ASSAY DEVELOPMENT
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Photo by Brian Pieters Photography
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Immunodiagnostic tests have experienced exponential growth over the past 30 years, all while maintaining a basis in the principles that guided their early development:
A surface binds a capture antibody which binds the analyte of choice, and a detector reagent—a second antibody coupled with an enzyme, or something else—is added to determine the concentration of the analyte in the biological matrix in question. This configuration, described as a sandwich immunoassay, has been the subject of multiple patents and of litigation relating to them.
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Figure 1. (click to enlarge) Antibody binding to a surface in an immunoassay.
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Other formats have evolved along with sandwich-type assays. These include competition assays, which use preferential binding to inhibit the antigen–antibody binding, and radioimmunoassays (RIAs), in which labels of radioactive material are employed to identify the antibody of choice. This is to name but two of many formats. The format of RIAs makes these tests unsuitable for use near patients. Also, technicians performing such tests must be regularly monitored for exposure to radioactivity.
However, essentially all immunodiagnostic development has involved some period of antibody binding to a surface (see Figure 1), with the surface of choice for antibody binding being the only significant area of changing focus in immunodiagnostic research over the past 30 years.
Candidate surfaces include:
- Microtiter wells composed of various polymers (PVC, polycarbonates, and polystyrene, and modified forms of these), which are used primarily in enzyme-linked immunosorbent assays (ELISAs).
- Various laminates of nitrocellulose, polyethyleneimine, and glass for lateral-flow tests, which provide qualitative or semiquantitative results for early diagnostic determination.
- Microparticles composed of latex, polystyrene, gold, or paramagnetic materials.2–6
All of these have played a role in the rapid evolution of immunodiagnostic test platforms.
This article focuses on the use of microparticles in near-patient IVD platforms and especially in immunodiagnostics. It addresses the utility of dyed, latex, polystyrene, modified, and paramagnetic microparticles as solid-phase and isolation materials for diagnostic and molecular biology.
Near-Patient Immunodiagnostics
Immunodiagnostics are now found in routine clinical use for the diagnosis and management of various specific medical conditions. Qualitative and quantitative assays detect pregnancy (for example, tests for human chorionic gonadotropin [hCG]), immunoglobulins, proteins (including secondary antibodies), drugs of abuse, viral diseases, Helicobacter pylori, and multiple disease states, including certain malignancies and cardiovascular disease. Indeed, the applications are so wide-ranging that, for many companies, manufacturing such tests is their main source of revenue.
Many immunodiagnostic assays are formatted as lateral-flow devices (LFDs) as the binding surface for the capture and reporter antibodies. Once the analyte in question is captured, the reporting antibody provides a method of specific analyte evaluation via qualitative and quantitative means. Biosite Inc. (San Diego), for example, started up by developing a point-of-care (POC) immunodiagnostic panel for the evaluation of drugs of abuse, but it rapidly evolved, optimizing a platform for the determination of cardiac events including myocardial infarction (MI). The company has introduced tests for creatinine kinase MB isomer (CK-MB) and, more important, troponin I (TnI) and ß-natriuretic peptide (BNP) for congestive heart failure. Specific to myocardial muscle, cardiac TnI is the confirmatory molecule of choice for MI diagnosis. It is easily differentiated from the skeletal muscle isoform by a significant variation in amino acid sequence and is not expressed in human fetal skeletal muscle or in adult human skeletal muscle in response to any pathological stimuli.7
Other companies present parallels. Spectral Diagnostics Inc. (Toronto), Abbott Point of Care Inc. (East Windsor, NJ), and Response Biomedical Corp. (Burnaby, BC, Canada) are examples. In addition to its cardiac immunodiagnostics panel that employs the principles of conductivity and amperometric analysis, Abbott’s i-STAT system offers a panel of 22 other analytes, including coagulation tests, that may be evaluated on the same POC platform. Response Biomedical, for its part, has a biodefense program and also a line of test cartridges for the detection of such viral diseases as West Nile virus and flu. Its system for evaluation of suspicious powders by the U.S. military is currently the only such system in use.
