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Carbamazepine (CBZ) is one of the most frequently detected pharmaceuticals in water samples. For the determination of this anthropogenic marker, various immunoassay formats were tested and evaluated in order to identify the most suitable one. For these direct competitive assays, the analyte was labelled with the enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP), and seven substrates with specific detection properties were used. The quality criteria for the standard curves were fulfilled by all HRP assays and the chemiluminescence AP format. Furthermore, intra- and inter-plate coefficients of variation as a measure of the achievable precision were determined for the samples. The application of the AP assays to surface water was unfeasible due to CBZ concentrations below the quantifiable concentration range. Surface as well as waste water samples could be analyzed with the HRP assays. Here, the HRP assay employing the chromogenic substrate 3,3',5,5'-tetramethylbenzidine yielded the best results.
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.
New monoclonal anti-carbamazepine antibody for application in fluorescence polarization immunoassays
(2014)
Homogeneous fluorescence polarization immunoassays (FPIAs) were developed and compared for the determination of caffeine in beverages and cosmetics. FPIAs were performed in cuvettes in a spectrometer for kinetic FP measurements as well as in microtiter plates (MTPs) on a multimode reader. Both FPIAs showed measurement ranges in the µg/L range and were performed within 2 and 20 min, respectively. For the application on real samples, high coefficients of variations (CVs) were observed for the performance in MTPs; the CVs for FPIAs in cuvettes were below 4%. The correlations between this method and reference methods were satisfying. The sensitivity was sufficient for all tested samples including decaffeinated coffee without preconcentration steps. The FPIA in cuvettes allows a fast, precise, and automated quantitative analysis of caffeine in consumer products, whereas FPIAs in MTPs are suitable for semiquantitative high-throughput screenings. Moreover, specific quality criteria for heterogeneous assays were applied to homogeneous immunoassays.
For the antiepileptic drug and anthropogenic marker carbamazepine (CBZ), a fast and cost-effective immunoassay based on fluorescence polarization (FPIA) was developed. The required fluorophore conjugates were synthesized from different fluorescein and CBZ derivatives. The most suitable tracer was CBZ–triglycine–5-(aminoacetamido)fluorescein. Additionally, the applicability of the assay in tubes and on microtiter plates was tested. The first format can be performed in a portable instrument and therefore can be applied in field measurements. The measurement of an individual sample can be carried out within 4 min. This assay shows a measurement range of 2.5–1000 µg L-1 and a test midpoint (or IC50) of 36 µg L-1. The FPIA performed on microtiter plates is useful for the assay development and is suitable for a very high throughput (up to 24 samples in 20 min). The test midpoint of this assay is 13 µg L-1 and the measurement range is 1.5–300 µg L-1. Furthermore, this assay requires smaller sample volumes and less reagents, including the crucial amount of antibody. The applicability of both assays to spiked surface water samples was evaluated. The recovery rates vary between 66–110% on microtiter plates and 81–140% in tubes.