Medical Device Link.

BEYOND CLINICAL DIAGNOSTICS

Samples are loaded into the Ruggedized Advanced Pathogen Detection System to screen for potential biological warfare agents using the Critical Reagent Program polymerase chain reaction reagents.

The significance of the effects of bioterrorism-associated category A agents (e.g., anthrax, plague, tularemia, and smallpox) on human health, public safety, and the economy makes the availability of rapid and accurate diagnostic testing of critical importance. Diagnostic technologies are an important first-line resource in identifying the agent in a timely manner, monitoring the geographic spread and impact of interventions, and facilitating clinical treatment by differentiating patients with infection from those with other but similar illnesses.

Diagnostics are relevant for determining how certain viral and bacterial agents or their products (e.g., toxins) will respond to antimicrobial therapy. They are also important in determining the likelihood of patient survival as well as the need for implementing special public health safety and containment measures. Thus, advances in the development of new and more-specific diagnostics will play a pivotal role in providing rapid and accurate analyses of both environmental and clinical specimens in the defense against bioterrorist events.

Immunoassays

The expanded use and performance characteristics of diagnostic assays based on the use of antibodies has progressed rapidly during the past several decades.¹ The replacement of radioisotopes with enzymes by researchers in the 1970s resulted in the introduction of enzyme-linked immunosorbent assays (ELISAs), which have become common tools for identifying various disease states, especially infectious diseases.^2,3

While ELISA technology is relatively rapid, sensitive, and specific, the need for even-more-rapid and easy-to-use assays continues to drive further improvement and format expansion of immunoassays. The development of single-use immunoassays, primarily for pregnancy testing, in the 1980s was based on the development of immunochromatographic assays that are rapid and simple to use.⁴

Immunoassay Formats

There are many variations on the application of immunoassays to the detection of potential biowarfare agents. As with all immunoassays, the primary limitations can include the sensitivity and specificity of the antibodies employed. While there are several different immunoassay formats, antigen-capture or competitive assays are the most common being used for detecting biological threat materials. The principle of the antigen-capture is the sandwich assay format in which an antibody binds and holds the target analyte and a second antibody is tagged in a manner that facilitates detection of the reaction. The analyte (usually an antigen) is sandwiched between the two antibodies where a signal is detected on a solid surface.

A variation of this format is based on competition between a known quantity of analyte and the sample analyte. For analytes that are made up of small molecules with limited binding sites, the competitive format is preferred. By including the analyte of interest into the test system as the labeled component rather than as a second antibody, a competition for binding with the antibody is created and the loss of signal indicates the presence of the unknown analyte.

From the use of rapid assays in providing first-line detection of agents of bioterrorism to the use of immunoassays as part of orthogonal testing, immunoassays are a very significant and established component in today’s diagnostic toolbox. Of particular importance is the role of immunoassays in orthogonal testing.

Orthogonal testing refers to tests that are statistically independent or nonoverlapping but, in combination, provide a higher degree of certainty of the final product. While orthogonal testing is not a standard perspective in the industry, the concept and application certainly needs to be. Any single detection technology has a set of limits with regard to sensitivity and, most importantly, specificity.

When it comes to the consequences of biothreat detection, there is little room for error, unlike those associated with over-the-counter pregnancy tests. In detection of a biothreat, the limited use of immunoassays solely as first responders is naïve and short-sighted. Immunoassays have long held a more definitive role in the clinical laboratory. Moving immunoassays beyond the role of the first responder in bioterrorism defense strategies is certainly within the technical application and practice of diagnostics.

Handheld Assays

Figure 1. (click to enlarge) Handheld assays with positive and negative results.

Handheld assays (HHAs) are immunoassays based on the principle of immunochromatography or lateral flow (see Figure 1). The basic premise of the test is that a sample is applied to the test cartridge and within a few minutes, an indicator line demonstrates the presence or absence of the analyte of interest. The test sample migrates through the filter paper matrix, which contains both fixed and impregnated antibodies. As the sample migrates, it solubilizes the tagged antibodies and initiates antibody binding to the target. The analyte of interest encounters the second set of antibodies and forms an antibody-analyte-antibody sandwich matrix.

While precursor HHAs incorporated enzymes as labels to yield a visible signal, advances in signal generation have eliminated the multistep enzyme immunoassay and replaced it with incorporated signals (tags) based on physical entities such as colloidal gold, carbon, or colored latex spheres. These signal generators rely on the aggregation of a large number of tagged antibody-analyte reactions to become visible to the naked eye or an instrument.

In general, the sensitivity of this signal is lower than that of an enzyme-amplified system, but due to its ease of use, it allows reagentless operation by nonlaboratory personnel. As is true with most analytical systems, HHAs have some inherent limitations in their use and interpretation.

One of the biggest limitations is decreased sensitivity compared with other immunoassay formats. Even the best HHAs differ from comparable ELISA formats by at least one order of magnitude, although this determination is not without controversy.

