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

Abstract

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 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.

Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

“…All incubation steps were performed at room temperature on a Titramax 101 plate shaker (Heidolph, Schwabach, Germany) at 750 rpm (Grandke et al, 2013).…”

Quality assurance in immunoassay performance – carbamazepine immunoassay format evaluation and application on surface and waste water

“…Buffers (phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane (TRIS), PBS-based and diethanolamine (DEA)-based washing buffers, sample buffer, and DEA substrate buffer), as well as calibrators were prepared and the same instruments (washer, shaker) were used as described in previous studies. 19,20 CBZ enzyme conjugate synthesis and coupling ratio analysis…”

“…Matrix-assisted laser desorption/ionization time-of-ight (MALDI-TOF) mass spectrometry measurements were carried out as described by Grandke et al 19 Masses of 45 290 Da and 44 210 Da m/z for the single-charged species were determined for the CBZ-HRP conjugate and the unmodied HRP, respectively. The analyte derivative minus water has an m/z of 390 Da.…”

“…19 The assessment of these criteria for direct competitive immunoassays for CBZ was extended to other luminescence detection techniques such as chemiluminescence. Additionally, the application to water samples is reported here.…”

Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

“…Satoshi Inouye reported that HRP had better precision but less linearity when compared with ALP . However, Julia Grandke's data supported another point of view that HRP was better than ALP . Thus, there was an interesting question as to which one system would be better when establishing a CLEIA method for a new target substance.…”