Propelled to the surface: Combining CE and ELISA for protein detection
- Published: Mar 20, 2017
- Author: Jon Evans
- Channels: Detectors
Advantages and disadvantages
Capillary electrophoresis (CE) and enzyme-linked immunosorbent assay (ELISA) are very different techniques for detecting whole proteins, with their own advantages and disadvantages. CE is much faster than ELISA, but ELISA tends to be more accurate and sensitive at detecting specific proteins. Whereas in CE a protein is identified by its migration time, which is not always sufficiently unique for a firm identification, in ELISA a protein is identified by exposing it to an antibody that only binds with that specific protein.
Despite these differences, a team of researchers from China and Japan, led by Heyun Shen at Beijing University of Chemical Technology, has now found a way to combine the two techniques, by essentially using ELISA as a CE detection method. In this way, they have combined the advantages of both techniques, producing a fast and sensitive system for detecting specific proteins.
The reason why conventional ELISA is so slow is because the protein sample first diffuses slowly past a surface covered in an antibody to the protein of interest, which captures any examples of this protein in the sample. Next, a fluorescently-labelled version of the antibody diffuses slowly past the surface, binding to any captured proteins, which are then detected by the emitted fluorescence. This reliance on diffusion ensures that the whole process usually takes a couple of hours.
Shen and his team realized that CE offered a way to speed up this process, by replacing the slow diffusion with rapid propulsion of the charged proteins and antibodies directly to the surface for binding. This led them to build a device comprising a glass cell separated by a semi-porous membrane made of cellulose acetate. They coated one side of this membrane with a layer of positive-charged molecules, which could then be coated with a layer of negatively-charged antibodies. As a first test of this combined technique, they used antibodies to the protein immunoglobulin G (IgG), which is itself an antibody.
They filled one half of the glass cell with a solution of IgG and the other half with a standard electrophoresis buffer; the side of the membrane coated with IgG antibodies faced into the half containing the IgG solution. They then attached electrodes to each end of the glass cell, such that the negatively-charged IgG molecules would migrate toward the membrane when a current was applied.
Applying a current for just two minutes proved sufficient to transport a large enough proportion of the IgG molecules in the solution to the membrane, where they were captured by the antibodies. Next, Shen and his team added fluorescently-labelled IgG antibodies to the IgG solution. The IgG antibodies were attached to polystyrene nanoparticles, as previous research had shown that nanoparticles aid binding and enhance the fluorescent signal. The nanoparticles were smaller than the pores in the membrane, so that any that didn’t bind with the captured proteins would pass through the membrane rather than clog its surface.
Again, two minutes was sufficient for the antibody-containing nanoparticles to migrate to the membrane, bind with the captured proteins and produce a detectable fluorescent signal. As a result, this technique could detect IgG within four minutes, compared to two hours for conventional ELISA. Furthermore, it was two orders of magnitude more sensitive, able to detect IgG at concentrations as low as 130 femtomoles. This enhanced sensitivity is due to the speed of the technique, which doesn’t give the fluorescently-labelled antibodies time to stick to other regions of the apparatus – as happens during the course of conventional ELISA – greatly reducing the background noise.
Scientific Reports, 2017, 7, 42562: "Dual electrophoresis detection system for rapid and sensitive immunoassays with nanoparticle signal amplification"
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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