Gotcha! New test stalks diseases early

Chemists adapt a DNA test to measure proteins that mark HIV, cancer and more
Apr 12, 2016 — 7:00 am EST
PCR machine

Chemists have created a powerful new test for detecting disease. It uses a common DNA-copying procedure, called PCR, to screen for antibodies in tiny test-tubes containing small amounts of blood or saliva.

vkovalcik/iStockphoto

This is one in a series presenting news on technology and innovation, made possible with generous support from the Lemelson Foundation.

Diseases sneak up on us. They begin triggering changes in the body long before we feel sick. But a new method has the potential to spot telltale signs early, before a disease gains a foothold. That could help doctors diagnose — and treat — disorders before they do too much damage.

The new technique detects antibodies. These are substances made by the immune system. They stick to foreign material, such as germs or cancer cells. This essentially brands the foreigners so the body knows to attack those cells. But occasionally antibodies will brand the wrong cells, triggering their destruction. That can lead to what is known as autoimmune disease. Such conditions include a type of diabetes that can occur in children.

Good or bad, antibodies serve as disease markers. Before symptoms show, these proteins lurk in the blood. But most lab tests can’t detect them early in the onset of a disease. That’s because there’s too little of these markers for a test to spot.

Peter Robinson and Cheng-ting Tsai are graduate students who work in a chemistry lab at Stanford University in Palo Alto, Calif. They were thinking about this problem when they got a flash of insight.

For years, researchers have been using tests to study a person’s DNA. These methods can measure super-small amounts of genetic material using a process known as PCR. Those initials stand for polymerase (Puh-LIM-ur-ace) chain reaction. This process makes millions of copies of DNA fragments in just a few hours. It offers a fast, cheap way to diagnose disorders caused by faulty genes. It also can identify the DNA fingerprint of disease-causing germs.

But antibodies are proteins. And measuring them is more cumbersome than studying DNA. Some tests don’t work well because they force proteins into unnatural shapes. Other tests keep proteins in their normal shape but require expensive chemicals or special equipment.

An “aha!” moment came while Robinson was reading about HIV. This is the virus that attacks immune cells, causing acquired immune deficiency syndrome, or AIDS. The immune system makes antibodies in response to HIV. And most HIV tests check for antibodies to this virus.

Those tests don’t actually scout directly for HIV. Upon learning that, Robinson got the idea to adapt the PCR test to detect HIV antibodies. This idea, he says, “just kind of clicked.”

The innovation

Typically, antibodies are fished out of blood samples by adding molecular “bait.” That’s some compound that the antibody will bind to. Robinson and Tsai did something clever: They attached short pieces of DNA to the “bait.” They used two DNA bits. Let’s call them “A” and “B.” For PCR to work, the “A” and “B” strands need to be side-by-side — but this would hardly ever occur when the bait floats randomly in a solution. However, when antibodies glom onto the bait, the “A” and “B” fragments are much more likely to come close together — close enough for the PCR to work.

PCR makes huge numbers of copies of a desired bit of DNA. If it made lots of the bait DNA, scientists would realize which antibodies had been present, even if there had been only tiny amounts — as would occur in the early stages of a disease.

Still, it wasn’t clear if the technique would work on real samples from sick people. That’s the most challenging part of developing a new medical test, known as an assay, Tsai says. “It’s scary and exciting to look at the results. They tell us whether to keep pursuing the project.”

Initially the team tested its assay on a marker for thyroid cancer. (Not all cancers have a reliable disease marker, but this one does. And it was easy to get patient samples.) Compared with four existing tests for the same marker, the new one was 1,000 to 10,000 times more sensitive. That means the new procedure required only a thousandth to a ten-thousandth as much marker to make a positive identification.

This sensitivity was dramatic. Indeed, Robinson notes, “We were pretty surprised.” The team described its new advance in a paper posted online February 16 in ACS Central Science.

