ABSTRACT
The construction and performance of an automated low-cost Spreeta-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 microRIU (refractive index units) and it could be operated in the range of 18-22 degrees C with a minimum baseline drift (5-10 microRIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 microm and a flow rate of 180 microl/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs.
Subject(s)
Endocrine Disruptors/analysis , Surface Plasmon Resonance , Benzhydryl Compounds , Phenols/analysis , Prealbumin , Thyroxine-Binding ProteinsABSTRACT
We investigated the effect of the pretreatment (sonification or centrifugation) of saliva samples on the concentration of several steroid hormones as measured with highly specific RIA after extraction and chromatography. It appeared that sonification of saliva resulted in significantly higher values for progesterone, cortisone, 17-hydroxyprogesterone, testosterone and oestradiol (10-49% increase), compared with the levels recorded after centrifugation. No differences were demonstrated for the concentrations of cortisol and androstenedione, except that a sex-dependent difference effect was observed in the values for androstenedione: concentrations measured in sonificated male saliva were lower than those measured in supernatant saliva.
Subject(s)
Saliva/chemistry , Steroids/analysis , Adolescent , Adult , Centrifugation , Chemistry, Clinical , Evaluation Studies as Topic , Female , Humans , Male , Middle Aged , Pregnancy , Radioimmunoassay , SonicationABSTRACT
Messenger RNA competition experiments were performed in an mRNA-dependent reticulocyte lysate using three kinds of mRNA: rabbit globin mRNA, calf eye lens mRNA and RNA of turnip yellow mosaic virus. Our results indicate that at supersaturating concentrations of mRNA preferential translation of certain mRNA species can be observed. Furthermore, the pattern of mRNA selection by the translational apparatus suggests that the rate of translation of different mRNA species is limited by different components of the reticulocyte lysate. Our observations n the cell-free system are strikingly different from our previously published mRNA competition experiments in Xenopus oocytes using the same preparations of lens and globin mRNA, in which no selective translation was observed [Asselbergs et al., Eur. J. Biochem. 94, 249-254 (1979)]. The restraints on mRNA translation in vitro are apparently different from those in vivo, i.e. in oocytes.
