ADVANCES IN SIMPLE IMMUNOASSAYS FOR DECENTRALIZED TESTING Ranald M. Sutherland and Barry Simpson Abbott GmbH Diagnostica, 6200 Wiesbaden-Delkenheim, Federal Republic of Germany I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Simple Immunoassays: Utility and Specifications .............................. 2.1. Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Assay Requirements sting . . . . . . . . . . . . 3. New Developments in Sim 3.1. Particle-Based Immunoassays. 3.2. Membrane-Support Immunoassays ..................................... 4. Discussion. . . . . . . . . . . . . References ........................................................
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1. Introduction In v i m testing based on immunological principles is currently one of the most important technologies for investigation and detection of disease. The reaction of an antiserum and an antigen to form a precipitated immune complex was originally described as the “precipitin” reaction in 1897 by Kraus (from Bl). This fundamental reaction is the basis of what is today a global, multibillion-dollar industry, characterized by leading-edge technology, multidisciplinarity, and the rapid development and worldwide introduction of the fruits of university-level research in one of the most rapid product cycles of any modem industry. A recent and visible example is the discovery of the link between the HTLV-111 (now HIV) retrovirus and the clinical disease of AIDS. From making the virus available in tissue culture to marketing of the first blood bank screening test there was only a delay of 9 months. The subject of this paper, simpler immunoassays, is another example of the rapid development and complexity of modem immunodiagnostics; it also demonstrates how new tests are being constructed to meet the changing needs of modem health care. Historically, quantitative immunoassays are centralized in hospital laboratories 93 Copynght 0 1990 by Academic Press, Inc. All rights of repduction in any form reserved.
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due in part to the need for highly qualified technical staff and also for complex instrumentation. Other reasons include problems with reagents and standardization variability and assay “nonruggedness.” The latter term is defined as meaning that a variety of calibrator and quality control materials must be included within each assay. Thus, practically and economically, most assays are run with batches of samples per analyte. However, batch-mode analysis is not the best solution for all clinical (and patient) needs. This is one of the driving forces that has resulted in simpler immunoassays, promoting a change in the requirements and aspirations of both the consumer and the providers of health care. A second driving force is technology per se, and a number of key innovations deserve further discussion here. First, the use of alternative labels should be considered. Quantitative immunoassays were originally constructed with radioisotopic labeling (Y1). Such radioisotopes have many analytically desirable characteristics, including high specific activity and low “environmental” background, and they can be detected in a very selective manner. However, the disadvantages are significant, the most important being short half-life, environmental undesirability (difficult handling, disposal, etc.), and the requirement for expensive capital equipment, laboratories, and specialized staff. The concept of alternative labels for immunoassays has been available since at least 1941 (C5), when fluorochrome-labeled antisera were used for the microscopic visualization of different cell types. Similarly, in 1966, Avrameas et al. (A4, A5) proposed the use of enzymes as labels for immunoassays, and many novel nonisotopic labels have since been developed (Cl, C2, El, L5), pushing back not only the boundries of analytical sensitivity, but also allowing the design of more rapid, simpler tests. The potential benefits in terms of sensitivity have been well discussed previously (e.g., Jl), but application of nonisotopic labels to simpler immunoassays is one focus of this paper. The second area of technological development that impacts modern immunoassays relates to the whole arena of biotechnology. Two major lines of development should be mentioned: the use and production of monoclonal antibodies (Kl) and the ability to analyze key epitopes on antigens, which allows synthetic gene construction and the expression and synthesis of desired proteins (e.g., M3) in effectively unlimited quantities for either immunization or for diagnostic test kit production. There is probably not a single immunoassay that has been introduced in the last 3 years that does not contain one or more of the reagents derived from these advances. The third major area of technology development that is impacting how immunodiagnostic tests are being perceived and used by the consumer is the production of assays with minimal operator intervention. This article will address the design of simple immunoassays as an illustration of how the three facets of evolving technology, vis-5-vis reagents, labels, and the physical execution of assays, are major driving forces in in vitro diagnostics.
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2. Simple Immunoassays: Utility and Specifications 2.1. UTILITY There are many potential clinical areas of application for measuring analytes using simple immunoassays. Some key issues relate to (1) the degree of urgency for the results, (2) requirements for privacy of the results, and (3) convenience of the results in terms of time and financial cost. Urgency is a factor when treatment has to be rapidly prescribed and when an in v i m test result is an important consideration. Although rapid results are not currently well addressed in product literature, areas of application include differential diagnosis of acute diseases, such as is found with large vessel disease and different forms of acute pancreatitis, and recognition and avoidance of allergic responses to drug regimens. The need for privacy of results is a factor with pregnancy and fertility tests that are currently for sale in pharmacies and even in some supermarket chains. In the future, this market may expand to include tests for detection of sexually transmitted diseases and into areas of fitness, or “wellness,” testing. The third factor, convenience, includes a broad range of applications wherein it may be advantageous to have a test result immediately available at the time the patient is being examined by the physician. Examples include therapeutic drug monitoring, longer term therapeutic regimes (e.g., diabetes, anticoagulant therapy, and antihypertensive therapy), and many instances wherein the result of a test can eliminate the need for a follow-up visit. In nearly all of the above examples, whether in a physician’s ofice, at home, or in a hospital intensive care unit, the in situ availability of appropriate technical skills to, carry out laboratory-type tests is minimal. This means that for the successful execution of simple immunoassay tests, a number of critical technical and performance requirements are imposed on test design. These will be addressed in the next section.
2.2. ASSAYREQUIREMENTS Assay requirements have been the subject of several reviews (H2, Ml), and are most readily illustrated as a list (Table 1). This background information demonstrates some of the challenges that face the design and fabrication of a simple immunoassay device that will achieve success in result quality and user acceptance. There is probably no product currently available that meets all of these requirements. However, there are a number of very successful devices that already have made significant strides in positioning testing in the hands of the nonskilled user. It is these devices that are the main subject of this article.
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RANALD M. SUTHERLAND AND BARRY SIMPSON TABLE 1 DESIGNGOALSFOR SIMPLE IMMUNOASSAYS Broad range of sensitivity Analyte specific Sufficiently rapid Operator independent with respect to chemistry, timing, and pipetting Reagents are stable Self- or factory calibrated Appropriate for mass manufacture Applicable for different assay formats Disposable Competitively priced
3. New Developments in Simple lmmunoassays for DecentraIized Testing
This review is directed at technically the simplest forms of immunoassay that are currently available and that do not require complex instrumentation. There are at least three basic technological approaches being used today (Table 2). Immunoassays, in which one of the immunological binding pair is particle bound, e.g., to latex or sensitized red blood cells, were proposed as early as 1951 (B2, 01). These qualitative assays are still in broad use, particularly in the field of pregnancy testing, bacteriology, and serology. This type of test has a drawback of being difficult to interpret and can thus have poor discriminatory power. However, many particulate immunoassays offer advantages in simplicity, fast results, adequate sensitivity for many qualitiative applications, good reagent
TABLE 2 A CLASSIFICATION OF SIMPLEIMMUNOASSAYS BASEDON TECHNOLOGY ~~
Class (A) Microparticle (B) Membrane/ filter-support (C) Inununosensors
Example Latex Hemagglutination Inununofiltration Immunochromatography TIRFa Surface plasmons
O T I R F , Total internal reflection fluorescence.
