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Diagnostic Tests Recognize Specific Disease Proteins

By LabMedica International staff writers
Posted on 19 Sep 2012
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An antibody-free, targeted mass-spectrometric approach has been used for the quantification of proteins at low levels in human plasma and serum.

The method uses high-pressure, high-resolution separations coupled with intelligent selection and multiplexing for sensitive selected reaction monitoring-based targeted protein quantification and is known as PRISM.

A team of scientists at the Pacific Northwest National Laboratory found that their PRISM technique performed as accurately as standard clinical tests known as enzyme-linked immunosorbent assays (ELISAs) in a side-by-side comparison using blood samples from cancer patients. The tests measure biomarkers, proteins whose presence identifies a disease or condition.

To get around the need for an antibody, the team concentrated the proteins in their samples by using the technique called high performance liquid chromatography (HPLC), which concentrated the proteins about 100 times more than in the initial sample. The next step was to find their protein of interest in their concentrated samples.

With a potential biomarker in mind, the team made a version that was atomically denser. They synthesized the protein using carbon and nitrogen atoms that contain extra neutrons. The unusual atoms added weight but did not change any other characteristics. The heavier versions are twins of the lighter proteins found within the blood, cells, or samples. Although the twins behave similarly in the analytical instruments, the heavier twin is easily found among the sample's many proteins.

The sample was passed through the instrument to concentrate the proteins. The instrument separates the sample, one concentrated fraction at a time. The fraction that contained the heavy biomarker was also the fraction that contained its twin, the lighter, natural protein. From this fraction, the team could quantify the protein. The team spiked blood samples from women with a biomarker called the prostate specific antigen (PSA) that is only found in men. The team found they could measure PSA at concentrations about 50 pg/mL. While typical of the sensitivity of ELISA tests, it represents about 100 times the sensitivity of conventional mass spectrometry methods.

The team then tested PSA in samples from male cancer patients and found PRISM performed as well as ELISA. Interestingly, PRISM measured three times the amount of PSA than the ELISA assay did. This result suggests that antibody-based ELISA tests fail to measure all of the forms of the biomarker. Wei-Jun Qian, PhD, the senior author of the study, said, "Clinical tests have almost always used antibodies to measure biomarkers, because antibodies can provide good sensitivity, but it often takes a year and a half to develop antibodies as tools. Antibody development is one of the bottlenecks for new biomarker studies in disease and systems biology research." The study was published on the September 5 2012, in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).

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