Biology Reference
In-Depth Information
3. The enzyme-antibody conjugate supplied is a 100× concen-
trate and must be diluted 1:100 with 1× diluent solution.
4. The rat Hb calibrator supplied is at a concentration of
178
g/mL and must be diluted to different concentrations
with 1× diluent solution. The concentration of diluted cali-
brators is the following: Calibrator 0 (0.0 ng/mL), Calibrator
1 (6.25 ng/mL), Calibrator 2 (12.5 ng/mL), Calibrator 3
(25 ng/mL), Calibrator 4 (50 ng/mL), Calibrator 5 (100 ng/
mL), and Calibrator 6 (200 ng/mL).
μ
2.4. Procedures
1. Samples are prepared as described in spectrophotometric assay.
2. Bring all reagents in the ELISA kit to room temperature before
use.
3. Pipette 100
L of calibrator 0, 1, 2, 3, 4, 5, or 6 in duplicate
into microtiter plate.
4. Pipette 100
μ
L of sample in duplicate into microtiter plate.
5. Incubate the microtiter plate at room temperature for 60 min.
Keep the plate covered and level during incubation.
6. Following incubation, aspirate the contents of the wells.
7. Completely fi ll each well with 1× diluted wash solution and
aspirate. Repeat three times, for a total of four washes.
8. Pipette 100
μ
L of 1× diluted enzyme-antibody conjugate to
each well. Incubate at room temperature for 30 min. Keep the
plate covered in the dark and level during incubation.
9. Wash and blot the wells as described in step 7.
10. Pipette 100
μ
L of TMB substrate solution into each well.
11. Incubate in the dark at room temperature for precisely 10 min.
12. After 10 min, add 100
μ
L of stop solution to each well.
13. Put the plate into the microplate reader and determine the
absorbance at 450 nm of the contents of each well. Zero the
plate reader to air.
μ
1. Subtract the average background value from the test values for
each sample.
2. Construct a calibration curve using the results of the calibrators.
3. Interpolate test sample values from the calibration curve.
Correct for sample dilution factor to calculate Hb concentra-
tion in original sample.
2.5. Calculation
2.6. Advantages
and Limitations
1. Very sensitive. This assay can be used to detect very low con-
centration of Hb in samples.
2. Large numbers of samples can be measured at the same time.
2.6.1. Advantages
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