Catching a disease in its earliest stages can lead to more effective therapies. Stanford chemists have increased the likelihood of detecting these diseases via a test that is thousands of times more sensitive than current diagnostics.
A common theme in medicine is that detecting a disease early on can lead to more effective treatments. This relies partly on luck that the patient gets screened at the right time, but more important is that the testing techniques are sensitive enough to register the minuscule hints that diseases leave in the blood stream.
Graduate students Cheng-ting “Jason” Tsai and Peter Robinson prepare a gel electrophoreresis experiment to analyze a DNA-tagged biomarker. (Image credit: L.A. Cicero)
A new technique developed by a team of chemists at Stanford has shown promise to be thousands of times more sensitive than current techniques in lab experiments, and it is now being put to test in real-world clinical trials.
When a disease – whether it’s a cancer or a virus like HIV – begins growing in the body, the immune system responds by producing antibodies. Fishing these antibodies or related biomarkers out of the blood is one way that scientists infer the presence of a disease. This involves designing a molecule that the biomarker will bind to, and which is adorned with an identifying “flag.” Through a series of specialized chemical reactions, known as an immunoassay, researchers can isolate that flag, and the biomarker bound to it, to provide a proxy measurement of the disease.
The new technique, developed in the lab of Carolyn Bertozzi, a professor of chemistry at Stanford, augments this standard procedure with powerful DNA screening technology. In this case, the chemists have replaced the standard flag with a short strand of DNA, which can then be teased out of the sample using DNA isolation technologies that are far more sensitive than those possible for traditional antibody detections.
“This is spiritually related to a basic science tool we were developing to detect protein modifications, but we realized that the core principles were pretty straightforward and that the approach might be better served as a diagnostic tool,” said Peter Robinson, a co-author on the study and graduate student in Bertozzi’s group.
The researchers tested their technique, with its signature DNA flag, against four commercially available, FDA-approved tests for a biomarker for thyroid cancer. It outperformed the sensitivity of all of them, by at least 800 times, and as much as 10,000 times. By detecting the biomarkers of disease at lower concentrations, physicians could theoretically catch diseases far earlier in their progression.
“The thyroid cancer test has historically been a fairly challenging immunoassay, because it produces a lot of false positives and false negatives, so it wasn’t clear if our test would have an advantage,” Robinson said. “We suspected ours would be more sensitive, but we were pleasantly surprised by the magnitude.”
Putting basic research to use in a clinical setting has been a focus of Bertozzi’s since she arrived at Stanford.
“I moved to Stanford with the anticipation that translation of my students’ innovations to clinically impactful products and technologies would be enabled,” said Bertozzi, who is a faculty fellow of Stanford ChEM-H, as well as a professor, by courtesy, of radiology and of chemical and systems biology. “That goal is being delightfully fulfilled.”
Based on the success of the thyroid screening, the group has won a few grants to advance the technique into clinical trials. One trial underway in collaboration with the nearby Alameda County Public Health Laboratory will help evaluate the technique as a screening tool for HIV. Early detection and treatment of the virus can help ensure that its effects on the patient are minimized and reduce the chance that it is transmitted to others. This effort is supported by a pilot grant from Stanford-Spectrum, funded by the National Center for Advancing Translational Sciences at the National Institutes of Health.
“Many of our collaborators are excited that the test can be readily deployed in their lab,” said co-author Cheng-ting “Jason” Tsai, a graduate student in Bertozzi’s group. “In contrast to many new diagnostic techniques, this test is performed on pre-existing machines that most clinical labs are already familiar with.”
The researchers are also pursuing tests for Type 1 diabetes, for which early detection could help patients manage the disease with fewer side effects.
Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers.
Detecting the onset of cancer at the cellular level
A chemical profiling technique that has potential for detecting the onset of cancer at the cellular level has been developed by scientists with the Lawrence Berkeley National Laboratory (Berkeley Lab) and the University of California at Berkeley.
