ELISA ASSY

1. ENZYME-LINKED IMMUNO
SORBANT ASSAY
(ELISA)
MISS. UTTARA L. JOSHI
1

2. ELISA
■ The ELISA assay is a widely used biochemical assay to
detect in a sample the presence of and quantity of proteins,
such as hormones and antibodies and bacteria or viruses.
■ The Elisa assay uses the coupling of antigens and antibodies
and relies on the specificity and affinity of antibodies for
antigens. Specificity is the ability to discriminate among
diverse proteins. Affinity is the ability to tightly bind to
molecules.
■ One can determine how much antibody is present by
starting with an antigen, or one can determine how much
antigen or hormone is present by starting with an antibody.[1]
2

3. What Are Antigens..?
■ Antigens are any foreign substance in the body.
■ Antigens include “not-self” molecules and cells, such as:
A. Foreign proteins
B. Viruses
C. Environmental pollutants and other foreign substances like
asbestos, tattoo ink, and cigarette smoke
D. Bacteria and parasites (Protista, Fungi, Plantae, and
Animalia cells)
E. Foreign transplanted tissue
F. cancerous cells.[1]
3

4. ANTIGEN (Ag):
Any molecule that induces the production of antibodies when
introduced in the body of an animal is called antigen.
ANTIBODY(Ab):
Proteins produced by the immune system which helps to defend
against antigens.
4

5. Depending on the antigen-antibody combination, the assay is
called as;
■ Direct ELISA
■ Indirect ELISA
■ Sandwich ELISA
■ Competitive ELISA
5
TYPES

6. TERMINOLOGIES
1.Testing sample
2. Antibody ( 1st,2nd)/Antigen
3. Polystyrene microtiter plate
4. Blocking buffer
5. Washing buffer
6. Substrate
7. Enzyme
6

7. TESTING SAMPLE
1. Serum
2. Sputum
3. Urine
4. Semen
5. Stool
6. Supernatant of culture
7

8. REAGENTS
■ WASHING BUFFER - 0.1M Phosphate buffer+ 0. 50M
NaCl + 0.1% Tween20
■ COATING BUFFER - 0.1M Phosphate buffer+ 0.15M
NaCl
■ BLOCKING BUFFER - Bovine Serum Albumin (BSA)
■ ENZYME - Horse Radish Peroxidase (HRPO)
■ CHROMOGENIC SUBSTRATE - Trimethyl benzidine
■ STOP SOLUTION - 0.5M H2SO4
8

9. LAB EQUIPMENTS
9
1. Micro plate strips
2. Pipettes
3. Pipette tips
4. Transfer pipette
5. Paper towels
6. Marking Pen
7. Absorbance Reader

10. ADVANTAGES OF ELISA
1. Reagents are long self life time.
2. Highly specific & sensitive.
3. Easy to perform & Quick procedure.
4. Used to detect a variety of infection.
10

11. DISADVANTAGES OF ELISA
1. Result may not be absolute.
2. Antibody must be available.
3. Concentration is not clear.
4. False positive possible.
5. False negative possible.
6. Requires trained person.
7. Labs requires a special license to handle radioactive materials.
11

12. DIRECT ELISA
 Primary labelled ab is directly binds to the ag.
 Perform with ag which is directly immobilized on assay plate.
Ag added to the plate
Add blocking buffer
Not require secondary ab.
Primary ab is added
Substrate added
Detect
12

13. ADVANTAGE OF DIRECT ELISA
13
1. Faster than other ELISA – the technique has fewer steps.
2. Less prone to error – As less reagents and fewer steps are
required
3. Only one Ab is needed.
4. Require less time.[1]

14. DISADVANTAGE OF DIRECT ELISA
1. Antigen immobilization is not specific - may cause higher
background noise than indirect ELISA.
2. Mainly because all proteins in the sample, including the
target protein, will bind to the plate.
3. Less flexible - each target protein needs a specific
conjugated primary antibody.
4. No signal amplification - reduces assay sensitivity.[1]
14

15. Ag added to the plate
Add blocking buffer
1° AB is added
2° AB is added
HRPO is Added (which recognize & bind to the 1°AB)
Substrate is added
Detect
15
INDIRECT ELISA

