Biotechnical Diagnostics

(From "Biotechnology at Work" by Industrial Biotechnology Association, Washington, DC 20006, Tel. 202/857-0244)

After five days of suffering a miserable sore throat, you find yourself in your doctor's office. Your doctor thinks you may have strep throat, a serious bacterial infection that, if left untreated, can lead to kidney and heart disease.

The doctor swabs your throat, sends the specimen to a laboratory for analysis, and three days later you know if you have strep throat. Meanwhile, the doctor is unsure whether or not to prescribe an antibiotic to fight the infection. But if the doctor could detect strep throat while you are still in the office, appropriate treatment could begin immediately.

Now, because of the diagnostic applications of biotechnology, doctors can identify strep throat, right in their offices, in a matter of minutes.

The first step in treating or curing any disease or infection is diagnosis, and the diagnostic applications of biotechnology extend far beyond strep throat. Heart disease, cancer, AIDS, cystic fibrosis, kidney disease and sickle-cell anemia are just some of the areas for which the biotechnology industry has been developing new diagnostic tools.

This article discusses the latest advances in diagnostics and looks at where applications of biotechnology are headed.

DISCOVERY OF DNA AND CELL FUSION TECHNOLOGIES

The origin of DNA technology can be traced to the mid-1800s and the work of Gregor Mendel, an Austrian monk and botanist. His work with pea plants uncovered the first evidence that genetic traits were passed from generation to generation.

In the early 1900s, biologists discovered that humans obeyed the same basic laws of heredity expressed in Mendel's work. THey found that conditions such as hemophilia, color blindness and baldness were passed from parent to child through chromosomes, the components of every living cell that contain genetic information.

By the early 1950s, scientists developed an understanding of the workings of DNA, or deoxyribonucleic acid, the molecule that carries the genetic information for all living systems.

In the early 1970s, genetic engineering entered a new frontier. Scientists created new genetic instructions by combining segments of DNA from different organisms. This process is called gene splicing, or recombinant DNA.

At the same time, other scientists focused their attention on monoclonal antibodies. Antibodies are produced in the body by white blood cells. They locate (and assist the body in attacking) bacteria, viruses, cancer cells and other foreign substances. Monoclonals are highly specific versions of the antibodies.

But it wasn't until the mid-1970s, when two scientists discovered how to mass produce monoclonals, that their use as diagnostic (and also therapeutic) tools began to take shape. By fusing, in a laboratory petri dish, an antibody-producing white blood cell with a cancer cell that produces unlimited generations of cells, the scientists developed a method to produce increased and consistent quantities of a particular monoclonal antibody. This manipulation is called hybridoma technology.

Using monoclonals in diagnostic tests requires scientists to produce the purest quality of these specific antibodies possible. At the same time, scientists also need mass quantities of the monoclonals. Hybridoma technology meets both of these needs.

The 1970s gave us yet another major contribution from the scientific world: DNA probes. Scientists developed the ability to extract single, small strands of DNA that could be used to seek their complementary matching strand.

These DNA probes can locate specific genetic material, information that is useful for both the detection and the treatment of various diseases.

IMPACT OF ADVANCES IN DIAGNOSTICS

The primary targets of research in the diagnostics field have been genetic and infectious diseases. Genetic diseases are those in which heredity plays either an exclusive or significant role. Infectious diseases are spread from person to person through exposure to a virus or bacterium.

Many Americans suffer from these conditions: Adult polycystic kidney disease - 300,000 to 400,000; Sickle-cell anemia - 50,000; Cystic fibrosis - 30,000; Huntington's disease - 25,000; Duchenne muscular dystrophy - 20,000 to 30,000; Hemophilia - 20,000; Alzheimer's disease - 2 to 4 million; and Manic depression 1 to 2 million. These data are reflective of the number of lives that are touched by inherited diseases.

To a large extent, the discovery of the genetic basis for these diseases has occurred in the last decade. Currently, there are more than 3,000 known genetic diseases. The development of biotechnology-based diagnostics will allow physicians to identify many of these illnesses more accurately and quickly.

Meanwhile, infectious diseases are among the most prevalent and dangerous threats to the health of the American public. Federal officials estimate that more than 1.5 million people have already been exposed to human immunodeficiency virus (HIV), which can lead to the acquired immune deficiency syndrome (AIDS). AIDS had already claimed the lives of more than 30,000 Americans by early 1988.

