Learn ELISA and ELECTROPHORESIS

1. ELISA AND
ELECTROPHORESIS
BY PRERNA BURATHOKI

2. INTRODUCTION
 ELISA(Enzyme linked immunoSorbent assay)
is a widely used technique for detection of
antigen or antibody.
 The technique was developed in 1971 by
Peter Perlmann and Eva Engvall at
Stockholm University Sweden.
 A technique to prepare something like
immunosorbent to fix antibody and antigen
to the surface of the container was published
byWide and Jerker Porath in 1966.

3. PRINCIPLE
 Principle is based on the formation
of Ag-Ab complex , which is
detected by chromogenic
detection using enzyme
conjugated secondary antibody.
 The conjugated enzyme acts on a
specific substrate and generates a
colored reaction product.
 This product is qualitatively and
quantitatively read using ELISA
plate reader.

4. ELISA KITS
KIT FOR REAGEN KIT FOR FORSIGHT

5. TYPES OF ELISA
 1. DIRECT ELISA
 2. INDIRECT ELISA
 3. SANDWICH ELISA
 4. COMPETITIVE ELISA

6. DIRECT ELISA
 It is used for detection of
antigen in a given biological
sample
 Microtiter wells are initially
coated with antigen to be
detected which is followed by
antibody linked to an enzyme
conjugate .This follows the
addition of substrate which
produces color detected using
ELISA detector

7. INDIRECT ELISA
 It is used to detect an
antibody in a given sample.
 Microtiter wells are initially
coated with antigen specific
for antibody to be detected,
followed by the addition of
sample. Enzyme
conjugated Secondary
Antibody is added followed
by the substrate which
forms a coloured reaction
product

8. SANDWICH ELISA
 It is used for detecting an antigen
in the given sample.
 Microtiter wells are initially
coated with monoclonal
antibodies(called capture
antibody) raised against antigen
to be detected, followed by
addition of sample.Any trace of
antigen is detected by adding
primary antibody (a
MAb),followed by enzyme
conjugated secondary Ab and a
chromogenic substrate; or by
directly adding an enzyme
conjugated primaryAb.

9. COMPETITIVE ELISA
 This variation of ELISA is used to
quantitatively estimate the amount
of antigen in the given sample.
 Ag -Ab are initially incubated so
that they formAg-Ab complex.This
mixture is then added to microtiter
wells coated with synthetic
analogue of antigen to be detected,
any free antibody binds to these
antigens .This complex is
estimated by enzyme conjugated
secondary antibody by
chromogenic detection .More the
amount of antigen in the sample,
lesser is the antibody available to
bind to microtiter wells.

10. ADVANTAGE DISADVANTAGE
 Sensitive assay Equipments
are widely available.
 No radiation hazards.
 Reagents are cheap with long
shelf life.
 Qualitative and quantitative.
 ELISA can be used on most
types of biological samples,
such as plasma, serum, urine,
and cell extracts
 Only monoclonal antibodies
can be used as matched pairs
 Monoclonal antibodies can
cost more than polyclonal
antibodies
 Negative controls may
indicate positive results if
blocking solution is
ineffective [secondary
antibody or antigen
(unknown sample) can bind
to open sites in well]
 Enzyme/substrate reaction is
short term hence color must
be read as soon as possible.

11. APPLICATIONS
 Since ELISA can detect both antigen and antibody it
is a useful tool for determining serum antibody
concentrations .
 It has also found applications in the food industry in
detecting potential food allergens, such as milk,
peanuts, walnuts, almonds, and eggs.
 The other uses of ELISA include:
 a. detection of Mycobacterium antibodies in
tuberculosis
 b. detection of hepatitis B markers in serum
 c. detection of enterotoxin of E. coli in feces
 d. detection of HIV antibodies in blood samples

12. INTRODUCTION
 It is a method based on differential rate of
migration of charged species in a buffer
solution on application of dc electric field.
 Developed by Swedish chemist Arne Tiselius
for study of serum proteins in 1930, was
awarded the Noble prize in 1948.
 It is been the principal method of separation
of proteins (enzymes, hormones, antibodies)
and nucleic acids.

