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Analytical Chemistry

PCR offers new way to detect disease antibodies

DNA-based method is 1,000 times as sensitive as current diagnostic techniques

by Erika Gebel Berg, special to C&EN
March 10, 2016 | A version of this story appeared in Volume 94, Issue 11

Schematic of PCR-based disease-detection method.
Credit: ACS Cent. Sci.

To diagnose some conditions, such as autoimmune disease, doctors often look for certain antibodies in patients’ blood. Unfortunately, these proteins can be few and far between.

Now, researchers have developed a polymerase chain reaction (PCR) method for detecting such antibodies that is 1,000 times as sensitive as enzyme-linked immunosorbent assay (ELISA), the gold-standard test (ACS Cent. Sci. 2016, DOI: 10.1021/acscentsci.5b00340).

In ELISA, antigen proteins sit on a plastic slide and bind to antibodies present in a sample, capturing them for detection. But “some antigen proteins unfold when bound to the ELISA plate,” which prevents antibodies from binding them, says Carolyn R. Bertozzi, a chemist at Stanford University and editor-in-chief of ACS Central Science. Also, ELISA isn’t usually sensitive enough to detect the low antibody levels in saliva or urine or in blood during the early stages of a disease.

In the new study, Bertozzi’s team wanted to detect antibodies associated with autoimmune thyroid disease. These antibodies bind the protein thyroglobulin.

The researchers first attached thyroglobulin (beige) to one of two different DNA sequences (red, green). Then they incubated these conjugates with a blood sample containing thyroglobulin antibodies (Y shapes).

Because the antibodies bind two thyroglobulin molecules, the antibodies bring the tethered DNA strands close to each other. The scientists added a piece of DNA bridging the two types of strands (shown). The team used PCR to amplify a piece of DNA that contained the two linked sequences. They detected it using gel electrophoresis.

The method detected 100 to 100,000 antibodies in 2 µL of serum, Bertozzi says .

Mark W. Pandori of the University of California, San Francisco, says this approach appears to be sensitive enough to detect antibodies in saliva, which would be helpful for diagnostics in resource-poor areas.


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