DRG EIA-2395 Instrucciones De Uso página 8

6.2 Test Procedure
Each run must include a standard curve.
1. Secure the desired number of Microtiter wells in the frame holder.
2. Dispense 15 µL of each Standard, Control and samples with new disposable tips into appropriate wells.
3. Dispense 100 µL Assay Buffer into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.
4. Incubate for 120 minutes at room temperature.
5. Briskly shake out the contents of the wells.
Rinse the wells 3 times with 300 µL diluted Wash Solution per well. Strike the wells sharply on absorbent paper to
remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of the washing
procedure!
6. Add 100 µL Antiserum to each well.
7. Incubate for 30 minutes at room temperature.
8. Briskly shake out the contents of the wells.
Rinse the wells 3 times with 300 µL diluted Wash Solution per well. Strike the wells sharply on absorbent paper to
remove residual droplets.
9. Dispense 100 µL Enzyme Complex into each well.
10. Incubate for 30 minutes at room temperature.
11. Briskly shake out the contents of the wells.
Rinse the wells 3 times with 300 µL diluted Wash Solution per well. Strike the wells sharply on absorbent paper to
remove residual droplets.
12. Add 100 µL of Substrate Solution to each well.
13. Incubate for 15 minutes at room temperature.
14. Stop the enzymatic reaction by adding 50 µL of Stop Solution to each well.
15. Determine the absorbance (OD) of each well at 450 ± 10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.
6.3 Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and patient samples.
2. Using linear graph paper, construct a standard curve by plotting the mean absorbance obtained from each standard
against its concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from the standard
curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a 4-Parameter
curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.) Other data reduction
functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples with concentrations higher
than that of the highest standard have to be further diluted or reported as > 100 ng/mL. For the calculation of the
concentrations this dilution factor has to be taken into account.
6.3.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time of assay.
Version 10.0 2017/05 - vk
Leptin Sandwich ELISA EIA-2395
Standard
Standard 0 (0 ng/mL)
Standard 1 (2 ng/mL)
Standard 2 (5 ng/mL)
Standard 3 (25 ng/mL)
Standard 4 (50 ng /ml)
Standard 5 (100 ng/mL)
-
7 -
Optical Units (450 nm)
0.02
0.07
0.16
0.74
1.41
2.30