INSTRUCTIONS FOR USE BINDAZYME Human Anti ... - inova
INSTRUCTIONS FOR USE BINDAZYME Human Anti ... - inova
INSTRUCTIONS FOR USE BINDAZYME Human Anti ... - inova
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
4. Sample dilution<br />
Dilute 10µL of each sample with 1000µL of sample diluent (1:100) and mix well.<br />
Note: Diluted sample must be used within 8 hours.<br />
5. Strip and frame handling<br />
Place the required number of wells in the strip holder. Position from well A1,<br />
filling columns from left to right across the plate. When handling the plate,<br />
squeeze the long edges of the frame to prevent the wells falling out.<br />
Note: Return unused wells to the foil bag immediately with the two desiccant<br />
pouches and re-seal tightly to minimise exposure to moisture. Take care not to<br />
puncture or tear the foil bag, see below.<br />
WARNING: Exposure of wells to moisture or contamination by dust or<br />
other particulate matter will result in antigen degradation, leading to poor<br />
assay precision and potentially false results.<br />
7.2 ASSAY METHOD<br />
Maintain the same dispensing sequence throughout the assay.<br />
1. Sample addition<br />
Dispense 100µL of each calibrator, control and diluted (1:100) sample into the<br />
appropriate wells of the plate provided.<br />
Note: Samples should be added as quickly as possible to the plate to minimise<br />
assay drift, and the timer started after the addition of the last sample.<br />
Incubate at room temperature for 30 minutes.<br />
2. Washing<br />
The washing procedure is critical and requires special attention. An improperly<br />
washed plate will give inaccurate results, with poor precision and high<br />
backgrounds.<br />
After incubation remove the plate and wash wells 3 times with 250-350µL wash<br />
buffer per well. Wash the plate either by using an automatic plate washer or<br />
manually as indicated below. After the final automated wash, invert the plate<br />
and tap the wells dry on absorbent paper.<br />
Plates can be washed manually as follows:<br />
a. Flick out the contents of the plate into a sink.<br />
b. Tap the wells dry on absorbent paper.<br />
c. Fill each well with 250-350µL of wash buffer using a multichannel pipette.<br />
d. Gently shake the plate on a flat surface.<br />
e. Repeat a-d twice.<br />
f. Repeat a and b.<br />
3. Conjugate addition<br />
Dispense 100µL of conjugate into each well, blot the top of the wells with a<br />
tissue to remove any splashes.<br />
Note: To avoid contamination, never return excess conjugate to the reagent<br />
bottle.<br />
Incubate at room temperature for 30 minutes.<br />
4. Washing<br />
Repeat step 2.<br />
5. Substrate (TMB) addition<br />
Dispense 100µL of TMB substrate into each well, blot the top of the wells with a<br />
tissue to remove any splashes.<br />
Note: To avoid contamination, never return excess TMB to the reagent bottle.<br />
Incubate at room temperature in the dark for 30 minutes.<br />
6. Stopping<br />
Dispense 100µL of stop solution into each well. This causes a change in colour<br />
from blue to yellow.<br />
7. Optical density measurement<br />
Read the optical density (OD) of each well at 450nm on a microplate reader,<br />
within 30 minutes of stopping the reaction.<br />
8 RESULTS AND QUALITY CONTROL<br />
1. Quality control<br />
In order for an assay to be valid, all the following criteria must be met:<br />
• Calibrators and positive and negative controls must be included in each run.<br />
• The values obtained for the positive and negative controls should be in the<br />
ranges specified on the QC Certificate.<br />
• The curve shape should be similar to the calibration curve, shown on the QC<br />
Certificate.<br />
If the above criteria are not met, the assay is invalid and the test should<br />
be repeated.<br />
2. Calculate mean optical densities (for assays run in duplicate only)<br />
For each calibrator, control and sample calculate the mean OD of the duplicate<br />
readings. The percentage coefficient of variation (% CV) for each duplicate OD<br />
should be less than 15%.<br />
3. Plot calibration curve<br />
The calibration curve can be plotted either automatically or manually by plotting<br />
the anti-β 2 GP1 autoantibody concentration on the log scale against the OD on<br />
the linear scale for each calibrator:<br />
• Automatic - use appropriately validated software, and the curve fit that best fits<br />
the data.<br />
• Manual - using log/linear graph paper, draw a smooth curve through the points<br />
