The presentation focuses on the ELISA and immunoblotting techniques like southern blotting, western blotting etc. It describes principle, method, advantages and disadvantages and applications of different types of ELISA, steps involves and applications of western blotting and southern blotting techniques.

1. IMMUNO BLOTTING TECHNIQUES
Dr. A. T. Sharma
Assist. Professor
Nanded Pharmacy College, Nanded

2. Introduction
Blotting: A powerful and sensitive technique for
identifying the presence of specific biomolecules within a
sample.
Subtypes:
• Southern blot (Detection of DNA sequence) – by Dr.
Edwin Southern in 1975
• Northern blot (Detection of RNA)
• Western blot (Detection of protein)
• Eastern blot (Detection of post translationally modified
proteins)
• Southwestern blot (Detection of DNA binding proteins)

3. ELISA
• Enzyme-linked immunosorbent assay – commonly used
biochemical assay
• First described by Engvall and Perlmann in 1972
• More rapid method than Western blot analysis
• Detect and quantify substances like peptides, proteins,
antibodies and hormones in a cell, tissue, organ, body fluid
• Based on property of proteins to bind to a plastic surface
• An enzyme conjugated with an antibody reacts with a
colourless substrate to give a coloured product
• Enzymes e.g. alkaline phosphatase, horse radish
peroxidase, beta galactosidase
• Chromogenic substrates e.g. ortho phenyl diamine
dihydrochloride, paranitro phenyl phosphate

4. Principle of ELISA
• Performed in 96-well polystyrene plates
• Wells surface are coated with antigen or antibody
• Test serum, positive control serum and negative control
serum incubated in different wells
• Antigen/antibody in serum are captured in wells
• Washing with buffer
• Secondary antibodies attached with an enzyme are added,
incubated
• Suitable substrate added – enzyme – substrate reaction –
coloured product
• Intensity of colour/ optical density at 450nm
• Useful in qualitative or quantitative detection of
antigen/antibody

5. 96-Well Polystyrene Plate

6. Direct ELISA:
• Detection of an antigen in a
sample
• Micro-titer wells are coated
with antigen to be identified
• Primary Antibodies attached
with enzyme is added –
incubated at 37⁰C, washed
• Suitable substrate is added
• Enzyme – substrate reaction
– coloured product

7. Indirect ELISA:
• Detection of an antibody in
a sample
• Micro-titer wells are coated
with the specific antigen
• Sample containing
antibodies (to be detected)
is added, incubated, washed
• Enzyme labeled secondary
antibodies are added
• Suitable substrate is added
• Enzyme – substrate reaction
– coloured product

8. Sandwich ELISA:
• Detection of an antigen in a
sample
• Micro-titer wells are coated
with monoclonal antibodies
(Capture antibodies)
• Sample containing antigen (to
be detected) is added,
incubated, washed
• Enzyme labeled secondary
antibodies for different
epitope are added -
incubated
• Suitable substrate is added
• Enzyme – substrate reaction
– coloured product

9. Competitive ELISA:
• Detection of concentration of antigen in a sample
• Antibodies incubated in a solution with a sample containing
antigen
• Micro-titer well coated with antigen
• Antigen-antibody mixture added to well - washing
• Enzyme liked secondary antibodies specific for primary
antibody are added
• Suitable substrate is added
• Enzyme – substrate reaction – coloured product
• Higher the concentration of the antigen in sample, less
antibodies available to bind with antigen in well – lower
absorbance

10. Competitive ELISA

11. Advantages & Disadvantages of Different Types of ELISA

12. Applications of ELISA
• Detection of antigen or an antibody
• Detection of potential food allergens in food industry
• In disease out-breaks to track the spread e.g. HIV, bird
flu, common colds, cholera, STD etc.
• Detection of certain drugs in toxicology
• Diagnosis of diseases like dengue, malaria, Chagas
disease (parasitic disease), Johne’s disease (fatal
infection in ruminants)
• Other uses: Detection of Mycobacterium antibodies in
TB, rotavirus in feces, Hepatitis B and C markers in
serum, enterotoxins of E. coli in feces

