The document discusses enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). ELISA involves coating an antigen or antibody on a solid surface, detecting it with a specific antibody or antigen linked to an enzyme. The enzyme then catalyzes a reaction producing a detectable color change. ELISA can be used to detect antibodies or antigens in samples. RIA uses a radioactive label instead of an enzyme. Both techniques are quantitative and can detect small amounts of target molecules in samples.

1. Guided by:
Dr.S.B.Bhanja
M.Pharm,Ph.D

2.  Dispersion system is defined as a
heterogenous two phase system in which
internal (dispersed,discontinous ) phase is
distributed or dispersed within the
continuous (external) phase or vehicle.
 The internal and external phase may be
solids, liquids and also gases.
Eg.suspensions,emulsions,aerosols

3. Continuous Phase
Disperse Phase
Solid Liquid Gas
Solid Glasses containing
finely dispersed
metals, e.g., ruby
glass containing
gold, pastes such
as toothpaste
Gels, suspensions
(Kaolin)
Smoke, dust
Liquid Solid emulsion
(mineral oil in wax),
Cold cream
Emulsions such as
milk, mayonnaise,
oil in water
Fog, mist, liquid
aerosol, throat and
nasal relief sprays
Gas Solid foam
(foamed plastic)
Foams
(carbonated soft
drinks)
none

4. A. Mixers
B. High speed dispersers
C. Rotor stator mixers
D. Combination mixers
E. In- line mixers
F. Non- mechanical disperse processing
G. Fine suspension and size reduction
equipment

5.  Propeller mixers
 Turbine mixers
 Anchor mixers
 Scraped surface agitators
 Counter rotation

6.  The most often used mixing implement is marine
propeller mixer.
 These machines use rounded,pitched,three blade
design that produces mostly axial flow
 They provide good flow and blending capabilities
in small batches of low to medium viscosities
 Propellers mixers can be installed on vertical
centerline or through the side wall of process vessel
 They can be operated at around 300-400rpm
 These are used mostly for liquid-liquid blending
applications in some easily producible suspensions.

7. Marine Propeller mixer
Clamp mounted
portable mixer

8.  The most versatile of all mixers in the entire span of
mixing equipments are the axial and radial flow
turbines
 Turbines mixers can be made to handle huge
batches,evenupto 5,00,00gal & suitable for
emulsification process.
Radial Flow turbine Axial flow turbine

9.  It is low speed and low
capability
 The anchor agitator is a slow
(50rpm) device whose sole
function is to rotate the
contents of a batch in a radial
direction without providing any
significant shear
 They are typically designed to
be able to withstand a
maximum viscosity beyond
which they might actually blend
or break.

10.  A flexible or movable
blades are attached to
the anchor for the
purpose of actually
scrapping the side
walls,they are known as
scraped surface
agitators
 These are definitely
required in
emulsification
equipment where heat
transfers are necessary

11.  It consists of anchor agitator
and counterrotating set of
cross bars provides excellent
blending
 These works well on
materials with viscosities
from 5000-25,000cps
 By installing a stationary
baffle,some mixing
capabilities are improved

12. It is also called as saw blade disperser  This machine consists of a variable speed
shaft connected to an impeller with a
serrated edge  The tip speed is set around 4000 ft/min  The diameter of impeller should be 1/3 of
diameter of vessel  The impeller should be located one impeller
diameter off the bottom of vessel  It can deagglomerating particles when the
viscosities between 10,000 to 20,000cps.
Application:
It is used for pigment dispersion,dye stuffs,
carbon dispersion and paints

13. Limitations:
 Air incorporation is
another problem so
it is best used for
suspensions and not
for emulsions

14.  This mixing machine uses an impeller that is
installed at a close tolerance to a stationary
housing,which baffles and restrict the flow
caused by the rotation of the impeller in the
liquid.
 Particles caught between rotor & stator are
crushed and separated by mechanical
action of the impeller.
 They are three types:
1. Radial flow with stator
2. Rotating stator
3. Axial flow rotor/stator mixers

15. RADIAL FLOW WITH
STATOR
ROTATING STATOR AXIAL FLOW ROTOR/STATOR
MIXER

16.  Better control of temperature and pressures
 Eliminates line loses caused by pumping from
tank to tank
 Saves floor space
 Simplifies the process, save labor costs
Types : Anchor plus rotor/stator
Anchor plus disperser
Counter rotating with coaxial
rotor/stator

