Polyclonal murine and rabbit antibodies for the bile acid isolithocholic acid.

Bile acids are relevant markers for clinical research. This study reports the production of antibodies for isolithocholic acid, the isomer of the extensively studied lithocholic acid. The IgG titer and affinity maturation were monitored during the immunizations of three mice and two rabbits. In both animal models, polyclonal antibodies with a high selectivity and affinity were produced. The development of a direct competitive ELISA with a test midpoint of 0.69 ± 0.05 µ g/L and a measurement range from 0.09-15 µg/L is reported. Additionally, the crystal structure of isolithocholic acid is described for the first time.

Immunoassays as high-throughput tools: monitoring spatial and temporal variations of carbamazepine, caffeine and cetirizine in surface and wastewaters.

Carbamazepine (CBZ), caffeine and cetirizine were monitored by enzyme-linked immunosorbent assays (ELISAs) in surface and wastewaters from Berlin, Germany. This fast and cost-efficient method enabled to assess the spatial and temporal variation of these anthropogenic markers in a high-throughput screening. CBZ and cetirizine were detected by the same antibody, which selectively discriminates between both compounds depending on the pH value used in the incubation step. To our best knowledge, this is the first dual-analyte immunoassay working with a single antibody. The frequent sampling with 487 samples being processed allowed for the repeated detection of unusually high concentrations of CBZ and caffeine. ELISA results correlate well with the ones obtained by liquid chromatography tandem mass spectrometry (LC-MS/MS). Caffeine concentrations found in surface waters were elevated by combined sewer overflows after stormwater events. During the hay fever season, the concentrations of the antihistamine drug cetirizine increased in both surface and wastewaters. Caffeine was almost completely removed during wastewater treatment, while CBZ and cetirizine were found to be more persistent. The maximum concentrations of caffeine, CBZ and cetirizine found in influent wastewater by LC-MS/MS were 470, 5.0 and 0.49 µg L(-1), while in effluent wastewater the concentrations were 0.22, 4.5 and 0.51 µg L(-1), respectively. For surface waters, concentrations up to 3.3, 4.5 and 0.72 µg L(-1) were found, respectively.

Non-invasive monitoring of immunization progress in mice via IgG from feces.

A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.

A highly sensitive caffeine immunoassay based on a monoclonal antibody.

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.