Reference H1 B2 A l , B3 A2, C4
s2 L4
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shelf-life, and reasonable costs. For these reasons alone such assay types will be in use for many years to come, and are currently the subject of development to improve the weaker performance characteristics. The membranelfilter-support systems, usually with a visual, colored, end point, represent the new wave of simple assays and were only recently introduced. These assay systems are being continually updated and improved in terms of sensitivity, speed, facility of use, and control of result quality. However, the third technological approach, the, “immunosensor” device, although the subject of considerable literature discussion in recent years, has yet to be developed into a successful product. The reader is referred to recent reviews (A3, M4, Pl), as immunosensor technology will only be briefly discussed herein. In the following sections are discussed the various successful approaches being used, with examples of each. 3.1. PARTICLE-BASED IMMUNOASSAYS
Two examples of recent developments in particle-base immunoassays will be discussed; the first two are based on latex particles. A novel latex agglutination assay has been described using colored latex particles (Hl). The reagent suspension is composed of two or more differently colored particles (red, blue, or green). In this example, each particle is coated with a different antibody for each color. Thus, on reaction with a sample, depending on the antigen present, latex particles of one color agglutinate and are visually more apparent due both to concentrated color of large complexes and to the reduction of the “dispersed” color in the background. The test is currently applied to the detection of three different Salmonella serogroup antigens, B, C, or D. The use of one sample for multiple testing in a single assay has several advantages, including reduced sample volume requirements and reduction in time to produce multiple results. However, results produced using this system will still suffer from highly subjective discrimination and the system is only appropriate for testing wherein the results are mutually exclusive. Without some objective reading device, it may be difficult to expand this technology into other areas of testing. The second example is based on attaching the antibodies to metal sol particles. The use of metal sol particles, especially gold sols on the order of 5 nm to 10 p,m in diameter, has been described, particularly for protein blotting assays (H4). Here, homogeneous immunoassays for urinary hCG (L2) and estrogens (L3) using sol gold-labeled monoclonal antibodies have been described based on the optical characteristics of the gold particle. The assay format is based upon reacting antibody-coated gold sol particles with a standard quantity of multivalent antigen. The antibodies bind the soluble antigen and thus the small particles form larger and larger particle aggregates. From classical physical theory, Rayleigh’s law relates light-scattering efficiency to the particle diameter, and to
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both the incident light wavelength and relative refractive index ratio. All else remaining constant, the effective light-scattering ability of the gold sol particle will increase with particle aggregation, and the optical dispersive properties change with aggregation, resulting in a visibly colored solution. However, on addition of a sample containing the same antigen, the formation of particle aggregates will be reduced, as the sample antigen will compete for available antibody-binding sites, and the resultant color will be visibly less. Such an assay system can obviously be designed to either increase or decrease in color when the target antigen is present in a sample. As discussed with the colored latex approach, this technology is also prone to highly subjective interpretation of the results. Unlike the colored latex approach, however, the reading of results is not carried out against a relatively high noise level (caused by other colored particles, which do not actively contribute to the signal but increase the background significantly), and it may be expected that this approach is less subjective.
3 . 2 . MEMBRANE-SUPPORT IMMUNOASSAYS The assay formats used with membrane immunoassays are very diverse, but are all primarily designed to reduce operator reliance and involvement. There are currently three basic assay formats, which have been called (1) immunoconcentration, or “pour-through devices,” (2) immunochromatography, and (3) the “dipstick” format. In all cases the assays are heterogeneous (i.e., requiring separation steps) and retain good sensitivity, generally by removing these key separation steps, which are arguably the main source of assay imprecision, from the operator’s hands.
3.2.1. Pour-Through Devices The types of devices used here essentially consist of two main parts; first, a filter or membrane pad in which the specific reactions take place, and second, an absorbent pad, which not only is the “waste sink” for excess liquids, but also acts as a wicking agent exerting a “sucking” force, causing solutions to be pulled through the reaction pad. Basic designs were proposed as early as 1980 (C3) for filtration devices to separate insoluble immune complexes generated by a doubleantibody precipitation radioimmunoassay (RIA). However, the new devices are more complex and can be typically represented as a “pour-through” system, with multiple layers of membranes and/or filters, and a visual end-point signal detection. A typical example is the Abbott TestPack (B3), which is used herein to illustrate some of the basic components and characteristics of such devices. An exploded view is given in Fig. 1 , where A and E are the exterior shell components. In this example the reaction pad is a flat circular fibrous support (D). The specific immunological binding reagents are attached to microparticles that have a diameter smaller than the pore size of this support, and so in a certain sense this
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FIG. 1. Exploded view of the Abbott TestPack single-use pour-through immunoassay device. See text for explanation.
also represents a microparticle-based assay. Underneath the reaction pad is the absorbent pad (B), and there can also be a third membrane (C) sandwiched between these two pads that can act as a barrier restricting fluid flow (e.g., to reduce the backflow of reagents). This particular example also has a removable filter (F) above the reaction pad, which helps prevent larger particulate materials from the sample from blocking the pores of the reaction pad. With such a device, the assay protocol is relatively simple and for most qualitative assays accurate pipetting and timing is deemphasized. The assay protocol for the detection of antibody to the two human immunodeficiency viruses (HIV-1 and HIV-2) in human serum or plasma is used for illustrative purposes (A 1). Here, the antigen consists of three recombinant proteins, HIV-1 core and envelope and HIV-2 envelope, which are precoated onto the microparticles (for a summary of the various HIV proteins and their use in assays, see Ref. F l , G1, M2). An initial dilution step is carried out by simply dipping a collection loop into the sample and transferring it to the diluent and swirling, avoiding the requirement for accurate pipetting steps. The reaction pad and filter are prewetted and the diluted sample is added by pouring it onto the filter and then allowing it to flow through completely. A wash solution is then immediately poured onto the filter and a solution of antihuman IgG (goat) coupled to alkaline phosphatase is added dropwise onto the filter. Following a 3-min incubation, the filter is removed, and a second wash solution is added to remove unbound materials from the reaction pad. Specifically bound antihuman IgG is then visualized by the dropwise addition of the enzyme substrate solution, a 2min incubation, and addition of the enzyme stop solution. This assay protocol is a good example of how systems are being designed with the aim of achieving simple immunodiagnostics. Reagents are prepackaged, assay protocols eliminate the necessity for accurate pipetting, there is minimal operator involvement, the assay is rapid (in this case less than 10 min to result), and the timing is generally controlled by the speed of solution uptake by the device. Other devices are
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RANALD M. SUTHERLAND AND BARRY SIMPSON TABLE 3 SOMETYPICAL APPLICATIONS OF SIMPLE IMMUNOASSAYTESTSON THE ABBOTTTESTPACK Analyte
Sample
Detection limit ~~
hCG hCG Strep A Rotavirus Respiratory syncytial virus Chlamydia
Urine Serum Throat swab Feces
HIV- 1 /2
Serum or plasma
Nasopharyngeal aspirates Endocervical swab
50 m IUlml 25 m IU/ml
NAa >2 x 107 particles NA >20 inclusionforming units NA
ONA, Not available.