13. PRINCIPLE
 Any charged ion or molecule migrates when
placed in an electric field. The rate of
migration depend upon its net charge, size,
shape and the applied electric current.
v = Eq F
 where F = frictional coefficient, which
depends upon the mass and shape of the
molecule.
 E = electric field (V/ cm)
 q = the net charge on molecule
 v = velocity of the molecule.

14. FACTORS AFFECTING THE
ELECTROFORETIC MOBILITY
 1. Charge – higher the charge greater the
electrophoretic mobility.
 2. Size – bigger the molecule, greater are the
frictional and electrostatic forces exerted on it by
the medium. Consequently, larger particles have
smaller electrophoretic mobility compared to
smaller particles.
 3. Shape – rounded contours elicit lesser frictional
and electrostatic retardation compared to sharp
contours.Therefore globular protein move faster
than fibrous protein

15. Apparatus
 Buffer tank to hold the buffer
 Buffer (depends on the nature of substrate to
be seprated)
 Electrodes made up of platinum or carbon
 Power supply
 Support media

17. Frontal Electrophoresis
 In this type of electrophoresis a free electrolyte is
taken in place of supporting media.
 It is mostly of two types―the micro-
electrophoresis which is mostly used in
calculation of Zeta potentials (a colloidal
property of cells in a liquid medium) of the cells
and moving boundary electrophoresis which for
many years had been used for quantitative
analysis of complex mixtures of macromolecules,
especially proteins.
 Nowadays this type of electrophoresis has
become outdated and mostly used in non-
biological experiments.

18. MICRO ELECTROPHORESIS
 Micro electrophoresis is the best-known method
for determination of zeta potentials.
 The apparatus includes a capillary cell, two
chambers that include electrodes, and a means
of observing the motion of particles.
 The apparatus is filled with very dilute
suspension and the chambers are closed.
 A direct-current voltage is applied between
electrodes in the respective chambers.

19.  One uses a microscope to determine the
velocity of particles.
 Zeta potential values near to zero indicate
that the particles in the mixture are likely to
stick together when they collide, unless they
also are stabilized by non-electrical factors.
 Particles having a negative zeta potential are
expected to interact strongly with cationic
additives.

21. MOVING BOUNDARY ELECTROPHORESIS
 Principle
 Allows the charged particles to migrate in a
free moving solution without the supporting
media.

22. Instrumentation
 Consist of U shaped of glass cell of rectangular
cross section , with the electrodes placed at one
on the each of the limbs of the cell.
 Sample solution is introduced at the bottom or
through the side arm, and the apparatus is placed
at a constat temperature ,bath at 40 degree
celsius.
 Detection is done by measuring the refractive
index throughout the solution

23. Application
1) Used to study the behavior of the
molecules in an electric field.
2) Analysis of complex biological mixtures.

24. Zone electrophoresis
 This is the most prevalent electrophoretic
technique of these days.
 • In this type of electrophoresis the
separation process is carried out on a
stabilizing media.
 •The zone electrophoresis is of following
types;
(a) Paper electrophoresis
(b) Cellulose acetate electrophoresis
(c) Capillary electrophoresis
(d) Gel electrophoresis

25. Paper electrophoresis
 It is the form of electrophoresis that is carried
out on filter paper.This technique is useful for
separation of small charged molecules such
as amino acids and small proteins.

26. Procedure
 While carrying out paper electrophoresis, a
strip of filter paper is moistened with buffer and
ends of the strip are immersed into buffer
reservoirs containing the electrodes.
 •The samples are spotted in the centre of the
paper, high voltage is applied, and the spots
migrate according to their charges.
 • After electrophoresis, the separated
components can be detected by a variety of
staining techniques, depending upon their
chemical identity.

27. Applications
• Peptides, proteins, DNA, viruses, organelles,
bacteria or cells can be separated at
resolutions of 3-5% of their electrophoretic
mobilities and a throughput of up to 50 mg
protein or 20 million cells per hour may be
achieved.
• Highly developed modern machines may be
operated continuously or at intervals with
segmented electrolyte .

28. CELLULOSE ACETATE
ELECTROPHORESIS
 It is a modified version of paper electrophoresis
developed by Kohn in 1958.
 In this type of electrophoresis bacteriological
acetate membrane filters are taken in place of
regular chromatography paper.