These and other companies have made advances toward moving immunodiagnostics, traditionally performed in the central laboratory with a long turnaround time (TAT), to the vicinity of the patient in a number of clinical settings. Test devices are being used in the patient’s home, en route before hospital admission, in the emergency room, and in the physician’s office.
Moreover, with the application of multiple-array analytic analyses, they will inevitably provide end-users with the means of making more-rapid diagnoses, which in turn leads to improved patient triage and better overall healthcare.
The sensitivity and specificity of a test varies with the type of binding surface and the detection modality used. Many of the early immunoassays were limited in these performance areas owing to the nature of the test signal. This prompted companies to move from spectrophotometric (colorimetric) means of determination to technologies based on agglutination, fluorescence, and then luminescence. Even more recently, chemiluminescence techniques have been developed for certain immunoassay systems, carrying detection sensitivity to a higher plane. Sensitivities in the pico and femto ranges (10–12 and 10–15 g/ml, respectively) have been achieved.
Higher sensitivity is just one of several factors that must be considered when developing a test platform optimized for POC applications.8,9 The test surface is another. More and more companies engaging in research and development involving ELISA technology are transitioning to alternative surfaces, such as microparticles, for the commercialization of their tests. This is because of the recognition that such surfaces may confer more-timely results (albeit of a merely qualitative or semiquantitative nature in the case of lateral-flow tests) and may also assist in conferring higher sensitivity and signal amplification. The latter is certainly the case with microparticles, owing to their increased surface areas and the kinetics of the assay.
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Table I. (click to enlarge)
Currently used immunodiagnostic detection modalities. Source: Bangs Laboratories Inc. (Fishers, IN) Latex Course. |
Currently used detection modalities are distinguished by individual sets of advantages and disadvantages (see Table I). Their sensitivities by label type are outlined in Table II.
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Table II. (click to enlarge) Detection sensitivities of current immunodiagnostic modalities according to the type of label employed. RIAs = radioimmunoassays; HRP = horseradish peroxidase; AP = alkaline phosphatase. Source: Bangs Laboratories Latex Course.
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It should be noted that some researchers consider the sensitivities listed in Table II as theoretical and not attainable with available systems.
Microparticles versus Traditional Surfaces
Microparticles can be used in the solid phase to enhance detection or separation of the target analyte, or both. They may be composed of many different materials, and they come in various sizes and densities.
Latex microparticles are considered the front-runner in providing innovative surfaces for antibody– antigen reaction. Many of them are modified with functionalized surface groups, including amino, acetyl, and chloromethyl groups, for the optimal covalent linking of antibody molecules. Such microparticles may be used in turbidimetric procedures or LFDs, which have both demonstrated good correlation with nephelometric immunoassay methodology.10 However, these particles are particularly prone to interference with other serum components, a circumstance that should be recognized prior to assay development. Furthermore, the variability among latex particles of different compositions suggests that it may be necessary to optimize the assay conditions for each type of particle in order to achieve maximum immunoreactivity and optimal analyte quantitation.6
Recent advances in the use of infrared laser technology have enabled the development of a system capable of two-photon excitation microparticle fluorometry (TPX). Polystyrene microparticles for this technology have demonstrated utility for immunoassays. The antibody-coated solid-phase particles are capable of capturing an antigen and then quantitating the capture with a fluorescently labeled tracer antibody.
The TPX technology makes possible very-low-volume homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. Indeed, the TPX assay system has been used both for reagent characterization and for the measurement of C-reactive protein (CRP) from plasma samples, targeting the assay range useful in infectious-disease diagnosis. The pentameric structure of CRP has allowed the optimization of an assay with the lower limit of detection at ≤1 µg/L (that is, 7.5 pmol) and an operating range of 1–150 mg/L by virtue of the same monoclonal antibody being used both for capture and as the tracer. This TPX method was observed to have an excellent correlation with the reference systems in a routine central-hospital laboratory, thereby demonstrating the feasibility of the technology for immunodiagnostic applications.11
Gold has been used extensively in immunoassay development, both in lateral-flow assays and for coating microparticles in order to enhance immobilized-antibody binding efficiencies and thus provide a better solid phase. In the latter application, gold-coated microparticles exhibit much lower nonspecific adsorption than tosylated polystyrene magnetic particles, along with an approximately 10-fold improvement in detection limit. This improved assay performance is attributable to the more favorable and specific binding orientation of the antibodies or their fragments on the gold-coated particles as compared with the seemingly random orientation on non-gold-coated surfaces. The binding behavior enhances sensitivity and promotes earlier detection of the target sample in a biological matrix.
