Relative Quantification of Na_V1.1 Protein in Mouse Brains Using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) Method

Background

Na_V1.1, also known as the alpha subunit of voltage gated sodium channel, type I, is a transmembrane protein encoded by the Scn1a gene (Meisler et al., 2010). Decreased expression of functional Na_V1.1 leads to Dravet syndrome (DS), a severe early-onset epileptic encephalopathy (Dravet et al., 2005). Expression of Na_V1.1 in biological samples has been used as a non-clinical pharmacological biomarker for DS and can be measured using densitometric analysis of immunoblots. Densitometry methods are often not as accurate and sensitive as standard immunoassays. In addition, some Na_V1.1 antibodies may cross react with other voltage gated sodium channels (VGSCs), including Na_V1.2, Na_V1.3 and Na_V1.8, due to homology of the protein sequences. We developed a specific and sensitive method to quantify Na_V1.1 protein in mouse brain tissue using a Meso Scale Discovery-Electrochemiluminescence (MSD-ECL) method. MSD-ECL method is ELISA (enzyme-linked immunosorbent assay) based and uses electrochemiluminescence to produce measurable signals based on labeled antibody binding to the target (Kuhle et al., 2016). Scheme of antibody binding in MSD-ECL assay described in this protocol is shown in Figure 1. The MSD-ECL method has advantages of low background and high sensitivity. The specificity of this assay was confirmed by using capture and detection antibodies that were validated using Scn1a knock-out (Scn1a^-/-) mouse brain tissue (Figure 2). Since Na_V1.1 is a transmembrane protein, reproducible production of a high-quality reference standard in cells could be challenging. Therefore, in this method, reference standards composed of mouse liver lysate spiked with mouse brain lysate was utilized. Mouse liver lysate was selected as a diluent to maintain equivalent protein load in each standard since it did not show detectable levels of Na_V1.1 protein expression (Figure 3). This “fit for purpose” MSD-ECL method was qualified and used for quantification of Na_V1.1 in mouse brain tissues with specificity, accuracy and precision. This method is limited, however, to have a relative narrow dynamic range of detection: The upper limit of quantification (ULOQ) is set to 100% of the Na_V1.1 expression in adult mouse brain. Further dilution of some sample lysate may be necessary if the Na_V1.1 expression in those samples exceed the ULOQ in the initial run.

Figure 1. Scheme of antibody binding in MSD-ECL assay described in this protocol. The electrode surface of each well is pre-coated with goat anti-mouse antibody (Ab). A mouse monoclonal antibody (mAb) is used as capture Ab for Na_V1.1. A rabbit polyclonal antibody (pAb) is used as detection Ab. The capture and detection antibodies bind to different epitopes within Na_V1.1. A Sulfo-tag labeled goat anti-rabbit Ab produces electrochemiluminescence signal.