16. 16

17. INDIRECT ELISA
■ ADVANTAGES :
1. High flexibility
2. Signal amplification
■ DISADVANTAGES :
1. Complex protocol.
2. Cross reactive from secondary antibody.[1]
17

18. SANDWICH ELISA
■ Ag in the sample bind with the capture ab become
immobilized.
■ The ab of the enzyme conjugate bind with the immobilized
ag to form sandwich of ab-ag-ab.
■ Antigens like tumor markers, serum, protein, hormones.[2]
18

19. PROCEDURE
Plate coated with ab (capture ab)
Blocking buffer added
Sample is added
Ag bounded to the capture ab
Enzyme linked ab is added
Substrate added
Color change observed
19

20. ADVANTAGE
■ High flexibility
■ High sensitivity
■ High specificity
DISADVANTAGE
■ Optimization in terms of antibody becomes problematic
due to cross-reactivity issues.[2]
20

21. COMPETITIVE ELISA
 Inhibition ELISA, blocking ELISA, plate/surface based
assay.
 Ab is first incubated in the solution with a sample containing
ag.
 Ag-ab mixture is added to the ag coated microtiter well.
 The more ag present in the sample , the less free ab is
available to bind to the ag coated well
21

22. PROCEDURE
Incubate unlabelled ab with ag
Bound ag-ab
Wash & remove unbound ab
2°ab with enzyme added
Substrate is added
Detect
22

23. COMPETITIVE ELISA
Advantages :
1. Negligible sample processing is required and can be
applicable to crude samples.
2. Less sensitive to experimental errors.
3. Good reproducibility and flexibility.
Disadvantages :
1. Basic ELISA limitations apply.[2]
23

24. APPLICATIONS OF ELISA
1. Hormones
2. Proteins
3. Infectious Agent ( Viral, Bacterial,
4. For Rapid Test Parasitic, Fungal
5. Drug Tests
6. IgG, IgM, IgA
7. Tumor Tests
8. Serum Proteins
9.In Clinical Research
10. HIV ,Hepatitis B & C test
11. Used to detect various biomarkers, like cytokinine,
prostaglandine, Insulin, many more. [2] 24

25. CELL CULTURE
Cell culture can be defined as the
process of cultivating cells and
tissues outside the body of an
organism(in vitro) in an artificial
environment, which stimulates
the in vivo conditions such as
temperature, nutrition and
protection from microorganisms.
25

26. Terminologies
 pH: (7.2 to 7.4) Phenol red is used as an internal indicator.
 Oxygen: Cells depend upon glycolysis for the supply of O2;
Selenium controls O2 diffusion; glutathione acts as free radical
scavenger
 Temperature: (37oC) Change of ± 5°C is acceptable.
 Humidity: For cell growth 100% humidity is essential to
reduce evaporation of the media.
 Antibiotics:
• Penicillin (100 mg/ml) for bacteria,
• Streptomycin (100 mg/ml) for bacteria,
• Gentamycin (50mg/ ml) for bacteria,
• Nystatin (50mg/ml) for fungi and yeast. [3]
26

27.  Primary cell culture: when cells are surgically removed from
an organism and placed into a suitable culture environment
they will attach, divide and grow.
 Cell line: when the primary culture is sub cultured and they
show an ability to continuously propagate.
 Anchorage dependency: cells grow as monolayers adhering to
the substrate (glass/ plastic)
 Passaging/ sub culturing: the process of splitting the cells.
 Finite cells: when the cells has finite life span.
 Continuous cell lines: when the cells can grow up to infinite
lifespan.[3]
27

28. Tissue Culture Media
■ Cells have complex nutritional requirements that must be met
to permit their propagation in vitro.
■ Previously, scientists employed chick embryo extract,
plasma, sera, lymph etc.
■ Today, a number of chemically-defined formulations have
been developed that support the growth of a variety of
established cell lines.
■ Eagle’s basal media
■ Eagles’s Minimum Essential Media (MEM)
■ Dulbecco’s Modified Essential Media (DMEM)
■ Iscove’s Modified Dulbecco’s Medium (IMDM)[4]
28

