The document discusses various immune blotting techniques including ELISA, Southern blotting, Northern blotting, and Western blotting. ELISA is an assay that uses antibodies to detect antigens or vice versa. Southern blotting detects DNA, Northern blotting detects RNA, and Western blotting detects proteins. All techniques involve separating molecules, immobilizing them, and using probes to detect specific targets. They are commonly used research tools in biochemistry and molecular biology.

1. Immune Blotting Techniques
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 1
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida
Abhijit Debnath
Asst. Professor
NIET, Pharmacy
Institute
Unit: 4
Subject Name: Biotechnology
(BP605T)
Course Details
(B. Pharm 6th Sem)

2. Immune Blotting
Techniques
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-1 2
 Basic Terms
 ELISA
 Introduction to Immune Blotting Techniques
 Southern Blotting
 Northern Blotting
 Western Blotting
CO1.1
Noida Institute of Engineering and Technology
(Pharmacy Institute) Greater Noida

3. • Antigen:
Any molecule that elicits the production of
antibodies when introduced into body.
• Antibodies:
Proteins produced in response to antigenic
stimuli.
• Enzyme conjugate:
An enzyme that is attached irreversibly to an
antibody. e.g: Horse-redish peroxidase
(HRPO).
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 3

4. • Chromogen:
A chemical alters color as a result of an enzyme
interaction with substrate (color reaction used as signal)
e.g Trimethyl benzidine (TMB).
• Stopping:
The process of stopping the action of an enzyme on a
substrate.
• Reading:
Spectrophotometric measurement of color developed in
ELISA.
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 4

5. • Adsorption:
The process of adding an antigen/antibody, diluted
in buffer, so it attaches to the solid phase on
incubation.
• Washing:
The simple flooding & emptying of wells with a
buffered solution to separate bound from un-
bound reagents in ELISA.
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 5

6. • Electrophoresis is an electrokinetic process which separates
charged particles in a fluid using a field of electrical charge.
• The procedures differ in some ways but all need a source for the
electrical charge, a support medium and a buffer solution.
• Electrophoresis is used in laboratories for the separation of
molecules based on size, density and purity.
• An electric field is applied to molecules and as they are
electrically charged themselves it results in a force acting upon
them.
• The greater the charge of the molecule the greater the force
applied by the electrical field and therefore the further through
the support medium the molecule will move relative to its mass.
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 6
 Electrophoresis

7.  Electrophoresis
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 7

8.  Electrophoresis
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 8

9.  Electrophoresis
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 9
Gel electrophoresis technique is used in separating protein
molecules of varying sizes,
• DNA fingerprinting
• DNA Analysis,
• Protein and Antibody Interactions,
• Testing Antibiotics,
• Testing Vaccines
Photograph of immunoelectrophoresis performed in
agar gel. Human serum is first separated by
electrophoresis following which troughs are cut in the
agar, and antibodies to whole serum, isolated isotypes,
or light chains are added.

10.  Electrophoresis
BASIC TERMS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 10

11. • The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is
used to measure the concentration of an analyte (usually antibodies or antigens) in solution.
ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 11

12. WORKING OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 12

13. WORKING OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 13

14.  Based on Basic Immunology Response
 Lock and Key Concept:
1) Antigen (key)
2) Antibody (lock):
Key fits into the lock
 Enzyme conjugate substrates
• Bound to a secondary antibody that binds with the antibody-antigen complex.
PRINCIPLE OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 14

15. 1) Microwell Plate: Flat bottom polystyrene plate,
contains 8 x 12 wells holding 350 μL each.
Equipment's CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 15

16. 2) Multipipette :
An 8-channel 100 μL pipette is a good help for even
small-scale work.
EQUIPMENT'S CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 16

17. 3) Washing Device:
• manually operated washing devices.
• may be of use particularly when there is a
risk that the samples tested in ELISA
contain infectious material, so must be
collected for subsequent disinfection.
EQUIPMENT'S CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 17

18. REAL EXPERIMENT OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 18

19. On the Basis of Detection:
1) Colorimetric ELISA:
Assay to Determine the Antibody Concentration.
TYPES OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 19