13. Western Blotting
• Developed by W. Neal Burnette in 1981
• Identification of proteins from a protein mixture
• Labelled antibodies used to identify a desired protein
(immuno-blotting)
• Blotting – To remove liquid from a surface by pressing soft
paper or cloth on it
• Protein mixture subjected for gel electrophoresis to
produce bands – transferred on to a nitrocellulose
membrane – stained with antibodies specific for target
proteins
• Used to confirm positive results of ELISA
• More difficult, time consuming, costly and requires more
skills than ELISA

14. Steps in Western Blotting
Extraction of Protein:
• Protein extracted from cell lysate (tissue
preparation) by mechanical or chemical lysis
• Protease inhibiter added to prevent denaturation
of proteins
• Sufficient concentration of protein (spectroscopy)
is obtained, diluted in loading buffer containing
glycerol – helps sample to sink in well
• Tracking dye (bromothymol blue) added to
monitor protein movement

15. Gel Electrophoresis:
• Sample loaded in well of SDS-PAGE consisting
of sodium dodecyl sulphate – polyacrylamide
gel and subjected for gel electrophoresis
• Proteins get separated on the basis of electric
charge, isoelectric point, molecular weight
• Proteins with low molecular weight move
faster
• Proteins are negative, so move toward
positive pole (anode) after application of an
electric current

16. Blotting:
• Nitro cellulose membrane placed on gel –
separated proteins transferred to membrane
by capillary action (Time consuming, 1-2 days)
• For fast and efficient transfer, electro blotting
is used
• Electro blotting nitro cellulose membrane
sandwiched between gel and a cassette of
filter paper and electric current applied

17. Blocking:
• As antibodies are proteins, may bind with nitro cellulose
membrane.
• Hence, membrane is masked with casein or bovine serum
albumin before adding primary antibody
Treatment with Primary Antibody:
• Primary antibody specific to desired protein added, forms
an antigen – antibody complex
Treatment with Secondary Antibody:
• An enzyme liked secondary antibody added (against
primary antibody, anti – antibody) – Antigen – Antibody
complex
• Enzymes e.g. alkaline phosphatase, horse radish peroxidase
Treatment with Substrate:
• Incubation with suitable substrate – band of colour can be
visualized

19. Applications of Western Blotting
• Determine size and amount of protein
• Diagnosis of viral and bacterial infections
• Confirmatory test for HIV
• Detection of defective proteins e.g. prions
disease
• Definitive test for Creutzfeldt-Jacob disease
(degenerative brain disorder), Lyme disease
(Tick borne disease), Hepatitis B and Herpes

20. Southern Blotting
• Developed by Edward M. Southern in 1975
• Hybridization technique for detection of particular size
of DNA from mixture of similar molecules
• DNA fragments separated on the basis of charge and
size by electrophoresis
• Transferred on nylon membrane
• Detection of specific DNA by marker labeled DNA
probe complimentary to desired DNA
• Probe – a short , single stranded DNA (100 – 500 bp)

21. Steps in Southern Blotting
Restriction Digestion:
• DNA fragmentation by suitable restriction enzyme – number of
fragments amplified by PCR
Gel Electrophoresis:
• Separation of DNA fragments by gel electrophoresis using SDS gel
Denaturation:
• SDS gel soaked in alkali (NaOH), or acid (HCl) to denature double
stranded DNA – single strands produced
Blotting:
• Separated strands transferred to positively charged nylon
membrane (Nitro cellulose paper)
Baking and Blocking:
• Membrane baked in an autoclave to fix DNA
• Membrane treated with casein or bovine serum albumin to mask
binding sites on it

22. Hybridization with Labeled Probe:
• DNA on membrane treated with labeled probe
(P32)
• Probe binds with desired DNA fragment
Visualization by Autoradiogram:
• Membrane bound DNA with probe is
visualized under autoradiogram as a pattern
of bands

23. Southern Blotting Technique

24. Applications
• Detection of DNA in a sample
• DNA finger printing
• For paternity test, criminal identification, victim
identification
• Isolation and identification of desired gene
• In Restriction Fragment Length Polymorphism
• Identification of mutation or gene rearrangement
in DNA sequence
• Diagnosis of disease caused by genetic defects

25. Thank You…!!!
(Disclaimer: The images and diagrams in this presentation
have been downloaded from the google source. I am grateful to
all the publishers & the google.)