A brief description of ELISA. Basic principle and mechanism. Mainly discussed: Direct, Indirect and Sandwich ELISA.

1. ELISA
Presenter: Dr Pranav Sopory
Junior Resident
Dept. of Pharmacology
All India Institute of Medical Sciences
New Delhi
Mob: 9999-491-690
Email: pranav.sopory@gmail.com

2. Contents
Antigen-Antibody Reactions
Introduction to ELISA
Non-competitive ELISA
Reporting

3. Antibody
Produced by:
B cells Plasma Cells Antibody
Chromosome:
2 Heavy Chains - Chrm. 14
2 Light Chains - Chrm. 2 (ϰ)
Chrm. 22 (λ)
Disulfide bonds
Fab region:
Fragment antigen-binding
Variable region
Paratope binds to Epitope (Ag)
Fc region:
Fragment crystallizable region
Interacts with - cell surface (Fc receptor)
- complement system
Activates the immune system
Fab region
Fc region

4. Antigen – Antibody Reaction
• General Features
1. Specific, but specificity is not absolute!
2. Requires
a. Specific pH
b. Correct temperature
c. Electrolyte balance
3. Bonds involved
a. Van der waal
b. Hydrogen
c. Ionic
4. Combination: firm and reversible

5. Types of Ag-Ab reactions
Sensitive for Ag
Soluble Ag
+
Ab
+
Electrolyte
=
PRECIPITATION
Particulate Ag
+
Ab
+
Electrolyte
=
AGGLUTINATION
Sensitive for Ab

6. ELISA
• Enzyme Linked Immunosorbent Assay
1. Plate based Immunoassay
2. Works on the principle of Ag-Ab binding
3. Based on enzymatic-color reaction
4. Detects and quantifies substances such as peptides, proteins, hormones and
antibodies.
5. Types of ELISA
a. Qualitative: Positive or negative results
b. Quantitative: Optical density interpolated into a standard curve, which is
typically a serial dilution of the target

7. Immunoassay History
Before 1970s:
Radioimmunoassay: Used radioactively-labeled antigens or antibodies.
Was the only test available.
Radioactivity provides signal indicating presence of Ag or Ab.
Radioactivity poses a health risk to the technician.
1971:
Peter Perlmann and Eva Engvall at Stockholm University invented ELISA
1986: Generation of the first monoclonal antibodies (George Kohler and Cesar Milstein)

8. Specimen sample for ELISA
Serum HIV
CSF Neurocysticercosis
Sputum Brucellosis
Urine Legionnaire’s disease
Stool Giardiasis

9. Microtiter Well
Characteristics:
1. Marked on top: Numerically
2. Marked on the side: Alphabetically
3. Generally: 96 wells

10. Indirect ELISA
Enzyme cleaves substrate to produce color
Add Substrate (chromogen) for the Enzyme
Wash – To remove unbound secondary Ab
Add secondary Ab (Enzyme Linked)
Wash – To remove unbound Antibodies
Add Serum (contains primary Ab)
Microtiter well coated with Antigen

11. Indirect ELISA
• The indirect ELISA detects the presence of antibody in a sample
• Advantages:
1. Wide variety of Enzyme linked secondary antibodies available
2. Versatile: many primary antibodies can be made in one species and the
same labeled secondary antibody can be used for detection.
3. Increased sensitivity: each primary antibody contains several epitopes that
can be bound by the labeled secondary antibody, allowing for signal
amplification.
• Disadvantages:
1. Cross-reactivity: might occur with the secondary antibody, resulting in nonspecific signal.
2. An extra incubation step is required in the procedure. (compared to Direct ELISA)

12. ELISA: materials required
1. Testing Sample
2. Antibody (1st and 2nd)/ Antigen
3. Microtiter well
4. Blocking Buffer
5. Washing Buffer
6. Substrate

13. Nomenclature
EL: The second antibody is attached to an enzyme (‘enzyme-linked’)
I: Antigen is recognized by specific antibody (‘immuno’)
S: Antigen of interest is absorbed on to plastic surface (‘sorbent’)
A: Substrate reacts with enzyme to produce a product, usually colored,
which can be assessed (‘assay’)

14. Direct ELISA
Enzyme cleaves substrate to produce color
Add Substrate (chromogen) for the Enzyme
Wash – To remove unbound Antibodies
Add Serum (contains primary Ab that is Enzyme Linked)
Microtiter well coated with Antigen

15. Direct ELISA
• The direct ELISA detects the presence of antigen in a sample
• Advantages:
1. Quick methodology since only one antigen is used
2. Cross reactivity of second antibody is eliminated
• Disadvantages:
1. Immunoreactivity of primary antibody is reduced because its enzyme linked
2. Lesser signal amplification than Indirect ELISA

16. Sandwich ELISA
Enzyme cleaves substrate to produce color
Add Substrate (chromogen) for the Enzyme
Add Enzyme linked Antibody
Wash – To remove unbound Antibodies
Add serum (contains Ag to be detected)
Microtiter well coated with Capture Antibody

17. Sandwich ELISA
• The sandwich ELISA detects the presence of antigen in a sample
• Advantages:
1. High specificity because the antigen is specifically captured and detected.
2. Suitable for crude/impure samples as the antigen does not require purification
prior to measurement.
3. Flexible and sensitive, both direct or indirect detection methods can be used.

18. Buffers
Blocking protein
• a.k.a. “Detergent”
• Prevents other proteins from adsorbing to the
plate.
• Non-reactive protein like
1. Bovine Serum Albumin
Washing
• Necessary to remove non-bound reagents
• E.g.
• 1. Phosphate-buffered solution (PBS)
• 2. Tris – buffered saline (TBS)

19. Enzymes used in ELISA
Enzyme Substrate Chromogen
Horseradish Peroxidase
(M/C)
H2O2 Tetra methyl benzidine
(TMB)
Alkaline Phosphatase Nitrophenyl Phosphate
(NPP)
Diethandamine + MgCl2

20. Results
• Absorbance from Microtiter wells detected via Microplate readers
Final plate of ELISA Microplate reader

21. Results
Source
Lens
Filter
MTP
Detector Calculation
REPORT

22. Calculation
• Data graphed between Optical density Vs Log concentration.
• Known conc. of Ag are used to produce a standard curve.
• This data is used to measure the conc. of unknown samples by
comparison to the linear portion of the standard curve.
Conc. of substance (pg/ml)
OpticalDensity
22

23. ELISA
Advantages
• Long shelf life
• Easy to perform
• Quick
• Widely available
Disadvantages
• Enzyme activity maybe
affected by plasma
constituents
• Expensive
Limitations
• Results may not be
absolute
• Ab may be not available
• False positives/negatives
(mutated antigen)

24. Thank You