FACULTY OF INDUSTRIAL SCIENCES AND TECHNOLOGY

BSB 2442 BIOANALYTICAL CHEMISTRY LABORATORY

SEMESTER 1 2014/2015
LAB REPORT

NAME OF EXPERIMENT : ELISA (Enzyme Linked Immuno Sorbent Assay)

DATE OF EXPERIMENT

: 20/11/2014

DATE OF SUBMISSION

: 27/11/2014

LAB INSTRUCTOR

: DR. SHAH SAMIUR RASHID

GROUP MEMBERS

NO.
1
2
3
4

GROUP MEMBERS
SIRATULAKMA BINTI ABDUL RAHMAN
NURUL SYAHIRAH BINTI NOR AZMI
MUHAMMAD ABDUL HAZIQ BIN ISMAIL
HILMI BIN KAMALUDIN

TITLE:
ELISA (Enzyme Linked Immuno Sorbent Assay)

ID NO.
SB13041
SB13042
SB13043
SB13044

OBJECTIVES:
1 To determine if a particular protein is present in a sample.
2 To determine the how much particular protein is present.
PRINCIPLE:

A sensitive immunoassay that uses an enzyme linked to an antibody or

antigen as a marker for the detection of a specific protein, especially

an antigen or antibody.
ELISA involves detection of analyte in a liquid sample using liquid

reagent ( wet lab) or dry strips (dry lab).

In dry analysis, strip can be read in reflectometry. The quantitative
reading usually based on detection of intensity of transmitted light by

spectrophotometry at specific wavelength.

The sensitivity of detection depends on amplification of signal during
the analytic reaction. In some enzymatic reaction, the signal generated
by enzyme which are linked to the detection reagent in fixed

proportions to allow accurate quantification (enzyme linked).

There are two main variations on ELISA method is
- ELISA can be used to detect the presence of antigens that are
-

recognized by antibody or
It can be used to test for antibodies that recognize an antigen.

INTRODUCTION:
Enzyme-linked immunosorbent assay (ELISA), also known as an
enzyme immunoassay (EIA), is a biochemical technique used mainly in

immunology to detect the presence of an antibody or an antigen in a sample.

The ELISA has been used as a diagnostic tool in medicine and plant
pathology, as well as a quality-control check in various industries,such
as ELISA application in food industry. In simple terms, in ELISA, an unknown
amount of antigen is affixed to a surface, and then a specific antibody is
applied over the surface so that it can bind to the antigen. This antibody is
linked to an enzyme, and in the final step a substance is added that the
enzyme can convert to some detectable signal, most commonly a colour
change in a chemical substrate.
Before the development of the ELISA, the only option for conducting
an immunoassay was radioimmunoassay,

technique

using radioactively labeled antigens or antibodies. In radioimmunoassay, the

radioactivity provides the signal, which indicates whether a specific antigen
or antibody is present in the sample. Radioimmunoassay was first described
in

scientific

paper

Berson published in 1960.

threat,

safer

by Rosalyn

Sussman

Yalow and Solomon

Because radioactivity poses a potential health

alternative

was

sought.

suitable

alternative

to

radioimmunoassay would substitute a nonradioactive signal in place of the

radioactive

signal.

When

enzymes

(such

as peroxidase)

react

with

appropriate substrates (such as ABTS or 3,3,5,5-tetramethylbenzidine), a

change in color occurs, which is used as a signal. However, the signal has to
be associated with the presence of antibody or antigen, which is why the
enzyme has to be linked to an appropriate antibody. This linking process was
independently developed by Stratis Avrameas and G. B. Pierce. Since it is
necessary to remove any unbound antibody or antigen by washing, the
antibody or antigen has to be fixed to the surface of the container; i.e., the
immunosorbent must be prepared. A technique to accomplish this was
published by Wide and Jerker Porath in 1966.

In 1971, Peter Perlmann and Eva Engvall at Stockholm University in

Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands
independently published papers that synthesized this knowledge into
methods

to

perform

EIA/ELISA.

Traditional

ELISA

typically

involves chromogenic reporters and substrates that produce some kind of

observable color change to indicate the presence of antigen or analyte.
Newer

ELISA-like

techniques

use fluorogenic,electrochemiluminescent,

and quantitative PCR reporters to create quantifiable signals. These new

reporters

can

have

various

advantages,

including

higher sensitivities and multiplexing. In technical terms, newer assays of this

type are not strictly ELISAs, as they are not "enzyme-linked", but are instead
linked to some nonenzymatic reporter. However, given that the general
principles in these assays are largely similar, they are often grouped in the
same category as ELISAs.
In 2012 an ultrasensitive, enzyme-based ELISA test using nanoparticles
as a chromogenic reporter was able to give a naked-eye colour signal from
the detection of mere attograms of analyte. A blue color appears for positive
results and red color for negative. Note that this detection only can confirm
the presence or the absence of analyte not the actual concentration.
Performing an ELISA involves at least one antibody with specificity for a
particular antigen. The sample with an unknown amount of antigen is
immobilized on a solid support (usually a polystyrene microtiter plate, see in
detail in the section of ELISA device) either non-specifically (via adsorption to
the surface) or specifically (via capture by another antibody specific to the
same antigen, in a"sandwich" ELISA). After the antigen is immobilized, the
detection antibody is added, forming a complex with the antigen. The
detection antibody can be covalently linked to an enzyme, or can itself be
detected by a secondary antibody that is linked to an enzyme through
bioconjugation. The part of antibody incubation of ELISA is similar with that

of western blot. Between each step, the plate is typically washed with a mild
detergent solution to remove any proteins or antibodies that are not
specifically bound. After the final wash step, the plate is developed by adding
an enzymatic substrate to produce a visible signal, which indicates the
quantity of antigen in the sample.

