ELISA (Enzyme-Linked Immunosorbent Assay) is a sensitive technique used to detect small quantities of antigens, antibodies, or other proteins in biological fluids like blood or urine. There are several types of ELISA including direct, indirect, sandwich, and competitive ELISA. ELISA works by using an enzyme-linked antibody or antigen to detect the presence of a target protein. This allows very small amounts of the target to be detected through the enzyme's catalytic activity.

3. ELISA
Enzyme Linked Immunosorbent Assay
ELISA is used for:
Quantitative Estimation of
Hormones
Amino Acids
Tumour Markers
Growth Factors
Other Biochemical analytes which are present in
small amount in biological fluids

4. Qualitative Estimation of:
Bacterial/Viral Antigens
Antibodies against microbes
Any other Antigen or Antibodies present
in biological fluid in small amount.

5. Advantages:
Very small amount(ng to pg) of Antigen or
Antibody can be detected in biological fluid
sample.
Highly economical, sensitive and time effective
method (2-3 hours)
Non-isotopic immunoassay in which labelled
enzyme is used.
More sensitive than RIA with no risk of Radiation
Hazard

6. Basic Principle:
ELISA is based on Immunochemical principle
of Antigen-Antibody reaction. The specific
interaction between Ag-Ab is used to
detect/quantitate the specific component
of biological fluid, using enzyme labelled
antibodies.

7. • The substrate which is suitable for enzyme is
added, coupled with coloured reagent
(Chromogen) which is colourless in
beginning but become coloured during
course of reaction. The intensity of the colour
formed predicts the concentration of
Antigen-Antibody in given biological sample.

8. Types of ELISA
1. Direct ELISA
2. Indirect ELISA
3. Immunometric/Sandwich ELISA
4. Competitive ELISA

9. Common terms used in ELISA:
• Enzyme Conjugate: indicator antibodies
For example:
HRP: Horse-Radish Peroxidase
ALP: Alkaline phosphatase

10. Substrate/Chromogen: indicator in Enzyme
and substrate reaction
Example of substrate:
pNPP: paraNitro phenyl phosphate
Hydrogen peroxide : 0.003-0.015%

11. Chromogen: light sensitive hence incubation
always done in dark
For example:
TMB: Tetramethyl benzidine
ABTS: Azino-benthiazoline sulphonic acid
OPD: Ortho-phenylenediamine
dihydrochloride

12. Most common wavelength studied is
450 nm
Other can be 490 nm /405 nm
depends on the kit manufacturer.

15. Direct ELISA:
This was the ELISA originally developed
by Perlmann and Engvall. Most simplest form of
ELISA.
The surface of the microtitre plate is coated
directly with the sample. An enzyme-tagged
antibody (Monoclonal Ab’s) is used for its
detection.
Incubation is done for reaction b/w Ag-Ab which
is followed by washing to remove unbound
antibodies from the medium.

16. The appropriate substrate is then added to the
medium to develop colour directly proportional
to the amount of antigen present in the sample.
Direct ELISA is suitable for determining the
amount of high molecular weight antigens.
Due to Few steps it is faster than other types of
ELISA.

18. Indirect ELISA (ELISA for Antibody Detection)
This is useful to detect small quantities of
Antibodies in the patient’s serum
Indirect ELISA is deemed to be highly sensitive
and more flexible than direct ELISA.
Indirect ELISA is a two-step binding process
involving the use of a primary antibody and a
labelled secondary antibody

19. In this method, the primary antibody is
incubated with the antigen-coated wells. Next, a
labelled secondary antibody that recognizes the
primary antibody is added.
This secondary antibody is often a polyclonal
anti-species antibody. A wide variety of labelled
secondary antibodies are readily available.

20. • This method is commonly utilized to
diagnose infection by bacteria, virus, or a
parasite s/a HIV Antibodies

21. Steps:
Antigen from HIV is coated in the well of microtitre
plate.
Then patients serum is added and incubated. (if it
contains antibodies for HIV it will get bound to
Antigen and fixed). This antibody for time being can
be called as Ab-1
Washing is done to remove unbound and excess Ab’s

22. Now second Ab (Ab-2 which is antibody
against antibody of humans) which is
conugated with HRP is added. Substrate and
Chromogen (Hydrogen peroxide-Diamino
benzidine) also added to wells
Incubation and washing done

23. Development of colour( brown) indicated
presence of Antibodies in patients serum.
Intensity of colour formation is directly
proportional to concentration Antibodies
present.

30. Immunometric/ Sandwich ELISA
Most commonly used ELISA for Antigen
Detection
Two Antibodies are used:
a) One which is coated in well (Ab to target
Analyte)
b) Second conjugated with Enzyme-chromogen
system

33. Sandwich ELISA Indirect ELISA
Microtitre plate is coated with
primary antibody
Microtitre plate is coated with
sample (Antigen)
Primary Ab-----Sample-----
Secondary Ab
Sample----Primary Ab------
Secondary Ab
Approx 2-5 times more sensitive
than Indirect ELISA

34. Competitive ELISA
Also c/a Inhibition ELISA or Competitive
Immunoassay
This ELISA is used to detect low molecular
weight Antigen becoz they have less
number of epitopes (Oxytocin, cAMP,
Corticosteroids)

37. Proteins bound to wells via:
a) Hydrophobic interaction : large molecules
b) Covalent Interactions : small molecules

38. Rapid Covid-19 Antibody Detection
This test detects patient generated Antibodies
against SARS-Cov-2 Virus that causes COVID-19
It detects IgM (First Ab to appear in Covid
Infection)and IgG ( Second to IgM)
This is basically a Lateral flow Immunoassay which
detect presence of Analyte in patient sample
Qualitatively.
Immunoassay and Chromatography together

41. Which of the following molecule(s) can be
detected by ELISA?
a) proteins
b) hormones
c) antibodies
d) all of the above

42. What does a weak color signal in competitive
ELISA represent?
a) more antigen in the sample
b) less antigen in the sample

43. What is a major advantage of ELISA in comparison
to other biological quantification techniques?
a) detection of a molecule at a low
concentration
b) inexpensive
c) low specificity
d) easily available

44. ELISA is designed for detecting and quantifying
substances such as peptides, proteins,
antibodies and hormones.
a)True
b)False