The new findings caught the attention of Mark Pandori. He is a microbiologist at the University of California, San Francisco. He also heads a county public-health lab. Each year, that lab screens thousands of people for HIV. Traditional methods measure HIV antibodies in blood. Nowadays, the lab also uses swab tests that scout for antibodies in saliva.

While more convenient and popular, oral HIV tests can be misleading. Antibodies in saliva are present at only a hundredth to one-thousandth the concentration that they can be found in blood. That means an oral test is only reliable if done at least 40 to 50 days after a person is infected, when lots of antibodies have developed. But people are most contagious 21 to 42 days (3 to 6 weeks) after getting infected, Pandori says. So during that important window of time, people can’t know they are HIV-positive because antibody counts are too low for common tests to pick up.

By comparison, blood tests can detect HIV antibodies just 20 to 21 days after a person first picks up the virus. With the new PCR-based test, “We’d have the sensitivity of the blood test with the convenience of oral fluid samples,” Pandori explains.

Next steps

The Stanford team is now working to validate its test with saliva from the public-health lab. If successful, the test could screen large populations of people for HIV infection at the most valuable time for starting treatment.

The researchers also are hoping to develop their assay for type 1 diabetes, the kind that can start during childhood. The immune systems in people with this disease make harmful antibodies. Those antibodies trigger the killing of cells in the pancreas. That’s the organ that makes the hormone insulin. (The other form of diabetes — known as type 2 — is not an autoimmune disease. People with type 2 diabetes make insulin, but their cells don’t use it as well as they should.)

Srinath Sanda is a pediatrician and researcher at the University of California, San Francisco. He sees a lot of kids with diabetes. He thinks the new assay looks promising. Having a fast, accurate assay could “help determine a patient’s type of diabetes and ensure they receive the right treatment,” he says.
 

 

 

Power Words

(for more about Power Words, click here)

AIDS    (short for Acquired Immune Deficiency Syndrome) A disease that weakens a body’s immune system, greatly lowering resistance to infections and some cancers. It is caused by the HIV virus. (See also HIV)

antibody    Any of a large number of proteins that the body produces as part of its immune response. Antibodies neutralize, tag or destroy viruses, bacteria and other foreign substances in the blood.

assay    A tests used to look for and measure the amount of some looked-for substance. It could be amount of some metal in an ore or perhaps the amount of some virus in a sample of blood.

autoimmunity   A process whereby the immune system turns against its host. This inappropriate reaction can cause disease instead of curing it. Autoimmune diseases can be quite severe and hard for doctors to treat. They include rheumatoid arthritis (affecting joints, such as knees), multiple sclerosis (targeting nerves and muscles), Crohn’s disease (affecting the gut), psoriasis and lupus (affecting skin) and the type of diabetes that typically develops in young children. In all of these cases, the immune system generates out-of-control inflammation.

cancer   Any of more than 100 different diseases, each characterized by the rapid, uncontrolled growth of abnormal cells. The development and growth of cancers, also known as malignancies, can lead to tumors, pain and death.

cell    The smallest structural and functional unit of an organism. Typically too small to see with the naked eye, it consists of watery fluid surrounded by a membrane or wall. Animals are made of anywhere from thousands to trillions of cells, depending on their size. Some organisms, such as yeasts, molds, bacteria and some algae, are composed of only one cell.

chemistry    The field of science that deals with the composition, structure and properties of substances and how they interact with one another. Chemists use this knowledge to study unfamiliar substances, to reproduce large quantities of useful substances or to design and create new and useful substances. (about compounds) The term is used to refer to the recipe of a compound, the way it’s produced or some of its properties.

clinical   (in medicine) A term that refers to diagnoses, treatments or experiments involving people.

diabetes   A disease where the body either makes too little of the hormone insulin (known as type 1 disease) or ignores the presence of too much insulin when it is present (known as type 2 diabetes).

diagnose    To analyze clues or symptoms in the search for their cause. The conclusion usually results in a diagnosis — identification of the causal problem or disease.

