Saliva is tested on the V-plex pro-inflammatory cytokine panel (Ref# K008074) using the Meso Scale Discovery (MSD)® multi-spot Assay system (MSD® www.mesocale.com). In this system, an array of capture antibodies against the different targets of interest is patterned on distinct spots in each well of a solid phase 96-well electrode surface plate. Standards and unknown samples are added to the plate and incubated while shaking at room temperature for 2 hours. The plate is washed and then a master mix containing detection antibodies (Abs) labeled with an electrochemiluminescent compound (MSD SULFO-TAG™) is added, and the plate undergoes another 2 hour shaking incubation. As in a traditional sandwich ELISA, analytes in the sample bind to the immobilized, arrayed, capture antibodies on the working electrode surface and recruitment of the labeled detection Abs by captured Abs completes the sandwich. A read buffer is added to provide the chemical environment for electrochemiluminescence and the plate is read in a MESO QUICKPLEX SQ 120 (MSD®) which applies a voltage to the plate electrodes causing the bound detection labels to emit light. The instrument measures the intensity of emitted light.
Each well of a 96-well plate is coated with capture-antibodies to TNF-α. The saliva samples are diluted 2-fold in MSD® Assay Diluent 2 prior to adding to the coated plate. Each standard and sample is tested in duplicate. Detection antibodies are coupled to SULFOTAG labels that emit light when electrochemically stimulated via carbon-coated electrodes in the bottom each microwell. The MSD 9-plex Multi-Spot Array assay curve has a four log dynamic range which allowed us to test saliva following the manufacturer’s recommended protocol without modification. Cytokine concentrations (pg/mL) are determined with MSD Discovery Workbench Software (v. 4.0) using curve fit models (4-PL with a weighting function option of 1/y2). MSD® determines the detection limit for each cytokine by interpolating the mean concentration minus 2.5 (SDs) (Avg Concentration blank + 2.5 (SDblank) over several kit lots.