Other infectious diseases do not share the headlines with AIDS, but their dangers persist. For example, hepatitis B is diagnosed in 300,000 patients every year. Influenza causes up to 50,000 death per year.

Advances in biotechnology-based diagnostics will afford improved and earlier detection of infectious and genetic diseases. Currently, some diseases are extraordinarily difficult to diagnose properly. What will these new advances mean for the patient? Early diagnosis of diseases can have a significant impact in three areas:

HIGHER SURVIVAL RATE. Breast cancer is one of the leading causes of death in women, and most Americans are aware of the value of monthly breast self-examinations. Finding a lump in a breast before it spreads to other parts of the body can save a woman's life. The theory is the same for biotechnology-based diagnostics. In fact, some of these diagnostics will be able to identify illnesses (cancer, alcoholism and others) before the appearance of any symptoms. Although early detection is not a guarantee of survival against all diseases, many patients will live longer if appropriate therapy begins as soon as possible.

IMPROVED QUALITY OF LIFE FOR THE PATIENT. By identifying a disease at its earliest stages, doctors can often prescribe treatments with the fewest side effects. For heart disease, it may mean a change in diet and increased exercise instead of surgery. For cancer, early diagnosis may mean surgical alternatives to chemotherapy are more feasible.

REDUCED HEALTH CARE COSTS. Again, by diagnosing a disease at its earliest stages, patients can often avoid surgery and hospitalization by undergoing less expensive treatments. Not only does this benefit the patient afflicted with the disease, but it can have an impact on health care and insurance costs throughout society.

APPLICATIONS OF BIOTECHNOLOGY-BASED DIAGNOSTICS

MONOCLONAL ANTIBODIES. As discussed earlier, monoclonal antibodies are highly specific. They are cloned, or duplicated, from a single white blood cell that produces a specific type of antibody. Because of their specificity, monoclonals can be used to diagnose infectious diseases and other conditions.

In order for a monoclonal antibody to be used in health care application, it must be linked to some sort of substance, such as a drug or an imaging agent. The monoclonal acts as a guided missile programmed to reach an exact location. When it hits its target, an imaging agent, such as a tiny radioactive particle, transmits information back to the doctor.

Many people are already using monoclonal technology in their homes to detect blood in the stool (an early warning sign of rectal cancer and other illnesses), to identify the time of ovulation, or to test for pregnancy. Diagnostic uses of monoclonal antibodies in laboratories include testing for sexually transmitted diseases (syphilis, gonorrhea, chlamydia), hepatitis B and cystic fibrosis.

Monoclonals are also used in the battle against AIDS. Current technology allows doctors to identify the existence of antibodies produced by the body when it is exposed to HIV. But scientists are trying to develop a monoclonal antibody-based diagnostic that will confirm when a patient has actually been infected with AIDS. They are also trying to find a way to treat AIDS using monoclonal antibodies.

Although not yet available for widespread use, clinical testing of monoclonal antibody-based technology for heart disease is underway. It is hoped these tests will locate dangerous blood clots, determine the severity of atherosclerosis (the hardening or narrowing of arteries, which is the underlying cause of most deaths from cardiovascular disease), and the extent of damage to a patient's heart following a heart attack.

Other diagnostic applications of monoclonal antibodies focus on cancer. One currently available diagnostic test identifies the continued presence of ovarian cancer in women who have already undergone initial treatment. This test helps doctors determine the necessity of follow-up exploratory surgery, and assists them in deciding to alter or discontinue therapy following this second look. Some 12,000 women die from ovarian cancer each year.

Clinical trials are underway for another monoclonal-based diagnostic, designed to help diagnose six cancer types (lung, colorectal, breast, pancreatic, stomach and ovarian). Together these cancers account for over 60 percent of the annual cancer deaths in the United States.

In this procedure, a radioactive substance is linked to a monoclonal antibody that can identify the presence of any one of these six types of cancer. The monoclonal transports the radioisotope to tumor sites, making their location visible through the use of an X-ray machine.

The test also confirms the malignancy of the tumors, and helps physicians determine which tumors can be successfully removed before surgery ever takes place. These distinctions were not possible with previous diagnostic methods.