20. 2) Chemiluminescent ELISA:
Assay for the Quantitation of an Antigen in a Biological Sample.
TYPES OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 20

21. 3) Competitive Fluorescence ELISA:
TYPES OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 21

22. • Reagents are relatively cheap & ‘ve long shelf life.
• It is highly specific & sensitive (<1pg/ml).
• No radiation hazards occur during labeling or disposal of waste.
• Easy to perform & quick procedures.
• Equipment is widely available.
• It can be used to a variety of infections.
• It can be used on most type of biological samples like plasma,
serum, urine, cell extracts.
ADVANTAGES OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 22

23. • Measurement of enzyme activity can be more complex than the measurement of activity of some type of
radioisotopes.
• Enzyme activity may be affected by plasma constituents.
• Kits are not cheap.
• Very specific to particular antigen but won’t recognize other antigens.
• False positive/ negative possible, especially with
mutated/ altered antigen.
DISADVANTAGES OF ELISA CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 23

24. • Results may not be absolute.
• Antibody must be available(poor producer, interference).
• Concentration may be unclear.
• False positive possible (Ab already present).
• False negative possible.
LIMITATIONS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 24

25. • Immunoblotting techniques use antibodies to identify target proteins among a number of unrelated protein species.
• They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
• Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows
the use of a gel electrophoresis.
• The Southern blot is used for transferring DNA,
• The Northern blot for RNA and
• The western blot for PROTEIN.
• The Eastern blot for PROTEIN, post-translational modifications including the addition of lipids, phosphates, and
glycoconjugates.
INTRODUCTION TO IMMUNE BLOTTING TECHNIQUES CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 25

26.  The mixture of molecules is separated.
 The molecules are immobilized on a matrix.
 The probe is added to the matrix to bind to the molecules.
 Any unbound probes are then removed.
 The place where the probe is connected correspondsto the location of the
immobilized target molecule.
PRINCIPLE CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 26

27. INTRODUCTION TO IMMUNE BLOTTING TECHNIQUES CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 27
Immune
Blotting
Techniques
Northern
Blot
Eastern
Blot
Southern
Blot
Western
Blot
Used to detect Protein
Used to detect RNA
Used to detect DNA
Used to detect Protein

28. WHAT IS BLOTTING CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 28

29. APPLICATIONS CO4.1
16 July 2021 Abhijit Debnath BP605T and Biotech Unit-4 29
 Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure
relative amounts in different samples.
 Southern blot is used to detect the presence of a particular bit of DNA in a sample
 analyze the genetic patterns which appear in a person's DNA.

30.  A Southern blot is a method used in molecular biology for
detection of a specific DNA sequence in DNA samples.
 Southern blotting combines transfer of electrophoresis-
separated DNA fragments to a filter membrane and
subsequent fragment detection by probe hybridization.
 The method is named after the British biologist Edwin
Southern, who first published it in 1975.
SOUTHERN BLOT CO4.1

31.  Other blotting methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar
principles, but using RNA or protein, have later been named in reference to Edwin Southern's name.
 As the label is eponymous, Southern is capitalised, as is conventional of proper nouns. The names for other
blotting methods may follow this convention, by analogy.
SOUTHERN BLOT CO4.1

32. APPLICATIONS OF SOUTHERN BLOT CO4.1
1. Southern blotting transfer may be used for homology-based cloning on the basis of amino acid sequence of the
protein product of the target gene.
2. Oligonucleotides are designed so that they are similar to the target sequence. The oligonucleotides are chemically
synthesized, radiolabeled, and used to screen a DNA library, or other collections of cloned DNA fragments.

33. APPLICATIONS OF SOUTHERN BLOT CO4.1
3. Sequences that hybridize with the hybridization probe are further analysed, for example, to
obtain the full length sequence of the targeted gene.
4. Southern blotting can also be used to identify methylated sites in particular genes.
5. Particularly useful are the restriction nucleases MspI and HpaII, both of which recognize and
cleave within the same sequence.

