1. Enzyme linked immuno sorbent assay (ELISA)

2. Introduction
Biochemical technique used mainly in immunology, medicine, plant pathology, quality check in industries, etc.
ELISA used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones.
Diagnosis of infectious agents such as viruses and other substances in blood
An immunological test that use an enzyme as a label to determine presence of target protein.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measureable product.

3. ELISA Assay - History
First demonstration on smallpox vaccination (Edward Jenner)
Demonstration of antibody activity against diphtheria and tetanus toxins. Beginning of humoral theory of immunity. (Emil von Behring & Shibasaburo Kitasato)
Antibody formation theory (Paul Ehrlich)
Antigen-Antibody binding hypothesis (John Marrack)
Antibody production in plasma B cells
Discovery of antibody structure
1971 – Invented ELISA (Peter Perlmann and Eva Engvall at Stockholm University, Sweden)
Generation of first monoclonal antibodies (George Kohler & Cesar Milstein)

4. Technique
Antigen is immobilized on a solid support (polystyrene microtiter plate)
Immobilization done either
Non-specifically (via adsorption to the surface) or
Specifically (via capture by another antibody specific to the same antigen - "sandwich" ELISA).
Detection antibody is then added to form a complex with the antigen.
Detection antibody can be
Covalently linked to an enzyme, or
Can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. (Enzyme conjugate)

5. Technique…
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 final wash, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.

6. Components - Antigen Any substance that stimulates an immune response
Antigen is our target protein which comes from the sample extract
Antigen binds to the antibody.
Epitope
Region on an antigen to which an antibody binds
Sites can be linear, non-epitope (needs digestion), conformational. denaturation

7. Components - Antibody
An antibody is a protein made in response to an antigen.
Each antibody binds only to its antigen.
Monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems.
Monoclonals used as detection antibody as they have monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen.

8. Producing Antibodies
Animals are injected over a period of weeks with purified antigen
Serum is drawn and antibodies are separated and purified

9. Components - Enzyme Conjugate
An enzyme conjugate is an antibody joined with an enzyme.
Joining of the enzyme to antibody is called conjugation
Detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody.
Most commonly used enzyme labels
Horseradish peroxidase (HRP)
Alkaline phosphatase (AP).

10. Enzyme Function
Enzymes are proteins that speed up the rate of a chemical reaction without being used up and usually react only to particular substrates.
Rate of this reaction is proportional to the amount of enzyme present
In the case of non-competitive ELISA, there is more binding of the enzyme conjugate to the antigen, the stronger will be the color development

11. Substrate Function
A substrate is a compound/ substance that undergoes change.
Substrates bind to active sites on the surface of enzymes and are converted or changed.
In ELISA, the specific substrate used changes the color.
Choice of substrate depends upon required assay sensitivity and instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer)

12. Chromogenic substrates
PNPP (p-Nitrophenyl Phosphate, Disodium Salt) - to detect alkaline phosphatase. Produces a yellow water-soluble reaction product that absorbs light at 405 nm.
ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) - to detect HRP . Produces a water-soluble green end reaction product which has two major absorbance peaks, 410 nm and 650 nm. ABTS is less sensitive than the OPD and TMB
OPD (o-phenylenediamine dihydrochloride) - to detect HRP and yields a water soluble yellow-orange reaction with an absorbance maximum of 492 nm.
TMB (3,3',5,5'-tetramethylbenzidine) - to detect HRP - yield a blue color with major absorbance maxima at 370 nm and 652 nm. Very sensitive

13. ELISA Methodology
ELISA methods are classified according to the way in which analytes are captured and detected
Capture
Antigen capture ELISA – Analyte is captured by the microplate
Antibody capture ELISA – An analyte is captured by an antibody attached to the microplate
Detection
Direct ELISA (Enzyme conjugate binds directly to the antigen)
Indirect ELISA (Enzyme conjugate binds indirectly to the antigen)