DNA PROBES. In the 1970s, scientists found ways to cut DNA into fragments at predictable points, using a kind of chemical scissors called restriction enzymes. After studying large groups of family members and their genetic makeup, they identified variations in the size of the DNA segments, called polymorphisms, that appeared along with certain diseases.

Using this knowledge, scientists devised DNA probes, short portions of DNA that are able to attach themselves to the polymorphism associated with a specific disease. The probes are labeled with a radioactive substance. They can be easily visualized by exposure on film.

DNA probes are used to diagnose a variety of genetic diseases, including Huntington's disease, Duchenne muscular dystrophy, and cystic fibrosis. Because they can often detect and identify diseases and infections in a matter of hours, DNA probe-based tests could replace current tests that take days to complete.

Dentists are also using DNA probes to diagnose periodontal (gum) disease, perhaps the most prevalent of all infectious diseases other than the common cold. According to the National Institute of Dental Research, more than 90 million Americans have periodontal disease. At least 23 million of them have severe cases. Gum disease accounts for 70 percent of all adult tooth loss.

Although this infection can be extremely painful, it often begins and progresses unnoticed. A test using DNA probe technology can now detect the various bacteria that cause the disease. This test establishes progression of the condition, helps dentists select appropriate therapy, and monitors treatment results.

Another important application of DNA probes is found in the food industry. DNA probe-based diagnostic tests can rapidly detect disease-causing microorganisms such as Salmonella, a bacterium that is a common cause of food poisoning.

The standard culture method for the detection of Salmonella in food requires a minimum of four days to identify negative samples. If the culture is positive, indicating the presence of the bacteria, an additional two to three days are required for confirmation,

This slow process causes a considerable expense to food processors, whose food must remain in quarantine during these diagnostic tests. Rapid detection of Salmonella in food products benefits the food industry by reducing inventory costs and response time in the event of a contamination problem.

A new DNA probe-based assay provides much quicker diagnosis of Salmonella contamination. When the probe is labeled with an identifiable "tag," it can determine the presence or absence of the bacteria. Nearly 100 samples can be analyzed in four to five hours following the growth of a culture in the laboratory. The test also provides confirmation of positive samples.

GENE MAPPING. Human genetics is in the midst of a revolution. In the mid-1970s, about all that could be done was study inherited diseases and track their frequency. Not it is possible to locate and identify those genes that cause hereditary diseases. As scientists learn more about defective genes, the role they play in disease, and their locations relative to each other, they are able to create a type of map. This process is called gene mapping.

Just as the explorers Lewis and Clark pieced together information into maps that guided settlers of the new American frontier, scientists are creating maps that will help lead medical researchers into the 21st century, and beyond.

Genetic mapping allows for the development of tests to diagnose diseases. Further study of the gene may provide new directions for treatment.

The complete genetic code of a human being is contained in 50,000 to 100,000 genes comprised of DNA. As discussed earlier, these genes are located in the 23 pairs of chromosomes that each of us possess.

Scientists are able to break the chromosomes into pieces called RFLPs, or restriction fragment length polymorphisms. RFLPs are also called genetic markers because they mark the location of a defective gene. Imagine you are looking for the public library, and someone tells you that it is next to a certain landmark, such as city hall. Now every time you try to find the library, you may look for the landmark and know that you will find it.

An RFLP is like city hall, a marker that helps scientists find the approximate location of a defective gene. Currently, genetic markers are useful for diagnosis in families in which specific inherited diseases are prevalent, such as cystic fibrosis.

Scientists have pinpointed the gene that causes cystic fibrosis, a disease that affects the digestive and respiratory systems so severely that, if not diagnosed early, premature death is often the result. With the discovery of the defective gene, the fetus of a woman who already has one child afflicted with cystic fibrosis can now be screened and diagnosed early in her pregnancy with 99 percent accuracy.

While there is no known cure for cystic fibrosis, early diagnosis can lead to therapy that can improve both the quality of life and the life expectancy of the patient.

Defective genes have been linked to other diseases as well, including Duchenne muscular dystrophy, adult polycystic kidney disease, a familial form Alzheimer's disease, a familial form of colon cancer, and a form of manic depression found among the Pennsylvania Amish.