available that have essentially similar design characteristics (H3, Vl). Examples of the areas of application of these devices are given in Table 3. One of the key elements in such simple immunoassays is their resistance to operator-induced error. One way this is addressed is by including a procedural control within the device. A binding agent that can capture the labeled antibody is preimmobilized at a site on the reaction pad, which receives exactly the same treatment as the preimmobilized HIV antigens, but is physically distinct from the antigen reaction area. In this fashion, addition of the reagents according to the correct assay protocol will result in the control area being colored, but no color will result if one of the steps is inadvertently missed. In the example, the procedural control is the horizontal line of a + ” sign and the specific binding signal is the vertical line. Similar approaches have been adopted with other designs, but the shapes are in the forms of rounded dots. “
3.2.2. Immunochromatography The principles of immunochromatography can be broadly summarized as allowing a liquid that acts as a solvent to move through a filter strip, to which one or more specific binding agents have been immobilized at predefined locations. These locations are serially hydrated by the moving solvent front and are also the basis of a sequential immunological reactions. Dissolved reagents and sample are carried in the aqueous phase through each location and can thus react with the immobilized specific binding reagents. For example, with a typical “sandwich” assay, the sample would be mixed with labeled antibody and the combined solution would be allowed to wick up the filter strip, to which an alternative antibody is immobilized at a specific site. Given appropriate reaction kinetics
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during sample migration, the labeled antibody will bind to the analyte, and subsequently this complex will then be bound to the second (alternative) antibody at the preimmobilization site. Unbound materials are then removed by the wicking solution and the specifically bound materials are visualized by dipping the complete strip into a substrate solution, or, alternatively, by wicking substrate up the strip. The first example is the Acculevel test (Sylva Corp.), which is based on two technologies, both the immunochromatography technique and the “enzyme-channeling immunoassay” (L6). Here specific signal effectively only occurs when two enzymes are physically in very close proximity, where the product of the first enzymatic reaction is the substrate for the second enzyme-catalyzed reaction, the closeness of the signal generators being achieved via immune complex formation. This basic concept is combined with the immunochromatographic strip format (C4) for a theophylline immunoassay. A monoclonal antibody to theophylline and the enzyme glucose oxidase are coimmobilized along the length of an immunochromatography strip. Sample is mixed with a solution containing the various enzyme substrates, a peroxidase-labeled theophylline conjugate, and an inhibitor to retard color formation until the wicking action is finished. The strip is dipped into this solution and the conjugate travels a distance along the length of the strip approximately directly proportionally to the concentration of sample antigen. The inhibitor, ascorbic acid, acts by reducing an oxidized (colorless) intermediate of the substrate-channeled enzyme reaction, thus preventing color formation. The ascorbic acid is consumed by this reaction and the concentration is selected such that color formation only occurs after the solution has traveled the full length of the strip (i.e., the immunoassay reaction has effectively finished). Results are visually read by comparison with a thermometer-like scale 30 min after starting the assay, and the detection limit is claimed to be at least 5 mg/liter. This assay is easy to use but is biochemically complex in design, and the requirement for multiple reagents to perform the procedure can result in a relatively high cost. An inhibitor is used to retard signal generation; this may indicate that there are limitations in the sensitivity of such a system, particularly with respect to background signals. The second example, equally elegant although considerably less complex in analytical principle, is a combination of the colored latex approach and immunochromatography (the Clearview test; Fig. 2) (UI). For the detection of urinary human chorionic gonadotropin (hCG), five drops of sample are put onto the end (A) of the immunochromatography pad (Fig. 2 I); these then wick up to the first zone (B), where a specific monoclonal antibody is predeposited. This first antibody is coated onto blue-colored latex particles and reacts with sample hCG to form the first part of the “sandwich.” The complex then continues along the pad, drawn by capillary and microfluidic forces (Fig. 2 11; C). This solution contacts the second preimmobilized anti-hCG monoclonal antibody, which is affixed in the form of a line across the pad (D). The final immune complex forms and the
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FIG.2. Schematic of an immunochromatography device. See text for details.
unbound materials are wicked up the pad to a third area (Fig. 2 111; D). Here a second antibody has been preimmobilized and binds the remaining (blue) latexlabeled anti-hCG antibody, forming a second blue line (Fig. 2 IV; E). One blue line represents a negative result and the presence of two blue lines represents a positive result. The assay only uses 0.5 ml of urine, is finished within 5 min, and all reagents are self-contained in the device. This device is currently the only example of a self-performing immunoassay generally available. However the use
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of this assay design has potential limitations. For example, using the colored latex (although avoiding the extra step of the enzyme-substrate reaction, with the extra reagent additions and incubations) rules out the possibility of signal enhancement inherent in using catalytic signal generators. Thus some questions must be posed as to the achievable sensitivity of this approach. Second, and more generally, the opportunity for the immune reaction to go to completion (i.e., maximized specific antibody-antigen complex formation) is limited by the short time that the sample and antibody physically have to react. This may mean that very high-affinity reagents have to be used and in higher concentrations than are normally required for traditional immunoassay methods. Another example of a self-performing assay is the TestPack +Plus. The device (Fig. 3) has a sample application area, a window to read the test result, and an “end-of-assay” window. The result is expressed as positive or negative, depending on the respective presence or absence of analyte. In the hCG urine test, three drops of urine are added to the sample application area and they wick along a nitrocellulose membrane. Any hCG that is present in the sample reacts with a colloid-labeled (Y2) monoclonal antibody to the a-chain of hCG. At the reading site, two sets of reagents have been applied in the shapes of vertical and horizontal bars. The vertical bar, which indicates the presence of antigen in the specimen, has a polyclonal antibody raised against the P-chain of hCG preimmobilized to the nitrocellulose. The horizontal bar, which indicates if the reagents are active, has a complex of anti-hCG : hCG preimmobilized to the membrane. Thus if the monoclonal antibody has been inactivated in some way, there will be no signal in the reading window. The end-of-assay indicator zone shows when the unreacted labeled monoclonal antibody has traveled the distance
FIG. 3. Schematic of the TestPack +Plus device. See text for details.
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of the membrane and that the assay is completed. The assay takes 5 min and has a minimum detection limit of 50 IU/liter. 3.2.3. Dipstick This format is very simple in concept and operation. Here, one of the immunological binding pair is immobilized onto a pad of absorbent material, which is physically fixed onto the end of a “stick.” The sample is mixed with the labeled conjugate and the pad is dipped into this solution, allowing either a competitivetype assay or sandwich-type assay to occur. The pad is washed and bound label is visualized by simply dipping the pad into the substrate solution. Usually the product of the enzyme-catalyzedreaction is insoluble and/or is bound to the pad (e.g., by electrostatic forces). A typical example is illustrated in Fig. 4 (Ll). This dipstick has both a sample pad (B) and a reference pad (A). In this case the reference pad is used to c o n f m that the assay procedure is correctly adhered to and also gives a reference color to which the sample signal can be compared. The example given with this device is for the measurement of morphine. Two labeled conjugates are mixed with the sample; one conjugate binds only to the reference pad and is a biotin-enzyme conjugate, and the second conjugate is a morphine-enzyme conjugate. Immobilized to pad B is a monoclonal antibody to morphine, and avidin is immobilized to the second pad. The presence of morphine in the sample results in a reduced amount of morphine-enzyme conjugate specifically bound to pad B, and the resultant color reaction is weaker than that seen in the control pad. The assay takes 30 min with a sensitivity of at least 30 ng/ml morphine. Limitations with these devices are probably most related to available surface area and the kinetics of the solution/solid-phase interface. With both the immunofiltration and immunoconcentration devices, the specific soluble reaction components are being actively moved close to the immobilized reagents in such a manner that there is a relatively high level of opportunity for reaction; they could be described as three-dimensional in nature. However, the dipstick-type devices are effectively static with respect to their liquid reaction kinetics, and are probably more diffusion limited; i.e., they could be described as two-dimensional. A second difference is that effectively all the sample is given an opportunity to react with
FIG. 4. A drawing of a typical “dipstick” immunoassay device. See text for details.
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the immobilized reagents within the other devices, unlike the dipstick devices, wherein the bulk of solution is not in direct contact with the specific immobilized reagents. Therefore, such a design is more reliant on “passive” diffusion. Such an approach could be significantly improved by using a reliable form of physical mixing, but it is not clear how this may be achieved in the type of testing environment envisaged for these devices.