34.  Northern blots are used to determine the identity, size, and
abundance of specific RNA sequences. Northern blot protocols begin
with RNA isolation, and separation techniques vary depending on
RNA size.
 Large RNAs are separated by electrophoresis on a formaldehyde
agarose gel or glyoxal agarose gel, which prevents normal base paring
and maintains RNA in a denatured state.
 Small RNAs are separated on a denaturing (urea) polyacrylamide gel.
NORTHERN BLOT CO4.1

35.  The RNA is then transferred from the gel to a nylon
membrane which is then incubated with a radioactively or
nonisotopically labeled RNA, DNA, or
oligodeoxynucleotide probe.
 The unhybridized probe is removed by washing with
buffer.
 Radiolabeled probes are visualized with X-ray film, and
enzymatically labeled probes are visualized with
chemiluminescence.
 The northern blot technique was developed in 1977 by
James Alwine, David Kemp, and George Stark at Stanford
University,
NORTHERN BLOT CO4.1

36.  Northern blotting allows one to observe a particular
gene's expression pattern between tissues, organs,
developmental stages, environmental stress levels,
pathogen infection, and over the course of treatment.
 The technique has been used to show overexpression
of oncogenes and downregulation of tumor-
suppressor genes in cancerous cells when compared
to 'normal' tissue, as well as the gene expression in
the rejection of transplanted organs.
APPLICATIONS OF NORTHERN BLOT CO4.1

37.  If an upregulated gene is observed by an abundance of mRNA on the northern blot the sample can then be sequenced
to determine if the gene is known to researchers or if it is a novel finding.
 The expression patterns obtained under given conditions can provide insight into the function of that gene. Since the
RNA is first separated by size, if only one probe type is used variance in the level of each band on the membrane can
provide insight into the size of the product, suggesting alternative splice products of the same gene or repetitive
sequence motifs.
 The variance in size of a gene product can also indicate deletions or errors in transcript processing. By altering the
probe target used along the known sequence it is possible to determine which region of the RNA is missing.
APPLICATIONS OF NORTHERN BLOT CO4.1

38. WESTERN BLOTING CO4.1
 Western blots are used to determine the identity, size, and abundance of specific proteins within a sample.
 The western blot protocol begins with sample lysate preparation from tissue or cell culture and separation on a
polyacrylamide gel via electrophoresis.
 The separated proteins are then transferred to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane.

39. WESTERN BLOTING CO4.1
 The membrane is incubated with a blocking agent to prevent nonspecific binding, followed by incubation with a
primary antibody to bind the protein of interest.
 There are two detection methods, direct and indirect.
 Direct detection relies on a labeled primary antibody.
 Indirect detection requires a primary antibody directed against the target protein, and a secondary antibody
directed against the immunoglobin class or subclass of the primary antibody’s species.

40. WESTERN BLOTING CO4.1
 Visualization methods include
colorimetric assays in which a
colored precipitate is produced,
chemiluminescence, and
fluorescence.

41. APPLICATIONS OF WESTERN BLOTING CO4.1
 The western blot is extensively used in biochemistry for the qualitative detection of single
proteins and protein-modifications (such as post-translational modifications).
 It is used as a general method to identify the presence of a specific single protein within a
complex mixture of proteins.
 A semi-quantitative estimation of a protein can be derived from the size and color intensity of a
protein band on the blot membrane.
 In addition, applying a dilution series of a purified protein of known concentrations can be used
to allow a more precise estimate of protein concentration.

42. APPLICATIONS OF WESTERN BLOTING CO4.1
 The western blot is routinely used for verification of protein production after cloning.
 It is also used in medical diagnostics, e.g., in the HIV test or BSE-Test.
 Also used as the definitive test for variant Creutzfeldt-Jakob Disease, a type of prion disease linked to
the consumption of contaminated beef from cattle with Bovine spongiform encephalopathy

43. APPLICATIONS OF WESTERN BLOTING CO4.1
 Another application is in the diagnosis of tularemia. An
evaluation of the western blot's ability to detect
antibodies against F. tularensis revealed that its sensitivity
is almost 100% and the specificity is 99.6%.
 Some forms of Lyme disease testing employ western
blotting. A western blot can also be used as a
confirmatory test for Hepatitis B infection and HSV-2
(Herpes Type 2) infection.