17.  It is combination of simple
anchor agitator with a
rotor/stator mixer
 It provides a high shear rates
for dispersion and
emulsification
 It is beneficial if there is
requirement to pull down
powders from the top
Limitation:
 It is difficult to design the
anchor such that it allows the
placement of the high shear
mixer close to the bottom of

18.  It is combination
of high speed
disperser with an
anchor agitator
 It works well on
medium to high
viscosity
suspensions

19.  This design is known as triple action mixer,combines
the high viscosity mixing capabilities of
counterrotating axial flow cross bars with a
standard anchor type scraped surface agitator
and also with a high speed rotor/stator mixer
 It is capable of generating fine dispersions and fine
droplets of the internal phase for stable emulsions
 It can operated at viscosity of 50,000cps
 They are always jacketed for heating and cooling
because it is necessary in case of preparation of
creams and ointments

20. Advantages:
 Handles wide range of
viscosities from water
thin to as high as
1million cps
 Disperse and emulsifies
very efficiently
 Provides shortest mixing
time
Disadvantages:
 Difficult to clean due to
complicated design

21. 1.Rotor/stator mixer disperser emulsifier
2.Colloidal mill
3.Piston homogenizer
4.Ultrasonic vibrating homogenizer
5.Micro fluidizer
6.Low pressure cyclone emulsifier
7.Homogenizer/Extruders
8.Static mixer

22.  These mixers acting as submerged pumps
design can be made that places the
rotor/stator in a pump housing and allows
for product to be pumped through itself.
 The product inside the rotor/stator mixing
pump,the droplets and particles subjected
to a wide variety of high shear rates.
 It is designed with fine tolerance rotor/stator
gaps that promotes high shear rates and
high amount of shear per pass through.

23. Stage 1
Stage 2
Stage 3

24.  It is used to disperse the solids into liquids
and to emulsify liquid-liquid systems.
 These generally used as polishing machines
for emulsions or suspensions because they
produce fine particle or droplet size
product to enhance a products stability.
 The rotor/stator gap is generally set
between 0.030 & 0.001 inch.
 They are operated at speed of 3600 rpm
 The rotor diameter is 10-30cm provides flow
rates in the area of 4000-6000L/hr
depending upon the viscosity.

26. Advantages :
 Shear rates high than in rotor/stator
machines
 Colloidal mill produces emulsions and
suspensions with particle size distribution
smaller than the particle sizes obtainable
using fixed gap rotor/stator mixers.
Limitations :
 Flow rates lower than in rotor/stator
machines
 Not used for abrasive products

27.  It is the most powerfull device for producing
emulsions and suspensions
 It uses high power positive displacement
piston type pump to produce pressure of
3000-10,000 psig and then force the
premixed product through a specially
designed restricting wall where a extremely
high shear forces are exerted
 Here turbulence and high shear are the
major parameters in size reduction

28.  It having continuous
Capabilities of 2500L/hr at
15hp to 50,000L/hr at
150hp
Limitations:
 They cannot handle the
product feed above
200cps
 High maintanence cost
and down time
 Lack product homogenity
and batch to batch
variability

29.  Has similarity with high pressure
homogenizer effective device for
dispersing and emulsifying
 It uses a positive displacement pump to
force the premixed liquid through an
elliptical opening at a speed of
100m/sec
 This high speed flow impinges on to the
edge of blade shaped obstacle called a
vibrating knife.

30.  Capacities and
pressures of systems
range from laboratory
units, producing 4-
10L/min at 1200-1700
psig requiring 2-5hp,to
full scale productions
units with capacities of
upto 450L/min at
350psig requiring 60hp.