4. Discussion The arena of simple immunoassay devices is the subject of intensive commercial activity and illustrates an area of development that requires multidisciplinarity, which can only usually be found in a commercial company. This, at least in part, explains the emphasis in this review on commercial developments. However, it should not detract from the fundamental scientific discoveries that are the underpinning of such commercial works. As alluded to in the introductory paragraphs of this paper, the current simple immunoassay devices would not be possible without the development and discovery of many of the current biotechnological skills and technologies such as hybridoma and recombinant DNA techniques. Similarly, the design and manufacturing of such devices are dependent on the developments in materials sciences and on precision liquid-handling techniques that allow depositing tiny volumes of reagent onto well-defined areas of such materials. This is only a partial list of some of the key elements in making and designing a simple immunoassay device. As reviewed above, current systems are well suited to the detection and even semiquantitation of many analytes, as illustrated in Table 3. Future developments will be aimed at designing even simpler devices, two of which were illustrated here-the self-performing TestPack +Plus and Clearview. It is probably inevitable that, as part of this process, simple instrumentation, such as that found for reading blood glucose strips, will be developed for immunoassays. An alternative approach to the above systems may lie in the successful design of a disposable immunosensor. Simple immunoassays are mainly heterogeneous enzyme immunoassays, combined with a device to allow ease of assay execution. Immunosensors are based on homogeneous types of immunoassays, with some of the most promising approaches being nonenzymatic. For example, one type of immunosensor is based on optical devices and was recently reviewed (Sl). In general, the approach taken here is to immobilize one of the binding pair to a continuous surface, such as an optical fiber. This surface has certain optical properties (e.g., transparent or reflective) and can be interrogated with visible radiation during and after the immunological reaction. Thus the growth of the antigen-antibody layer can be detected as modulations of the radiation that impinge on the continuous surface. Such modulations can be chosen to react to
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the interfacial layer thickness and refractive index changes, or equally to a change in specific fluorescence, wherein one of the binding pair is fluorochrome labeled. Application of these basic physical phenomena to monitoring interface immunassays has been described. For example, the use of surface plasmon resonance (L4)can detect minute changes in the relative interfacial refractive index. Similarly, monitoring of fluorescenceat an optical interface can be canied out using total internal reflection fluorescence (TIRF) (S2). The design goals for such immunosensor devices are to be self-contained, rapid, and effectively operator independent. A typical example is proposed in Ref. S 1: the whole device is made from injection-molded plastic and is totally self-contained in terms of reagents. Sample is allowed to wick into the reaction area via capillary action, and the sample volume is defined by the walls of the reaction area. All reagents are preimmobilized or coated onto the interior of the device in a stable and hydrophilic form. The device is inserted into a simple photometer, which can monitor the kinetic immunoassay reaction in real time. In summary, the current developments in simplifying immunoassays have demonstrated the design of many devices that allow performance of these complex techniques in nonlaboratory environments. The current devices are very adequate for many quantitative and qualitative applications in clinical diagnostics. In the future, there may be a role for alternative technologies such as immunosensors, and a short-term goal is for simple instrumentation such as optical reading devices. REFERENCES Al. Abbott TestPack, HIV-l/HIV-2 package insert. BlA839 ref no. 82-2995/R2 (1989). Abbott Laboratories, Abbott Park, Illinois. A2. Abbott TestPack +Plus package insert. B2A991 ref no. 83-5533/Rl (1990). Abbott Laboratories, Abbott Park, Illinois. A3. Andrade, J. D., Van Wagenen, R. A,, Gregonis, D. E., Newby, K.,and Lin, J.-N., Remote fibre-optic biosensors based on evanescent excited fluoroimmunoassay: Concept and progress. IEEE Trans. Electron. Devices 32, 1175-1 179 (1985). A4. Avrameas, S., and Uriel, J., MCthode de marquage d’antigtnes et d’anticorps avec des enzymes et son application en immunodiffusion. C.R. Hebd. Seances Acud. Sci. 262,25342535 (1966). A5. Avrameas, S., and Berson, S. A,, Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochimie 54, 837-842 (1972). BI. Barrett, J. T., “Textbook of Immunology.” Mosby, St. Louis, Missouri, 1988. B2. Boyden, S. V., The adsorption of proteins on erythrocytes treated with tannic acid and subsequent haemagglutination by antiprotein antisera. J . Exp. Med. 93, 107-1 15 (1951). B3. Brown, W. E., Devereaux, DS. M., Knigge, K. M., Clemens, J. M., Hofler, J. G., and Safford, S. E., Solid-phase analytical device and method for using same. Eur. Pat. Appl. 86 6,113,675 (1987). C1. Cambiaso, C. L., Leek, A. E., De Steenwinkel, F., Billen, J., and Masson, P. L., Particle
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counting immunoassay (PACIA). 1. A general method for the determination of antibodies, antigens and haptens. J. Immunol. Methods 87, 18, 33-44 (1977). C2. Canico, R. J., Christner, J. E., Boguslaski, R. C., and Yeung, K.K., A method for monitoring specific binding reactions with cofactor labeled ligands. Anal. Biochem. 72, 271 1-282 (1976). C3. Chalkey, S. R., and Renshaw, A., Microfiltration as a means of separating free antigen from antigen-antibody complexes in immunoassay. In “Methods in Enzymology” (H. V. Vunakis and I. J. Langone, eds.), vol. 70, pp. 305-314. Academic Press, New York, 1980. C4. Chen, R., Li, T. M., Memcck, H., Parrish, R. F., Bruno, V., Kwong, A,, Stiso, C., and Litman, D. J., An internal clock reaction used in a one-step enzyme immunochromatographic assay of theophylline in whole blood. Clin. Chem. (Winston-Salem, N.C.)33, 1521-1525 (1987). C5. Coons, A. H., Creech, H. I., and Jones, R. N., Immunological properties of an antibody containing a fluorescent group. Proc. SOC. Exp. Biol. Med. 47, 200-210 (1941). E l . Engvall, E., and Perlmann, P., Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8, 871-880 (1971). F1. Fresner, G. G., Erfle, V., Mellert, W., and Helmam, R., Diagnostic significance of quantitative determination of HIV antibody specific for envelope and core proteins. Lancet 1, 160 (1987). G I . Gelderblom, H. R., Hausmann, E. H. S., Winkel, T., Pauli, G., and Koch, M. A,, Fine structure of HIV and immunolocalisation of structural proteins and virus-cell relations. Virology 156, 171-176 (1987). HI. Hadfield, S. G., Lane, A., and McIllmurray, M. B., A novel coloured latex test for the detection and identification of more than one antigen. J. Immunol. Methods 97, 153-168 (1987). H2. Hicks, J., In situ monitoring. Clin. Chem. (Winston-Salem, N.C.) 31, 1931-1935 (1985). H3. Hossmom, M. G . , Transverse flow device. U.S. Pat. 4,693,834 (1987). H4. Hsu, Y. H., Immunogold for detection of antigen on nitrocellulose paper. Anal. Biochem. 142, 221-225 (1984). J I . Jackson, T. M., and Ekins, R. P., Theoretical limitations on immunoassay sensitivity. Current practise and potential advantages of fluorescent Eu chelates as non-isotopic tracers. J . Immunol. Methods 87, 13-20 (1986). K1. Kohler, G., and Milstein, C., Derivation of specific antibody producing tissue culture and tumour lines by cell fusion. Eur. J. Immunol. 6, 511-520 (1976). L1. Lee, T. T.-T., and Katz, D. H., Apparatus for use in detecting ligands in solution. Eur. Pat. Appl. 85 3,007,785.7 (1986). L2. Leuvering, J. H., Goverde, B. C . , Thal, P. J., and Schuurs, A. H., A homogeneous sol particle immunoassay for human chorionic gonadotropin using monoclonal antibodies. J . Immunol. Methods 60, 9-23 (1983). L3. Leuvering, J. H., Thal, P. J., White, D. D., and Schuurs, A. H., A homogeneous sol particle immunoassay for total oestrogens in urine and serum samples. J . Immunol. Methods 62, 163174 (1983). LA. Liedberg, B., Nylander, C., and Lundstrom, I . , Surface plasmon resonance for gas detection and biosensing. Sens. Actuarors 4, 299-304 (1983). L5. Litchfield, W. J., Freytag, J. W., and Adamich, M., Highly sensitive immunoassays based on the use of liposomes without complement. Clin. Chem. (Winston-Salem, N.C.) 30, 144-145 (1984). L6. Litman, D. J . , Hauboon, T. M., and Ullman, E. F., Enzyme channeling immunoassay: A new homogenous enzyme immunoassay technique. Anal. Biochem. 106, 223-229 (1980).
