Methods for determining the potency, specificity, and toxicity of hematopoietic prostaglandin d2 synthase
A specificity and potency technology, applied in biochemical equipment and methods, microbial measurement/inspection, instruments, etc., can solve problems such as inability to simulate complex biological conditions of cells
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Embodiment 1
[0027] Example 1: Cells were treated with calcium ionophores and incubated with test and control compounds
[0028] In this example, RBL-2H3 cells (obtained from American Tissue Cell Culture Center) were trypsinized (0.05% trypsin EDTA in premade MEM medium (Gibco 11095-080) supplemented with 10 % fetal bovine serum, L-glutamine, penicillin and streptomycin supplementation) into a single cell suspension. RBL cells in accordance with 2 × 10 4 Cells / well were plated in a 96-well plate. The plate was incubated overnight at 37°C.
[0029] HBSS buffer (Gibco / Invitrogen 14025-092) was diluted with DMSO (Sigma D-8779) to the highest concentration corresponding to the DMSO contained in the reference or test compound. Thaw compound plates and reference controls and mix well before use. In this example, the COX-1 inhibitor SC560 was used as a reference, which was purchased from Cayman Chemical.
[0030]
EIA control
standard s...
Embodiment 2
[0037] Example 2: Quantification of Prostaglandin D2 Levels: Enzyme Immunoassay
[0038] In this example, the concentration of PGD2 was measured using an enzyme immunoassay (EIA). EIAs that measure PGD2 are commercially available. For example, the PGD2 EIA kit is commercially available from Cayman Chemical.
[0039] This assay is based on the binding of a limited amount of PGD2-MOX-specific rabbit antisera between PGD2-methyloxime (MOX) and a PGD2-MOX-acetylcholinesterase (AchE) conjugate (PGD2-MOX tracer) site competition. Because the concentration of PGD2-MOX tracer is held constant and the concentration of PGD2-MOX is varied, the amount of PGD2-MOX tracer that can bind to the rabbit antiserum will be inversely proportional to the concentration of PGD2-MOX in the well . This rabbit antiserum-PGD2-MOX complex binds to mouse monoclonal anti-rabbit IgG previously adsorbed to the wells. After washing the plate, Ellman's reagent (which contains a substrate for AchE) was adde...
Embodiment 3
[0043] Example 3: Quantitative prostaglandin E2 levels
[0044] In this example, the concentration of PGE2 was measured using an enzyme immunoassay (EIA). EIAs that measure PGE2 are commercially available. For example, the PGE2EIA kit is commercially available from Assay Designs. PGE2EIA uses a mouse monoclonal antibody specific for PGE2. Free PGE2 in solution competes with a known amount of PGE2 tracer for binding of a limited amount of anti-PGE2 antibody. The PGE2 tracer is a conjugate of PGE2 and acetylcholinesterase. Then, the anti-PGE2 antibody was immobilized with goat anti-mouse Ig antibody. The immobilized antibody is then treated with Ellman's reagent containing the acetylcholinesterase substrate. The product produced by this reaction is yellow in color and strongly absorbs at 412 nm. If the concentration of PGE2 is high, PGE2 will win the competition with the tracer, and the obtained sample has a weak absorbance at 412nm. Conversely, if the concentration of PG...
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