In an effort to improve the sensitivity of HHAs while retaining overall simplicity, IVD manufacturers such as Response Biomedical Corp. (Burnaby, BC, Canada) are incorporating different signal-generation methods such as fluorescent microspheres. This format requires the use of Response’s RAMP system to detect the signal. Paramagnetic particles serve as the antibody tag, with detection of the signal accomplished using the Magnetic Assay Reader by Quantum Design (San Diego). Both technologies offer improved sensitivity while providing a quantitative assessment of the amount of analyte present.

Other limitations of HHAs include the potential for false positives as a result of “dirty” environmental samples that form a dirt line in the zone of antibody capture. This line can easily be misinterpreted as a positive signal by personnel who are not well trained in the use of the assay.

As is evident with most immunoassays, there is a point at which a false-negative reaction will occur if too much analyte is tested. This prozone effect is well known and has been overcome in some HHAs, including the RAMP system, but the effect must nonetheless be considered when using HHAs for direct analysis.

Furthermore, the amount of sample required for HHAs, and the need for additional retesting, can be detriments to law-enforcement agencies that require additional samples for first responders conducting an investigation at the scene. Combined with the lack of overall validation of sample preparation and testing by HHAs, many agencies are opposed to the use of HHAs by first responders.

While HHAs have limitations, their overall use does have some significant advantages in the detection of biological threats. When used in conjunction with air-sampling systems, they can provide a relatively rapid automated screening of samples. This promotes the efficient application of labor and resources to the definitive analysis of only those samples that exceed the screening.

The inherent advantage of an HHA’s use by nonlaboratory persons is the ability to perform a quick assessment of samples at the scene which allows incident commanders to make more-informed decisions without the delay associated with off-site laboratory analysis. However, even in these situations, the advantages and limitations of any assay must be considered when responding to biological threat incidents. This is why all HHAs are regarded and marketed as screening tests and not as confirmatory tests.

Time-Resolved Fluorescence

Although a seldom-used older technology, time-resolved fluorescence (TRF) employs the basic analyte sandwich capture format. The strength of TRF over conventional ELISAs is increased sensitivity and the potential for multiplexing.

The principle of TRF lies in the differential fluorescence life span of lanthanide chelate labels compared with background fluorescence. The long-lived fluorescence signal and a large Stokes shift (difference in wave- length between absorbed and emitted quanta) results in a very high signal-to-noise ratio and excellent sensitivity.^5,6 The long fluorescence decay time allows the measurement of immunoassay fluorescence after any remaining background fluorescence decays. The TRF signal is wavelength-dependent and is produced by excitation of the lanthanide chelate. By pulsing light repeatedly at 1-second intervals, the fluorescent material can be excited over 100 times with an accumulation of the generated signal.

TRF assays are particularly useful in clinical immunoassays but have limitations when applied to environmental samples. Such samples have the potential to be contaminated with europium or other lanthanides commonly found in soils. These contaminating compounds behave like the labeled lanthanides by prolonging background fluorescence and lowering TRF sensitivity comparable to that of conventional assays.

Electrochemiluminescence

Figure 2. (click to enlarge) Electrochemiluminescence principle.

Electrochemiluminescence (ECL) is another adaptation of ELISA-based technology (see Figure 2). While there are several formats for applying this technology, the preferred format is the analyte-sandwich capture. In this format, an antibody is tagged with a defined chemical that emits light and the signal is triggered by the application of an electrical current.

The ECL chemical moiety may comprise any of the metal-containing organic compounds consisting of ruthenium, osmium, rhenium, iridium, rhodium, platinum, palladium, molybdenum, technetium, or tungsten. Ruthenium is the most commonly used element, especially in the form of tri (2,2'-bipyridine) ruthenium (Ru) because of its relatively small size and because it is easily conjugated to any antibody using standard chemistries without affecting the immunoreactivity or solubility of the antibody.

The technology is comprised of two components: the ECL-label (Ru), which is coupled to an antibody, and the tripropylamine (TPA) present in the reaction buffer. When voltage is applied to an electrode, both components are activated by oxidation. The oxidized TPA is transferred into a highly reducing agent, which activates the Ru to create an excited-state form of Ru. This form returns to its ground state with emission of a photon at 620 nm. An advantage of the Ru-TPA methodology is that the measurement of a single sample can be repeated multiple times because the electron transfer photon-release reaction regenerates the Ru, resulting in signal amplification.

The use of paramagnetic beads to capture the reaction also provides a greater surface area than conventional ELISAs, and the reaction kinetics are faster since there is less surface steric hindrance and diffusion distances. The bead suspension allows for shorter incubation times. Detection limits of 200 fmol/L are feasible with a linear dynamic range spanning six orders of magnitude.⁷

Figure 3. BioVeris Corp. (Gaithersburg, MD) M1M analyzer.

One common ECL platform for biothreat agent detection, the M1M analyzer by BioVeris Corp. (Gaithersburg, MD), incorporates a flow cell with a photon detector above the electrode and a capture magnet positioned below the electrode (see Figure 3). The magnet captures the magnetic bead–Ru-tagged immune complex and concentrates the reactants in a small area. The magnetic-capture flow cells add the benefits of washing away extraneous materials and reducing background noise. In addition to the BioVeris system, ECL technology has been incorporated into the Elecsys Immunoanalyzer by Roche Diagnostics (Indianapolis) and the Meso Scale Discovery (Gaithersburg, MD) system.

