Std level (μU/mL)
Volume of 100 μU/mL mixed calibrator (μL)
Volume of serum pool ( μL)
2.5
12.5
487.5
5
25
475
10
50
450
25
125
375
50
250
250
(b)
Insulin Regular calibrator in SFS: Add 20 μL of 10 mU/mL Insulin Regular working solution to 1.98 mL of steroid free serum to prepare the 100 μU/mL (high) Insulin Regular calibrator. Mix well. Dilute as shown in Table 2 to prepare calibrators.
Table 2
Dilution scheme for insulin regular calibrators
Std level (μU/mL) | Volume of 100 μU/mL calibrator (μL) | Volume of SFS (μL) |
|---|---|---|
2.5 | 12.5 | 487.5 |
5 | 25 | 475 |
10 | 50 | 450 |
25 | 125 | 375 |
50 | 250 | 250 |
2.5 Analytical Equipment and Supplies
1.
Eppendorf LoBind tubes, 2.0 mL (Eppendorf, Mississauga, ON).
2.
2 mL Nunc® 96 DeepWellTM round-bottom well plates (Thermo Scientific, Waltham, MA).
3.
BSA-treated 2 mL 96 deep well plates (see Note 6 ):
(a)
Add 0.5 mL of 100 mg/mL BSA to each well of a 96-well plate. Seal with capmat.
(b)
Attach on rotator or rocker to and rotate/rock for 2 h.
(c)
Remove from rocker or rotator and let sit at room temperature for an additional 22 h.
(d)
After 24 h, discard BSA from plate(s).
(e)
Add ~1 mL PBS to each well of plate(s), mix, and discard.
(f)
Repeat PBS wash two times, for a total of three PBS washes.
(g)
Centrifuge plates upside-down at 1100 × g for 10 min to remove all residual PBS from the plate.
4.
Vacuum manifold or positive pressure manifold, installed in robotic liquid handler or manually controlled.
5.
Acroprep Advance 2 mL 1 μm glass fiber filter plates (PALL Life Sciences, Ville St Laurent, Quebec).
6.
Nunc® capmats for round bottom plates (Thermo Scientific, Waltham, MA).
7.
AB Sciex5500 QTRAP® triple quadrupole mass spectrometer (AB SCIEX, Concord, ON) or other mass spectrometer capable of reaching the required limit of detection, equipped with appropriate software (e.g., Analyst®).
8.
Shimadzu 20 AC LC System with pumps, column oven, degasser, autosampler.
9.
Analytical column: ACE C18-300, 50 × 2.1 mm, 5 μm (ACE HPLC Columns, Aberdeen, Scotland).
10.
Guard cartridge: C18 4 × 3.0 mm ID (Phenomenex, Torrance, CA).
3 Methods
3.1 Incubation Procedure
1.
Aliquot 500 μL of standards and samples to BSA-treated 96-well plate.
2.
Add 25 μL of 540 μU/mL Bovine Insulin (Internal Standard) to each standard and sample.
3.
Add 5 μL of delipidation reagent.
4.
Seal plate with capmat, and vortex mix at high speed for approximately 20 s.
5.
Remove capmat, and add appropriate volume of antibody-coated paramagnetic bead slurry. In our case, this has been determined to be 250 μL (see Note 3 ).
6.
Seal the plate with a capmat, swirl plate manually until beads are evenly distributed in all wells.
7.
Let sit at room temperature for 1 h.
3.2 Extraction Procedure
1.
Tape off unused positions of filter plate for future use if desired.
2.
Remove capmat from incubation plate, and transfer all standards and sample to filter plate.
3.
Apply a weak vacuum—eluent goes to waste.
4.
Wash each well with 3 × 1 mL of PBS, applying weak vacuum after each addition to elute the PBS wash to waste.
5.
After the last PBS wash, apply vacuum for 1 min to remove all wash from plate. Remove filter plate, tap on paper towel to remove residual wash drips from the filter plate.
6.
Place a BSA-treated 96-well plate in the collection position of the vacuum manifold.
7.
Replace filter plate on vacuum manifold.
8.
Add 75 μL of 1 % acetic acid to standard and sample positions in the filter plate.
9.
Let sit for 2 min.
10.
Apply vacuum to elute acetic acid to the collection plate.
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