Development of a novel electrochemical immuno-assay using a screen printed electrode for the determination of secretory immunoglobulin A in human sweat

This paper reports on the steps involved in the development of a novel electrochemical immunoassay for secretory immunoglobulin A (sIgA) determinations in human sweat. This novel immunoassay involves an initial step whereby sweat sIgA is adsorbed onto a polyvinylidene fluoride (PVDF) membrane (sweat patch). Following a wash step, a solution containing bovine serum albumin (BSA) is used to block any sites at which non-specific binding might occur. After a second wash step, the PVDF sweat patch is transferred to a second tube to which is added diluent and anti-sIgA antibody conjugated to horseradish peroxidase (HRP). Following an incubation step, an aliquot of supernatant containing unbound antibody is transferred to a well in a 96 well plate format which had been previously coated with sIgA. After a second incubation step and wash step, 3,3′,5,5′-tetramethylbenzidine (TMB) is added to the same well and left to undergo enzymatic oxidation. Finally a screen-printed carbon electrode (SPCE) is inserted into the well and any oxidised TMB is detected by chronoamperometry using an applied potential of +50 mV. The resulting reduction current is then referred to a calibration plot to deduce the unknown sIgA concentration. The optimisation of the steps involved in this assay is described in detail. Some preliminary data is presented on sweat sIgA levels collected from human volunteers who had undergone a controlled exercise regime on a bicycle (Ergociser). To our knowledge this is the first report where a sweat patch and a SPCE have been successfully incorporated into an immunoassay for sIgA.