Addition of substrate and measurement of absorbance
15.
Add 200 ul of IX TMB substrate to each well.
16.
Incubate at room temperature for 10 minutes.
17.
Add 100 of Stop Solution to each well.
18.
Transfer the contents of each well to individual tubes containing 2 ml of Stop Solution.
19.
Prepare substrate blank by adding 200 ul of IX substrate solution to 2.1 ml of Stop Solution.
20.
Read the absorbance at 450 nm after blanking the spectrophotometer with substrate blank.
Flowchart:
RUAS
Department of Biotechnology (2021-22)
Immunology and Molecular
Biology Laboratory
Observation:
Look for the development of blue colour in the wells at the end of the experiment. Read the
absorbance at 450 nm after blanking the spectrophotometer with substrate blank and record the
readings as follows:
Sample
Concentration
(ng/mi)
A450
Average
A45D
a
c
d
h
Blank (IX
wash
buffer
Calculation of antigen concentration in test sample:
Calculate the average A450 for each of the samples (standard and test) and plot A450 of standards
on Y axis (linear scale) versus the concentration of antigen in ng/ml on X axis (log scale) on a semi-
log graph sheet:
RUAS
Department of Biotechnology (2021-22)
Immunology and Molecular Biology Laboratory
Result:
Particulars
Marks
Maximum
Actual
Conduction of Experiment
10
Write up
05
Observation and Result
05
Viva
05
Total
25
Signature of Staff Incharge
