Meso Scale Discovery-Electrochemiluminescence: A Better Way to Quantify Na_V1.1

By Alomone Team • January 2022

Put aside your densitometric analysis – MSD-ECL is here.

No doubt you’ve used some form of densitometric analysis (measuring how big and dark a band is) to quantify something like Na_V1.1 protein on an immunoblot. The bottom line though is that densitometry methods can lack the accuracy and sensitivity of standard immunoassays. Beyond just our day-to-day work, getting especially good measurements can have real-world consequences. The expression levels of Na_V1.1, for example, are important because they can be used as a non-clinical pharmacological biomarker of Dravet syndrome, a severe early-onset epileptic encephalopathy. Thankfully, a more accurate and sensitive approach has been devised.

The MSD-ECL method

Researchers from Stoke Therapeutics (MA, USA) have come up with a mesoscale discovery-electrochemiluminescence (MSD-ECL) method to quantify Na_V1.1 that comes with low background and high sensitivity.¹ Their MSD-ECL method is based on a standard ELISA (enzyme-linked immunosorbent assay) that uses electrochemiluminescence to generate signals from a labeled antibody binding to the target.

The electrode surface of each well on a plate is first pre-coated with a goat anti-mouse antibody. Here the researchers used a mouse monoclonal antibody as the Na_V1.1 capture antibody. They then used our rabbit polyclonal Anti-SCN1A (NaV1.1) Antibody (#ASC-001) as the detection antibody. In their assay, the capture and detection antibodies each bind to different epitopes on the NaV1.1 protein, while a sulfo-tag labeled goat anti-rabbit antibody generates the electrochemiluminescence signal.

Validating the Assay

With the aid of Scn1a knock-out (Scn1a-/-) mouse brain tissue, the researchers were able to validate both capture and detection antibodies, going a long way to help confirm the specificity of their assay. As a measure of assay precision, the researchers suggest the %CV between duplicates should be ≤20%.

To overcome the difficulty of reproducibly producing robust reference standards with a transmembrane protein like Na_V1.1, they relied on mouse liver lysate spiked with mouse brain lysate. Mouse liver lysate, which had no detectable levels of Na_V1.1 protein expression, served as a diluent to ensure an equivalent protein load in each standard.

Figure 1. An example of MSD standard curve fitting from the paper. Curves have been fit to a four-parameter logistic curve (in green) or a 2nd order polynomial curve (in red). The upper limit of quantification (ULOQ) is defined by STD12, while the lower limit of quantification (LLOQ) is defined by STD2.

Accurate and Precise

After a good amount of analysis and example of data output, the researchers conclude that their MSD-ECL method is “qualified and used for quantification of Na_V1.1 in mouse brain tissues with specificity, accuracy, and precision.”

It’s worth noting that the method does have a narrow dynamic range of detection, with the upper limit of quantification (ULOQ) is set to 100% of the Na_V1.1 expression in the adult mouse brain.

To see the full data set and a step-by-step protocol, head over to bio-protocol.

Featured Product

Anti-SCN1A (NaV1.1) Antibody (#ASC-001) as the detection antibody

Reference

Han, Z., Christiansen, A., Meena, M. & Liau, G. Relative Quantification of NaV1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method. Bio-Protocol 11, 1–14 (2021).

Photo by Terry Vlisidis.

Navigation

Order Summary