32.  This device uses a high pressure positive
displacement pump operating at a pressure of
500-20,000psig which accelerate the process
flow upto 500m/min through the interaction
chamber
 The interaction chamber consists of small
channels known as micro channels having
diameter narrow as 50μm and cause the flow
of product to occur as very thin sheets.
 The configuration of micro channels within the
interaction chamber ressembles Y-shaped flow
streams in which the process stream divides
into these micro channels,creating two
separate micro streams

33.  The micro streams are then brought
together in an impingement area through
which all of the product must flow
 At the impingement area the collision of
the two high speed flow streams in a very
tight spot creates various droplet size
reduction and different mixing mechanisms
such a cavitation,implosion,shears and
turbulence
 The micro fluidizer technology satisfy the
requirements for producing finest emulsions
known as micro emulsions

35.  It is used for formation of emulsions and
suspensions
 the shear arises from the difference in the
velocity of the fluid,as the fluid travels in a spiral
towards the center
 They operates in the 200psig and capacities of
7.5-225L/min.
 The recommended viscosity limit is 1-2000cps.
 They are capable of producing emulsions in
the
2-10μm range.

36. Working :
 Product enters through
tangential entry port
 Product is forced to
circulate in concentric
layers towards the
center and ends of
the chamber
 Shear arises from the
difference in the
velocity of fluid,as the
fluid travels in a spiral
towards the center
Cross section of flow path
through the interaction
chamber

37.  It is a high pressure homogenizer with an
adjustable valve having production
capacities from 8mL/hr to 12000mL/hr
 A positive displacement pump produces
pressure up to 30,000psig.
 The apparatus capable of producing
fine emulsions and liposomal dispersions

38.  A true low shear and low energy
requirement device for emulsifying
immiscible liquid mixture is the static
mixture.
 It is also called as pipe line mixture,this
device is actually a series of specially
designed baffles in a cylindrical pipe.

39.  These simple devices are extensively used
for the preparation of unstable emulsions
for liquid-liquid extraction purposes
 Size distributions obtainable range from 100-
1000μm

40. Critical fluids liposome process:
 Near critical or super critical fluid solvents
with or without polar co-solvents for the
formation of uniform and stable liposomes
having high encapsulation efficiences.
 Super critical fluids can be uniquely used to
encapsulate very hydrophobic molecules
 Super critical fluids are gases such as
carbon dioxide & propane

41.  When these fluids compressed at
conditions above their critical
temperature and pressure,these
substances become fluids and ability to
dissolve other materials.
 The gaseous characteristics increases
mass transfer rates,thereby significantly
reducing processing time.
 Small added amounts of visible polar co-solvents
such as alcohol can be used to
adjust polarity and to maximize the
selectivity and capacity of the solvents

43. Types:
1. Triple roll mill
2. Ball mill
3. Agitated bead mill

44. Triple roll mill:
• Disperse small tightly bound
agglomerates and hard discrete particles
 Particles are subjected to
High shear
Mechanical crushing
Smearing

45.  It is used for size reduction fine solid
discrete particles or for
deagglomeration of very tightly bound
agglomerates.
 The machine consists of cylindrical drum
into which a charge of heavy spherical
balls usually metal or ceramic is loaded
along with the components of the
dispersion.

46. Ball mill
Limitations
Typically time consuming process,milling time
are often measured in days.

47.  A Modernized improvement of the ball mill is the
agitated bead mill
 The bead mill uses a charge of inert small balls to
2-8mm diameter
 Media mill- if beads are ceramic
 Shot mill- if beads are steel
 Sand mill- if grains of sand are used
 The Design consist of a cylinder which can be
either vertical or horizontal,that has a high speed
agitator,which is capable of fluidizing the charge
of beads,causing them to collide at very high
speed.

48.  The premix is pumped through the
housing.There is a high probability that
each particle must be repeatedly
subjected to the high stresses that result
when the beads collide with each other or
the high speed impeller
 Agitated bead mill is used in pigment
dispersion industry due to its fine solids
grinding and dispersing capabilities.It is not
often used in pharmaceutical industry
except when particle size requirements falls
below 10μm.

49.  Pharmaceutical dosage forms,disperse
systems volume 3 by
Lackman,Lieberman,Marcel
Dekker,NY.pg no:291-362
 Pharmaceutical engineering(principles &
practices) by C.V.S.
Subrahmanyam.pg.no:155,161,229.

