2. RIA involves competition between radiolabelled and
unlabelled antigen test sample for the limited
binding sites on specific antibody molecules.
After the reaction reaches equilibrium, the free and
bound fractions of labelled antigen area separated
and radioactivity associated with each fraction is
counted using a gamma counter.
Ab +Ag*+Ag ↔Ab-Ag* +Ab-Ag

3.  A antigenic molecule is first coupled with a radio
labelled isotope to form a radio radioactive hapten.
 The labelled antigen is then mixed with a known volume
of specific antibody, due to which they chemically bind
with each other.
 A small amount of standard blood sample containing a
known quantity of same antigen is added and incubated.
 Since labelled and unlabelled are identical they compete
with each other for antibody binding sites, this resilts in
displacement of some labelled antibodies.

4.  Upon further addition of unlabelled antigen to reaction mixture,
the bound fraction of labelled antigen decreases
due to competitive binding to antibody molecule.
 The bound antigen is separated from free antigen by addition
of second antibody which precipitates antigen-antibody
complexes.
 The radio labelled antigens are collectd in the supernatant
and non labelled antigens as precipitate.
 The radioactivity is measured by gammacounter.
 From these data, a standard binding curve, like this one shown
in red can be drawn.

6. Gamma Counter

7.  The samples to be
assayed (the unknowns)
are run in parallel.
 After determining the
ratio of bound to free
antigen in each unknown,
the antigen
concentrations can be
read directly from the
standard curve.

8. Types of RIA:
LIQUID PHASE RIA:
It includes a) Direct RIA
b)Indirect RIA
c)Competitive Inhibition RIA
SOLID PHASE RIA:
It includes a) Direct RIA
b)Indirect RIA

9.  A)Direct RIA
 In this method antigen to be determined is
prepared in different dilutions and is incubated
with a radiolabelled antibody
 Bound one’s are separated by centrifugation
 End concentration of the antigen specimen
is the dilution which precipitates 50% of the
labelled antibody.

10.  In this method neither of the primary
reactants(antigen and antibody) are radiolabelled.
 Species antiglobulin specific to primary antibody is
radiolabelled, it reacts with antigen antibody
complexes.
 The primary reaction is indirectly
measured by secondary immunological reaction.
 The unbound radiolabelled indicator is removed
by rinsing.

11.  Sample containing antigen is pre-incubated with
specific antibody.
 During this time complexes formed is directly
proportional to concentration of antigen in sample.
 Labelled antigen is then added and re-incubated,
it occupies sites not occupied by unlabelled one.
 The amount of labelled antigen bound will be
inversely proportional to amount of antigen in the
sample.

12.  In this method specimen is attached to an inert
insoluble material and the immunological reactions
takes place on solid surface.
 Solid supports are various polymers mostly
polystyrene is utilised in form of beads, plastic tubes
and small discs, these are activated by heat
treatment to produce protein binding.
 It includes
A)Direct RIA
B)Indirect RIA

13.  In this method antigen is attached to the solid
phase which is then incubated with radiolabelled
antibody.
 After complex formation quantification is
done by Gamma counter.

14.  In this method neither of the primary reactants are
radiolabelled.
 Species antiglobulin specific to primary antibody is
radiolabelled,it reacts with antigen-antibody
complexes.
 The primary reaction is indirectly
measured by secondary immunological reaction.
 The unbound radiolabelled indicator is removed
by rinsing.

15.  RIA is used in the assay of drugs like
amphetamine, barbiturate, digitoxin, morphine.
 In analysis of vitamins like riboflavin, folicacid.
 In analysis of homones like aldosterone, insulin,
growth hormone, thyroxine.
 To analyse anti-DNA antibodies in Systemic
Lupus Erythematosus.

16.  Pregnancy diagnosis in dairy cows using milk
progesterone.
 Plotting total counts over bound vs. ligand
concentration, helps in establishing
optimum assay conditions.
 Oncology
◦ Carcino embryonic Antigen
◦ Early Cancer Detection and Diagnosis

18.  DEFINITION:
The term Polarimetry may be defined the study of the
rotation of polarised light by optically active substance.
 PRINCIPLE :
It is a non-destructive technique involving measurement of
angle of rotation of linearly polarised light. This rotation is
caused by asymmetric chemical structure of the compound
through which light is passed and angle is a linear function
of concentration.

