A highly sensitive caffeine immunoassay based on a monoclonal antibody

Abstract

A new immunoassay has been developed based on a commercially available anti-caffeine monoclonal antibody and a de novo synthesized tracer, using horseradish peroxidase and UV-visible detection. Caffeine, which is frequently found in surface waters, can be quantified with a relative error lower than 20% for concentrations above 0.025 microg L(-1) (limit of quantitation, direct analysis). The limit of detection is 0.001 microg L(-1) and can be reduced by solid-phase extraction (SPE). Moreover, with minor adaptations, the assay can be used to quantify caffeine in several beverages, shampoo, and caffeine tablets. The results obtained by ELISA correlate well with those from liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the tested matrices. Several surface waters from Berlin were analysed and all tested positive for caffeine, with concentrations higher than 0.030 microg L(-1). In one run 66 samples can be analysed within 2 h.

Monitoring Caffeine in Human Saliva Using a Newly Developed ELISA

“…A C18 reversed-phase column, 250 mm  2.1 mm, 5 mm (Phen, UltraSep ES, SepServ, Berlin, Germany) was used and the ionization performed in electrospray positive ion mode (ESIþ). The chromatographic method was previously described in detail (Carvalho et al 2010).…”

“…The cross-reactivity was calculated as the ratio of mass concentrations at the inflection points (midpoints, parameter C in the 4-parameters sigmoidal curve % half maximum inhibitory concentration, IC50) of the corresponding calibration curves and expressed in percent relative to the caffeine midpoint, as previously described (Bahlmann et al 2009;Carvalho et al 2010). …”

“…The secondary (capture) antibody, a polyclonal anti-mouse IgG ''whole molecule'' (R12569, Lot 20243, 2.2 mg=mL), from sheep, was obtained from Acris Antibodies GmbH (Herford, Germany). The enzymatic tracer (horseradish peroxidase conjugate) was synthesized in-house based on a novel coupling hapten (Carvalho, Emmerling, and Schneider 2007) as previously described (Carvalho et al 2010). 3,3 0 ,5,5 0 -Tetramethylbenzidine (TMB), research grade, and Tween 20, pure, were purchased from Serva (Heidelberg, Germany).…”

“…We developed an ELISA, based on a monoclonal antibody, for measuring caffeine in several non-biological matrices with a low detection limit and relatively low cross-reactivities (Carvalho et al 2010). Herein, we describe the application of the assay to saliva samples as a useful tool for single-point caffeine measurements (Tarantino et al 2006), doping control (Chambaz et al 2001;Hogervorst et al 2008), veterinary medicine (Golden et al 1994), and cognitive and behavioral studies (de Leon et al 2003;Rogers et al 2005;Hewlett and Smith 2007).…”

Advances in Water Quality Monitoring of Inorganics: Current Trends

“…Immunoassays have been widely applied in other areas of science, in particular clinical analyses [56]. However, despite the first studies on using immunoassays to detect pharmaceutical in surface waters showing up around 10 years ago [57] for the detection on Diclofenac, they have received limited further attention [58][59][60]. This may be due Copyright © 2013 SciRes.…”