easily measured decrease in the fluorescence of the fluorescein donor which was reversed when unlabeled albumin was present (22, 23). By replacing this competitive immunoassay format with a sandwich format, the assay sensitivity could be improved. Competitive assays depend upon competition of the sample antigen and a labeled antigen for binding to a limited amount of antibody, and are limited in sensitivity by the affinity of the antibody. In sandwich immunoassays two antibodies simultaneously bind to separate regions of the antigenic analyte. Since the antibodies can be used in excess, the sensitivity limitation due to affinity is circumvented (see paper by R. Ekins on pages 769–780). When we labeled a portion of our polyclonal anti-albumin antibody with the rhodamine acceptor and another portion with the fluorescein donor, energy transfer occurred when both antibodies bound to the same molecule (Fig. 4). This new homogeneous fluorescence energy transfer immunoassay, FETI (now usually referred to as fluorescence resonance energy transfer or FRET), permitted the assay of proteins at concentrations down to 100 pM, and similar sensitivity was possible for small molecules using competitive immunoassays when sufficiently high-affinity antibodies were used. Unfortunately, the ability to detect only decreases in fluorescence instead of the appearance of a fluorescent signal limited further improvements in sensitivity, a barrier that has since been overcome with the advent of new donor–acceptor pairs (24). Competition Arrives One of the interesting dynamics of companies is the fear of replacing a very successful product with an untried improved product. This can so paralyze a company that even when it becomes evident that a competitor will introduce the improvement first, it becomes impossible to act. FETI was long delayed because of these fears. It was fully nine years from the demonstration of FETI until the introduction in 1982 of the Syva Advance® system, a fully automated immunochemical analyzer that could handle all but the lowest-concentration

Figure 4. Fluorescence energy transfer immunoassay (FETI). Energy transfer occurs when a fluorescent donor (D) and an acceptor (A) become bound in an immune complex. In the sandwich assay an excess of antibody is used and binding to antigen increases energy transfer (fluorescence of D decreases and A increases). In the competitive assay, antigen competes with labeled antigen for a limited amount of labeled antibody. Binding to antigen therefore decreases energy transfer (fluorescence of D increases and A decreases).



analytes. Numerous redesigns over the years, driven by the ambivalence of the company to support the project, resulted in a disastrously unreliable instrument, and despite reliable chemistry, the overall system was a failure. This proved to be a major turning point in the field of clinical immunodiagnostics. After the introduction of EMIT, Abbott Laboratories initiated a program that would take advantage of Dandliker’s FPIA using modern fluorometric methods. However, Abbott was apparently hesitant to launch a product in the face of Syva’s dominance in therapeutic drug monitoring. As soon as it became clear that Advance was a failure, Abbott quickly introduced their TDx system for the assay of therapeutic drugs using FPIA. The Abbott system did not provide increased analytical capabilities over EMIT and unlike FETI it was not readily adaptable for proteins. However, major improvements in fluorometry had occurred since Dandliker’s original studies and Abbott’s user-friendly instrumentation quickly took over much of the therapeutic drug monitoring market that Syva had pioneered. Only the drugs-of-abuse screening remained dominated by EMIT, because the Abbott instrument was not designed for highvolume screening. The power of homogenous immunoassays became widely appreciated following the introduction of EMIT. Not only did Abbott develop FPIA but Ames Diagnostics developed a variation in which an enzyme cofactor served as a label in place of the enzyme label in EMIT (25). When antibody bound to the cofactor-labeled antigen the enzyme could not turn over. However, the presence of free cofactor in clinical samples and the need for high enzyme concentrations limited the utility of the method. Microgenics commercialized another variant, CEDIA, in which the EMIT enzyme was replaced by a peptide derived from β-galactosidase (26 ). Antigen conjugates of the peptide were designed that were able to bind to and activate a recombinant fragment of an enzyme that lacked the peptide. Binding of the conjugate by antibody reduced the enzyme activity by interfering with the assembly of the enzyme fragments. Several companies also began developing more advanced versions of latex agglutination, which took advantage of laser-light-scattering and particle-counting techniques to overcome light scatter from the sample. However, all of these homogeneous methods were affected to some degree by serum components, and none was able to achieve the sub-picomolar sensitivities available by ELISA and RIA. We therefore turned our attention to designing a homogeneous immunoassay that was insensitive to the size of the analyte and at least as sensitive as the best heterogeneous immunoassay. For this purpose it would be necessary to find a more sensitive method of detecting an antibody–antigen–antibody sandwich structure. Alternative Immunoassay Concepts One approach to a homogeneous sandwich enzyme immunoassay required an enzyme that would produce a product that would serve as the substrate for a second enzyme. Mosbach (27 ) had previously shown that when such enzymes are both attached to the same particles in a particle suspension the final product is produced more rapidly than when the enzymes are segregated on separate particles. This occurs because the second enzyme is localized exactly where the concentration of its substrate is highest. David Litman applied this principle to an immunoassay by using a peroxidase-labeled antibody