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| Carolyn Bertozzi was recently named Director of Berkeley Lab’s Molecular Foundry. She is a chemist with Berkeley Lab’s Materials Sciences and Physical Biosciences Divisions, a UC Berkeley professor, and an investigator with the Howard Hughes Medical Institute. | |
In a paper published in the Proceedings of the National Academy of Sciences (PNAS) that is now on-line, a team of researchers, led by chemist Carolyn Bertozzi, has reported a technique for rapidly profiling O-linked glycoproteins in living animals. Changes in O-linked protein glycosylation – the attachment of sugars to proteins through an oxygen atom on the protein – are known to correlate with cancers and other diseases, such as inflammations and bacterial infections. Until now, however, scientists have lacked a practical means of monitoring such changes in a physiological setting.
“With our profiling technique, we can take pictures over time of the sugars that coat a cell’s surface or are released by the cell into the bloodstream and monitor any changes that occur,” said Bertozzi. “We can then compare the sugars produced by cells that become cancerous with the sugars from normal cells. Ultimately, the idea would be to use this information to create a simple blood test that would diagnose a patient for cancer.”
Bertozzi is the Director of Berkeley Lab’s Molecular Foundry, a faculty scientist with Berkeley Lab’s Materials Sciences and Physical Biosciences Divisions, the T.Z. and Irmgard Chu Distinguished Professor of Chemistry and professor of Molecular and Cell Biology at UC Berkeley. She is also an investigator with the Howard Hughes Medical Institute (HHMI). Co-authoring the PNAS paper with her were Danielle Dube, Jennifer Prescher and Chi Quang, who were all members of her research group when this work was done.
The key to successful cancer treatment is detection at an early stage of development – the sooner the better. While detecting an over-abundance of an antigen has been used to create an effective blood test for prostate cancer, it is believed that even more effective blood tests for a large number of epithelial cancers, also known as carcinomas, could be realized if a practical means of detecting the sugars attached to blood-borne proteins were available. Most proteins are modified post-translationally (i.e., after a protein’s polypeptide chain has been formed), and one of the most common of these events is glycosylation, which can be either oxygen or nitrogen-linked. Glycoproteins are ubiquitous on the surfaces of most cells and help a cell communicate with its neighbors.
“Studies of cells in culture have suggested that monitoring changes in O-linked glycoproteins would be an effective biomarker for cancer, but for the past three decades if you wanted to know which glycans were present on a cell, you had to isolate the surface proteins one at a time,” said Bertozzi. “Our profiling technique lets you quickly scan all the proteins at once. Once we attach our tags and probes, the proteins either light up due to the presence of sugars or they don’t.”
The profiling technique developed by Bertozzi and her team starts by tagging certain glycoproteins with a metabolic label called N-azidoacetylgalactosamine (GalNAz). An over-secretion of the labeled glycoproteins, which form the lubricant that protects the surfaces of cells, is known to increase the potential of cancer to spread.
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| Profiling mucin-type O-linked glycoproteins by metabolic labeling with an azido GalNAc analog (Ac4GalNAz) followed by Staudinger ligation with a phosphine probe (Phos-FLAG). R and R’ are oligosaccharide elaborations from the core GalNAc residue. |
GalNAz has an azide group, which can be chemically tagged with probes that can be visualized. The probes react with the azide by virtue of a phosphine group, a process developed in Bertozzi’s laboratory called the Staudinger ligation, named for the German synthetic-organic chemist and Nobel laureate Hermann Staudinger, who first described the reaction between azides and a phosphines almost 100 years ago.
“After injection of mice with GalNAz, azide-labeled glycoproteins were observed in a variety of tissues,
including liver, kidney, and heart, and also in serum and on isolated splenocytes,” Bertozzi and her co-authors state in their PNAS paper. “B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism.
“Furthermore, GalNAz-labeled B cells could be selectively targeted with a phosphine probe by Staudinger ligation within the living animal. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of posttranslational modifications and for identifying O-linked glycoprotein fingerprints associated with disease.”
Berkeley Lab is a U.S. Department of Energy national laboratory located in Berkeley, California. It conducts unclassified scientific research and is managed by the University of California. Visit our Website at www.lbl.gov.
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