ELISA

These slides made by Miss Khunsha Fatima. This presentation will cover mainly ELISA, its Basic Principle, Steps and its related topics discussed in detail.

1. ELISA
Presented by: FATIMAH
SINDH BIOTECHNOLOGY
ASSOCIATON

2. ELISA
ELISA (Enzyme linked immuno-sorbent assay) is one
of the immunoassay method used to detection of
1)ANTIBODIES
2)PROTEINS
3)PEPTIDES
4)BIOMOLECULES

3. WHAT IS IMMUNOASSAY?
•The term “immunoassay” is a combined
term of “immuno”=(practically antigen-
antibody reaction) and
•“Assay”=(Determination of purity of
substance or the amount of any constituent
of a mixture.

4. WHY KNOWN AS…?
Enzyme Linked Immuno-sorbent Assay
1.Antigen/Antibody of interest is absorbed on to plastic
surface (‘Sorbent’)
2.Antigen is recognized by specific antibody (‘Immuno’)
3.This antibody recognized by second antibody
(‘immuno’) which has enzyme attached(‘enzyme
linked’)
4.Substrate react with enzyme to produce product usually
colored.

5. HISTORY OF ELISA
Radioimmunoassay was first described in a scientific
paper by Rosalyn Sussman Yalow and Solomon
Berson Published in 1960.
In 1971, Peter perlmann and Eva Engvallstockhlom
University in sweeden,and anton schuurs and Bauke
van Wccmen in the Netherlands independently
published papers that synthesized this knowledge into
methods to perform EIA/ELISA.

6. Important components in
immunoassay
1.AntiBody(Anti-Serum)
2. Antigen
3. Labeling Materials

7. 1.ANTIBODY(ANTISERUM)
Antibody:proteins produced by the immune system
which help defend against antigens
Symbol for antibody
The variable regions are though to be the place for
recognition and binding with antigen.

8. 2.ANTIGEN
•Any molecules that induces production of antibodies
when itroduced in the body is called antigen,
OR
•Any “thing” foreign to the immune system eg.bacteria
viruses,(or their parts), pollen..etc.

9. 3.LABELLING MATERIALS
In immunoassay, it is necessary to use any marker to
know the antigen-antibody binding.for such purpose,
we label either antigen or antibody with some
materials that do not interfere with the binding.
eg.Horseradishperoxidase ezyme Substrate
:trimethylbenzidine

10. ELISA….
ELISA KIT
ELISA
READER

11. Basic Principle of ELISA
Use an enzyme to detect the binding of antigen(Ag)
antibody(Ab)
The enzyme convert a colourless substrate
(chromogen)to coloured product,indicating the
presence of Ag-Ab binding.
An elisa can be used to detect either the presence of
antigen or antibodies in a sample depending how the
test is designed
Elisa was developed in 1970 and become rapidly
accepted

13. ELISA Quantitative/Qualitative
Qualitative
determines antigen or antibody is present or absent
Quantitative
odetermine the quantity of antibody
otiter
othe highest dilution of the specimen usually
serum which gives positive reaction in the test

15. ADVANTAGES OF ELISA
Reagents are relatively cheap and have long shelf life
ELISA is highly specific and sensitive
No radiations hazards occur during labelling or
disposal of waste.
Easy to perform and quick procedures
Equipment can be inexpensive and widely available.
ELISA can be used to variety of infections.

16. DISADVANTAGES OF ELISA
Measurement of enzyme activity can be more
complex than measurement of activity of some type of
radioisotopes.
Enzyme activity may be affected by plasma
constituent.
Kits are commercially available ,but not cheap
Very specific to a particular antigen. Won’t recognize
any other antigen.
False positive/negative possible, especially with
mutated/altered antigen

17. TYPES OF ELISA
1.COMPETATIVE ELISA:
2.NON COMPETATIVE ELISA:
a)SANDWICH ELISA
b)DIRECT ELISA
c)INDIRECT ELISA

18. Common Steps…
1.Collection and processing of serum
2.Coating of wells antibodies/antigens
3.Washing
4.Incubation with the test sample
5.Washing
6.Incubation with enzyme conjugated antibodies
7.Washing again
8.Colour development by adding chromogenic substrate
9.Stopping the color development
10.Reading of results

20. APPLICATIONS OF ELISA..
1.To detect Viral Contamination (HIV,Hepatitis C)
2.Hormone levels (HGH,Testosterone,ACTH)
3.Various Infections (Dengue,Human Papilloma Virus)
4.Specific disease factors (Clotting factor VII,
Lyme’s disease)
5.Drugs (Narcotics, Antipsychotics, Antidepressants)
6.Allergens in food (egg protein, milk protein) Residues in food
(Antibiotic, lupine)
7.Toxins in food (Pesticides, botulinum)
8.Others (Pregnancy Test)
9.Preclinical screening

21. THANK YOU
FOR YOUR
PATIENCE