The document describes the immunological assay ELISA (enzyme-linked immunosorbent assay). It discusses how ELISA uses the antigen-antibody reaction to detect the presence of antigens or antibodies. There are four main types of ELISA: direct ELISA, indirect ELISA, competitive ELISA, and sandwich ELISA. ELISA has become a popular technique due to its high sensitivity, safety, low cost, and simple instrumentation requirements. It has various applications such as detecting HIV antibodies, microorganisms, food allergens, and markers for diseases.

1. IMMUNOLOGICAL ASSAY
(ELISA)
PRESENTED BY:
Prasanta deka
M pharm 1st semester
Pharmaceutical analysis
Sub: Modern pharmaceutical analysis
Krupanidhi college of pharmacy,bangalore

2. IMMUNOASSAY
 Immunoassay means the method to measure any particular substance in a mixture using its
specific-binding antibody.
 One of the quality of the immunoassay is that we can measure a substance that is present in a
mixture of various contaminants.
 Immunoassay have become very popular in view of their high sensitivity, safety economy, and
simple instrument requirements.
Antigen: Substance that capable of stimulating an immune response, specially activating
lymphocytes, which are the bodies infection-fighting WBC.
Antibody: A protective protein produced by the immune system in response to the presence of the
foreign substance(antigen).

4. ELISA
Enzyme Linked Immune Sorbent Assay
 Antigen of interest is absorbed on to plastic surface “sorbent”.
 Antigen is recognized by the specific antibody “immune”.
 Antibody is recognized by the secondary antibody, which has attached with enzyme
“enzyme linked”.
Substrate linked with the enzyme to produce a product, usually colored.

5. ELISA
 ELISA is a bio chemical technique uses mainly in the immunology to detect the presence of
antibody or an antigen in the sample.
 It is a plate based assay designed for detecting and quantifying substances such as peptide,
protein, antibody, and hormone.
 In an ELISA, an antigen must be immobilized on the solid surface and then complexed with
an antibody that is linked to an enzyme.
 The detection is accomplished by assessing the conjugated enzyme activity via incubation
with a substrate to produce a measurable product.
 The test can be done by polystyrene tube( macro-ELISA) or polyvinyl microtiter( micro-
ELISA).

6. Principle
 ELISA is based on the antigen-antibody reaction, which is representing the chemical interaction between
antibodies producing by beta cell of lymphocyte and antigens.
 The specific immune system produced an important role in protecting the body from invaders such as
pathogen and toxins.
 Hence, by exploiting the this reaction, ELISA permits the highly sensitive quantitative/qualitative analysis of
antigens.
 To detect the molecule, an antigen or antibody is labeled using enzymes.
 An antigen in the fluid phase is immobilized to the solid surface, such as microtiter plate constituting
polystyrene, polyvinyl chloride and poly propylene.
 Subsequently the antigen is allowed to react with the specific antibody, which is detected by enzyme labeled
secondary antigen.

7.  The development of the color by using a chromogenic substance, indicating the presence of Ag: Ab binding.
 There fore ELISA can be used to detect either the presence of Ag or Ab in the sample depending upon how the
test is designed.

8. Steps involves in the ELISA
Step 1: Antigen binding
 Sample antigen is binding or immobilized to the microplate via absorption to the surface.
 Binding is achieved by the incubating the wells with a solution containing antigen (Ag) for 2 hrs. at
room temperature or overnight at 4˚C.
 The protein adheres due to the hydrophobic interaction between protein and plastic.
 Coating is done using carbonate/ bicarbonate buffer at pH 9.4
Step 2: Blocking
 All unbound site on the solid support are block to prevent non specific binding of the antibody.
 Blocking buffers like BSA, non fat dry milk powder in PBS or TBC at pH 7.4
 Protein in blocking solution will attach to the membrane in plate where the target protein have not
attached.
 Excess blocking agent is removed by washing the plate membrane with washing buffer.

9. Step 3: Primary antibody
The primary antibody is added and will be bound only if there is any recognized epitope with in the
sample antigen.
Step 4: Secondary antibody
o An enzyme linked secondary antibody is added with suitable dilution which will bind to any available
primary antibody.
o Secondary antibodies are linked to the enzyme through bi-conjugation.
o Plate is washed with buffer or mild detergent to remove any unbound antibody or protein.
Step 4: Detection/ development
o After all final wash step, the plate is developed by adding an enzymatic chromogenic substrate to give color.
o The entire plate is placed into a plate reader and OD is determined for the each well.
o The intensity of the color reflects the amount of the specific secondary antibody bound to the target.