While ECL assays are simple, rapid, and sensitive, assay sensitivities may vary depending on the sample tested. The sample matrix will influence the interpretation of positive cutoff values. As a result, matrix-specific positive- and negative-control samples are used to establish standard curves and cutoff values.

Microarrays

Figure 4. (click to enlarge) Microarray processing of genomic data.

Microarrays, or DNA chips, are one of the recent methods that have been applied to potential biowarfare agent diagnostics (see Figure 4). Microarrays are a recent adaptation of Northern blots.^8,9 The ability to tag nucleotide sequences with fluorescent tags, much like fluorescent antibody technology, has led to their increased use in diagnostics. Microarrays are small, solid supports onto which DNA sequences are attached or spotted, at fixed orderly locations. Evolving development of microarrays has also included the application of this technology to protein analysis.

Supports are usually glass slides, the size of a typical microscopic slide, but may also be silicon chips or nylon membranes. DNA, typically comprised of short, single-stranded fragments (5–50 nucleotides) commonly known as oligonucleotides, is printed, spotted, or actually synthesized directly on the support. Microarrays can be designed to accommodate tens of thousands of spots, allowing for the collection of a large quantity of data from a single sample.

Microarrays rely on the hybridization properties of nucleic acids. Hybridization refers to the annealing of two nucleic acid strands following base-pairing rules. When sample DNA is prepared, usually by polymerase-based amplification, fluorescent dyes, or other molecules that can accommodate binding of fluorescent dyes, are incorporated into the sample so that hybridization can be detected.

In gene expression studies that use microarrays, two different DNA samples, a test and a control, are typically prepared. During reverse transcription into cDNA, mRNA samples are labeled with different fluorochrome dyes, usually Cy5 (red) and Cy3 (green), and the samples are mixed together into one single sample. After the sample is incubated with oligonucleotides on the microarray slide, the DNA hybrids are created. In most diagnostic applications, a single dye is used noncompetitively. After a washing phase, the slide is scanned with a laser and the intensity of the fluorescence signal at each spot is detected. The fluorescence is proportional to the amount of labeled DNA that has hybridized at that spot. The intensity of fluorescent dyes can also yield quantitative information about the genes in the sample.

The kind of information that is desired drives how microarrays are used. Microarrays can be spotted with known sequences of a variety of oligonucleotides for use in basic genomic investigations.¹⁰ Another genomic approach that is gaining wide acceptance is the use of microarrays to resequence organisms. By using known sequences from previously sequenced microorganisms, the identity of unknown organisms can be determined on the basis of differences in sequences. With nearly 10,000 known sequences to match against (a number that is expanding as automated systems improve), variation in genomic sequences can provide accurate species and subspecies determination.

James W. Snyder, PhD, department of pathology and laboratory medicine, University of Louisville School of Medicine and Hospital. He can be reached at jwsnyd01@ gwise.louisville.edu.

One of the earliest applications of microarrays is their use in transcriptomics or gene-expression studies. Gene-expression-based measurements of mRNA levels, and the differences between these levels in various states of organism growth (i.e., aerobic versus anaerobic), have provided significant insights into gene regulation of various organism functions.

Mark J. Wolcott, PhD, is chief of field operations and training and chief of special pathogens at U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID; Fort Detrick, MD). He can be reached at mark.j.wolcott@ us.army.mil.

While microarrays have the demonstrated potential for diagnostics, general use is hampered by the unavailability of high-quality, validated, and standardized arrays and processes. A major limitation is identification of appropriate targets. While ribosomal RNA gene targets are widely used, the ability to resolve bacteria below the species level is limited. Other bacterial target genes, including housekeeping genes, are potentially useful, but the limitations of data across the wide spectrum of organisms is limited or nonexistent. Even when specific targets are identified, efforts to provide optimal hybridization conditions for all of the probes on an array are challenging. Redundant variations in probes are of some value but are far from optimal.

Another challenging issue in the use of microarrays is the sensitivity of most systems. To obtain appropriate sensitivity for the majority of samples, polymerase amplification is necessary. In most systems, this requires a multitude of primers specific for the genes of interest. Because multiplexed polymerase chain reaction (PCR) is limited to a dozen or so reactions, several hundred iterations on PCR could be required to completely cover all of the potential probes contained on an array, which is not practical for routine use. The use of this technology in routine testing will only occur when an acceptable on-chip amplification or signal detection method is developed.

Conclusion

Developing more-sophisticated diagnostic systems and addressing the current limitations of immunoassay, TRF, ECL, and microarray technologies are necessary steps toward bringing diagnostic testing into even greater prominence in biothreat defensive strategies. The continued development, improvement, and application of these diagnostics, as well as the search for other technologies for the detection of bioterrorism-associated agents, will require the extensive efforts of scientists from academia, state and federal agencies, and private enterprise with the support of national and international assets.

---