14. Direct vs Indirect Detection ELISA Formats
DIRECT ELISA
Uses a labeled primary antibody that reacts directly with the antigen.
Performed with antigen that is directly immobilized on the assay plate or with the capture assay format.
Not widely used in ELISA.
Used in immuno histochemical staining of tissues and cells.
INDIRECT ELISA
Uses a labeled secondary antibody for detection
Most popular format for ELISA.
Secondary antibody has specificity for the primary antibody not capture antibody

15. Direct Antigen Capture
Analyte is captured by the microplate
Enzyme conjugate binds directly to
the antigen

16. Indirect Antigen Capture
Analyte is captured by the microplate
Enzyme conjugate binds indirectly to
the antigen

17. Direct Double Antibody Sandwich (DAS) Direct antibody capture
An analyte is captured by an antibody attached to the solid phase or microplate
Enzyme conjugate binds directly to
the antigen

18. ELISA Formats - (Sandwich - DAS)
Direct sandwich ELISA
Antibodies (Ab) are coated to micro wells.
Antigen (Ag) is added and it binds with antibody.
Excess antigen is washed away.
Enzyme conjugate (Ab-E) is added and it binds with antigen to form the double antibody sandwich.
Wells are washed to remove any excess (Ab-E).
Substrate is added and color development is observed.
The enzyme conjugate binds ‘directly’ to the antigen (Ab + Ag + Ab-E)

19. Sandwich ELISA The most powerful ELISA assay format
It is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody.
It is more sensitive and robust
Screening viruses in potato tuber seeds

20. Indirect Triple Antibody Sandwich (TAS) Indirect antibody capture
An analyte is captured by an antibody attached to the solid phase or microplate
Enzyme conjugate binds indirectly to
the antigen

21. Indirect Sandwich ELISA - TAS
The secondary antibody will be specific for the detection primary antibody only and not the capture antibody.
Hence this assay will not be specific for the antigen.
This is achieved by using capture and primary antibodies from different host species
(e.g., mouse IgG and rabbit IgG, respectively).

22. Competitive ELISA
Used to assay small molecules (antigen with one epitope) eg. hormones in blood
Competition between natural antigen (hormone) and enzyme conjugated antigen
Mix together test sample and defined amt of enzyme conjugated antigen in well
Both compete for available space
More the natural antigen, more it displaces the conjugated form leading to their reduction
Relationship of substrate colour development is therefore inverted.
More the natural antigen bound lower the signal
Used routinely to test thyrozine in blood

23. PLATE READER Machine which measures color density in plate well.
A specific wave-length of light is chosen
Light is cast from the bottom of the well through sample.
No color change wells absorb very little light
Wells with color change absorb more light
This is measured as the optical density or OD.

24. Reading plates Select proper wave-length on machine.
Carefully wipe bottom of plate to remove excess moisture.
Plates that are wet may diffuse light source, giving inaccurate readings.
Mix the plate. This will distribute color evenly. Some machines have a ‘mix’ setting.

25. OD Values – good results A B C D
E Buffer + EC
F Buffer + Known pos no EC
G Known positive
H Known negative
A1 – H1 are expected negatives

26. Applications
Determining the concentrations of serum antibody during the test of HIV or West Nile Virus.
Detection of allergens in the foods like milk, peanut, eggs, almonds and walnuts and almost all the foods which are rich with proteins.
Certain drugs can be analyzed by using the method of ELISA in the field of toxicology.

27. ELISA Applications (Agricultural)
Greenhouse/ornamental growers - Detect symptomatic plant material
Port-of-entry sites - Detect plant pathogens being imported or exported. – USDA-APHIS
Large commercial plant propagators - Check mother stock; screen plants before marketing
Seed producers - Seed/leaf purity testing ; Determine trait purity percentages
AP screens (Organics) - Screen for presence of unintended transgenic events
Plant breeders - Determinations of trait presence and determine segregation
Alternate means of testing - Confirm other assay results or second-party results.

28. Other labels used in plate based assays
Fluorescent tags
Detectable probes
Specific protein binding interactions