Figure 5. Enzyme channeling immunoassay using an antibody– horseradish peroxidase (HRP) conjugate. (A) Antigen (circles) from the sample binds the HRP conjugate at a glucose oxidase (GO) coated surface. The bound HRP catalyzes the oxidation of a chromogenic substrate by hydrogen peroxide produced by GO-catalyzed oxidation of glucose. Hydrogen peroxide that diffuses into the bulk solution is diluted and destroyed by catalase. (B) Solution phase formation of an immune complex containing GO and HRP is detected by creating a GO coated surface in situ. A sample is combined with an antibody–GO conjugate and an antibody–HRP conjugate. A complex forms when antigen is present. Addition of anti-glucose oxidase antibodies and GO produces a finely divided polymeric suspension that catalyzes the formation of a colored product only if a complex has formed.

and latex particles coated with a second antibody and glucose oxidase (28). When antigen was present the peroxidase-labeled antibody bound to the particles. In the presence of glucose, hydrogen peroxide was produced at the particle surfaces and the bound peroxidase catalyzed the formation of colored product more efficiently than when it was free in solution. By also including catalase, hydrogen peroxide that accumulated in the bulk solution was destroyed and a nearly linear assay response was achieved (Fig. 5A). This “enzyme channeling immunoassay” could be set up as either a competitive or a sandwich assay. Ian Gibbons subsequently found that the particles could be replaced by fully soluble reagents that formed a dispersed immunoprecipitate during the assay (Fig. 5B), and a sandwich assay could be constructed that detected down to 20 attomol (200 fM) of polyribose phosphate, a capsular antigen from Haemophilis influenzae (29). However, the effect of serum components on the enzyme activities could not be adequately controlled and the method was never used clinically. With the recurrent problem of sample interference in enzyme immunoassays, we decided to turned our attention to latex agglutination assays. In this method antibodies are bound to latex particles and changes in light scattering signal


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Figure 6. Fluorescence particle quenching immunoassay for IgE. Fluorescent latex particles coated with anti-IgE are quenched when mixed with IgE and an excess of charcoal that is coated with a second less specific anti-immunoglobulin antibody.

antigen-modulated particle aggregation. Although light scattering by sample components was a major problem, exceptional sensitivity was reported when the analyte was in an ultra-clean particle-free solution (30). Thus it seemed possible that detection by some method other than light scattering might avoid the sample interference problem without compromising sensitivity. To this end a homogeneous assay was constructed using fluorescent latex particles coated with a monoclonal antibody specific to IgE and carbon particles coated with antiimmunoglobulin polyclonal antibodies (Fig. 6) (31). As little as 10 pM IgE in serum produced detectable quenching. Microphotographs showed that the quenching was due to embedding of the fluorescent particles into a light-absorbing carbon matrix. These results seemed to be in the right direction, but the large amount of agglutination required to produce quenching seemed unlikely to provide high sensitivity. Moreover, a decrease in an initially strong signal is much less readily detectable than a signal that is produced over a negative background. The ideal approach would be to use chemiluminescence for detection. Chemiluminescence can be generated with very low background signals and by the late 1980s was proving to be more sensitive than fluorescence, at least in heterogeneous immunoassays. However, despite the potentially high sensitivity of chemiluminescence, interference by the presence of the clinical sample in homogeneous chemiluminescence assays was a daunting problem. During early studies of energy transfer immunoassay, Ed Maggio, a Syva scientist, had demonstrated energy transfer from chemi-excited luminol to a fluorescent acceptor located in the same immune complex (32), but inconsistent quenching by different serum samples was uncontrollable. Despite subsequent reports of homogeneous chemiluminescent immunoassays (33), this problem presented a barrier to practical implementation of this opportunity (34).

