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IMMUNO BLOTTING TECHNIQUES
Dr. A. T. Sharma
Assist. Professor
Nanded Pharmacy College, Nanded
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)
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
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
96-Well Polystyrene Plate
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
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
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
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
Competitive ELISA
Advantages & Disadvantages of Different Types of ELISA
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
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
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
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
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
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
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
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)
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
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
Southern Blotting Technique
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
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.)

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PRINCIPLE & APPLICATIONS OF IMMUNO BLOTTING TECHNIQUES.pptx

  • 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
  • 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
  • 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
  • 18.
  • 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
  • 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.)