The use in immunoassays of particles with magnetic properties has increased exponentially in recent years. Researchers consider this technology to offer some optimal properties for immunoassay development.
Many IVD manufacturers that employ magnetic and paramagnetic particles in their immunoassay systems now improve the binding and separation characteristics of the particles for their own specific needs through chemical modification or refinement of the antibody–particle binding conjugation. Still other companies now develop and manufacture particles of varying size and composition specified for their particular platforms. This enables them to maintain their proprietary intellectual property in-house and provides the option of patent application to ensure further protection.
In addition, this practice has prompted the spin-off of sister companies that not only make and supply the specific particles the parent company needs but also may serve as a particle vendor to other research-and-development and commercial diagnostics organizations. The secondary company thus can provide revenue for the parent company and support it in its other activities. (In fact, many copy machine manufacturers have expressed an interest in the magnetic particles the IVD suppliers produce, seeing in them a potential to improve on their own technologies.)
Paramagnetic Microparticles in Application
Cleveland Biosensors (Brisbane, Australia) has developed a handheld POC test platform, the BioFiniti cartridge, that utilizes tosyl-activated magnetic particles 2.8 µm in size from Invitrogen Corp. (Carlsbad, CA) coupled with detector-filled liposomes. Both the particles and the liposomes have been conjugated to biological receptors.12 These microparticles are mixed with the sample matrix and flow onto the capture magnet where the electroactive marker is released from the liposomes. Quantitation of the analyte is then achieved by means of amperometric analysis of the amplified signal. The system has demonstrated an ability to detect d-dimer, BNP, and microcystin that compares favorably with traditional methods.
This platform might be an alternative for use in commercial POC systems. It offers the advantage of low-concentration targets in difficult sample matrices such as blood and urine, which is due to the integration of complex microfluidic characteristics with the electronic sensors. Moreover, it is suitable for assay development or conversion of immunodiagnostics.12,13
Some think that paramagnetic microparticles are potentially the preferable surface for antibody binding, for separation of analytes from the media milieu, and for optimal light collection.14 One company to capitalize on this technology is CardioGenics Inc. (Mississauga, ON, Canada), which has developed in-house microparticles that are patent protected and proprietary for the company’s diagnostic platform. These are beads composed of white-colored paramagnetic microparticles of sizes varying between 1 and 50 µm. Their surface layer is silver coated with a hydrophilic polymer and equipped with a variety of functional groups of linking chemistry. CardioGenics’ manufacturing process encapsulates or inserts the paramagnetic pigment in a polymer matrix. It involves a robust—that is, stable—multilayer chemical deposition of polymer. Further, numerous active components are integrated between layers for nanoreactor functionality. These microparticles additionally maximize light collection by color conversion to the silver-white surface coupled with a high magnetic moment, are easy to manipulate, and provide a hydrophilic polymer surface for optimal (very-low-background) specific and nonspecific binding.15
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Table III. (click to enlarge) The test sensitivity of silver microparticles of 25 µm compared with that of black microparticles
of similar size, in relative light units (RLU). Source: CardioGenics Inc. (Mississauga, ON, Canada). |
CardioGenics’ microparticles have demonstrated a consistent fivefold increase in sensitivity over that of black paramagnetic particles (see Table III). The table gives readings for 25-µm particles, but these increased sensitivities are realized with all sizes of microparticle manufactured by this proprietary process.