MATERIALS:
1. Antigen 1: BSA, 10 mg/ml (for the standard curve)
2. Antigen 2: the albumin fraction
3. Antibody: rabbit anti-BSA (will be provided by the assistant)
4. Conjugate: goat anti-rabbit IgG-ALP diluted 1/1000 (will be provided by the assistant)
5. ELISA plate: plastic plates with high binding properties
6. Coating buffer: 50 mM sodium carbonate, pH9.6
7. Incubation buffer: PBS (phosphate buffered saline), pH7.2 with 0.05% Tween 20 (v/v) =
PBST
8. Washing solution: PBST
9. Enzyme substrate: p-nitro phenyl phosphate 1.25mg/ml (dissolved in water)
10. Substrate buffer (1M dietanolamine buffer, pH9.8
made (1:1) by the assistants immediately prior to use.
11. Multi-channel pipettes

containing 0.5 mM MgCl2) will be

METHODS:
1. 100 l of all samples are transferred to a microtiter plate. Do not use the wells closest to
the edge of the plate. The plate allowed to stand in room temperature overnight (Dont let
the well dry).
2. The coated ELISA plate is washed 3 times in washing solution (PBST).
3. Blocking solution 200 l/well (0.5% gelatin in PBS) is added to all coated wells.
4. Incubate in room temperature for 30 minutes.
5. The coated ELISA plate is washed 3 times in washing solution (PBST).
6. 100 l anti-BSA antibody diluted 1/2000 is added in incubation buffer (PBST) to all
treated wells.
7. Incubate in room temperature for 30 minutes.
8. The coated ELISA plate is washed 3 times in washing solution.
9. 100 l Alkaline-Phosphatase conjugated goat anti-rabbit antibody diluted 1/1000 times is
added in incubation buffer (PBST) to all treated wells.
10. Incubate in room temperature for 30 minutes.
11. The coated ELISA plate is washed 3 times in washing solution.
12. 100l enzyme substrate solution, p-nitro phenyl phosphate, are added to all treated wells.
13. Read the absorbance at 405 nm in the ELISA plate reader.
Calculations: (Use an MS Excel sheet to plot the graph)
1. Standard curve: Plot the Absorbance to log dilution of the BSA on a graph sheet. The plot
is made for all the time points, value from the Elisa result.
2. The best time point with smooth negative sigmoidal curve is chosen and the graph is
made for its respective absorbance to log concentration values. This graph ends up in a
smooth positive sigmoidal curve.
3. The slope is calculated for the resulting Absorbance Vs Log concentration and this slope
should be used to calculate the concentration.
4. The time point used to plot Abs. Vs Log conc. for standard curve is used for the
experiment sample also.

5. The mean concentration of all dilutions in the particular time point are calculated.

FLOW CHART:
Flow Chart:
The BSA was diluted in coating
Incubate in room temperature.
buffer to a final concentrations of 1
mg/ml, 100 g/ml, 10 g/ml, 1
g/ml, 500 ng/ml, 100 ng/ml, 50
ng/ml, 10 ng/ml, 5 ng/ml, 0 ng/ml.
Wash the coated ELISA plate 3 times
in washing solution.
The albumin was diluted in coating
buffer which includes the dilutions
of 1/10, 1/50. 1/100. 1/500. 1/103.
1/5x103, 1/104, 1/5x104, 1/105,
Add 100 l Alkaline-Phoshatase
1/5x105, 1/106, 1/5x106, 1/107,
conjugated goat anti-rabbit
1/5x107, 1/108, 1/5x108.
antibody diluted 1/1000 times in
incubation buffer to all treated
wells.
Transfer 100 l of all samples to
a microtiter plate
Incubate in room temperature.
Wash the coated ELISA
plate 3 times in washing
solution.
Wash the coated ELISA plate
3 times in washing solutions.
Add blocking solution of
200 l to all created wells.

Incubate in the room

temperature.

Add 100 l enzyme

substrate solutions, p-nitro
phenyl phosphate, to all
treated wells.

Wash the coated ELISA plate 3

times in washing solution.

Read the absorbance at 405 nm in the

ELISA plate reader.

Add 100 l of anti-BSA antibody diluted

1/2000 in incubation buffer to all treated
wells.

CONCLUSION:
In this experiment, we have been shown how to conduct an experiment
of ELISA in a right way. From the demonstration of ELISA, any foreign
substances could be detected by the primary antibodies that bind to it. The
primary antibodies were then being detected by secondary antibodies that
are coupled to an enzyme such as peroxidase. This enzyme will react and
convert the reagent that being added later on. This will result in coloured
mixture and hence, will be easy for us to analyse the foreign substances.

REFERENCES:
1 What is ELISA? Enzyme-linked immunosorbent assay (ELISA). (n.d.).
Retrieved

November

22,

2014,

from

http://www.elisa-

antibody.com/ELISA-Introduction
2 ELISA. (2014, November 19). Retrieved November 22, 2014, from
http://en.wikipedia.org/wiki/ELISA