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Researchers Demonstrate New PCR-Based Method More Sensitive Than ELISA in Detecting Antibodies

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NEW YORK (GenomeWeb) – A group at Stanford University has shown that proximity-ligated amplification (PLA), which has classically been used as a way to detect proteins, can also be used for ultrasensitive detection of antibodies.

The team is now establishing a company and foresees clinical applications such as detection of autoantibodies for early diabetes diagnosis and thyroid cancer treatment monitoring.

The method, called antibody detection by agglutination-PCR (ADAP), was described recently in ACS Central Science, and was developed in the chemistry lab of Carolyn Bertozzi. It requires no washing or blocking, is a single-tube reaction, and can be modified to detect multiple analytes using DNA barcoding.

In comparison, traditional enzyme-linked immunosorbent assays and Western blots are notoriously fussy. They require antibodies that don't always work in every context, plus a lot of incubation time and numerous washing steps. Radioimmunoassays are more sensitive and are the "gold standard" to detect antibodies that denature easily, but they demand extensive washing and centrifuging. In addition, the use of radioactive reagents limits the kinds of labs that can run them.

On the other hand, Bertozzi said her group began realizing that proximity-ligation PCR — which usually uses antibodies conjugated to DNA in order to detect antigen proteins — was really underused, "even though it is so simple, so sensitive and robust, and so much easier."

In fact, in her lab it seems to work quite readily and reliably, and even "undergrads can get it to work the first time," Bertozzi told GenomeWeb.

Traditionally, the PLA method is used to detect proteins by conjugating DNA to a commercial antibody. "You need multiple antibodies to bind to the same antigen at the same time, so you can get multiple DNAs into proximity."

The group's key insight was the fact that antibodies are bivalent, even sometimes multivalent, and could therefore also be used to bring DNA strands close enough together for ligation. "It was just one extra little leap to realize that we could flip it around, and use the antigen to detect whether an antibody is present."

With a known antigen, such as insulin or thyroglobulin, the antigen protein can be chemically conjugated to two PCR-incompetent half-amplicons that are essentially two different single-stranded DNA sequences. This complex is then added to as little as 2 microliters of patient sample, and if an antibody is there it will bind at least two antigens.

"That brings the single-stranded DNAs into proximity," Bertozzi explained. "We put in a splint oligonucleotide that's half complementary to each end of the two DNAs, and then some ligase, and we ligate those two DNAs to make a single amplicon, do PCR amplification, and quantify on a qPCR machine."

The ADAP method thus yields a quantitative measure of how much antibody was in the patient sample. And it is so sensitive that the group is able to detect antibodies in saliva. "It's 1,000 times more sensitive than a totally optimized ELISA assay," Bertozzi said.

The method was also further developed to employ free DNA to protect the antigen-DNA conjugates from being aggregated by anti-DNA autoantibodies — which patients with certain autoimmune disorders might have — and yielding a false-positive result.

In examination of patient samples for anti-thyroglobulin autoantibodies — a biomarker of thyroid cancer recurrence after thyroidectomy — the researchers found three to four orders of magnitude lower detection limits compared to other FDA-approved methods reliant on radioimmunoassay or electrochemiluminescence.

They also found insulin-DNA conjugates could be used to detect anti-insulin antibodies, an early biomarker of type I diabetes that typically requires clinical labs to use radioimmunoassays. The limit of detection for this assay was 170 zeptomoles of antibody in a 2 microliter sample, an 865-fold improvement over a direct ELISA assay, according to the study.

The ADAP method was also shown to be effective in detecting small molecule-binding antibodies that are not so amenable to immunoassays.

Two of the authors on the study are now forming a company, called Enable Biosciences, in conjunction with a clinician and are seeking Small Business Innovation Research grants and other funding, Bertozzi said. It is a bit on the early side to consider partnerships, but she said the group is collaborating on a project to see if the ADAP method can be used to screen saliva samples for HIV antibodies.

"Saliva antibodies are very low abundance, which is why people generally prefer blood tests using blood draws and finger pricks," she said. But the sensitivity of the ADAP assay is so high, she thinks it will likely be able to detect anti-HIV antibodies from oral swabs.

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