Diagnosis: Medicine

Diagnostic tests are also critical to the drug discovery and development industry, where they serve as research tools.For example, a drug candidate might be identified in high-throughput screening by its ability to block a diagnostic binding site, signaling its presence by interfering with the assay.

Traditional testing
The worldwide clinical diagnostics industry is valued at approximately $19 billion, with a growth rate of nearly 5% per year (1). In the worldwide market, the biggest areas of bulk sales are for immunoassays ($7.2 billion yearly), clinical chemistry ($3.1 billion), hematology and blood gases (>$2.0 billion), and routine microbiology ($1.3 billion). Although a biochemical assay exists for almost every disease, diabetes is the largest single disease diagnostic category at $2.8 billion yearly.

The modern popularity of the immunoassay is almost directly related to the development of monoclonal antibody technology in 1975. Hybridoma-derived or bacterially cloned monoclonal antibody technology has enabled the mass production of highly specific probes for antigenic sites, whether on enzymes, receptors, hormones, or microbial products.The great utility of such antibody assays is in their ability to be easily automated and standardized, primarily through an adaptation of the enzyme-linked immunosorbent assay (ELISA). By linking an enzyme that can produce a color reaction with the appropriate substrate to the diagnostic antibody, the presence of a disease-related antigen can be rapidly determined through antibody binding and colorimetric screening.

Kits for research or clinical diagnostics exist for almost every imaginable disease or physiological condition. Immunomonitoring of 6-sulfoxymelatonin provides diagnostics for depression, a variety of sleep disorders, and even premenstrual syndrome. ELISA kits are used to detect the allergic status of patients by detecting the presence of IgE-mediated versus non-IgE-mediated allergies. Several neuropathologies, including Guillain–Barré syndrome, can be diagnosed and monitored for therapeutic purposes by an anti-myelin-associated glycoprotein immunoassay.

Radioimmunoassays (RIAs) also have a long history of diagnostic use, but they are becoming less popular than colorimetric assays because of worker safety and hazardous waste disposal issues.³H and ¹²⁵I, however, provide relatively safe and highly sensitive signals of antibody binding for assays ranging from the detection of vitamin B₆—associated with the local recurrence and metastasis of breast cancer—to the detection of angiotensin II, which is a useful marker of essential hypertension.

Genomics meets diagnostics
Analysts see a particularly bright future in the diagnostics market for nucleic acid and genetic testing. The traditional sequence-matching probes (currently “only” a $500 million market worldwide) and the newer single nucleotide polymorphism (SNP) approach are likely to become ever more important because of the tide of genetic information obtained from the completion of sequencing projects, from that of the human genome to those of various microbial parasites.As more of this information is processed and made usable, new and better sites for genetic probe diagnostic assays will inevitably present themselves. Immunoassays will undoubtedly also benefit as knowledge of the genome is translated through proteomics and into diagnostic antibody probes.

The most routinely used nucleic-acid-based diagnostic test has been that for HIV testing,often used as a follow-up to antibody-based tests. Recently, assays for the hepatitis C virus have become more available, as new therapies have spurred diagnosis. Many companies are developing or have recently begun marketing nucleic acid probe kits for various cancers, including those of the breast and prostate, as well as for numerous bacterial infections.

Home is where the drug test is

Have children? Worried about their dealing with a world rife with illicit narcotics, liquor, and tobacco? Perhaps it is time you instituted your own Family Drug and Alcohol Policy, as have so many employers across the nation. Numerous companies offer home diagnostic kits that detect drug and alcohol use, often with the same methods as those used for workplace testing. Intrinsic to such a program, suggests one such company, Home Health Testing, is for you to “consider making random drug and alcohol testing a part of the family policy.” According to the company’s Parent’s Info Center (www.home-drugtest.com/link07.htm), “Letting your children know at an early age that they may be subject to random testing will make it easier in the future.”

There’s no place like home?
One benefit of the reproducibility and ease of use of modern diagnostic kits is the development of home versions of these tests. From monitoring your own insulin or cholesterol level to determining whether you are pregnant; from diagnosing HIV, diabetes, or colorectal cancer to whether you test positive for alcohol or cocaine—the home assay is coming into its own. (See box at right). The key requirement for home test diagnostic kits—ease of use and interpretation—usually makes them more difficult to develop than assays designed for laboratory-trained professionals, where sensitivity issues can outweigh such considerations.

However, significant dangers and dilemmas are associated with the development and use of home tests. Among these is still the issue of mistakes and improper use, despite the improvements—false positives or false negatives confusing and perhaps even endangering the patient’s life.But more frequently, especially in cases of the diagnosis of life-threatening conditions, the biggest concern is the risk of psychological trauma that a positive diagnosis can have on what are essentially unprepared, and quite frequently, solitary patients without a doctor or support group. When the list of home test kits includes those for HIV and prostate cancer, problematic outcomes can easily be imagined. Even home pregnancy tests can have a significant negative psychological impact, especially for minors and rape victims.