10. Types of ELISA
There are four common ELISA test- based on the binding structure between antibody and
antigen.
1. Direct ELISA
2. Indirect ELISA
3. Competitive ELISA
4. Non-competitive ELISA ( sandwich ELISA)

11. Direct ELISA
 In 1971, Engvall, Perlmann, Van Weemen and Schuurs were 1st to develop direct ELISA, which
was the base style for other type of ELISA.
 Primarily, an antibody or antigen is immobilized on the surface of the microtiter plate.
 After the surface is blocked with the other protein to avoid the non specific absorption of the
other protein.
 The corresponding enzyme linked Ab or Ag is allowed to react with the immobilized target.
 Followed by color development with appropriate substrate, with increasing amount of the target,
the signal increases.
 Direct ELISA is suitable for the qualitative analysis of macromolecule.

12. E
E
E
E
E E
E E
E E
E E
E E
a)
b)
d)
C
)
antigen
antibody
E
Enzyme
labelled
antibody
Attach Ag/Ab
to solid
surface
Incubate
with enzyme
label Ag/Ab
Wash the
unbound
Ag/Ab out
Develop the
color with
substrate
Direct ELISA

13. Advantages of direct ELISA
 Quick methodology since one antibody is used.
 Cross reactivity of secondary antibody is eliminated.
Disadvantages of direct ELISA
 Immuno-reactivity of primary antibody may be reduced as a result of labelling.
 Labelling of energy primary antibody label from one experiment to another.
 Little signal amplification.

14. Indirect ELISA
 Development on the basis of the direct ELISA to evaluate the presence of antibody in antisera
(1˚ antibody)
 The key step of this system is the two binding process of the primary antibody and enzyme
linked secondary antibody, which is labelled with the enzyme, called indirect ELISA.
 Antigen is primarily immobilized on the surface of the microtiter plate, which block the
surface with blocking protein.
 Primary antibody binding to the immobilized antigen is allowed to react with the enzyme
linked secondary antibody, followed by development of the color.
 Signal increases with increasing amount of the immobilized target antigen.

15. E E
E E
a) Attach antigen to solid phase
b) Incubate with primary antibody
C) Wash unbound antibody out
d) Incubate with enzyme labelled secondary antibody
e) Develop color with substrate
Indirect ELISA

16. Advantages of indirect ELISA
 High sensitivity: more than one labelled antibody id bound per antigen
molecule
 Flexible: different primary detection antibodies can be used with a single
labelled secondary antibody
 Cost saving: fewer labelled antibodies are required.

17. Competitive ELISA (CE)
1. In 1973, Belanger develop competitive ELISA.
2. The key event of the competitive ELISA is the competitive reaction between the target
(Ag/Ab) in the sample and enzyme labelled target (Ag/Ab) against corresponding immobilized
Ag/Ab.
3. To detect the antigen in CE, an enzyme labelled antigen is used to compete with the target
antigen against the immobilized antibody.
4. Hence, the higher amount of the antigen in the sample, lower the amount of the enzyme linked
antigen that bind to the antibody.
5. This is with increasing amount of target antigen, signal decreases.

18. Advantages of competitive ELISA
 High specificity, since two antibodies are used the antigen/analyte is specifically
captured and detected.
 Suitable for complex samples, since the antigen does not require purification prior to
measurement.
 Flexibility and sensitivity, since both direct and indirect detection method can be used.

19. Sandwich ELISA
 The target antigen is detected via anchoring between two antibody, which recognized
different epitope, so it is called as sandwich.
 Sandwich ELISA, starts from the immobilized of an antibody, called captured antibody on
microtiter plate.
 Sample is added and any antigen is present bind to the captured antibody.
 Antigen bound to the captured body is then sandwich with the enzyme linked antibody for
color development.
 Signal increases with the increases the amount of the antigen.

20. EE
EE
Attach capture antibody to the
solid surface
Incubate with target
antigen
Wash unbound target out
Incubate with enzyme linked
antibody
Develop the color with substrate
Sandwich ELISA

21. Advantages of sandwich
 High specificity
 Suitable for complex sample
 Flexibility and sensitivity

22. Application of ELISA
 Detection of HIV antibodies
 Detection of microorganisms and the toxins produced by them
 Food allergens: it has also found applications in the food industry in detecting potential food
allergens.
 Serological blood test for coeliac disease.
 ELISA can be used as a detection method for detection of mycobacterium antibodies in
tuberculosis, detection of rotavirus in faeces, detection of hapatitis B markers in serum, detection
of HIV antibodies in blood samples.