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MI. Marks, V., and Alberti, G., “Clinical Chemistry Nearer the Patient.” Churchill-Livingstone, Edinburgh, 1985. M2. Modrow, S.,Hoflacher, B., Giirtler, L., Deinhardt, F., and Wolf, H.,Carrier bound synthetic oligopeptides in ELISA test systems for distinction between HIV-I and HIV-2 infection. AIDS 2, 141-148 (1989). M3. Morrison, S. L., Canfield, S. Porter, S., Tan, L. K., Tao, L. 0.-H., and Wims, L. A,, Production and characterisation of genetically engineered antibody molecules. Clin. Chem. (Winsron-Salem, N . C . ) 34, 1668-1675 (1988). M4. Mosbach, K., Mandenius, C. F., and Danielsson, B., New biosensor devices. I n “Biotech 83,” pp. 665-670. Online Publications, London, 1984. 0 1 . Oreskes, I . , and Singer, J. M., The mechanism of particulate carrier reactions. J. Immunol. 86, 338-345 (1961). P1. Place, J. F., Sutherland, R. M., and Dahne, C., Optoelectronic immunosensors: A review of optical immunoassays at continuous surfaces. Biosensors 1, 321-353 (1985). S1. Sutherland, R. M., and Dahne, C., IRS devices for optical immunoassays. I n “Biosensors: Fundamentals and Applications” (A. F. P. ’nuner. 1. Karube, and G. S. Wilson, eds.), pp. 655-678. Oxford Univ. Press, London, 1987. S2. Sutherland R. M., Dahne, C., Bregnard, A., Hybl, E., and Maystre, J.-L., Evanescent wave immunoassays. In “Complimentary Immunoassays” (W. P. Collins, ed.), pp. 241-260. Oxford Univ. Press, London, 1988. UI. Unilever Oxoid, One Step Package Insert. Bedford, G.B. VI. Valkirs, G. E., and Barton, R., Immunoconcentration-A new format for solid-phase immunoassays. Clin. Chem. (Winston-Salem. N.C.)31, 1427- 1435 (1985). Y1. Yalow, R. S., Immunoassay of endogenous plasma insulin in man. J . Clin.Invest. 39, 11571175 (1960). Y2. Yost, D. A. Nonmetal colloidal particle immunoassay. U.S. Pat. Appl. 072,084 (1987).
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1. Introduction In v i m testing based on immunological principles is currently one of the most important technologies for investigation and detection of disease. The reaction of an antiserum and an antigen to form a precipitated immune complex was originally described as the “precipitin” reaction in 1897 by Kraus (from Bl). This fundamental reaction is the basis of what is today a global, multibillion-dollar industry, characterized by leading-edge technology, multidisciplinarity, and the rapid development and worldwide introduction of the fruits of university-level research in one of the most rapid product cycles of any modem industry. A recent and visible example is the discovery of the link between the HTLV-111 (now HIV) retrovirus and the clinical disease of AIDS. From making the virus available in tissue culture to marketing of the first blood bank screening test there was only a delay of 9 months. The subject of this paper, simpler immunoassays, is another example of the rapid development and complexity of modem immunodiagnostics; it also demonstrates how new tests are being constructed to meet the changing needs of modem health care. Historically, quantitative immunoassays are centralized in hospital laboratories 93 Copynght 0 1990 by Academic Press, Inc. All rights of repduction in any form reserved.
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due in part to the need for highly qualified technical staff and also for complex instrumentation. Other reasons include problems with reagents and standardization variability and assay “nonruggedness.” The latter term is defined as meaning that a variety of calibrator and quality control materials must be included within each assay. Thus, practically and economically, most assays are run with batches of samples per analyte. However, batch-mode analysis is not the best solution for all clinical (and patient) needs. This is one of the driving forces that has resulted in simpler immunoassays, promoting a change in the requirements and aspirations of both the consumer and the providers of health care. A second driving force is technology per se, and a number of key innovations deserve further discussion here. First, the use of alternative labels should be considered. Quantitative immunoassays were originally constructed with radioisotopic labeling (Y1). Such radioisotopes have many analytically desirable characteristics, including high specific activity and low “environmental” background, and they can be detected in a very selective manner. However, the disadvantages are significant, the most important being short half-life, environmental undesirability (difficult handling, disposal, etc.), and the requirement for expensive capital equipment, laboratories, and specialized staff. The concept of alternative labels for immunoassays has been available since at least 1941 (C5), when fluorochrome-labeled antisera were used for the microscopic visualization of different cell types. Similarly, in 1966, Avrameas et al. (A4, A5) proposed the use of enzymes as labels for immunoassays, and many novel nonisotopic labels have since been developed (Cl, C2, El, L5), pushing back not only the boundries of analytical sensitivity, but also allowing the design of more rapid, simpler tests. The potential benefits in terms of sensitivity have been well discussed previously (e.g., Jl), but application of nonisotopic labels to simpler immunoassays is one focus of this paper. The second area of technological development that impacts modern immunoassays relates to the whole arena of biotechnology. Two major lines of development should be mentioned: the use and production of monoclonal antibodies (Kl) and the ability to analyze key epitopes on antigens, which allows synthetic gene construction and the expression and synthesis of desired proteins (e.g., M3) in effectively unlimited quantities for either immunization or for diagnostic test kit production. There is probably not a single immunoassay that has been introduced in the last 3 years that does not contain one or more of the reagents derived from these advances. The third major area of technology development that is impacting how immunodiagnostic tests are being perceived and used by the consumer is the production of assays with minimal operator intervention. This article will address the design of simple immunoassays as an illustration of how the three facets of evolving technology, vis-5-vis reagents, labels, and the physical execution of assays, are major driving forces in in vitro diagnostics.
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2. Simple Immunoassays: Utility and Specifications 2.1. UTILITY There are many potential clinical areas of application for measuring analytes using simple immunoassays. Some key issues relate to (1) the degree of urgency for the results, (2) requirements for privacy of the results, and (3) convenience of the results in terms of time and financial cost. Urgency is a factor when treatment has to be rapidly prescribed and when an in v i m test result is an important consideration. Although rapid results are not currently well addressed in product literature, areas of application include differential diagnosis of acute diseases, such as is found with large vessel disease and different forms of acute pancreatitis, and recognition and avoidance of allergic responses to drug regimens. The need for privacy of results is a factor with pregnancy and fertility tests that are currently for sale in pharmacies and even in some supermarket chains. In the future, this market may expand to include tests for detection of sexually transmitted diseases and into areas of fitness, or “wellness,” testing. The third factor, convenience, includes a broad range of applications wherein it may be advantageous to have a test result immediately available at the time the patient is being examined by the physician. Examples include therapeutic drug monitoring, longer term therapeutic regimes (e.g., diabetes, anticoagulant therapy, and antihypertensive therapy), and many instances wherein the result of a test can eliminate the need for a follow-up visit. In nearly all of the above examples, whether in a physician’s ofice, at home, or in a hospital intensive care unit, the in situ availability of appropriate technical skills to, carry out laboratory-type tests is minimal. This means that for the successful execution of simple immunoassay tests, a number of critical technical and performance requirements are imposed on test design. These will be addressed in the next section.
