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 NaV1.1 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 NaV1.1, 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 NaV1.1 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 NaV1.1 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 NaV1.1, they relied on mouse liver lysate spiked with mouse brain lysate. Mouse liver lysate, which had no detectable levels of NaV1.1 protein expression, served as a diluent to ensure an equivalent protein load in each standard.
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 NaV1.1 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 NaV1.1 expression in the adult mouse brain.
- 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.