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Enzyme Linked Immunosorbent Assay


(ELISA)
Tanurjyoti Bala 12MS066
Aim
To find out the antigen concentration in an unknown sample using ELISA.

Principle
Enzyme linked Immunosorbent Assay (ELISA) is a technique mostly used in clinical labs
to detect the presence of an antigen in a liquid sample. First, the antigen is allowed to
attach to the surface of the well. Then primary antibodies are allowed to attach to the
antigen and then the secondary antibodies tagged with enzyme are allowed to attach
to primary antibody at the Fc region (Secondary Igs are used because tagging the
primary with enzymes is too much expensive). The substrate usually produces a
coloured product on being acted upon by the tagged enzyme. A series of known
concentrations of antibody is used to generating a standard curve by plotting A 450 of
antibodies vs. concentration, the concentration of the unknown sample can be
calculated from this plot.

Materials Required
Antigen (Goat IgG), Blocking solution (BSA in PBST), Primary antibody (Rabbit anti-goat
IgG), Secondary antibodies ( Goat anti-rabbit tagged with HRP), 1X Assay Buffer,
Blocking Solution, 1X Stop Solution, Substrate (TMB/H2O2).

Procedure
200 L of antigen was added to each well of the micro plate strip and incubated
overnight at 4oC.
Liquid was tapped out and wells were washed with 1X Assay buffer twice to
remove unbound antigen.

200 L of blocking solution was added to each well and incubated for 1hour at
room temperature. The blocking solution prevents non-specific interactions
between the antibodies and the well.
Liquid was tapped out and wells were washed with 1X Assay buffer twice.
Standard antibody dilutions were prepared having the following concentrations :
Concentration
(ng/mL)
1. 40,000
2.
3.
4.
5.
6.

10,000
2500
625
156
39

Dilution
6.7 L of standard antibody (3 mg/mL) +
493.3 L of Assay Buffer
125 L of (1) +375 L of Assay Buffer
125 L of (2) +375 L of Assay Buffer
125 L of (3) +375 L of Assay Buffer
125 L of (4) +375 L of Assay Buffer
125 L of (5) +375 L of Assay Buffer

Test sample was also prepared by taking 2 L of test antibody + 2mL of 1X Assay
Buffer.
In one well 200 L of Assay buffer was added, in one well 200 L of the test
sample was added and in the rest of the wells 200 L each of the standard
dilutions of antibody were added and incubated for 30 mins at room temperature.
Liquid was tapped out and the wells were washed 4 times with 1X Assay buffer
for 2mins each to remove unbound primary antibody.
Secondary antibody dilution was done. 2 L of secondary antibody + 2mL of 1X
Assay buffer.
200 L of secondary antibody tagged with HRP was added to each well and
incubated for 30 mins.
Liquid was tapped out and the wells were washed 4 times with 1X Assay buffer
for 2mins each to remove unbound secondary antibody.
Substrate was diluted just before use. 0.1mL of TMB 20X concentrate + 1.9mL
distilled water.
200 L of diluted substrate was added to each well.
After about 10 mins 100 L of 1X Stop solution was added to each well.
The contents were then transferred into tubes containing 1.7mL of 1X Stop
solution.
Absorbance of the samples were measured at 450nm using a spectrophotometer.

Observations
Concentration
(ng/mL)
Control
39
156
625
2500
10,000
Test

A450
0.001
0.032
0.077
0.15
0.214
0.239
0.167

Results
Absorbance vs. log (concentration) graph was plotted which came out to be
linear with adj. R2 value is 0.97333.
The concentration of test sample was found to be 1180.864 ng/mL (from the
graph).

Graph: Absorbance vs. Log (Concentration)

Sources of Error
Enough time for specific interactions (Antigen-Antibody) has to be provided
else faint colour will be produced.
Washing has to be done carefully for removing the loosely bound antibodies.
Non-specific interactions will cause error to increase in the experiment. So,
here also sufficient time has to be given.