29. APPLICATION
 Clinical investigation such as separation of
glycoproteins, lipoproteins and haemoglobin
from blood.

30. GEL ELECTROPHORESIS
 It is a technique used for the separation of
DNA, RNA or protein molecules according to
their size and electrical charge using an
electric current applied to a gel matrix.
 What is a gel?
> Gel is a cross linked polymer whose
composition and porosity is chosen based on
the specific weight and porosity of the target
molecules.

31. TYPES OF GEL
 Agarose gel.
 Polyacrylamide gel

32. AGAROSE GEL
 A highly purified uncharged polysaccharide
derived from agar.
 Used to separate macromolecules such as
nucleic acids, large proteins and protein
complexes.
 It is prepared by dissolving 0.5% agarose in
boiling water and allowing it to cool to 40°C.
It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds.

33. Materials
Electrophoresis
chamber
Agarose gel
Gel Casting tray
Buffers-TRIS,
boric/acetic acid,EDTA
Staining agent(dye)-
ETBR
A comb
DNA ladder
Sample to be separate

34. Procedure
Prepare agarose gel (melt,cool and add etbr mix
throughly)
Pour into casting tray with comb and allow to solidify
Add running buffer, load samples and marker
Run gel at constant voltage until band separation
occurs
View DNA on UV light box and show results

35. Running the gel
 Since the DNA has a
negative charge, it will
move toward the positive
end of the gel tank when
electricity run through
the solution.
 Smaller fragments move
faster and further than
larger fragments,
allowing for separation

37. ADVANTAGE AND DISADVANTAGE
 The advantages are that the gel is easily
poured, and does not denature the samples.
The samples can also be recovered.
 The disadvantages are that gels can melt
during electrophoresis, the buffer can
become exhausted, and different forms of
genetic material may run in unpredictable
forms

38. POLYACRYLAMIDE GEL
 Commonly used components: Acrylamide
monomers, Ammonium persulphate,
Tetramethylenediamine (TEMED), N,N’-
methylenebisacrylamide.
 These free radicals activate acrylamide
monomers inducing them to react with other
acrylamide monomers forming long chains.
 Used to separate most proteins and small
oligonucleotides because of the presence of
small pores.

40. SDS PAGE ELECTROPHORESIS
 Sodium Dodecyl Sulphate (SDS)
polyacrylamide gel electrophoresis is
mostly used to separate proteins
accordingly by size.
 This is one of the most powerful
techniques to separate proteins on the
basis of their molecular weight.

41. PRINCIPLE
 This technique uses anionic detergent Sodium
Dodecyl Sulfate (SDS) which dissociates
proteins into their individual polypeptide
subunits and gives a uniform negative charge
along each denatured polypeptide.
 • SDS also performs another important task. It
forces polypeptides to extend their
conformations to achieve similar charge: mass
ratio.
 •The rate of movement is influenced by the
gel’s pore size and the strength of electric field.

42.  In SDS- PAGE the vertical gel apparatus is
mostly used.
 • Although it is used to separate proteins on a
routine basis, SDS-PAGE can also be used to
separate DNA and RNA molecules

43. PROCEDURE
 Protein sample is first boiled for 5 mins in a
buffer solution containing SDS and β-
mercaptoethanol.
 Protein gets denatured and opens up into
rod-shaped structure.
 Sample buffer contains bromophenol blue
which is used as a tracking dye, and sucrose
or glycerol.
 Before the sample is loaded into the main
separating gel a stacking gel is poured on top
of the separating gel.

44.  Current is switched on.
 The negatively charged protein-SDS
complexes now continue to move towards
the anode.
 As they pass through the separating gel, the
proteins separate, owing to the molecular
sieving properties of the gel.
 When the dye reaches the bottom of the gel,
the current is turned off.
 Gel is removed from between the glass plates
and shaken in an appropriate stain solution.
 Blue colored bands are observed under UV
rays

46. APPLICATIONS
 1. Establishing protein size
 2. Protein identification
 3. Determining sample purity
 4. Identifying disulfide bonds
 5. Quantifying proteins
 6. Blotting applications