However innovative in design and applicable for immunoassay development and commercialization these particles may be, they must be associated with an optimal mode of signal production and detection, and a platform suitable for use in the POC clinical environment. CardioGenics has developed both. The company uses biochemiluminesence derived from natural sources for the former. And for the latter, it devised an inexpensive cartridge-based POC system with no moving parts. This platform provides fully automated testing, requires no membranes, uses unadulterated whole-blood samples needing no additional handling or dilution, and has a TAT of no more than 15 minutes. In addition to other analytical tests now in late-stage development (proteins, TnI, troponin T, and myoglobin), this system lends itself to markers for infectious diseases, zymogens of the coagulation cascade, and malignancy.
Clinical Utility of Microparticles in Assays
Magnetically responsive particles are used in a variety of biomedical applications, including optimized biological separation; high-sensitivity bacterial binding in the preparation of foodstuffs; clinical laboratory test products; cell isolation; nucleic acid separation; protein purification; and sample enrichment. Some say that they have potential to greatly simplify biological separations and reduce assay times.15,16
The clinical relevance of this is suggested by the fact that the rate of use of POC tests in acute clinical settings to improve patient triage and outcomes continues to increase and will likely do so for the foreseeable future. While glucose testing continues to lead the POC market, much of the rest of the market is made up of tests that incorporate immunochemistry detection systems.
However, what is required are POC test systems with better sensitivities than are currently available, and that can be used at the site where the patient is being triaged in order to determine the disease state earlier in the pathology process. Such systems allow the physician to initiate the appropriate therapy in a more timely fashion. This is especially important in those diseases where time is muscle, as the saying goes, which include MI literally and stroke metaphorically. Besides more-sensitive detection technology, new POC tests should include simple, inexpensive, user-friendly platforms that can minimize the time investment in both technician handling and the acquisition of diagnostic results.
Although the fluorescence assays from Biosite and Response Biomedical and the i-STAT electrochemical platform from Abbott POC that were mentioned earlier in this article are striving to meet these needs, biochemiluminescence may offer additional features that can optimize POC devices.
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Table IV. (click to enlarge) A comparison of currently available microparticles with those developed by CardioGenics for possible future applications.
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In this connection, the paramagnetic modified microparticles offered by CardioGenics and described just above offer superior characteristics (see Table IV). The patent-protected proprietary electroless silver-coating process moderates the amount of deposited silver to control reflectivity. This process has no, or negligible, effects on magnetic properties. The effects on chemical and mechanical stability have been assessed. And finally, batch sizes are adaptable; the manufacturing process can be scaled up to satisfy larger needs.
These microparticles for CardioGenics’ biochemiluminescence assay lend themselves well to both POC platforms and laboratory batch analyzers, adapting to the latter easily. They have demonstrated utility in the determination of TnI, myoglobin, and certain coagulation proteins using the current platform, with sensitivities in the picogram range and below.
Although paramagnetic particles have been used in central-laboratory batch analyzers for more than a decade, there have been no major advances in that application, and the long TAT still impedes the critical decision-making process. Examples of these immunoassay systems are the Centaur and ACS180 from Bayer Diagnostics (Tarrytown, NY), and the Access system offered by Beckman Coulter Inc. (Fullerton, CA).
The Centaur is a traditional-capture sandwich immunoassay using direct chemiluminescence technology. The antibody of choice in the patient sample is captured by monoclonal mouse antihuman A antibody covalently coupled to paramagnetic particles in the solid phase. While its manufacturer claims high throughput and a capability of performing analyses in 18 minutes, the actual TAT for this assay is in excess of 45 minutes from sample to decision due in part to the time for sample delivery to the central laboratory for evaluation.
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David Carville, PhD,
is cofounder, chief executive officer, and chief scientific officer at Clinical Solutions and Innovations (South Bend, IN). He can be reached at dcarvill@iusb.edu. |
The Access system also makes paramagnetic particles and chemiluminescence integral components of its immunoanalytical tests. This system, like the Centaur, has numerous moving parts. Both systems require sample dilution and reagent preparation, and users need special training to ensure proper implementation.
