A Three-Barbed Protein Hook - Bioscience Technology Online

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A Three-Barbed Protein Hook

by Mike May

Edith Schallmeiner

Sensitivity alone is not always enough when it comes to detecting proteins. With prostate cancer, for example, PSA levels increase in patients with prostate cancer or with benign prostatic hyperplasia. PSA, however, forms complexes with antichymotrypsin (ACT) and alpha 1–protease inhibitor (API), and measuring the ratio of complexed to free PSA appears to help clinicians distinguish between a cancerous and an enlarged prostate. So Schallmeiner and her colleagues tweaked 3PLA to pick out PSA complexes. With PSA-ACT complexes, for example, two of the binding antibodies attach to PSA and one binds ACT. This configuration of 3PLA identified as few as 300 molecules of the PSA-ACT complex in a buffer.

Cells and body fluids are packed with proteins. Just one lymphocyte can contain one billion protein molecules. Despite the high concentration of these molecules, their range of distribution within a single cell can cover orders of magnitude. To identify the less-common proteins and to apply them as biomarkers for diseases, scientists need more-sensitive assays, such as one developed by Edith Schallmeiner, a doctoral student in Ulf Landegren’s laboratory at Uppsala University in Sweden.

A crucial step in a protein-finding assay arises in locking onto the desired protein. In many conventional assays, binding molecules often pick out the wrong protein. To reduce that problem, Schallmeiner and others in Landegren’s lab looked for ways to turn multiple binding molecules into teams. In particular, Schallmeiner is developing a proximity-ligation strategy called 3PLA. This assay relies on three binders working together. “The advantage of the 3PLA assay,” says Schallmeiner, “is the high sensitivity of the assay, but additionally the extended biological specificity that enables the detection of protein complexes.”

Here’s how it works. First, Schallmeiner finds three antibodies that attach to the target protein in three distinct spots. Then, she modifies two of the antibodies with dangling strings of oligonucleotides. She also adds blocking oligonucleotides that prevent other strands of DNA from hybridizing with the pieces attached to those two antibodies. The third antibody gets an oligonucleotide string that can hybridize to the oligonucleotides on the other two antibodies so they are connected but not touching—literally forming a bridge with the oligonucleotides from the other two antibodies acting as the “land” on either side of the bridge.

Previously, researchers in Landegren’s lab developed a similar assay, 2PLA, based on just two binding molecules. Schallmeiner says, “I invented the 3PLA design using a third antibody as a ligation template combined with blocking oligonucleotides to eliminate non-target mediated background.”

Here’s what happens when all of the pieces get together. The three antibodies bind to the target protein such that the strings from the first two antibodies come close together. With the third antibody also bound to the target, its string of oligonucleotides out-competes the blocking oligonucleotides and forms the bridge. Then, a cassette oligonucleotide fills in the space between the ends of the two oligonucleotide strings of the first two antibodies. That makes a DNA strand composed of part of the third antibody’s oligonucleotide strand and the cassette oligonucleotide that can be identified with real-time PCR. So if the PCR finds that strand, the target protein was in the sample; if the strand does not turn up in PCR, the target protein was not there.

A report on 3PLA’s efficacy appeared in Nature Methods, published online December 17, 2006. In that report, Schallmeiner and her colleagues used this assay to pick out three different proteins: prostate-specific antigen (PSA), tropinin I, and vascular endothelial growth factor (VEGF). With VEGF, 3PLA proved 100 times more sensitive than 2PLA. In fact, 3PLA picked out VEGF when only 60 molecules of it existed in a buffer. For PSA, 3PLA proved 200,000 times more sensitive than a sandwich ELISA assay that is currently used when searching for this protein to detect prostate cancer.

The increase in sensitivity obtained by adding a third binder makes one wonder how much more sensitivity could be obtained with even more binders. “The extension to a 4 or 5PLA assay has been discussed,” says Schallmeiner. “It may have some advantages where even higher biological specificity is needed for example, in the detection of targets with repetitive epitopes or large protein complexes.”

In 3PLA, three antibodies get modified with strings of oligonucleotides (yellow), and two of them also get blocking oligonucleotides (red). As shown in the center, the three antibodies bind the target protein (grey), the third antibody's oligonucleotide out competes the blocking oligonucleotides and forms a bridge between the strands on the other antibodies. At right, a cassette oligonucleotide (green) hybridizes to the bridge, and that new piece of DNA can be identified with PCR, which indicates the presence of the target protein. (Courtesy Edith Schallmeiner.)Click to enlarge.

The key to how far this technique can go depends on how difficult it is to come up with three binders for a particular protein. “In the 3PLA publication,” says Schallmeiner, “we show that it is possible to use both matched monoclonal antibody triplets and polyclonal batches of antibodies.” She adds, “Polyclonal antibodies exist for a wide range of target proteins, and these binders will give the method a very wide range of possible targets.”

Moreover, Schallmeiner believes that she can extend 3PLA to a wide range of applications. “The assay can also be applied to the detection of complexes of up to three proteins or protein modifications, such as phosphorylation,” she says. She points out that 3PLA could also be used to study complexes formed in signaling pathways, such as those involved in oncogenesis. Overall, Schallmeiner’s three-barbed technique adds new specificity and breadth to protein assays.

Improving Prostate Diagnostics