29. TYPES OF CELL CULTURE
Cell culture is classified into three:
1. Primary cell culture:
• Adherent cell culture
• Suspension cell culture
2. Secondary cell culture
3. Cell line:
• Finite cell line
• Continuous cell line
29

30. PRIMARY CELL CULTURE
30
■ When cells are surgically removed from an organism and
placed into a suitable culture environment they will attach,
divide and grow.
■ Most of the primary culture cells have a finite lifespan of 50-
60 divisions in vitro.
■ Due to their limited lifespan, one cannot do long-term
experiments with these cells.[4]

31. Suspension Cell CultureAdherent Cell Culture
■ Appropriate for most cell types,
including primary cultures
■ Requires periodic passaging, but
allows easy visual inspection
under inverted microscope
■ Growth is limited by surface
area, which may limit product
yields
■ Requires tissue-culture treated
vessel
■ Used for cytology, harvesting
products continuously, and many
research applications
■ Appropriate for cells adapted to
suspension culture and a few other
cell lines that are non adhesive (e.g.,
hematopoietic)
■ Easier to passage, but requires daily
cell counts and viability
determination to follow growth
patterns; culture can be diluted to
stimulate growth
■ Growth is limited by concentration
of cells in the medium, which allows
easy scale-up
■ Can be maintained in culture vessels
that are not tissue-culture treated, but
requires agitation (i.e., shaking or
stirring) for adequate gas exchange
■ Used for bulk protein production,
batch harvesting, and many research
applications 31

32. Finite cell lines
 Limited life span
 Go through a limited
number of cell
generation
 Properties:
• Contact inhibition
• Density
• Limitation
• Anchorage dependence
• Less growth rate
• Doubling time (24-
92hrs.)
Continuous cell lines
 Grow indefinitely
 Grows either in
monolayer or in
suspension
 Properties:
• Contact inhibition
• Absence
• Anchorage dependence
• High growth rate
• Doubling time (12-
24hrs.)
32

33. Application
33
 Mass culture of animal cell line manufacture of viral vaccine &
other product of biotechnology.
 Stem cell differentiate to various somatic cell type for
transplantation.
 Stem cell culture used for therapeutic development.
 Biological products produced by recombinant DNA (rDNA)
technology in animal cell cultures include enzymes,
synthetic hormones, immunobiologicals (monoclonal
antibodies, interleukins, lymphokines), and anticancer agents.

34. 34
 The cost of growing mammalian cell cultures is high, so
research is underway to produce such complex proteins in
insect cells or in higher plants, use of single embryonic cell
and somatic embryos as a source for. direct gene transfer
via particle bombardment, transit gene
expression and confocal microscopy observation is one of
its applications
 Cell culture is also a key technique for cellular agriculture,
which aims to provide both new products and new ways of
producing existing agricultural products like milk, (cultured)
meat, fragrances, and rhino horn from cells and
microorganisms. [5]

35. Reference
1. Stephanie D. Gan, and Kruti R. Patel, “Enzyme Immunoassay and
Enzyme-Linked Immunosorbent Assay”, Journal of Investigative
Dermatology (2013) 133.
2. Seiichi S.,Waraporn P. ,SornkanokV.,Waranyoo P.,Yukihiro S.,
HiroyukiT. ,Satoshi M., “Enzyme-linked immunosorbent assay
for the quantitative/qualitative analysis of plant secondary metabolites;
Journal of Natural Medicines (2018) 72:32–42
35

36. 3. Baksh, D., Song, L., &Tuan, R. S. “Adult mesenchymal stem
cells: characterization, differentiation, and application in cell and
gene therapy” Journal of cellular and molecular medicine, (2004).
8(3), 301-316
4. Butler, M. “Animal cell cultures: recent achievements and
perspectives in the production of biopharmaceuticals”. Applied
microbiology and biotechnology, (2005). 68(3), 283-291.
5. Freshney, R. I. “Culture of animal cells”. Hoboken. Gangal, S.
Principles and Practice of AnimalTissue Culture.(2010)
36

37. Thank you.
37