ELISA protocols for biomarker quantification in plasma samples

Potential systemic reactions to repeat MAP application were evaluated by quantification of a range of key biomarkers before and after the completion of the application/wear protocol. The biomarkers studied were C-reactive protein (CRP), interleukin 1-β (IL-1β), tumour necrosis factor-α (TNF-α), immunoglobulin G (IgG) and immunoglobulin E (IgE). Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used (Human total IgG Platinum ELISA quantification kit, Affymetrix, Austria; Human IgE Platinum ELISA quantification kit, Affymetrix, Austria; Human CRP instant ELISA quantification kit, eBioscience, Austria; Human TNF-α ultrasensitive ELISA quantification kit, Invitrogen Corporation, USA; Human IL-1β Platinum ELISA quantification kit, Affymetix, Austria), according to the manufacturers’ instructions. Plasma samples were diluted using the sample diluents provided with the kits. Plasma samples for the relevant ELISAs were diluted as follows: CRP, 1:500; total IgG, 1:500,000; IgE, 1:10; IL-1β, 1:2 and TNF-α, 1:2.

The ELISA protocols for the kits were broadly the same. Briefly, 96-well microplates were washed with the requisite washing buffer before addition of the samples. Standards were reconstituted, diluted and added, with the diluted plasma samples, to the plates. Plates were incubated at either room temperature or 37 °C for 1 or 2 h to allow the binding of the biomarker to the primary antibody. The wells on the plates were then washed to remove any unbound antigen. Detection antibody (100 μL) was added to the wells and the microplates were incubated at room temperature with gentle agitation for 1–2 h. The washing step was repeated and aliquots (100 μL) of diluted streptavidin-horse radish peroxidase (HRP) were added to the wells, followed by incubation with shaking for 30 min at room temperature. The washing step was repeated. Substrate (100 μL) was then added to the wells and the colorimetric reaction was terminated by the addition of acid. Sample absorbance readings were recorded at 450 nm on a Microplate spectrophometer (FLOUstar™ Omega, BMG Labtech, Ortenberg, Germany).