Dimethylsulfoxide (DMSO) was selected as the sample diluent and internal standards were used to minimize signal variations due to the preparative step.
Components of the assay selected for optimization were adjuvant dissolution, ultracentrifuge conditions including ultracentrifuge model, sample diluent, mobile phase and column oven temperature.
The mixture was centrifuged at 3000×g, the supernatant was diluted 1 8 in provided sample diluent and 20 μl was used for the assay.
Briefly, 10 μL serum of peripheral blood from positive rhLZ transgenic chickens of 20 weeks old was mixed with 990 μL sample diluent, then 100 μL mixed liquor was added to 96-well plate astest group, meanwhile, some wells were only added 100 μL sample diluent as control group, and 100 μL standard sample was added to other wells as standard group.
The cells were terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 μL per specimen) conjugate diluted at 1 50 in sample diluent for 30 min.
For the determination of the serum concentration of PICP, serum was diluted 5 times with sample diluent.
All samples were then diluted to 1 mL with sample diluent (HBS-EP that contains 0.1% tween 20).
To make a calibration curve we diluted PICP with sample diluent to 600, 300, and 150 ng/ml; and these were used as samples.
Prior to assay, urine samples were diluted between 1∶2 and 1∶12 (depending on the C-peptide level and amount of urine available) with IBL sample diluent (Art.
Sample diluent for serum was 0.8x SB17 with 0.6 mM MgCl2, 2 mM EGTA, 2 µM Z-Block_2, 0.05% Tween and for EDTA-plasma was 0.8x SB18 with 0.8 mM MgCl2, 2 mM EGTA, 2 µM Z-Block_2, 0.05% Tween.
A 20% sample solution was prepared by transferring 16 µL of thawed sample into 96-well plates (Hybaid Omnitube 0.3 mL, ThermoFisher Scientific) containing 64 µL per well of the appropriate sample diluent at 4°C.