Figure 7. Luminescence oxygen channeling immunoassay (LOCI). Antibody-coated particles containing a photosensitizer (S) and antibody-coated particles containing a chemiluminescent olefin (CL) form particle pairs in the presence of an antigen (Ag). Irradiation of the mixture leads to photosensitized formation of singlet oxygen (1∆ gO2). Only CL that is associated with particle pairs can react with the 1∆ gO2 before it decays back to ground state oxygen. Light emitted by the CL- 1∆ gO2 reaction product is detected after cessation of irradiation.

Figure 8. Concentration of 1∆ g O2 as a function of distance from a 180-nm-diameter sensitizer particle at a typical light intensity. Surface coatings cause a bound chemiluminescent particle to be positioned at a distance from the surface depicted by the vertical lines.

LOCI—Scientific Success, Commercial Question The insight that led to a successful particle-based homogeneous chemiluminescence immunoassay arose from studies related to photodynamic therapy for cancer. In this procedure a patient is treated with a photosensitizer that becomes localized at the tumor. Upon irradiation of the tumor the sensitizer is excited and transfers its energy to oxygen. The resulting excited singlet state of oxygen (1∆gO2) is highly reactive. Because of its short lifetime in water (4 µs) it can migrate only a short distance, but if it diffuses into a nearby cell it causes cell death. Levy had shown that photosensitizers could be specifically delivered to a target tissue by attaching them to antibodies 786

Figure 9. LOCI response when streptavidin-labeled sensitizer particles were titrated into 109 biotin-labeled chemiluminescer particles/mL (u) and when biotin-labeled chemiluminescer particles were titrated into 109 streptavidin-labeled sensitizer particles/ml (+).



specific to the tissue (35). Further, she was able to construct an in vitro immunoassay for cell surface antigens in which cells were selectively killed upon irradiation in the presence of sensitizer-labeled antibodies. These developments set the stage for an idea that might permit homogeneous quantitative detection of subfemtomolar concentrations of both large and small molecules. Two types of latex particles would be used. One would carry a large number of photosensitizers and the other a large number of molecules designed to chemiluminesce upon reaction with singlet oxygen. Antigen would modulate binding of the particles to each other as in latex agglutination, but detection of binding would be by chemiluminescence instead of light scatter (Fig. 7). We calculated that during irradiation a halo of singlet oxygen should extend on the order of 200 nm about each of the sensitizer particles, much as the product of the first enzyme in enzymechanneling immunoassays. Chemiluminescer particles that become bound to the sensitizer particles would be exposed to singlet oxygen, but the effect on particles farther away than about 300 nm would be negligible (Fig. 8). Since the chemiluminescent decay would not be instantaneous, the signal could be detected after a short irradiation period and would be proportional to the number of particle pairs formed. Hrair Kirakossian carried out the initial experiments on this luminescence oxygen channeling immunoassay (LOCI), and quickly demonstrated the remarkable sensitivity of the method (36 ). Small latex particles (~220 nm) in which a phthalocyanine photosensitizer was dissolved were decorated on their surface with biotin, and similar particles containing N-methyl-9-benzalacridan were coupled to streptavidin, a protein that binds strongly to biotin. After addition of as few as 1000 of one type of particle into a 1-mL suspension of a large excess of the other particle, irradiation (680 nm) induced detectable chemiluminescence of the acridan. The response remained linear upon addition of up to 108 particles per milliliter (Fig. 9). In this model assay, a rosette of about eight particles forms for each particle added. By contrast, in a sandwich immunoassay only two particles become glued together for each antigen molecule added. This plus the need to dilute serum samples about 10-fold reduces the relative sensitivity of LOCI clinical immunoassays somewhat. However, detection of 12.6 femtomolar thyroid stimulating hormone in serum (400,000 molecules in the assay mixture) (37 ) and 2 pg/mL of hepatitis B surface antigen exceed by up to an order of magnitude the sensitivity of currently used methods. These ultrasensitive assays can be carried out in 15–30 minutes and only about a factor of four in sensitivity is sacrificed using times as short as 3 min. Interference from components in the sample is remarkably low. Both formation and reaction of singlet oxygen occur in polymer matrices, out of molecular contact with sample components, and the short time required for it to diffuse through the sample medium only permits interception by high concentrations of very reactive compounds, which are not found in serum. Simple mix-and-read LOCI assays for antibodies, nucleic acids, drugs, proteins, and even cell surface receptors have recently been demonstrated and experiments have been carried out suggesting that the sensitivity can be further increased. Additionally, because the reagents are thermally stable, it has been possible to amplify DNA by PCR and detect the product by LOCI with no fluid-handling steps other than the initial

