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Quality assurance in immunoassay performance - comparison of different enzyme immunoassays for the determination of caffeine in consumer products

  • Enzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format isEnzyme immunoassays with optical detection are amongst the most widely used bioanalytical tools. We defined seven parameters for the quality assessment of immunoassays that were addressed in a systematic study of direct and indirect immunoassays, using the enzymes horseradish peroxidase (HRP) and alkaline phosphatase (AP), the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) and para-nitrophenyl phosphate, and the fluorescent substrates 3-(4-hydroxyphenyl)propionic acid and 4-methylumbelliferyl phosphate. The same monoclonal antibody against caffeine was used throughout the study. The four quality parameters regarding the standard curve were the test midpoint (sensitivity), the measurement range, the relative dynamic range of the signal, and the goodness of fit of the adjusted four-parameter logistic function. All HRP immunoassays showed a higher sensitivity compared to the AP assays. On the basis of all four criteria, it was established that the direct assay format is superior to the indirect format, the immunoassay using HRP TMB fulfilling all requirements best. In a second step, caffeine concentrations in 24 beverage and cosmetics samples were determined and three more quality parameters were assessed with this application. The direct HRP TMB assay showed one of the best intra- and inter-plate precisions and the best accuracy, defined by the correlation of results with those from the chosen reference method liquid chromatography tandem mass spectrometry (LC-MS/MS). Considering all criteria, HRP TMB seems to be the enzyme substrate system of choice preferably used in the direct assay format.zeige mehrzeige weniger

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Metadaten
Autoren/innen:Julia Grandke, Lidia Oberleitner, Ute Resch-Genger, L.-A. Garbe, Rudolf Schneider
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):Analytical and bioanalytical chemistry
Jahr der Erstveröffentlichung:2013
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik
1 Analytische Chemie; Referenzmaterialien / 1.2 Biophotonik
Verlag:Springer
Verlagsort:Berlin
Jahrgang/Band:405
Ausgabe/Heft:5
Erste Seite:1601
Letzte Seite:1611
Freie Schlagwörter:Assay format; Caffeine; Competitive assay; Direct; Enzyme immunoassay; Enzyme substrate; Indirect
DOI:https://doi.org/10.1007/s00216-012-6596-0
ISSN:1618-2642
ISSN:1618-2650
Verfügbarkeit des Volltexts:Papiergebundenes Belegexemplar in der Bibliothek der BAM vorhanden ("Hard-copy Access")
Bibliotheksstandort:Sonderstandort: Publica-Schrank
Datum der Freischaltung:20.02.2016
Referierte Publikation:Ja
Datum der Eintragung als referierte Publikation:01.02.2013