Suicide is only the most obvious of the possible responses. Despair, apathy, or rage at what the home-tester may (mistakenly) consider a death sentence can transform a patient’s life, keeping him or her from seeking appropriate medical care, while overshadowing all routine life events, even before the onset of debilitating physical symptoms.For this reason, most home kits for significant diseases include information on obtaining counseling, often including hotline numbers provided at company expense.

Doctor, you can tell me . . .
Historically, diagnostic assays have usually been done by specially designed clinical laboratories, either on-site or, more typically, outsourced. One recent trend in diagnostics, however, made possible by improvements in assay technology, has been the popularity of the “point-of-care” concept.

In point-of-care, the assay is performed on-site and interpreted immediately. This procedure can be as simple as a nurse or doctor taking a sample and then testing it with a home diagnostic kit such as those described above. Or it can be as complex as using colorimetric or fluorometric assay kits (sometimes complete with minichromatography columns), whose results are read by relatively inexpensive, portable spectrometers or fluorometers that have been purchased or leased on-site to quantify results. In either case, the results can be immediately interpreted and quickly related to the patient for the establishment of an appropriate care or drug regimen.

The growth of e-noses
A unique approach to diagnostic kits is the development of e-nose systems. E-nose technology has been around for almost 10 years, but medical applications, especially in diagnostics, are relatively recent. Table 1 gives a sample of the medical areas being “e-nosed” by various research groups.

Who nose what evil lurks . . .

Your head is pounding, your mouth feels pasty, and your stomach is doing its rendition of The Perfect Storm. A uniformed man hands you a straw into which to blow and then shakes his head when he reads the machine’s monitor. No, you’re not standing on a rain-slick roadside awaiting your just deserts. You’re actually in a doctor’s office, and your physician is about to say that there is a chance you have tuberculosis (TB). Really? Well, maybe not yet. But if Joseph Stetter of Chicago’s Illinois Institute of Technology has anything to say about it, there will soon come a day when a simple breath test will tell physicians a lot about your medical condition. Stetter and colleagues have designed an e-nose that directly senses the presence of TB bacteria in the lungs, much as a Breathalyzer approximates the amount of alcohol in the bloodstream by measuring the alcohol content of exhaled air. Unlike the common Mantoux test, which measures only anti-TB antibodies, the e-nose system measures chemicals produced by the bacteria and should therefore be more accurate.

In most cases, e-noses that are being developed for medical diagnostics measure chemicals released by bacteria, either natural metabolites or compounds generated by the organisms in response to the chemistry of their environment. These compounds are often captured in the headspace of a sample vial and then blown across the surface of a biochip, which might contain 30 or more sensors that measure different chemical constituents (see box at right).

Most e-noses used for medical diagnosis are based on the principle of the disruption of an electrical current as it passes through a chip. For e-noses like those made bythe aptly named Cyrano Sciences in Pasadena, CA (the Cyranose), this disruption is mediated by the interaction of volatile organic molecules with conductive composite materials housed in a polymer matrix. As the molecules interact, the composite material expands and its electrical resistance changes. This change reflects the concentration and identity of the organic molecules.

Similar in concept is another group of e-noses that use metal oxides instead of composite materials and polymers. This category includes the KAMINA e-nose, which was designed at the Karlsruhe Research Center in Germany. In this device, the chip’s surface, typically SnO₂ or WO₃, is heated, and atmospheric oxygen becomes bound to the surface. The oxygen moleculesstrip electrons from the metal, reducing its conductivity. As oxidizable organic molecules contact the surface, their combustion releases the oxygen and returns the electrons, and thus conductivity, to the metal surface.

Another category of e-noses now undergoing medical application testing is based on the quartz microbalance. This system measures the beat frequency between two quartz-controlled oscillators, one of which is coated with metalloporphyrins. As organic molecules interact with the coated oscillator, its resonant frequency decreases relative to that of the reference unit, and this difference provides the signal. The composition of the metalloporphyrin coating provides the selectivity for specific compounds.

Regardless of the mechanism, e-nose technologies are expected to make great strides in the field of medical diagnostics. It may not be too much longer before a Breathalyzer does more thanhelp revoke your driver’s license.

Future diagnosis
If the concept of genetically tailored drugs is to become viable, diagnostic kits will be the key to identifying and monitoring appropriate patient populations. The breakthroughs needed are not in the methodology of diagnostics. Whether ELISA, RIA, a DNA probe, or an e-nose is used, efficient and easily automated technologies are available.

The real problem, as always, will be in that ever-elusive area of target identification. What antigenic site, gene, or gene product is truly diagnostic for disease X or metabolic condition Y? The discovery of such information is potentially the greatest benefit of genomics and proteomics. Because such information is also required for rational drug design and rapid screening and identification of drug candidates, it is obvious that diagnostics and drug discovery will grow functionally ever closer, to the benefit of both.

Reference

1. Hasulo, S. Biobusiness: Trends and Developments in Canadian Life Science, Winter 2000/2001, pp 1, 4–5.

Randall C. Willis is an assistant editor and Mark S. Lesney is a senior editor of Modern Drug Discovery. Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.