Lecturer: Dr.

Yunfei Xie

ELISA METHOD AND AFLATOXINS

DETECTION

Fulfilled by Do Thi Van Thanh

 OUTLINE
Introduce to ELISA
Principle of ELISA method
Types of ELISA
Aflatoxins Detected by ELISA method
Advantages
Disadvantages
Application
Conclusion
Introduce to ELISA - What is ELISA test?
Enzyme-linked immunosorbent assay (ELISA)
test is the most widely used type of
immunoassay and uses antibodies and color
change to identify a substance.

In 1971, ELISA was introduced by Peter

Perlmann and Eva Engvall at Stockholm
University in Sweden.

ELISA is a plate based assay technique which is

used for detecting and quantifying substances
such as peptides, proteins, antibodies,
hormones, toxins, and viruses.
 Principle of ELISA method
ELISA methods developed for the detection of antigen or
antibody consist of use of corresponding antibody or
antigen.
+ Test sample is added in the microtitre plate, if there is
presence of antigen or antibody in the test sample, there
will be antigen- antibody reactions (with immobilized
antigen or antibody ).
+Enzyme labelled antibody is added in the reaction mixture
+ In the final step, Substrate is added after the antigen-
antibody reaction. The enzyme catalyses (usually hydrolyses)
the substrate to give a color end point.
PRINCIPLE OF ELISA METHOD

Example for indirect ELISA

 Principle of ELISA method
The enzyme system consists of;
An enzyme: horseradish peroxidase, alkaline
phosphatase which is labelled or linked, to a specific
antibody.
A specific substrate:
O-Phenyl-diamine-dihydrochloride for peroxidase
P Nitrophenyl Phosphate- for Alkaline Phosphatase

The intensity of the color is proportional to the

amount of antibody or antigen present in the test
sample, which can be quantified using ELISA reader.
TYPES OF ELISA

Indirect ELISA

Direct ELISA

Sandwich ELISA

Competitive ELISA
MATERIALS NEEDED IN ELISA METHOD

ELISA Readers: Readers need to have

appropriate filter (650 nm and 450 nm).
Pipette: Are available as fixed as well as
adjustable volume as well as single channel
and multi-channel.
Washing system:
It can be manual system that washes one row
or column at a time or
semi automated systems that wash one strip
or plate at a time
fully automated systems that can process
multiple plates.
MATERIALS NEEDED IN ELISA METHOD
Reagents needed for the testing
Concluded in the kit
Coated plates: The 96-well plates are
made of polystyrene.
Controls

Conjugates

Wash Concentrate

Stop solution
AFLATOXINS DETECTED BY ELISA METHOD

A set for the determination of aflatoxins B1.

Consisted of a 96-well plate coated with antibodies to

aflatoxin B1.
AFLATOXINS DETECTED BY ELISA
METHOD
2g of sample was extracted + 6 ml of methanol-
water mixture (4:1) and homogenised in 10
min resultant solution.

An aliquot (100 l) of the supernatant was

diluted with 700 l of phosphate buffer + the
resultant solution the standard solution for
determinations.

Analiquot (100 l) of the standard solution and

50 l of the aflatoxin- peroxidase conjugate
solution were added to each plate well.
AFLATOXINS DETECTED BY ELISA METHOD

The rest was stirred and incubated at room

temperature for 1 h.
Next, the wells were emptied and washed 5 times
with 0.1 mol/l phosphate buffer (pH = 7.2).
Then, 150 l of the chromogen -substrate mixture
was added to each well incubated at room
temperature for 30 min in darkness.
The reaction was terminated by adding the stop
reagent.
AFLATOXINS DETECTED BY ELISA METHOD
AFLATOXINS DETECTED BY ELISA METHOD

The absorbance was measured at the wavelength of

450 nm by using the ELISA reading apparatus.

The aflatoxin B1 content was calculated from the

reference curve.
ADVANTAGES OF ELISA METHOD
- 96-well assay platform, a large number of tests can
be done at one time.
- Highly specific and sensitive.
- No radiation harzards
- Reagents used for ELISA are stable and relatively
cheap.
- Easy to perform and quick procedures.

- ELISA kits are cheap and easy to use.

DISADVANTAGES OF ELISA METHOD
- Requires multiple washing steps, at times prove
not only laborious but also time consuming.
- Very specific to a particular antigen. Wont
recognize any other antigen.
- Falso positives/ negatives possible, especially
with altered antigen.
Application of ELISA
1. Presence of antigen or the presence
of antibody in a sample can be
evaluated.

2. Determination of serum antibody

concentrations in a virus test.

3. Used in food industry when

detecting potential food allergens.

4. Applied in disease outbreaks-

tracking the spread of disease e.g.
HIV, bird flu, common, colds,
cholera, STD etc.
CONCLUSION
The Enzyme-linked immunosorbent assay (ELISA)
method can be successfully used for the determination
of contaminants such as aflatoxin in food.
The method makes it possible to determine
aflatoxin M1 at a level of 2.5 ng/kg, aflatoxin B1
12.5 ng/kg and total aflatoxin 25 ng/kg.
Aflatoxins were found in some samples at a very
low level.