EXPERIMENT: DATE:
Aim: To perform ELISA (Enzyme Linked Immuno Sorbent Assay)
Reagents required:
a) Phosphate buffer saline; pH – 7.0
Mol wt. Element 10X 1X
(g/mol)
1L 500 mL 100mL 500mL
58.4 NaCl 8g 4g 0.8g 40g
141.96 Na2HPO4 1.16g 0.58g 0.116g 5.8g
136.08 KH2PO4 0.2g 0.1g 0.02g 1g
74.551 KCl 0.2g 0.1g 0.02g 1g
b) Coating Buffer (Carbonate – Bicarbonate buffer); pH – 9.5
20mL 100mL
Solution A 0.2 M Na2CO3 0.33gm 1.68gm
Solution B 0.2M NaHCO3 0.42gm 2.114gm
Working (1:2 ratio of solution A and B)
Solution A Solution B DDW
30mL 2.4mL 4.8mL 22.8mL
10mL 0.8mL 1.6mL 7.6mL
c) Washing Buffer
PBST: 0.05% Tween-20 in PBS
(150mL: 149.625 mL DDW + 375µL Tween 20)
d) Blocking buffer
3% BSA in PBS
(100mL – 3gm / 20mL – 0.6gm)
e) Antibody diluents:
1% BSA in PBS
(100mL in 1gm)
f) Citrate Buffer; pH – 5.0
(50mL)
Citric acid anhydrous – 0.365gm / 365mg
NaH2PO4.2H2O – 593.5gm / 593.5 mg
DDW – 50mL
g) Substrate solution:
15mL citrate buffer + 10mg orthophenylene diamine (OPD) + 8 µL
Hydrogen peroxide
h) Stop Solution:
1M H2SO4
Theory:
ELISA (which stands for enzyme-linked immunosorbent assay) is a
technique to detect the presence of antigens in biological samples. An
ELISA, like other types of immunoassays, relies on antibodies to detect a
target antigen using highly specific antibody-antigen interactions.
In an ELISA assay, the antigen is immobilized to a solid surface. This is
done either directly or via the use of a capture antibody itself immobilized
on the surface. The antigen is then complexed to a detection antibody
conjugated with a molecule amenable for detection such as an enzyme or
a fluorophore.
An ELISA assay is typically performed in a multi-well plate (96- or 384-
wells), which provides the solid surface to immobilize the antigen.
Immobilization of the analytes facilitates the separation of the antigen
from the rest of the components in the sample. This characteristic makes
ELISA one of the easiest assays to perform on multiple samples
simultaneously.
Types of ELISA:
There are four main types of ELISA: direct ELISA, indirect ELISA, sandwich
ELISA and competitive ELISA. Each has unique advantages, disadvantages and
suitability.
Direct ELISA
In a direct ELISA, the antigen is immobilized to the surface of the multi-well
plate and detected with an antibody specific for the antigen The antibody is
directly conjugated to HRP or other detection molecules.
Indirect ELISA
Indirect ELISA is a technique that uses a two-step process for detection,
whereby a primary antibody specific for the antigen binds to the target, and a
labeled secondary antibody against the host species of the primary antibody
binds to the primary antibody for detection. As for direct ELISA assays, the
antigen is immobilized to the surface of the multi-well plate.
The method can also be used to detect specific antibodies in a serum sample by
substituting the serum for the primary antibody.
Sandwich ELISA
Sandwich ELISA (or sandwich immunoassay) is the most commonly used
format. This format requires two antibodies specific for different epitopes of the
antigen. These two antibodies are normally referred to as matched antibody
pairs. One of the antibodies is coated on the surface of the multi-well plate and
used as a capture antibody to facilitate the immobilization of the antigen. The
other antibody is conjugated and facilitates the detection of the antigen.
Competitive ELISA
Also known as inhibition ELISA or competitive immunoassay, competitive
ELISA assays measure the concentration of an antigen by detection of signal
interference. Each of the previous formats can be adapted to the competitive
format.
The sample antigen competes with a reference antigen for binding to a specific
amount of labeled antibody. The reference antigen is pre-coated on a multi-well
plate and sample is pre-incubated with labeled antibody and added to the wells.
Depending on the amount of antigen in the sample, more or less free antibodies
will be available to bind the reference antigen. This means the more antigen
there is in the sample, the less reference antigen will be detected and the weaker
the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody.
The labeled antigen and the sample antigen (unlabelled) compete for binding to
the primary antibody. The lower the amount of antigen in the sample, the
stronger the signal due to more labeled antigen in the well.
Procedure:
1. The 96 well plate is coated with antigen and incubated overnight at
4oC.
2. The wells are washed thrice with PBS.
3. Blocking buffer (250µL) is added to each well and incubated at 37oC
for 2 hours.
4. The wells are then washed four times with PBST.
5. 50 µL of primary antibody is added and incubated at 37oC for 1 hour.
6. The wells are then washed five times with PBST.
7. Secondary antibody (50µL) is added and incubated at 37oC for 1 hour.
8. The wells are then washed with PBST six times.
9. 50 µL of substrate is added and incubated at 37oC for 15 minutes.
10. The absorbance is measured at 492 nm.
11. 50 µL of stop solution is then added to the wells and again the
absorbance is measured at 492 nm.
Result:
Precautions:
1. Ensure assay accuracy
2. Avoid contamination
3. Produce reliable standard curves
4. Guarantee accurate sample analysis
5. Be sure to include all background and control well suggested in the
protocol
6. Use the provided/recommended diluents and buffers
7. Follow the recommended incubation times, temperatures and conditions
8. Obtain optimal detection and data analysis