51. ENZYME LINKED IMMUNO SORBENT
ASSAY(ELISA)&
RADIO IMMUNOASSAY (RIA)
P.SAHITHI REDDY
M.PHARMACY
H.T.NO:256213886024
Under the guidance of
Mr Utham prasad

52. Contents
ELISA
OVERVIEW PRINCIPLE PROTOCOL DIFFERENT TYPES MATERIAL REQUIRED MERITS&DEMERITS APPLICATIONS

53. OVERVIEW:
Enzyme Linked Immuno sorbent
Assay (ELISA)
Term Was Coined By Engvall and
Pearlmann in 1971
is a quantitative technique used in
immunology to detect the presence of
an antibody or an antigen in sample

54. • Antigen of interest is absorbed on to
surface sorbent
• Antigen is recognised only by the specific
antibody immuno
• This antibody is recognised by second
antibody (immuno) which has enzyme
attached (enzyme linked)
• Substrate reacts with enzyme to produce
product usually depicted by coloured

55. PRINCIPLE:
 ELISA is based on specific interaction between Ag and
their corresponding Ab. One of the immuno reactant is
coated to a solid phase support such as polycarbonate,
polyvinyl polyacrylamide tubes or microwells.  To the respective antibody or antigen is added  To antigen-antibody complex so formed an enzyme
linked antibody is added. A colourless substance is
added to well. This substance is a substrate for the
enzyme which catalyses the conversion of colourless
substance to coloured product.

56. MATERIALS REQUIRED
• Test sample
• Antibody /Antigen
• Polystyrene microtiter plates
• Blocking buffer
• Washing buffer
• Substrate
• Enzyme

57. Enzymes Used in ELISA
 Horseradish peroxidase(very
commonly used )  Alkaline phosphatase  Beta –galactosidase  Lacto peroxidase  Tetra Methyl Benzidine (substrate)

59. The enzyme substrate should be colourless
After degradation by the enzyme it should be strongly coloured
fluorescent

60. TYPES:
a)Indirect ELISA
b)Sandwich ELISA
c) Competitive ELISA

62. Sandwich ELISA direct method
 2 Antibodies Required
 Must Recognize Different Epitopes
 1st Antibody Is Referred To As Capture Ab
 2nd Antibody Detection Ab
 2nd Antibody Is Biotinylated
 Enzymes Commonly Used: HRP (Horse Radish
Peroxidase) And AKP (Alkaline Phosphatase)
 Substrate is TMB (Chromogen)

63. The ELISA plate is coated with Antibody to detect
specific antigen

64. Protocol for sandwich
ELISA
Prepare a surface to which a known quantity of capture
antibody is bound Block any non specific biding sites on the surface Apply the antigen – containing sample to the plate Wash the plate ,so that unbound antigen is removed Apply enzyme linked primary antibodies as detection
antibodies which also bind specifically to the antigen

65. Wash the plate, so that the unbound antibody-enzyme
conjugates are removed Apply a chemical which is converted by the enzyme
into a coloured product Measure the absorbency of the plate wells to
determine the presence and quantity of antigen

67. Indirect ELISA:
• The protein antigen to be tested for is added to each
well of ELISA plate, where it is given time to adhere
to plastic through charge interactions
• A solution of non-reacting protein is added to block
any plastic surface in the well that remains uncoated
by the protein antigen
• Then the serum is added, which contains a mixture of
the serum antibodies, of unknown concentration,
some of which may bind specifically to the test
antigen that is coating the well.

68. Conti….
• Afterwards, a secondary antibody is added, which will bind to
the antibody bound to the test antigen in the well. This
secondary antibody often has an enzyme attached to it
• A substrate for this enzyme is then added. Often, this
substrate changes colour upon reaction with the enzyme. The
colour change shows that secondary antibody has bound to
primary antibody, which strongly implies that the donor has
had an immune reaction to the test antigen.
• The higher the concentration of the primary antibody that was
present in the serum, the stronger the colour change. Often a
spectrometer is used to give quantitative values for colour
strength
•

70. COMPETIVE ELISA
• The labelled antigen competes for primary antibody
binding sites with the sample antigen (unlabelled).
The more antigen in the sample, the less labelled
antigen is retained in the well and the weaker the
signal).

73. RESULTS
 After reading the results the standard curve is drawn were the
concentration is plotted on the X-axis and the absorbance on
the Y-axis.

