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Reverse Capture Autoantibody Array Probes For Prostate-Cancer Proteins
by Mike May
Oncologists need a new test for prostate cancer the most common form
of cancer diagnosed in American males over the age of 50. Any male tested for
prostate cancer, though, knows that an oncologist can measure the level of prostate-specific
antigen, or PSA. Despite its name, this protein is not specific to prostate cancer
alone. It also rises with benign prostatic hyperplasia, also known as BPH, or
simply an enlarged prostate. In BPH, PSA levels can surpass 4 nanograms per milliliter
of blood. The problem is that 35 percent of men with prostate cancer have PSA
levels below 4 nanograms per milliliter. In other words, PSA alone cannot determine
the difference between a patient with prostate cancer and a patient without it.
Brian Liu, director of translational research in urology at the Brigham and Women’s
Hospital in Boston, wanted some way to tell BPH from prostate cancer all
with just a sample of blood.
Brian Liu |
Today’s oncologists turn to biopsies to distinguish these two conditions. “Even
if you do biopsies,” says Liu, “you might not catch the cancer because it could
be small compared to the size of the prostate.” It can take up to half a dozen
biopsies or more to ensure that a needle does not miss a potential cancer. “Think
of the prostate like an apple,” says Liu, “and if there’s something wrong inside
it, you can’t see it from the outside. If the disease is only in one portion of
the ‘apple,’ you can imagine how easy it is to miss that part when just probing
with a needle.”
In search of some way to amplify disease-related proteins, Liu turned to the immune system. “The immune system is a really smart mechanism,” explains Liu. “It can tell you that something abnormal is going on inside the body.” For example, cancer often generates autoantibodies, or antibodies that act against a person’s own proteins, or antigens. So, Liu wanted to build a system that could find those autoantibodies, which probably exist in much higher quantities than, say PSA. “Then, we have a way of finding the really important players, instead of just the noise,” Liu says. If he could find those relevant proteins, he says, “any one of them that goes up or down could be indicative of something such as cancer.”
In the past, when scientists used microarrays to detect cancer, they spotted the devices with recombinant proteins or peptides. The trouble is that such artificial molecules might not catch the autoantibodies, which could be modified, say with post-translational modifications. As a result, the recombinant hooks might not snag all of the disease-related proteins. Instead, Liu wanted a system that used native antigens as the bait for the autoantibodies. These native proteins would be just right for grabbing autoantibodies. Liu’s team including Shuzhen Qin, one of his postdoctoral colleagues, and Joshua R. Ehrlich, then a research assistant in Liu’s lab and now a medical student at Weill Cornell Medical College of Cornell University (New York, NY) developed a reverse-capture autoantibody microarray. This works like an ELISA assay, making an antibody-antigen-autoantibody sandwich.
Robert Caiazzo, Jr., a research assistant
in Brian Liu’s lab, examining microarray data from the reverse-capture technique.
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Liu’s team started with a BD Clontech (Mountain View, CA) AB Microarray, spotted
with 500 antibodies in duplicate. This microarray can capture antigens down to
the level of picograms per milliliter. Liu’s team used this microarray to capture
native antigens from cells and fluids. Next, they purified autoantibodies
or IgGs from patients with prostate cancer or BPH and labeled them with different-colored
CyDyes for example, red for prostate-cancer IgGs and green for BPH IgGs.
The native antigens captured by the antibodies on the microarray serve as bait
for the autoantibodies. The labeled autoantibodies can be washed over the array
and allowed to hybridize with the native antigens. Then, Liu’s team could measure
the differential fluorescence from the autoantibodies to look for distinguishable
patterns between the patients with prostate cancer and BPH. It works. “We have
a paper submitted,” says Liu, “and our data show that we can separate patients
with prostate cancer or BPH most of the time percentages in the high 80s.”
This technique will not replace PSA testing, but the reverse-capture approach could be combined with PSA to get a deeper level of diagnosis, all without a biopsy. “To prove this concept,” says Liu, “we need to do multi-center trials and do it blinded. That will give some confidence in terms of using this test.”
Moreover, the reverse-capture technique using native antigens could also provide other benefits. For example, it could be used to find potential targets for drugs. The same technique could also be used to find biomarkers and therapeutic targets for other forms of cancer. By using native antigens, this reverse-capture method takes advantage of the immune system’s powerful capabilities as a sentry against disease.
In 2003, Jian-Ying Zhang of the W.M. Keck Autoimmune Disease Center at The Scripps Research Institute (La Jolla, California) and his colleagues described autoantibodies in the serum of people with several forms of cancer, including prostate cancer. Consequently, Brian Liu thought that he might be able to use these autoantibodies to diagnosis specific forms of cancerous disease. Using body fluids, though, presents an obstacle. As Liu says, “Blood, for example, contains lots of proteins in high abundance, and these may not tell us very much.” The disease-related proteins usually exist in tiny amounts like nanograms per milliliter of blood. Using a combination of microarray technology and antibodies created by the immune system in response to cancer, Liu suspected that he might find some way to zero in on the proteins of interest.
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