The CardioGenics system, by contrast, is a closed-cartridge-based platform with no moving parts. The operator simply inserts the cartridge and waits for the result, which arrives typically in 10 minutes. All reagents are already in the cartridge, which is bar coded to remove the possibility of technical error. In addition, this system costs much less than the central-lab analyzers.
Conclusion
Microparticles represent an important advance in immunoassay development. In light of the clinical need for improved POC platforms and better assay chemistries, they should maintain their position as an important component of immunoassay systems.
References
1. AM Campbell, Monoclonal Antibody Technology: The Production and Characterization of Rodent and Human Hybridomas, vol. 13 in Laboratory Techniques in Biochemistry and Molecular Biology, ed. RH Burdon and PH van Knippenberg (New York: Elsevier, 1984), 1–30.
2. MP Sanz Izquierdo et al., “Amino, Chloromethyl and Acetal-Functionalized Latex Particles for Immunoassays: A Comparative Study,” Journal of Immunological Methods 287, no. 1/2 (2004): 159–167.
3. D Dosev et al., “Spray Pyrolysis Synthesis of Particles Possessing Magnetic and Luminescent Properties: Application of Magnetic/Luminescent Particles in Immunoassays,” in Proceedings of SPIE, vol. 6036: BioMEMS and Nanotechnology II (Bellingham, WA: SPIE, 2006), 60360T.
4. M Meza, “Applications of Magnetic Particles in Immunoassays,” in Scientific and Clinical Applications of Magnetic Carriers, ed. U Häfeli et al. (New York: Plenum, 1997), 303–309.
5. RC Birkmeyer et al., “Application of Novel Chromium Dioxide Magnetic Particles to Immunoassay Development,” Clinical Chemistry 33, no. 9 (1987): 1543–1547.
6. H Zhan and ME Meyerhoff, “Gold-Coated Magnetic Particles for Solid-Phase Immunoassays: Enhancing Immobilized Antibody Binding Efficiency and Analytical Performance,” Analytical Chemistry 78, no. 2 (2006): 609–616.
7. D Lozano et al., “Analytical Performance of the Sanofi Access Cardiac Troponin-I Procedure,” Clinical Chemistry 43 (1997): 1088–1089.
8. DGM Carville and KE Guyer, “Coagulation Testing, Part 1: Current Methods and Challenges,” IVD Technology 4, no. 4 (1998): 59–66.
9. DL Reich, “Monitoring Hemostasis in the Perioperative Period: Anticoagulation Control,” Journal of Cardiothoracic and Vascular Anesthesia 6 (1991): 4–7.
10. JL Tonkinson et al., “Smaller Is Better,” IVD Technology 9, no. 2 (2003): 29–34.
11. ME Waris et al., “Two-Photon Excitation Fluorometric Measurement of Homogeneous Microparticle Immunoassay for C-Reactive Protein,” Analytical Biochemistry 309, no. 1 (2002): 67–74.
12. C Robillot et al., “A Handheld Immunosensing Device Based on Low-Cost Self-Contained Microfluidic Cartridges and Ferromagnetic Actuation. Refining Point of Care Testing Strategies for Critical and Emergency Care” (poster presented at the American Association for Clinical Chemistry Critical and Point-of-Care Testing Div. 21st International Symposium, Quebec, September 28–30, 2006).
13. A Hatch et al., “A Ferrofluidic Magnetic Micropump,” Journal of Micro-Electromechanical Systems 10, no. 2 (2001): 215–221.
14. CH Self and DB Cook, “Advances in Immunoassay Technology,” Current Opinion in Biotechnology 7, no. 1 (1996): 60–65.
15. D Sezholtz et al., “Paramagnetic Particles for Optimized Biological Separations,” IVD Technology 11, no. 2 (2005): 39–43.
16. SI Tu et al., “Factors Affecting the Bacterial Capture Efficiency of Immuno-Beads. A Comparison between Beads with Different Size and Density,” Journal of Rapid Methods and Automation in Microbiology 11 (2003): 35–36.
