2. Introduction
Word derived from Greek word, Nephele
means’ cloud’ + Meter
Definition :
A. In analytical chemistry The measurement of
the concentration of
a solution, suspension or dispersion based
upon its light-scattering properties.
In immunology A technique used to determine
the levels of antibodies or antigens in a
suspension, based on its light-scattering
properties
3. Introduction
It is performed by measuring the turbidity in a
water sample by passing light through the
sample being measured.
In nephelometry the measurement is made by
measuring the light passed through a sample
at an angle
It is based on the principle that a dilute
suspension of small particles will scatter light
(usually a laser) passed through it rather than
simply absorbing it.
The amount of scatter is determined by
collecting the light at an angle (usually at 30
and 90 degrees)
4. Introduction
.Antibody and the antigen are mixed in
concentrations such that only small
aggregates are formed that do not quickly
settle to the bottom
. The amount of light scatter is measured
and compared to the amount of scatter
from known mixtures.
The amount of the unknown is determined
from a standard curve.Nephelometry can
be used to detect either antigen or antibody
5. Introduction
Purpose :
Nephelometry is a technique used
in immunology to determine the levels of
several blood plasma proteins.
For example the total levels of antibodies or classes
It is important in quantification of free light
chains in diseases such as multiple myeloma.
Quantification is important for disease classification
and for disease monitoring
once a patient has been treated (increased skewing
of the ratio between kappa and lambda light
chains after a patient has been treated is an
indication of disease recurrence).
6. Types
In immunology two types of test run
End point nephelometry
tests are run by allowing the
antibody/antigen reaction to run through to
completion (until all of the present reagent
antibodies and the present patient sample
antigens that can aggregate have done so
and no more complexes can form).
the large particles will fall out of the solution
and cause a false scatter reading, thus
kinetic nephelometry was devised.
7. Types
kinetic (rate) nephelometry
In kinetic nephelometry, the rate of scatter is
measured right after the reagent is added.
As long as the reagent is constant the rate
of change can be seen as directly related to
the amount of antigen present
8. Principle
The principle of nephelometry is based on
the scattering or absorption of light by solid
or colloidal particles suspended in solution.
When light is passed through the
suspension, part of incident radiant energy
is dissipated by absorption, reflection, and
reaction while remainder is transmitted.
In nephelometry light is passed through
the sample solution (suspended particles)
directly
the amount of scattered radiation is
measured generally at 90°C.
9. principle
The measurement of intensity of scattered light as
a function of concentration of dispersed phase is
the basis of analysis of nephelometry.
It is very important to note that in nephelometry
incident and scattered light are of same
wavelength whereas in fluorimeter (in fluorimetry)
scattered light is of longer wavelength than
incident light.
10.
11. instrumentation
1- light source:
Tungsten its relatively low intensity makes
it less useful for samples with low light
scattering. Alternatives are:
quartz halogen lamp, xenon lamp and
laser which have higher intensities than
tungsten lamp.
2-lens assembly:
Light enter the sample holder through lens
assembly.
12. Instrumentation
3-there is provision for the insertion of
filter between the sample and source
of light(monochromator).
4- detector (photo –cell):
It is shielded to minimize interference
from stray light.
5-Read out device:
Light intensity is converted to an
electrical signal by the detector .
13. Factors affecting the scatting
property
1. Particle size & shape
2. Wavelength dependence
3. Distance of observation (Set-up)
4. Polarization of incident light
5. Concentration of particles
6. Molecular weight of particles
14. Measurement procedure
1-plug the instrument into ground outlet.
2- choose desirable scale from 0-10
starting with the highest conc. (for std
1=scale 10)
3- turn power switch on.
4-selecte desirable range by range
selector at desirable position .
5- select filter required.
15. Measurement
6- transfer your standards in the cleaned
cell and place them in cell holder.
7- remove the standards.
8- fill the second cell with blank to set zero
.
9- check the reading of the standards
again.
10- measure your unknown.
11- draw calibration curve.
16. Precautions
-Number and size of the particles
should remain constant if repeated
preparation are made
-clean cell & filter
- avoid air bubbles (high reading).
-dilute sample if there is need.
-prepare the blank ,standards, Sample
at the same time.(to avoid ppt)
17. Clinical applications
Widely used to determine concentrations
of unknowns where there is antigen-
antibody reactions such as
Determination of immunoglobulin's (total,
IgG, IgE, IgM, IgA) in serum and other
biological fluids
Determination of the concentrations of
individual serum proteins; hemoglobin,
haptoglobin, transferring, c-reactive
protein, !1-antitrypsin, albumin (using
antibodies specific for each protein)
Determination of the size and number of
particles
18. Advantage & disadvantage
Advantage
Very rapid procedure
Simplicity in measurement
High accurate
Disadvantage
High cost
Easily damaged
Require high power supply
Used for lower concentration
