Nephlometry

1. NEPHLOMETRTRY
Praveen Kumar
MSC MLT clinical biochemistry

2. NEPHLOMETRTRY
• Its is a directed method of measuring
light, scattered by the particles
suspended in the solution
• Quantitation of specific protein by
nephlometery is accurate, precise and
fast.
• And easy to do
• Fully automated

3. • Good specificity
• Good sensitivity
• Most commonly used for measuring the
plasma proteins
– IgG
– Complement proteins
– Acute phase proteins
• Ceruloplasmin
• Haptoglobulin
– Etc.

4. Princple
• When a beam of light is focused on
the solution the particles present in
the solutions results in scattering of
the incident light
• Scattered light is not visible to naked
eye
• Sensitive detector arranged at an
angels will detect the scattered light
and measures directly

5. Ag excessequivalence
Ab excess
Kinetics of fluid phase
precipitation.
• Interaction of Ag and Ab in a solution
depends on many factors, in that the
important factor is their relative
concentration
Ab/Ag
pt
Antigen concentration

6. • The light is scattered more either in
Ab excess or Ag excess zone.
• Often used to quantitate Ag
concentration in the presence of
excess Ab so that the amount of
lattice aggregates and light scattering
or proportional to the Ag
concentration

7. • Ag and Ab concentration is greatly
enhanced by the presence of
– Nonionic
– Hydrophobic polymers
• Poly ethylene glycol is used in
nephlometry assays to speed up the
reaction rate, increases the slope of the
precipitation curve in Ab excess zone,
and pushes the zone of equivalence to
the higher Ag concentration

8. • This made the nephlometry popular
with greater sensitivity, wider
detection range, and faster assay.

9. Instrumentation
• Light source
• Collimating light system
• Mono chromator
• Cuvette
• Photo multiplier tube

10. Recorder
sensor
cuvette
Light
4.789
Columinating
sysytem
slit monochromator

11. Light source varies from
• Simple tungsten lamp
• Mercury lamp
• Xenon arc lamp
• High intensity lasers

12. Methods of nephlometric assays
• End point nephlometry
• Kinetic or rate nephlometry

13. End point nephlometry
• In immunoprecipitation reaction, the
time to reach the plateau light
scattering may vary from few min to
1hr depending on reaction conditions

14. Kinetic or rate nephlometry
• Speed of the reaction depends on the
concentration of Ag & the affinity &
titer of the anti sera

15. Applications
• Ig G
• Complement proteins
• Acute phase proteins
• And many other serum proteins
• Kinetic laser nephlometry is used for
specific serum proteins, clotting
factors, prothrombin,
antiprothrombin, and many abnormal
proteins.

16. Disadvantages
• High cost of optical clear
• Potent anti sera of uniform
specifications
• Lipids and Hb are interfering
substances