19. POLARISATION OF LIGHTPOLARISATION OF LIGHT
OrdinaryOrdinary
(nonpolarized)(nonpolarized)
light consists oflight consists of
many beamsmany beams
vibrating invibrating in
different planesdifferent planes
Plane-polarizedPlane-polarized
light consists oflight consists of
only those beamsonly those beams
that vibrate in thethat vibrate in the
same planesame plane

20. Homogenous liquids and gases transmit radiation at equal velocities in
all directions. Such substances are called ISTROPIC SUBSTANCES.
EX: solids that crystallise in cubic form and Glass.
Substances that transmit light in different directions with
different velocities are called ANISOTROPIC SUBSTANCES.
This is due to non distribution of atoms in crystal.
EX: Non-cubic crystals
When light is passed through an anisotropic crystal, it is divided into two
rays viz. Ordinary ray and extraordinary ray. Ordinary ray travels with
in all Same velocity in all directions where as extraordinary ray travels
at a faster rate or slower rate.
In Calcite extraordinary travels at a faster rate than ordinary ray.

22.  Calcite crystal is cut into a suitable shape in which one of the ray
is totally reflected internally while other is plane polarised, such
calcite crystal permitting passage of polarised light in a particular
plane is called NICOLPRISM.
 If a beam of light ordinary is made to pass through a nicol prism,it
is called polariser, if second nicol prism is placed in path of beam
with its optic axis parallel to first one, light remains visible.
 If second prism is rotated at right angles field of vision will be
dark since no light will pass through it, since vibrations of plane
polarised light is perpendicular to optic axis of analyser.


24. If a solution is placed in between two prisms plane of polarisation
is rotated, such substances are said to be optically active.
Substances which rotate light towards right are called
DEXTRO ROTATORY(+).
Substances which rotate light towards left are called
LEAVO ROTATORY(-).
If a molecule posses both plane of symmetry as well as
centre of symmetry it is said to be optically inactive.

27. If a molecule of a substance possess no plane of symmetry
and no centre of symmetry it is said to be optically active.
such a compound can be resolved into two optical isomers.
If two optical isomers are mirror images of each other
then they are called ENANTIOMORPHS, they have same
physical and chemical properties.
Isomers which are not mirror images of each other are
called DIASTEREOMERS, they have different physical
and chemical properties.
If a substance has right and left handed enantiomers it
is called RACEMICMIXTURE, it has no optical activity.

29. The Rotatory power of a substance is given by
SPECIFIC ROTATION, it is defined as number of degrees
of rotation of plane polarised light produced by a solution
in sample tube of one decimeter in length having one gram
substance per 100ml of the solution.
[ ]
mL100
g
c
decimetersinpathlength
lc
α10
α
2
=
=
=
l
t

30. MAGNITUDE OF ROTATION DEPENDS ON :
a)Nature of Substance
b)Nature of solvent
c)Concentration of solution
d)Temperature
e)Presence of other optical substances in traces

31. PARTS OF POLARIMETER
ARE:
1. Light source
2. Polariser and Analyser
3. Sample tube
4. Detector

33. o The sodium vapour lamp and mercury vapour
lamp are used as sources.
o Sodium vapour lamp which emits radiation at
5890A0
and 5896A0
is considered for source.
o Mercury vapour lamp which emits radiation
at 4368A0
, 4916A0
,
5461A0
, 5776A0
is considered for source.
o Each line is isolated by proper filters.

34.  Polariser is used for producing polarised light.
 Analyser measures the amountof rotation.
 Glan-Thomson prism and Nicol prism are generally used.

35. SAMPLE TUBE:
Sample in polarimeter is taken in sample
tubewhich are cylindrical in shape and it is
5-25cms inlength.
For accurate measurements sample tube is
surrounded by jacket for constant
temperature.
DETECTOR:
Eye serves as detector

36. A tube containing optically active liquid in path of light between
two nicol prisms.
Field will appear uniform bright since the optically active substance
has certain angle.
 The analyser is rotated till the field becomes one of uniform
illumination as before. Reading is noted.
The difference between analyser and polariser readings gives angle
of rotation.

37.  QUALITATIVE ANALYSIS
Polarimetry is used in identification of aminoacids, steroids,
alkaloids.
 QUANTITATIVE ANALYSIS
If specific rotation of sample is known its concentration can
be determined.
 Polarimetry is used in assay of Anticoagulent Citrate dextrose
injection, Iron Dextran injection.

38.  SACCHARIMETRY
It is an practical application of Polarimetry, in which solutions
containing high concentration of sugars can be determined.
In this method quartz wedge having same roatary dispersion as
that of sucrose is taken. Wedge is adjusted such that it
compenstes rotation, Quartz wedge compensation is
calibrated in sugar degrees.

39. The measurement of optical rotation against time is called Rotography.
In this method substrates are added to sample tube and then
Enzyme is added and change in rotation with time is recorded,
Slope of the curve in degrees per minute is directly proportional to
concentration of enzyme.

40.  Instrumental methods of
Chemical Analysis
by B.K.SHARMA
 Principles of Biochemistry
by KEITH AND WILSON