2.2. ASSAYREQUIREMENTS Assay requirements have been the subject of several reviews (H2, Ml), and are most readily illustrated as a list (Table 1). This background information demonstrates some of the challenges that face the design and fabrication of a simple immunoassay device that will achieve success in result quality and user acceptance. There is probably no product currently available that meets all of these requirements. However, there are a number of very successful devices that already have made significant strides in positioning testing in the hands of the nonskilled user. It is these devices that are the main subject of this article.
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RANALD M. SUTHERLAND AND BARRY SIMPSON TABLE 1 DESIGNGOALSFOR SIMPLE IMMUNOASSAYS Broad range of sensitivity Analyte specific Sufficiently rapid Operator independent with respect to chemistry, timing, and pipetting Reagents are stable Self- or factory calibrated Appropriate for mass manufacture Applicable for different assay formats Disposable Competitively priced
3. New Developments in Simple lmmunoassays for DecentraIized Testing
This review is directed at technically the simplest forms of immunoassay that are currently available and that do not require complex instrumentation. There are at least three basic technological approaches being used today (Table 2). Immunoassays, in which one of the immunological binding pair is particle bound, e.g., to latex or sensitized red blood cells, were proposed as early as 1951 (B2, 01). These qualitative assays are still in broad use, particularly in the field of pregnancy testing, bacteriology, and serology. This type of test has a drawback of being difficult to interpret and can thus have poor discriminatory power. However, many particulate immunoassays offer advantages in simplicity, fast results, adequate sensitivity for many qualitiative applications, good reagent
TABLE 2 A CLASSIFICATION OF SIMPLEIMMUNOASSAYS BASEDON TECHNOLOGY ~~
Class (A) Microparticle (B) Membrane/ filter-support (C) Inununosensors
Example Latex Hemagglutination Inununofiltration Immunochromatography TIRFa Surface plasmons
O T I R F , Total internal reflection fluorescence.
Reference H1 B2 A l , B3 A2, C4
s2 L4
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shelf-life, and reasonable costs. For these reasons alone such assay types will be in use for many years to come, and are currently the subject of development to improve the weaker performance characteristics. The membranelfilter-support systems, usually with a visual, colored, end point, represent the new wave of simple assays and were only recently introduced. These assay systems are being continually updated and improved in terms of sensitivity, speed, facility of use, and control of result quality. However, the third technological approach, the, “immunosensor” device, although the subject of considerable literature discussion in recent years, has yet to be developed into a successful product. The reader is referred to recent reviews (A3, M4, Pl), as immunosensor technology will only be briefly discussed herein. In the following sections are discussed the various successful approaches being used, with examples of each. 3.1. PARTICLE-BASED IMMUNOASSAYS
Two examples of recent developments in particle-base immunoassays will be discussed; the first two are based on latex particles. A novel latex agglutination assay has been described using colored latex particles (Hl). The reagent suspension is composed of two or more differently colored particles (red, blue, or green). In this example, each particle is coated with a different antibody for each color. Thus, on reaction with a sample, depending on the antigen present, latex particles of one color agglutinate and are visually more apparent due both to concentrated color of large complexes and to the reduction of the “dispersed” color in the background. The test is currently applied to the detection of three different Salmonella serogroup antigens, B, C, or D. The use of one sample for multiple testing in a single assay has several advantages, including reduced sample volume requirements and reduction in time to produce multiple results. However, results produced using this system will still suffer from highly subjective discrimination and the system is only appropriate for testing wherein the results are mutually exclusive. Without some objective reading device, it may be difficult to expand this technology into other areas of testing. The second example is based on attaching the antibodies to metal sol particles. The use of metal sol particles, especially gold sols on the order of 5 nm to 10 p,m in diameter, has been described, particularly for protein blotting assays (H4). Here, homogeneous immunoassays for urinary hCG (L2) and estrogens (L3) using sol gold-labeled monoclonal antibodies have been described based on the optical characteristics of the gold particle. The assay format is based upon reacting antibody-coated gold sol particles with a standard quantity of multivalent antigen. The antibodies bind the soluble antigen and thus the small particles form larger and larger particle aggregates. From classical physical theory, Rayleigh’s law relates light-scattering efficiency to the particle diameter, and to
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both the incident light wavelength and relative refractive index ratio. All else remaining constant, the effective light-scattering ability of the gold sol particle will increase with particle aggregation, and the optical dispersive properties change with aggregation, resulting in a visibly colored solution. However, on addition of a sample containing the same antigen, the formation of particle aggregates will be reduced, as the sample antigen will compete for available antibody-binding sites, and the resultant color will be visibly less. Such an assay system can obviously be designed to either increase or decrease in color when the target antigen is present in a sample. As discussed with the colored latex approach, this technology is also prone to highly subjective interpretation of the results. Unlike the colored latex approach, however, the reading of results is not carried out against a relatively high noise level (caused by other colored particles, which do not actively contribute to the signal but increase the background significantly), and it may be expected that this approach is less subjective.
3 . 2 . MEMBRANE-SUPPORT IMMUNOASSAYS The assay formats used with membrane immunoassays are very diverse, but are all primarily designed to reduce operator reliance and involvement. There are currently three basic assay formats, which have been called (1) immunoconcentration, or “pour-through devices,” (2) immunochromatography, and (3) the “dipstick” format. In all cases the assays are heterogeneous (i.e., requiring separation steps) and retain good sensitivity, generally by removing these key separation steps, which are arguably the main source of assay imprecision, from the operator’s hands.
3.2.1. Pour-Through Devices The types of devices used here essentially consist of two main parts; first, a filter or membrane pad in which the specific reactions take place, and second, an absorbent pad, which not only is the “waste sink” for excess liquids, but also acts as a wicking agent exerting a “sucking” force, causing solutions to be pulled through the reaction pad. Basic designs were proposed as early as 1980 (C3) for filtration devices to separate insoluble immune complexes generated by a doubleantibody precipitation radioimmunoassay (RIA). However, the new devices are more complex and can be typically represented as a “pour-through” system, with multiple layers of membranes and/or filters, and a visual end-point signal detection. A typical example is the Abbott TestPack (B3), which is used herein to illustrate some of the basic components and characteristics of such devices. An exploded view is given in Fig. 1 , where A and E are the exterior shell components. In this example the reaction pad is a flat circular fibrous support (D). The specific immunological binding reagents are attached to microparticles that have a diameter smaller than the pore size of this support, and so in a certain sense this
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FIG. 1. Exploded view of the Abbott TestPack single-use pour-through immunoassay device. See text for explanation.
