2. Types of ELISA

3. Direct ELISA Antigen Antibody-enzyme Antigen substrate Antibody-enzyme

4. Sandwich ELISA ("Indirect" ELISA)
substrate
Antibody-enzyme
Antibody
Antigen
Antibody
Antigen
Antibody
Antigen
Antibody
Antibody
Antibody

5. Competitive ELISA
Unlabeled antibody is incubated in the presence of its antigen (sample).
These bound antibody/antigen complexes are then added to an antigen-coated well.
The plate is washed, so unbound antibody is removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence "competition".)
The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme.
A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
The reaction is stopped to prevent eventual saturation of the signal.

7. (conjugated) to horseradish peroxidase (HRP)
Microplates: Polystyrene – proteins absorb (bind)
by hydrophobic bonds to the polystyrene
Secondary antibody: anti-human antibody linked
(conjugated) to horseradish peroxidase (HRP)
Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns yellow. A substrate is a compound or 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 color.
Substrate Solution:
chromogen A and
chromogen B should be mixed together in equal volumes up to 15 minutes before use.
STOP SOLUTION:
(sulphuric acid solution (H2SO4).

8. Horseradish peroxidase
The enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in biochemistry 
Applications
ability to amplify a weak signal and increase detectability of a target molecule.
Horseradish peroxidase is also commonly used in techniques such as ELISA  
it is smaller, more stable and less expensive than other popular alternatives such as alkaline phosphatase

9. Horseradish peroxidase cont….
Applications
Horseradish peroxidase is a
44,173.9 dalton glycoprotein with
4 lysine residues for conjugation to a labelled molecule.
It produces a coloured, fluorimetric or luminescent derivative of the labeled molecule allowing it to be detected and quantified.
HRP is often used in conjugates 
conjugates (molecules that have been joined chemically) to determine the presence of a molecular target. For example, an antibody conjugated to HRP may be used to detect a small amount of a specific protein in a ELISA.

10. Substrates
Alone, the HRP enzyme, or conjugates there presence must be made visible using a substrate that when oxidized by HRP using hydrogen peroxide as the oxidizing agent, yields a characteristic change that is detectable by spectrophotometric methods
HRP
Enzyme + substrate + H2O2
DIIMINE
chromogen A: contains hydrogen peroxide
chromogen B: containes tetramethylbenzidine (TMB)

11. Numerous substrates for the horseradish peroxidase enzyme have been described commercialized to exploit the desirable features of HRP.
These substrates fall into several distinct categories.
HRP catalyzes the conversion of chromogenic substrates (e.g. TMB, ) into colored molecules, and produces light.

12. 3,3’,5,5’-Tetramethylbenzidine
TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.
The resulting diimine causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at a wavelength of 650 nm.
The reaction can be halted by addition of acid or another stop reagent. Using sulfuric acid turns TMB yellow. The colour may be read at 450 nm.

14. Material Safety
TMB should be kept out of direct sunlight as it is photosensitive.
On that evidence, TMB has been used as a replacement for carcinogenic compounds such  o-phenylene diamine.
Carcinogenic is a physical or chemical agent that having the potential to cause cancer.
mutagen is a physical or chemical agent that changes the genetic material, usually DNA

15. Conjugated protein simple proteins
Many proteins contain only amino acids and no other chemical groups, and they are called simple proteins.
conjugated protein
A conjugated protein is a protein that functions in interaction with other (non-polypeptide) chemical groups attached by covalent bonding or weak interactions.
The nonamino part of a conjugated protein is usually called its prosthetic group.
Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups.
Some examples of conjugated proteins are lipoproteins, glycoproteins, phosphoproteins, hemoproteins, flavoproteins, metalloproteins, phytochromes, cytochromes and opsins.

16. Spectrophotometry 
Spectrophotometry involves the use of a spectrophotometer.
A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength.
It is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It deals with visible light, near-ultraviolet, and near-infrared,

17. Bovine serum albumin (also known as BSA or "Fraction V")
a serum albumin protein that has numerous biochemical applications including ELISA and  immunohistochemistry.
It is also used as a nutrient in cell and microbial culture.
In restriction digests,
BSA is used to stabilize some enzymes during digestion of DNA 
prevent adhesion of the enzyme to reaction tubes and other vessels.
This protein does not affect other enzymes that do not need it for stabilization.

18. Sample diluents PBS: Phosphate buffered saline –
provides stable buffered environment to maintain antibody structure
A PBS buffer containing
0.02% Tween 20, protein as stabilizer and
0.05% Kathon CG as preservative.

19. Wash Buffer Wash Buffer(Tween 20)
detergent – removes non-specifically bound proteins
to reduce background and blocks protein binding sites on the polystyrene
If a smaller volume of Wash Buffer is desired,
add 1 volume of Wash Buffer (20X) to
19 volumes of distilled or deionized water.
Wash Buffer is stable for 1 month at 2-8°C. Mix well before use.

20. How we will detect: read absorbance at 450 nm

21. Absorbance 450 nm 1 2 3 4 5 6 7 8 9 10 11
NPTII Std W E L A B C D F G H

24. Anti-HEV IgM is the serologic marker of choice for diagnosis of acute HEV infection.