DNA   (short for deoxyribonucleic acid) A long, double-stranded and spiral-shaped molecule inside most living cells that carries genetic instructions. It is built on a backbone of phosphorus, oxygen, and carbon atoms.In all living things, from plants and animals to microbes, these instructions tell cells which molecules to make.

germ   Any one-celled microorganism, such as a bacterium, fungal species or virus particle. Some germs cause disease. Others can promote the health of higher-order organisms, including birds and mammals. The health effects of most germs, however, remain unknown.

gland   A cell, a group of cells or an organ that produces and discharges a substance (or “secretion”) for use elsewhere in the body or in a body cavity, or for elimination from the body.

HIV    (short for Human Immunodeficiency Virus) A potentially deadly virus that attacks cells in the body’s immune system and causes acquired immune deficiency syndrome, or AIDS.

hormone    (in zoology and medicine)  A chemical produced in a gland and then carried in the bloodstream to another part of the body. Hormones control many important body activities, such as growth. Hormones act by triggering or regulating chemical reactions in the body. 

immune system   The collection of cells and their responses that help the body fight off infections and deal with foreign substances that may provoke allergies.

infection   A disease that can spread from one organism to another.

insulin   A hormone produced in the pancreas (an organ that is part of the digestive system) that helps the body use glucose as fuel.

marker    (in biomedicine) The presence of some substance that usually can only be present because it signals some disease, pollutant or event (such as the attachment of some stain or molecular flag). As such, this substance will serve as a sign — or marker — of that related thing.

microbiology    The study of microorganisms, principally bacteria, fungi and viruses. Scientists who study microbes and the infections they can cause or ways that they can interact with their environment are known as microbiologists.

pancreas     A gland found in animals with backbones that secretes the hormone insulin and enzymes that help break down foods in the gut.

pathogen    An organism that causes disease.

pediatrics    A field of medicine that has to do with children and especially child health. A doctor who works in this field is known as a pediatrician.

polymerase chain reaction (PCR)    A biochemical process that repeatedly copies a particular sequence of DNA. A related, but somewhat different technique, copies genes expressed by the DNA in a cell. This technique is called reverse transcriptase PCR. Like regular PCR, it copies genetic material so that other techniques can identify aspects of the genes or match them to known genes.

proteins    Compounds made from one or more long chains of amino acids. Proteins are an essential part of all living organisms. They form the basis of living cells, muscle and tissues; they also do the work inside of cells. The hemoglobin in blood and the antibodies that attempt to fight infections are among the better-known, stand-alone proteins.Medicines frequently work by latching onto proteins.

screening    A health test that is done early, before any symptoms are present. That can help find disease when it is easiest to treat. Screenings can include blood tests (such as for HIV, diabetes or high cholesterol), X-rays or scans (such as mammograms for breast cancer).

sequencing    Technologies that determine the order of nucleotides or letters in a DNA molecule that spell out an organism’s traits.

thyroid    A gland in the neck that releases hormones, which play a pivotal role in directing development and metabolism (the use of food as fuel). The gland is relatively small, with two lobes separated by a bridge-like structure. Some therefore refer to its shape resembling a butterfly.

virus    Tiny infectious particles consisting of RNA or DNA surrounded by protein. Viruses can reproduce only by injecting their genetic material into the cells of living creatures. Although scientists frequently refer to viruses as live or dead, in fact no virus is truly alive. It doesn’t eat like animals do, or make its own food the way plants do. It must hijack the cellular machinery of a living cell in order to survive.

NGSS: 

  • MS-LS1-2
  • MS-ETS1-2
  • HS-LS1-1
  • HS-LS1-3
  • HS-ETS1-4

Further Reading

S. Perkins. “Speedy cancer detector.” Science News for Students. May 23, 2012.

S. Ornes. “Poop-sniffing mice to the rescue.” Science News for Students. September 7, 2010.

Original Journal Source: C. Tsai, P. Robinson et al. Ultrasensitive antibody detection by agglutination-PCR (ADAP). ACS Central Science. Vol. 2, February 16, 2016, p. 139. doi: 10.1021/acscentsci.5b00340.