filling of a tube with sample and reagents. Assay volumes as low as 20 µL have been used without serious loss in sensitivity (~10,000 molecules), and the detection instrumentation required is little more than a luminometer equipped with a laser light source and shutter. With the demonstration of the remarkable sensitivity, speed, and simplicity of LOCI, the holy grail of immunodiagnostics, a universally applicable supersensitive homogeneous assay seems to be in hand. However, no LOCI assays are commercially available and its future prospects remain uncertain. Unfortunately, superior technology does not necessarily translate into commercial products. The current efforts to cut medical costs and the resultant merger frenzy are discouraging companies from taking risks with new technologies despite the potential cost savings and improved competitive position. Syva has not been exempt from this phenomenon and in the past four years has been sequentially owned by Syntex, Roche, Behring, and Dade Behring. It remains to be seen whether the surviving companies in the ongoing dance will have any interest in further pushing the frontiers beyond currently available technology. Epilogue Many of the remarkable scientists that made this story possible went on to numerous additional accomplishments. Carl Djerassi won the Priestley Medal and has become one of the foremost writers of science in fiction; Harden McConnell founded Molecular Devices; William Little founded MMR; Alejandro Zaffaroni founded Alza, DNAx, Affymax, and Affymetrix; Richard Leute founded Abaxis; Prithipal Singh founded Chemtrak and LNX; Fred Gamble became director of research at Exxon; David Boocock is professor of chemical engineering at University of Toronto; Edward Maggio founded Symbiotics, Immunopharmaceutics and Bioinformatics; Ronald Breslow was elected president of the American Chemical Society; Avram Goldstein founded the Addiction Research Center; Richard Schneider became president of Liposome Technologies and later joined a venture capital company; David Litman is VP and Director of R&D at Becton Dickinson. Many others not mentioned here also hold leadership positions in academia or in the diagnostic industry. I owe a great debt of gratitude to all these able collaborators who helped in the creation of Syva and in many ways created the foundation of today’s immunodiagnostic industry. Literature Cited 1. Little, W. A. Sci. Am. 1965, 212(2), 21. 2. Gamble, F. R.; Osiecke, J. H.; Cais, M.; Pisharody, R.; DiSalvo, F. J.; Geballe, T. H. Science 1971, 174, 493. 3. Boocock, D. G. B.; Darcy, R.; Ullman, E. F. J. Am. Chem. Soc. 1968, 90, 5945. 4. Boocock, D. G. B.; Dorchai, R. O.; Osiecke, J. H.; Ullman, E. F. 4,5-Substituted N-Oxy and Hydroxyhydroimidazoles; U.S. patent 3,799,942, 1974. 5. Kopf, P. W.; Kreilick, R.; Becher J.; Ullman E. F. J. Am. Chem. Soc. 1969, 91, 5121. 6. Leute, R.; Ullman, E. F.; Goldstein, A.; Herzenberg, L. A. Nature New Biol. 1972, 236, 93. 7. Leute, R.; Ullman, E. F.; Goldstein A. J. Am. Med. Assn. 1972, 221, 1231. 8. Bechhold, H. Z. Phys. Chem. 1905, 52, 185. 9. Wassermann, A.; Neisser, A.; Bruch, C. Dtsch. Med. Wochenschr. 1906, 32, 745.


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Homogeneous Immunoassays: Historical Perspective and Future

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