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Printed For Final Review: 10/10/03
ECO Number: 27341
9.5 Add 200 µL of
125
9.6 Vortex the tubes gently without foaming and incubate for 2 hours (±15)
minutes at 2-8°C (refrigerator or crushed ice bath).
9.7 Vigorously mix the DAG-PPT; add 500 µL to all the tubes except the total
count tubes.
9.8 Vortex the tubes gently without foaming and incubate for 15 (±5) minutes at
20-25°C.
9.9 Centrifuge the tubes at a minimum of 760 x g* for 20 minutes at 20-25°C.
9.10 Immediately decant the supernatant from all the tubes except the total count
tubes by inverting them for a maximum of 2 minutes. Blot the tubes with an
absorbent paper to remove any drops of supernatant that may be remaining on
the rims before turning the tubes upright.
9.11 Using a gamma scintillation counter, count the precipitate of each tube and the
total count tubes for a sufficient time to achieve statistical accuracy. (See
Results section-Limitations of the Procedure.)
10. PROCEDURAL COMMENTS
10.1 Add each aliquot of reagent to the lower third of the assay tube to ensure
complete mixture of reagents.
10.2 Some manufacturers' disposable borosilicate glass tubes yield elevated
nonspecific bindings.
10.3 If you choose to aspirate the supernatant from the precipitate, be careful not to
disturb the precipitate.
10.4 To completely monitor the consistent performance of an RIA there are
additional factors which may be checked. DiaSorin suggests a check of the
following parameters to assure consistent kit performance.
a.
Total Counts
b.
Maximum Binding
Average counts per minute (CPM) of 0 Calibrator Tube / Average CPM of
Total Count Tubes.
c.
Nonspecific Binding
Average CPM of NSB Tube / Average CPM of Total Count Tubes.
d.
Slope of Calibrator Curve
For example, monitor the concentrations at 80, 50 and 20% suppression
points of the calibrator line.
11. QUALITY CONTROL
Each laboratory should include at least two control sera in every assay to ensure the
validity of each assay's results. A mean and standard deviation should then be
determined for each control using a minimum of ten assays. An acceptable range of
values may then be obtained for these controls using ±2 standard deviations of the
values previously determined. The DiaSorin Quality Control Laboratory has determined
a range for the control serum included in this kit.
12. CALCULATION OF RESULTS
There are many methods in existence for calculating results of RIAs. Each is based on
obtaining a calibration curve by plotting the extent of binding against stated
concentrations of the calibrators. This graph may be either a linear or logarithmic scale.
Each of these methods gives essentially the same values for controls and samples,
although certain assays may "fit'' better into one particular method versus another. The
calculation method of choice for DiaSorin laboratories is %B/B
concentration.
12.1 Calculate the average CPM for each calibrator, control and unknown sample.
12.2 Subtract the average CPM of the NSB tubes from all counts.
* g = (1118 x 10
10:45 AM
I PTH-MM II to all tubes.
) (radius in cm) (rpm)
-8
2
5
versus log
0
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