Detection of GDF-8 modulating agents
A technology for GDF-8 and regulators, which is applied to the detection of exogenous GDF-8 regulators in biological samples and the detection of GDF-8 inhibitors in biological samples, which can solve the disadvantages of cumbersome GDF-8 regulators, introduction, and direct detection And other issues
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0199] To detect MYO-029 in human serum, an ELISA assay was performed as follows. First, add streptavidin coating solution (100 μL / well) (5 μg / mL immunopurified streptavidin (Pierce) in 0.1M carbonate / bicarbonate buffer, pH 9.6) to absorb streptavidin. Parafilm-covered plates were incubated overnight at 2-8°C. Using an automatic plate washer, wash with THST buffer (300 μL / well) (50 mM Tris-HCl, pH 8.0, containing 1.0 mM glycine, 0.5 M NaCl and 0.05% (v / v) Tween 20 (J.T. Baker)) wash the plate four times and invert the plate after the second wash. For blocking, 200 μL of blocking buffer (1% bovine serum (Sigma), 0.02% sodium azide in PBS (Dulbeccos)) was added to each well. Cover the plate with parafilm and incubate at room temperature for 1-2 hours, then wash as above. Add biotinylated GDF-8 solution (biotin:GDF-8 molar ratio between 0:1 and 3:1) (100 μL / well) (0.5 μg / mL in THST in the buffer). The plate was sealed and incubated with a shaker at room temperature for 2 ...
Embodiment 2
[0215] Dilution linearity: The dilution linearity of the method was evaluated by analyzing human serum samples spiked with MYO-029 in 11 different dilutions. Samples at 54000 ng / ml were first diluted 8-fold in THST buffer + 4% skim milk powder, and then serially diluted (1:2) in THST + 4% skim milk powder + 10% normal human serum. The dilution is intended to drop the concentration above, within or below the assay range. The dilution bias was determined to be between -9.7% and -0.4% for samples within the quantitative range of the assay. No trend of bias was found. The observed decrease in concentration was as expected and there was no evidence of a prozone effect.
[0216] Specificity: Incorporation / recovery experiments were performed using 10 different batches of human serum (individual donors) at MYO-029 incorporation concentrations of 0, 135, and 540 ng / mL to study potential incorporation from the sample matrix (or matrix effect). non-specific interference. Endogenous m...
Embodiment 3
[0226] Biotinylated GDF-8 was as follows. Expression of full-length GDF-8 in fed-batch CHO cell culture bioreactor processing provides potential complex forms of GDF-8. Cell culture harvests were clarified using normal flow microfiltration followed by concentration and diafiltration using tangential flow ultrafiltration. The retained mixture was then loaded onto the Ni that captures the GDF-8 complex 2+ - NTA-immobilized metal affinity chromatography (IMAC). with 50mM Na 2 HPO 4 , 300mM NaCl, 20-500mM imidazole for linear gradient elution over 5 column volumes. The resulting peak was then buffer exchanged by dialysis to remove the imidazole from the IMAC and replace it with the appropriate buffer for the biotinylation reaction.
[0227] The potential complex preparation is then biotinylated. The molecular molar ratio of target sulfo-NHS-LC-biotin and GDF-8 complex used in this reaction was 14:1. For example, reagent to substrate ratios of 10:1, 15:1 and 20:1 were also t...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More