USES IN AGRICULTURE. Have you ever noticed a house plant that has sagging leaves? Or maybe they have turned yellow, or have fallen off their stems. When it comes to their health, plants are a little like people. Infectious diseases can make them sick. The same is true with farm animals. That's why biotechnology-based diagnostics will play an important role in agriculture.

Some of the most promising aspects of new diagnostics are their potential to reduce the use of certain chemicals, and to better target the application of some necessary chemicals in the fields. By quickly identifying a crop disease, a farmer can use a more specific type of herbicide or fungicide in a smaller dose. This can help a farmer increase the yield and reduce the cost of raising crops. To the consumer, it might mean lower food prices. It can also mean a cleaner environment, including fewer chemicals in groundwater.

Diagnostics for conditions that cause rotting in stored vegetables can also prevent tremendous losses, as can tests for diseases common among expensive fruit trees.

Monoclonal antibody-based diagnostics can identify fungal diseases affecting many plants. An example of a test already in use involves turf grass. It is being marketed to golf courses and will soon be available to home gardeners.

The turf grass diagnostic kit detects three highly destructive fungal diseases (pythium blight, dollar spot and brown patch) before visible symptoms appear. As with early diagnosis of diseases in humans, early identification of turf grass problems means appropriate treatment can begin at a time when it can be the most beneficial.

The disease can be diagnosed by using a dipstick. A plastic stick is coated with the diagnostic material. The stick is dipped into the soil, and if a disease is present, the tip of the dipstick is turns purple. The severity of the disease is determined by the depth of the color.

Monoclonals will also provide quick and definitive diagnoses of animal diseases. Now, when an animal gets sick, the farmer or veterinarian often can only treat the symptoms. But many diseases can produce similar symptoms, so without a quick and accurate diagnosis, the farm animal -- or the domestic companion animal -- may not receive proper treatment.

ETHICAL CONSIDERATIONS

As the advances in diagnostics expand our knowledge of the human genetic code, society must ensure that this information is used properly. The biotechnology industry must be careful to protect the rights and safety of people; it does not take this responsibility lightly. This is one of the roles of government regulation, and various federal agencies are working with the scientific community to ensure that our health and the environment are protected.

While biotechnology-based diagnostics may confirm the presence of some diseases for which there are no life saving treatments at this time, the ability to use the tests to study these diseases enables scientists to develop new approaches for prevention and cure.

THE FUTURE OF DIAGNOSTICS

Have you ever wondered why some people smoke two packs of cigarettes a day and live to be 90 years old, while others develop lung cancer at the age of 45? Or why an apparently healthy person dies of a heart attack at 40, while someone who is overweight and has bad eating habits seems to be immune to heart disease?

The answer may lie in their genes. It appears some people are more likely than others to develop high blood pressure, heart disease, cancer, diabetes, arthritis, alcoholism and other conditions. These people are said to have a genetic predisposition to certain diseases.

Scientists hope that gene mapping will lead us into a new era of diagnostics. Much of the scientific community is concentrating its efforts on mapping the genome, the entire genetic material of humans. The project, which is being worked on by government and private scientists, is expected to take years to complete. It will probably cost hundreds of millions of dollars to pay for this research.

Through the mapping of defective genes and their markers, many diseases could be diagnosed just a few weeks after conception. In some instances, gene mapping may lead to effective treatments where currently there is no cure.

In late 1987, several judges around the country allowed the results of biotechnology-based tests to be used as evidence in criminal cases. A Florida court convicted a man of rape and assault on the basis of a DNA test.

Some scientists and law enforcement officials believe that DNA probes and monoclonal antibody-based tests will be used more extensively in the future. The tests, which can analyze blood and other body fluids, may provide more accurate identification of both suspects and victims. As the use of these tests becomes more widespread, prosecutors and defense attorneys may turn to biotechnology to support their cases.

Biotechnology-based diagnostics that have been approved by federal regulatory agencies involve in vitro (in the laboratory) techniques. But researchers are developing diagnostics that are used in vivo, or in the body. In vivo diagnostics will allow doctors to "see" diseases as they appear within our bodies. This will provide doctors with greater insight into diseases that have confounded us for centuries, leading to improved treatment for all of us.

But the most promising potential result of advances in diagnostics goes beyond merely treating the diseases that affect our lives. The understanding that biotechnology- based diagnostics will provide may help scientists find the true causes of these diseases and provide them with the information necessary to prevent and cure them.