Calibrator concentration pg/mL
Intermediate 50 ng/mL std (mL)
Stripped serum (mL)
0
0
250
50
0.25
249.75
100
0.50
249.5
300
1.50
248.5
600
3.0
247.0
1000
5.0
245.0
2000
10.0
240.0
2.
Dissolve 500 μg 15NPTH in 1 mL internal standard diluent to make a high stock concentration of 500 μg/mL. (Aliquot and store unused high stock at −80 °C.) Dilute the 500 μg/mL appropriately to achieve a final concentration of 10 ng/mL in 500 mL charcoal-stripped serum. Store at −80 °C for 2 years.
3.
Three levels of quality control samples (Low: 50–100 pg/mL; Medium: 150–200 pg/mL; High: 450–550 pg/mL) are prepared by pooling patient samples at appropriate concentrations. Samples can be aliquoted and stored at −80 °C for 2 years.
2.4 Supplies and Equipment
1.
Transfer pipettes, vortex and microtiter plate shaker.
2.
Incubator shaker that can heat to 37 °C and can shake 250 rpm.
3.
Positive pressure manifold capable of handling a 96 well plate.
4.
Analytical column: Higgins Analytica, TARGA C18, size 50 × 3.0 mm, 5 μm particle size (Chromtech).
5.
IMMULITE™ ¼ inch diameter polystyrene beads, IMMULITE™ 2000 PTH assay (Siemens L2KPP6).
6.
Isolute PPT+ filter plate, 96-well (Biotage).
7.
Thermo-Cohesive HPLC system (Thermo Scientific).
8.
Applied Biosystems API 5000 triple quadrupole mass spectrometer.
3 Method
3.1 Sample Preparation
1.
Thaw the patient samples, calibrators, controls, blanks, and IS (15NPTH) on ice and allow PTH antibody beads to reach room temperature.
2.
Transfer 1 PTH antibody bead per sample into each well of the 96 well filter plate (Fig. 1).
Fig. 1
Work flow for quantifying PTH(1–84) in serum and plasma
3.
Pipette 1.0 mL of each of patient samples, calibrators, controls or blanks into individual well of the filter plate.
4.
Add 50 μL IS (15NPTH) into each well. Cover the plate with an adhesive plate seal and incubate at ambient temperature on the plate shaker for 4 h with constant shaking.
5.
Push serum waste into reservoir by placing the plate on a positive pressure manifold and applying pressure. Blot bottom of plate on paper towels to remove residual liquid.
6.
Wash the contents of the 96 well plate twice with 1–2 mL PBS. Allow all the liquid to pass through the filter plate using positive pressure manifold between washes.
7.
Add 300 μL trypsin buffer (1 μg in 300 μL 50 mM ammonium bicarbonate) to each well.
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