also represents a microparticle-based assay. Underneath the reaction pad is the absorbent pad (B), and there can also be a third membrane (C) sandwiched between these two pads that can act as a barrier restricting fluid flow (e.g., to reduce the backflow of reagents). This particular example also has a removable filter (F) above the reaction pad, which helps prevent larger particulate materials from the sample from blocking the pores of the reaction pad. With such a device, the assay protocol is relatively simple and for most qualitative assays accurate pipetting and timing is deemphasized. The assay protocol for the detection of antibody to the two human immunodeficiency viruses (HIV-1 and HIV-2) in human serum or plasma is used for illustrative purposes (A 1). Here, the antigen consists of three recombinant proteins, HIV-1 core and envelope and HIV-2 envelope, which are precoated onto the microparticles (for a summary of the various HIV proteins and their use in assays, see Ref. F l , G1, M2). An initial dilution step is carried out by simply dipping a collection loop into the sample and transferring it to the diluent and swirling, avoiding the requirement for accurate pipetting steps. The reaction pad and filter are prewetted and the diluted sample is added by pouring it onto the filter and then allowing it to flow through completely. A wash solution is then immediately poured onto the filter and a solution of antihuman IgG (goat) coupled to alkaline phosphatase is added dropwise onto the filter. Following a 3-min incubation, the filter is removed, and a second wash solution is added to remove unbound materials from the reaction pad. Specifically bound antihuman IgG is then visualized by the dropwise addition of the enzyme substrate solution, a 2min incubation, and addition of the enzyme stop solution. This assay protocol is a good example of how systems are being designed with the aim of achieving simple immunodiagnostics. Reagents are prepackaged, assay protocols eliminate the necessity for accurate pipetting, there is minimal operator involvement, the assay is rapid (in this case less than 10 min to result), and the timing is generally controlled by the speed of solution uptake by the device. Other devices are
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RANALD M. SUTHERLAND AND BARRY SIMPSON TABLE 3 SOMETYPICAL APPLICATIONS OF SIMPLE IMMUNOASSAYTESTSON THE ABBOTTTESTPACK Analyte
Sample
Detection limit ~~
hCG hCG Strep A Rotavirus Respiratory syncytial virus Chlamydia
Urine Serum Throat swab Feces
HIV- 1 /2
Serum or plasma
Nasopharyngeal aspirates Endocervical swab
50 m IUlml 25 m IU/ml
NAa >2 x 107 particles NA >20 inclusionforming units NA
ONA, Not available.
available that have essentially similar design characteristics (H3, Vl). Examples of the areas of application of these devices are given in Table 3. One of the key elements in such simple immunoassays is their resistance to operator-induced error. One way this is addressed is by including a procedural control within the device. A binding agent that can capture the labeled antibody is preimmobilized at a site on the reaction pad, which receives exactly the same treatment as the preimmobilized HIV antigens, but is physically distinct from the antigen reaction area. In this fashion, addition of the reagents according to the correct assay protocol will result in the control area being colored, but no color will result if one of the steps is inadvertently missed. In the example, the procedural control is the horizontal line of a + ” sign and the specific binding signal is the vertical line. Similar approaches have been adopted with other designs, but the shapes are in the forms of rounded dots. “
3.2.2. Immunochromatography The principles of immunochromatography can be broadly summarized as allowing a liquid that acts as a solvent to move through a filter strip, to which one or more specific binding agents have been immobilized at predefined locations. These locations are serially hydrated by the moving solvent front and are also the basis of a sequential immunological reactions. Dissolved reagents and sample are carried in the aqueous phase through each location and can thus react with the immobilized specific binding reagents. For example, with a typical “sandwich” assay, the sample would be mixed with labeled antibody and the combined solution would be allowed to wick up the filter strip, to which an alternative antibody is immobilized at a specific site. Given appropriate reaction kinetics
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during sample migration, the labeled antibody will bind to the analyte, and subsequently this complex will then be bound to the second (alternative) antibody at the preimmobilization site. Unbound materials are then removed by the wicking solution and the specifically bound materials are visualized by dipping the complete strip into a substrate solution, or, alternatively, by wicking substrate up the strip. The first example is the Acculevel test (Sylva Corp.), which is based on two technologies, both the immunochromatography technique and the “enzyme-channeling immunoassay” (L6). Here specific signal effectively only occurs when two enzymes are physically in very close proximity, where the product of the first enzymatic reaction is the substrate for the second enzyme-catalyzed reaction, the closeness of the signal generators being achieved via immune complex formation. This basic concept is combined with the immunochromatographic strip format (C4) for a theophylline immunoassay. A monoclonal antibody to theophylline and the enzyme glucose oxidase are coimmobilized along the length of an immunochromatography strip. Sample is mixed with a solution containing the various enzyme substrates, a peroxidase-labeled theophylline conjugate, and an inhibitor to retard color formation until the wicking action is finished. The strip is dipped into this solution and the conjugate travels a distance along the length of the strip approximately directly proportionally to the concentration of sample antigen. The inhibitor, ascorbic acid, acts by reducing an oxidized (colorless) intermediate of the substrate-channeled enzyme reaction, thus preventing color formation. The ascorbic acid is consumed by this reaction and the concentration is selected such that color formation only occurs after the solution has traveled the full length of the strip (i.e., the immunoassay reaction has effectively finished). Results are visually read by comparison with a thermometer-like scale 30 min after starting the assay, and the detection limit is claimed to be at least 5 mg/liter. This assay is easy to use but is biochemically complex in design, and the requirement for multiple reagents to perform the procedure can result in a relatively high cost. An inhibitor is used to retard signal generation; this may indicate that there are limitations in the sensitivity of such a system, particularly with respect to background signals. The second example, equally elegant although considerably less complex in analytical principle, is a combination of the colored latex approach and immunochromatography (the Clearview test; Fig. 2) (UI). For the detection of urinary human chorionic gonadotropin (hCG), five drops of sample are put onto the end (A) of the immunochromatography pad (Fig. 2 I); these then wick up to the first zone (B), where a specific monoclonal antibody is predeposited. This first antibody is coated onto blue-colored latex particles and reacts with sample hCG to form the first part of the “sandwich.” The complex then continues along the pad, drawn by capillary and microfluidic forces (Fig. 2 11; C). This solution contacts the second preimmobilized anti-hCG monoclonal antibody, which is affixed in the form of a line across the pad (D). The final immune complex forms and the
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FIG.2. Schematic of an immunochromatography device. See text for details.
unbound materials are wicked up the pad to a third area (Fig. 2 111; D). Here a second antibody has been preimmobilized and binds the remaining (blue) latexlabeled anti-hCG antibody, forming a second blue line (Fig. 2 IV; E). One blue line represents a negative result and the presence of two blue lines represents a positive result. The assay only uses 0.5 ml of urine, is finished within 5 min, and all reagents are self-contained in the device. This device is currently the only example of a self-performing immunoassay generally available. However the use
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of this assay design has potential limitations. For example, using the colored latex (although avoiding the extra step of the enzyme-substrate reaction, with the extra reagent additions and incubations) rules out the possibility of signal enhancement inherent in using catalytic signal generators. Thus some questions must be posed as to the achievable sensitivity of this approach. Second, and more generally, the opportunity for the immune reaction to go to completion (i.e., maximized specific antibody-antigen complex formation) is limited by the short time that the sample and antibody physically have to react. This may mean that very high-affinity reagents have to be used and in higher concentrations than are normally required for traditional immunoassay methods. Another example of a self-performing assay is the TestPack +Plus. The device (Fig. 3) has a sample application area, a window to read the test result, and an “end-of-assay” window. The result is expressed as positive or negative, depending on the respective presence or absence of analyte. In the hCG urine test, three drops of urine are added to the sample application area and they wick along a nitrocellulose membrane. Any hCG that is present in the sample reacts with a colloid-labeled (Y2) monoclonal antibody to the a-chain of hCG. At the reading site, two sets of reagents have been applied in the shapes of vertical and horizontal bars. The vertical bar, which indicates the presence of antigen in the specimen, has a polyclonal antibody raised against the P-chain of hCG preimmobilized to the nitrocellulose. The horizontal bar, which indicates if the reagents are active, has a complex of anti-hCG : hCG preimmobilized to the membrane. Thus if the monoclonal antibody has been inactivated in some way, there will be no signal in the reading window. The end-of-assay indicator zone shows when the unreacted labeled monoclonal antibody has traveled the distance
FIG. 3. Schematic of the TestPack +Plus device. See text for details.