(not a straight line or point to point).<br />
4. Treatment of anomalous points<br />
If any one point does not lie on the curve, it can be removed. If the absence of<br />
this point means that the curve has a shape dissimilar to that of the sample<br />
calibration curve, or more than one point appears to be anomalous, then the<br />
assay should be repeated.<br />
5. Calculation of autoantibody levels in controls and samples<br />
Read the level of the anti-β 2 GP1 autoantibody in the controls and diluted<br />
samples directly from the calibration curve. The control values should fall within<br />
the ranges given on the QC Certificate.<br />
Note: The calibrator values have been adjusted by a factor of 100 to account<br />
for a 1:100 sample dilution. No further correction is required.<br />
6. Assay calibration<br />
The assay is calibrated in U/mL against an internal arbitrary reference<br />
calibrator, as no internationally recognised standard is currently available.<br />
7. Limitations<br />
• The results obtained from this assay are not diagnostic proof of the presence or<br />
absence of disease.<br />
• Where results produce a negative serum anti-β 2 GP1, in the presence of clinical<br />
indications, an anti-cardiolipin, lupus anti-coagulant or additional test is<br />
required.<br />
• Patient treatment must not begin on the basis of a positive anti-β 2 GP1 result<br />
alone. Clinical indications must also be present.<br />
9 PER<strong>FOR</strong>MANCE CHARACTERISTICS<br />
9.1 PRECISION<br />
The intra-assay precision was measured using six samples tested 20 times<br />
within one assay across the range of the calibration curve. The concentration<br />
and % C.V. for each sample are given below:<br />
INTRA-ASSAY PRECISION<br />
n=20 Concentration (U/mL) % CV<br />
Sample 1 6.4 5.8<br />
Sample 2 16.0 2.9<br />
Sample 3 20.5 2.9<br />
Sample 4 37.4 4.6<br />
Sample 5 48.7 3.6<br />
Sample 6 66.4 4.1<br />
The inter-assay precision was measured using six samples tested in duplicate<br />
six times over three days. The concentration and % C.V. for each sample are<br />
given below.<br />
INTER-ASSAY PRECISION<br />
n=6 Concentration (U/mL) % CV<br />
Sample 1 6.1 10.7<br />
Sample 2 15.8 3.6<br />
Sample 3 19.5 5.1<br />
Sample 4 37.9 4.4<br />
Sample 5 49.3 3.7<br />
Sample 6 65.4 2.6<br />
9.2 NORMAL RANGE<br />
<strong>Anti</strong>-β 2 GP1 autoantibody levels were measured in serum from 100 male and<br />
100 female normal blood donors. The results are displayed in the graph below.<br />
Based on a positive cut-off of 20.0U/mL, the two positive samples obtained<br />
were confirmed positive using an alternative manufacturer’s anti-β 2 GP1 IgA kit.<br />
Number of samples<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
This normal range is provided as a guide only. It is recommended that each<br />
laboratory determine its own normal range.<br />
9.3 RELATIVE SPECIFICITY, SENSITIVITY, AGREEMENT<br />
The relative specificity, sensitivity and agreement have been determined<br />
against an alternative anti-β 2 GP1 IgA kit using 114 clinical and normal samples.<br />
The samples were from 46 SLE patients who had previously tested positive for<br />
anti-phospholipid antibodies, 8 from patients with primary anti-phospholipid<br />
syndrome, 20 RA patients, 20 SLE patients with unknown APS status and 20<br />
normal sera from healthy blood donors. The status of the samples was<br />
determined applying a positive cut-off of 20.0U/mL with a borderline region of<br />
15-20U/mL for the BINDAZYME IgA S assay and that recommended in the<br />
alternative kit.<br />
EIA<br />
BINDAZYME<br />
<strong>Anti</strong>-β 2 GP1 IgA S EIA<br />
Alternative EIA<br />
+ *Borderline -<br />
+ 18 2 1<br />
Borderline 3 3 3<br />
- 1^ 5 78<br />
Relative sensitivity 94.7%<br />
Relative specificity 98.7%<br />
Relative agreement 98.0%<br />
*Samples in the borderline range were excluded from the analysis<br />
^The discrepant sample tested negative in an alternate kit<br />
9.4 ANALYTICAL SENSITIVITY<br />
Confirmation that the anti-β 2 GP1 IgA S assay can distinguish between two<br />
samples with values close to the bottom of the measuring range (173 and 199%<br />
of the lowest calibrator) was obtained by statistical analysis (Student’s t-test) of<br />
the results obtained from testing multiple replicates of each sample.<br />
9.5 MEASURING RANGE<br />
0<br />
20.0<br />
IgA <strong>Anti</strong>-B2GP1 U/mL<br />
The measuring range of the assay is 3.7-300U/mL.<br />
Insert Code: E141, Version: 17 January 2008, Page 2 of 13