these slides will let you know about the ELISA and its types and how it is preformed in the laboratory

1. PRESENTATION
ELISA(ENZYME LINKED IMMUNOSORBENT ASSAY )
 PRESENTED TO:
DR. MUHAMMAD ISMAIL
 GROUP MEMBERS :
JONATHAN JAVID (80002)
SHAHRUKH HASSAN (80003)
HAFIZ ABDUL HANNAN (80008)
BS- BIOINFORMATICS (EVENING)

2. Contents
 Introduction to ELISA
 History of ELISA
 Principle of ELISA
 Materials
 Types of ELISA
 Competitive
 Non-competitive
Direct ASSAY
Indirect ASSAY
Sandwich ASSAY
• Advantage and Disadvantage of ELISA
 Limitations
 Application

3. Introduction to ELISA
The Enzyme Linked Immunosorbent Assay
(ELISA) is a common laboratory technique which
is used to measure the concentration of an
analyte (usually antibodies or antigens) in
solution.
The term ELISA was first used by Engvall &
Perlma in 1971.
The ELISA test, or the enzyme immunoassay
(EIA), was the first screening test commonly
employed for HIV. It has a high sensitivity.

4. History of ELISA
 Radioimmunoassay was first described in a
scientific paper by Rosalyn Yalow and Solomon
Berson published in 1960.
 In 1971, Peter perlmann and Eva Engvall in
Sweden, and Anton & Bauke van Weemen in
Netherlands independently published papers that
synthesized this knowledge into methods to
perform EIA/ELISA

5. Principle of ELISA
Based on Immunology Response
Lock and Key concept :
Antigen (Key) Antibody (Lock)
Use an enzyme to detect the binding of antigen Y
(Ag) antibody (Ab).
 The enzyme (HRP) converts a colorless substrate (chromogen) to
a colored product e.g.TMB(Trimethyl benzidine), indicating the
presence of Ag: Ab binding
 An ELISA can be used to direct either the presence of antigens or
antibodies in a sample depending how the test is designed.

6. MATERIALS
 Antibody-coated 96-well microplate
 Detection antibody (usually biotinylated)
 Standard
 HRP(Hosre-redish peroxidase)conjugate
 Diluent buffers
 Wash buffer
 Chromogenic substrate (usually TMB (trimethyl
benzidine) )
 Stop solution
 Plate covers

7. Types of ELISA
Competitive ELISA
 Non-competitive ELISA
Direct Assay
Indirect Assay
Sandwich Assay

8. COMPETITIVE ELISA
 Antibody coated microwell.
 Serum antigen & labeled antigen added together.
 Used to determine small molecules like
T3(triodothyroxin), T4(thyroxin), & progesterone.
 Increased serum antigen result in reduced binding of Ag-
enzyme conjugate with the antibody producing less
enzyme activity & (yellow) color formation.
 It is used to detect Ag (Free testosterone)

9. NON-COMPETITIVE ELISA
DIRECT ASSAY
 Apply a sample of known antigen to a surface.
 Enzyme linked primary antibody is applied to the plate.
 Washed, After this wash, only the antibody-antigen complexes remain
attached.
 Apply a substrate which is converted by the enzyme to dicit a
chromogenic signal.
 It is used to detect Ab (HIV, HCV )

10. INDIRECT ASSAY
 Antigen is added to plate.
 Adding blocking buffer.
 Suitable primary antibody is added.
 TMB substrate is added, is converted to detected
(Yellow) form.

11. SANDWICH ASSAY
 1. The plate is coated with suitable antibody
2. Blocking buffer is added
 Sample is added to plate so antigen is bounded by capture antibody.
 A suitable biotin labeled detection antibody is added to plate.
 Enzyme HRPO is added and bind the biotin labeled detection
antibody.
 TMB substrate is added and converted by HRPO to colored product.
 It is used to detect Ag (Tumor Markers, Hormones )

12. ADVANTAGES of ELISA
 Reagents are relatively cheap & have long shelf life.
 It is highly specific & sensitive.
 No radiation hazards occur during labeling or
disposal of waste.
 Easy to perform & quick procedures.
 Equipment is widely available.
 It can be used to variety of infections.
 It can be used on most type of biological samples like
plasma, serum, urine, cell extracts.

13. DISADVANTAGES of ELISA
Measurement of enzyme activity can be more
complex than the measurement of activity of
some type of radioisotopes.
Enzyme activity may be affected by plasma
constituents.
Very specific to particular antigen but won't
recognize other antigens.
False positive/negative possible, especially with
mutated/altered antigen.

14. LIMITATIONS
Results may not be absolute.
Antibody must be available (poor
producer, interference).
Concentration may be unclear.
False positive (Ab already present).
False negative possible.

15. APPLICATIONS
 Screening donated blood for evidence of viral contamination by
HIV-1 and HIV-2 (presence of anti-HIV antibodies)
Hepatitis C (presence of antibodies)
Hepatitis B (testing for both antibodies and a viral antigens)
 Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of ovulation)
TSH, T3 and T4 (for thyroid function)
 Detecting infections
Sexually-transmitted agents like HIV, syphilis and chlamydia
Hepatitis B and C
Toxoplasma gondii
 Detecting illicit drugs
 Detecting allergens in food and house dust

16. REFERENCE WEBSITES AND BOOKS
 www.Healthline.com/health/elisa
 https://www.bio-rad-antibodies.com/an-introduction-to-elisa.html
 www.elisa-antibody.com/ELISA-Introduction
 www.enzolifesciences.com › Platforms › Immunoassay and Assay
Development
 The ELISA Guidebook-second Edition (John R.Crowther)
 ELISA: Theory and Practice (John R.Crowther)
 The Anxiety Book, Blood River, Glasswings,etc