74. Advantages
• Reagents are relatively cheap& have a long shelf life
• Easy to perform and quick procedures
• No radiation hazards during handling
• Has wide usage to variety of infections

76. Limitations
 To carry out the test antibody must be
available  Concentration may be unclear  Possibility of False positive  Possibility of False negative

77. Applications
The various sections devoted to the uses are
 Detection of mycobacterium antibodies in tuberculosis  Detection of rotavirus in faeces  Detection of hepatitis B markers in serum  Detection of HIV antibodies in blood sample  Hormones  Proteins  Drug markers  Serum proteins  Vaccine quality control  Tumour markers

79. CONTENTS
RADIO IMMUNOASSAY (RIA)
OVERVIEW PRINCIPLE MATERIAL REQUIRED PROTOCOL INSTRUMENTAION MERITS&DEMERITS APPLICATIONS

80. Overview:
 Radio immuno assay was developed by  Berson&yalow(1956) for the quantitative
measurement of insulin in human plasma  RIA principles have found wide application in the
field of drug analysis kinetic studies, immuno
diagnosis  RIA is specific, sensitive &rapid

81. An immunoassay is a test that uses antibody and antigen
complex as means as a mean of generating a measureable
result
An antibody :antigen complex is known as a immune
complex.
Immunoassays are different from other types of laboratory
tests, such as colorimetric tests, because they use antibody:
antigen complexes to generate a signal that can be measured .
In contrast, most routine clinical chemistry tests utilize
chemical reaction between the reagent and sample to generate
a test result

82. Principle
RIA is a competitive binding assay.
The antibody & labelled antigen are always present as
limiting factors and the concentrations of unlabelled
antigens(sample) under examination is increased
continually
Uses an immune reaction
(Antigen –antibody reaction ) to estimate ligand

83. Unbound Ag* and Ag washed
Radioactivity of bound residue measured
Ligand conc is inversely related to radioactivity
[Ag: ligand to be measured ;Ag* radiolabelled ligand ]

85. Materials required
a) Preparation &characterisation of Antigen [Ligand to be
analysed ]
b) Radiolabelling of the Antigen
c) Preparation of the specific antibody
d) Development of Assay system

86. PREPARATION AND RADIOLABELLING
OF THE ANTIGEN
Antigens prepared by
 synthesis of the molecule  isolation from natural sources
Radiolabelling [Tagging procedure ]
 3H,14C,125I are used as radioactive tags  Antigens are tagged to 3H14c125I  tagging should NOT affect antigenic specificity and activity

87. PREAPRATION OF SPECIFIC
ANTIBODY
Antigen injected intradermal into rabbit
antibody production Antibodies recovered from the serum Some ligand are not antigenic
hormones ,steroids drugs HAPTENS
E.g.: castrin morphine

88. ASSAY Procedure
Add known amounts of the test sample+ labelled
antigen into the microtitre wells
 incubate – allow the reaction to reach completion Decant and wash the contents of the well-removes
all unbound antigens Radioactivity remaining in the mocrotitre wells
measured by a counter [GM counter, scintillation
counter]

89. Conti…
Intensity of radioactivity is inversely correlated
with the conc of the antigens in the test sample

90. INSTRUMENTATION:
CENTRIFUGE:
 swing bucket rotor : 1200-2500 rpm  Fixed angle head rotor :3500-4000 rpm
RADIOACTIVE counter
 gamma counter : which is used for a gamma energy
emitting isotopes. E.g. 125I. Scintillation counter : it is used for beta energy emitting
isotopes.eg. Tritium 3H and 14C isotopes

92. The results obtained by plotting a graph
antibody bound to labelled antigen Unlabelled antigen

93. Merits and demerits
Merits :
highly specific : immune reactions are specific
high sensitivity : immune reaction are sensitive
Demerits:
radiation hazards :uses radiolabelled reagents
Requires specially trained personnel
Labs require special license to handle radioactive
material
Requires special arrangements for :requisition,
storage of radioactive material
radioactive waste disposal.

94. APPLICTIONS :
Used in the estimation of pharmaceutical
drugs like Morphine Clonazepam Barbiturates Neobentine Flurazepam
And others Insulin Gastrin Glucogon Growth hormones

95. Limitations :
 The limitations of the RIA are  Its expensive  Being hazardous Handling the radioactive material  The radio isotopes used 125I or 131I emit gamma
radiation these requires a special counting machine

96. References :
A textbook of pharmaceutical analysis
Biotechnology and its applications in pharmacy