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INSTRUCTIONS FOR USE BINDAZYME Human Anti ... - inova

INSTRUCTIONS FOR USE BINDAZYME Human Anti ... - inova

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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

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