Ensure that no fingerprints are present on the bottom of the microtiter plate before OD measurement. Fingerprints could lead to false positive results.
Patient sample dilution 1 + 20 (v/v) i.e 50 µl serum + 1000 µl sample diluent (C) after the addiction of the sample the color of the sample diluent (C) changes from olive green to dark blue
The samples are applied horizontally into 4 cavities of a module according to the pipetting scheme:
In case of use of the kit controls (P, N), those controls can replace two samples in the above scheme.
1. Bring all reagents and the required number of test cavities to room temperature (18-25 °C) before use. Mix gently without causing foam.
2. Dispense into 4 horizontal wells of one module: 200 µl diluted samples. alternatively 200 µl of Negative control (N) and Positive control (P) instead of 2 samples
3. Add 50 µl of Start reagent (G) to all wells.
4. Cover plate, shake for 30 seconds, incubate 45 minutes at 37 °C.
5. Decant, then wash each well 5 times using 350 µl wash solution (made of B), use a soak time of 20 seconds each.
6. Add 100 µl of conjugate (D) solution to all wells.
7. Cover plate, incubate 45 minutes at 37 °C.
8. Repeat wash step 5. 9. Add 100 µl of substrate (E) to each well.
10. Incubate 15 min protected from light at room temperature (18…25 °C). 11. Add 100 µl of stop solution (F) to each well and mix gently.
12. Read the OD at 450 nm versus 620 (630 nm) within 20 min after adding the stop solution.
If the washing process cannot be carried out with a soak time of the washing buffer, the washing process must be extended by one step.
The test results are evaluated by calculating a cut-off from the mean value of the Negative control row (well N).
Cut-off (Co) = 0.250 + OD well N
The binding index BI is calculated by the ratio of OD values of samples to the Cut-off:
BI = OD sample / Co
Interpretation is done according to the following table:
Patients with borderline results should be retested after a period of 1-2 weeks using a freshly collected sample.
A confidence index of the confirmation test defines the degree of reliability of the given result and is listed in the table below:
This index is under further evaluation for “low” interpretation. A low index does not mean that the patient is negative and antibodies are not confirmed.
The test run is valid if:
- OD 450/620 nm of Negative control < 0.200
- OD 450/620 nm of Positive control > 0.500
- OD 450/620 nm of row N < 0.250
If the above mentioned quality criteria are not met, repeat the test and make sure that the procedure is followed correctly (incubation times and temperatures, sample and wash buffer dilution, wash steps etc.). In case of repeated failure of the quality criteria contact your supplier.