Receptor capture assay
An analysis method and analyte technology, applied to the analysis of materials, sugar derivatives, material inspection products, etc., can solve expensive and time-consuming problems
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Embodiment 1
[0148] Reagent preparation
[0149] A. Preparation of Ah receptor
[0150] Heteromers containing AhR are obtained from mammalian hepatocytes by standard methods. These cells include Hepa 1c1c7 hepatoma cells and rodent liver. Hepa 1c1c7 was obtained from the American Type Culture Collection ATCC, Accession No. CRL 2026. Rodents include C57BS / 6J mice, Long-Evans rats and Hartley guinea pigs obtained from Jackson Laboratories or Charles Rivers Laboratories.
[0151] Hepa 1c1c7 cells were treated as follows:
[0152] The composition of MENDG buffer is 25 mM MOPS, 1 mM EDTA, 0.02% NaN3 (weight / volume ratio), 1 mM DTT, 10% glycerol (volume / volume ratio), 1X protease inhibitor (SigmaP2714), pH 7.5.
[0153] Briefly, plates of Hepa 1c1c7 cells were grown to confluence and washed with cold PBS. Cells were scraped into phosphate buffered saline (PBS) and centrifuged at 1000 xg for 5 minutes. Cell pellets were resuspended in MENDG and 8 ml were prepared for approximately 50 x 150 ...
Embodiment 2
[0230] Detection of Dioxins
[0231] A. Measurement of dioxins by real-time PCR
[0232] Prepare a set of 2,3,7,8-tetrachlorodibenzo-p-dioxyl containing 5000, 2500, 1250, 626, 313, 156 and 78 parts per trillion (ppt or pg / ml) in methanol A standard product of TCDD. In addition to TCDD, a set of standards for the AhR agonist β-naphthoflavone (BNF) was also prepared and analyzed in the assay. In addition, 25 μl of 10 μM nucleic acid tracer stock solution diluted 50,000 times was added to every 1 ml of rodent cell fluid as the source of AhR, and then divided into equal parts and placed in glass tubes. Dilute the standard 20-fold with 50-100 μl of rodent cell fluid, and incubate at room temperature to 37°C for 1 to 2 hours. After incubation, 40 to 100 μl of the reaction solution was transferred to each well of the antibody-coated capture strip and incubated at room temperature for 30 minutes to 1 hour on a horizontal shaker. Capture strips were washed 5 times with Wash Buffer ...
Embodiment 3
[0274] Detection of activated Ah receptors
[0275] In order to confirm that activated Ah receptors can be detected in cells (or tissues) that have been previously exposed to dioxin-like compounds, the following experiments were performed. Hepa 1c1c7 cells were grown in Dulbecco's modified Eagle's medium and 8% fetal calf serum until near confluence. Cells were treated with 10 [mu]M β-naphthoflavone (BNF) in DMSO for 1 hour. Wash twice with ice-cold PBS, and then harvest the cells by centrifugation at 1000 g. The cells were then suspended in 10 mM HEPES (pH 7.5) at 5 times the volume of the cell pellet and left for 10 minutes.
[0276] Suspended cells were collected again by centrifugation, and then resuspended in 2 times the volume of the cell pellet in 25 mM HEPES (pH 7.5), 3 mM MgCl 2 , 1 mM DTT, followed by 10 shock lysis in a Kontes all-glass Dounce homogenizer with type B pestle. The resulting homogenate was examined microscopically to confirm cell lysis and then cen...
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