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of the membrane and that the assay is completed. The assay takes 5 min and has a minimum detection limit of 50 IU/liter. 3.2.3. Dipstick This format is very simple in concept and operation. Here, one of the immunological binding pair is immobilized onto a pad of absorbent material, which is physically fixed onto the end of a “stick.” The sample is mixed with the labeled conjugate and the pad is dipped into this solution, allowing either a competitivetype assay or sandwich-type assay to occur. The pad is washed and bound label is visualized by simply dipping the pad into the substrate solution. Usually the product of the enzyme-catalyzedreaction is insoluble and/or is bound to the pad (e.g., by electrostatic forces). A typical example is illustrated in Fig. 4 (Ll). This dipstick has both a sample pad (B) and a reference pad (A). In this case the reference pad is used to c o n f m that the assay procedure is correctly adhered to and also gives a reference color to which the sample signal can be compared. The example given with this device is for the measurement of morphine. Two labeled conjugates are mixed with the sample; one conjugate binds only to the reference pad and is a biotin-enzyme conjugate, and the second conjugate is a morphine-enzyme conjugate. Immobilized to pad B is a monoclonal antibody to morphine, and avidin is immobilized to the second pad. The presence of morphine in the sample results in a reduced amount of morphine-enzyme conjugate specifically bound to pad B, and the resultant color reaction is weaker than that seen in the control pad. The assay takes 30 min with a sensitivity of at least 30 ng/ml morphine. Limitations with these devices are probably most related to available surface area and the kinetics of the solution/solid-phase interface. With both the immunofiltration and immunoconcentration devices, the specific soluble reaction components are being actively moved close to the immobilized reagents in such a manner that there is a relatively high level of opportunity for reaction; they could be described as three-dimensional in nature. However, the dipstick-type devices are effectively static with respect to their liquid reaction kinetics, and are probably more diffusion limited; i.e., they could be described as two-dimensional. A second difference is that effectively all the sample is given an opportunity to react with
FIG. 4. A drawing of a typical “dipstick” immunoassay device. See text for details.
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the immobilized reagents within the other devices, unlike the dipstick devices, wherein the bulk of solution is not in direct contact with the specific immobilized reagents. Therefore, such a design is more reliant on “passive” diffusion. Such an approach could be significantly improved by using a reliable form of physical mixing, but it is not clear how this may be achieved in the type of testing environment envisaged for these devices.
4. Discussion The arena of simple immunoassay devices is the subject of intensive commercial activity and illustrates an area of development that requires multidisciplinarity, which can only usually be found in a commercial company. This, at least in part, explains the emphasis in this review on commercial developments. However, it should not detract from the fundamental scientific discoveries that are the underpinning of such commercial works. As alluded to in the introductory paragraphs of this paper, the current simple immunoassay devices would not be possible without the development and discovery of many of the current biotechnological skills and technologies such as hybridoma and recombinant DNA techniques. Similarly, the design and manufacturing of such devices are dependent on the developments in materials sciences and on precision liquid-handling techniques that allow depositing tiny volumes of reagent onto well-defined areas of such materials. This is only a partial list of some of the key elements in making and designing a simple immunoassay device. As reviewed above, current systems are well suited to the detection and even semiquantitation of many analytes, as illustrated in Table 3. Future developments will be aimed at designing even simpler devices, two of which were illustrated here-the self-performing TestPack +Plus and Clearview. It is probably inevitable that, as part of this process, simple instrumentation, such as that found for reading blood glucose strips, will be developed for immunoassays. An alternative approach to the above systems may lie in the successful design of a disposable immunosensor. Simple immunoassays are mainly heterogeneous enzyme immunoassays, combined with a device to allow ease of assay execution. Immunosensors are based on homogeneous types of immunoassays, with some of the most promising approaches being nonenzymatic. For example, one type of immunosensor is based on optical devices and was recently reviewed (Sl). In general, the approach taken here is to immobilize one of the binding pair to a continuous surface, such as an optical fiber. This surface has certain optical properties (e.g., transparent or reflective) and can be interrogated with visible radiation during and after the immunological reaction. Thus the growth of the antigen-antibody layer can be detected as modulations of the radiation that impinge on the continuous surface. Such modulations can be chosen to react to
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the interfacial layer thickness and refractive index changes, or equally to a change in specific fluorescence, wherein one of the binding pair is fluorochrome labeled. Application of these basic physical phenomena to monitoring interface immunassays has been described. For example, the use of surface plasmon resonance (L4)can detect minute changes in the relative interfacial refractive index. Similarly, monitoring of fluorescenceat an optical interface can be canied out using total internal reflection fluorescence (TIRF) (S2). The design goals for such immunosensor devices are to be self-contained, rapid, and effectively operator independent. A typical example is proposed in Ref. S 1: the whole device is made from injection-molded plastic and is totally self-contained in terms of reagents. Sample is allowed to wick into the reaction area via capillary action, and the sample volume is defined by the walls of the reaction area. All reagents are preimmobilized or coated onto the interior of the device in a stable and hydrophilic form. The device is inserted into a simple photometer, which can monitor the kinetic immunoassay reaction in real time. In summary, the current developments in simplifying immunoassays have demonstrated the design of many devices that allow performance of these complex techniques in nonlaboratory environments. The current devices are very adequate for many quantitative and qualitative applications in clinical diagnostics. In the future, there may be a role for alternative technologies such as immunosensors, and a short-term goal is for simple instrumentation such as optical reading devices. REFERENCES Al. Abbott TestPack, HIV-l/HIV-2 package insert. BlA839 ref no. 82-2995/R2 (1989). Abbott Laboratories, Abbott Park, Illinois. A2. Abbott TestPack +Plus package insert. B2A991 ref no. 83-5533/Rl (1990). Abbott Laboratories, Abbott Park, Illinois. A3. Andrade, J. D., Van Wagenen, R. A,, Gregonis, D. E., Newby, K.,and Lin, J.-N., Remote fibre-optic biosensors based on evanescent excited fluoroimmunoassay: Concept and progress. IEEE Trans. Electron. Devices 32, 1175-1 179 (1985). A4. Avrameas, S., and Uriel, J., MCthode de marquage d’antigtnes et d’anticorps avec des enzymes et son application en immunodiffusion. C.R. Hebd. Seances Acud. Sci. 262,25342535 (1966). A5. Avrameas, S., and Berson, S. A,, Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochimie 54, 837-842 (1972). BI. Barrett, J. T., “Textbook of Immunology.” Mosby, St. Louis, Missouri, 1988. B2. Boyden, S. V., The adsorption of proteins on erythrocytes treated with tannic acid and subsequent haemagglutination by antiprotein antisera. J . Exp. Med. 93, 107-1 15 (1951). B3. Brown, W. E., Devereaux, DS. M., Knigge, K. M., Clemens, J. M., Hofler, J. G., and Safford, S. E., Solid-phase analytical device and method for using same. Eur. Pat. Appl. 86 6,113,675 (1987). C1. Cambiaso, C. L., Leek, A. E., De Steenwinkel, F., Billen, J., and Masson, P. L., Particle
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