Last Month's Most Accessed Feature: ELISA meet NLISA: A novel immunosorbent assay with gel electrophoresis

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  • Published: Dec 1, 2017
  • Categories: Electrophoresis
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ELISA meet NLISA: A novel immunosorbent assay with gel electrophoresisFalse detections

Using gel electrophoresis and DNA, a team of US researchers has developed a novel form of enzyme-linked immunosorbent assay (ELISA) that is faster, more sensitive and less prone to false detections than existing versions. Termed a nanoswitch-linked immunosorbent assay (NLISA), it could form the basis for quick, cheap and effective medical diagnosis devices, say the researchers, led by Wesley Wong at Harvard Medical School in Boston, US.

Conventional ELISAs use antibodies targeted to a protein of interest, which are attached to a flat glass surface, to capture any examples of that protein in a sample flowing over them. A solution of the same antibody, but this time with an attached fluorescent label, is then passed over the surface. The fluorescently-labelled antibodies will bind to any captured proteins, which are then detected by the emitted fluorescence.

Although ELISAs are widely used for detecting specific proteins, including disease biomarkers, they suffer from a number of shortcomings. For a start, they’re slow, primarily due to the time taken to pass first the sample and then a solution of fluorescently-labelled antibodies over a surface. They’re also prone to false detections, especially when used with complex biological fluids such as blood or urine, because antibodies aren’t foolproof and can bind with similar, non-target proteins. What is more, proteins in the sample can also bind directly to the flat surface, which can also interfere with the detection process.

In the loop

By contrast, the NLISA doesn’t suffer from any of these shortcomings. Rather than attach the antibodies to a glass surface, Wong and his team attach them to a strand of DNA. In fact, they attach two antibodies that bind to the same protein to each end of a long strand of DNA, producing what they call a nanoswitch. The idea is that when these antibodies encounter the protein of interest, they both bind to it, causing the formerly straight DNA strand to curl into a loop.

This change in shape can be spotted by gel electrophoresis, because straight, unbound DNA migrates faster than bound, looped DNA, offering a clear way to detect the protein. The protein concentration can also be determined from the intensity of the band produced by the looped DNA in the gel, which Wong and his team showed could be imaged with an iPad.

This detection process takes just 45 minutes, around half the time of even the fastest form of ELISA. Preparing the nanoswitch takes much longer – over six hours – but this can be done in advance as the nanoswitches are quite stable, able to stay usable for around six months if stored in a refrigerator. More importantly, the risk of false detection is far less: in part, this is because there’s no glass surface for proteins to bind to, but it’s also due to the gel electrophoresis step.

Dislodged proteins

Although antibodies can, by mistake, bind to proteins with a similar amino acid sequence to the target protein, they don’t tend to bind as firmly. If exposed to some kind of force, such as the pull of an electric field or movement through a gel, then the similar protein can be dislodged. Wong and his team demonstrated this by testing NLISA on the similar proteins chorionic gonadotropin (CG) and luteinizing hormone (LH), using a nanoswitch with antibodies against LH.

Whereas LH produced a clear band, CG merely produced an indistinct smear in a different region of the gel, probably as a result of CG temporarily binding to the nanoswitches before being dislodged. The researchers then went on to show that NLISA could distinguish between other similar proteins, such as the same protein from two different serotypes of dengue virus.

NLISA also proved to be much more sensitive than ELISA. Wong and his team found that it could detect prostate-specific antigen (PSA) in 100μL of blood serum at concentrations as low as 5.6fM, compared with 220fM for ELISA. NLISA could thus offer a fast, accurate way to detect biomarkers for infectious diseases such as Ebola or an imminent heart attack at a much earlier stage than currently possible.

Proceedings of the National Academy of Sciences, 2017, 114, 10367–10372: "Nanoswitch-linked immunosorbent assay (NLISA) for fast, sensitive, and specific protein detection"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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