CONTENT :Nephelometry. Turbidimetry. Reflectance photometry

1. PHOTOMETRY II
Presenter: Dr.Anurag Yadav
Moderator: Mr.Arun Kumar

2. CONTENT :
 Nephelometry.
 Turbidimetry.
 Reflectance photometry.

3. NEPHELOMETRY AND TURBIDIMETRY
 These are the analytical techniques used to measure scattered
light.
 Principle of nephelometry – intensity of light scattered by a
suspension is measured at 90 degrees angle.
Intensity of scattered light α concentration of suspension
 Principle of turbidimetry- measurement of decrease in light
transmitted through a turbid solution is measured .

4. FACTORS INFLUENCING LIGHT SCATTER
1. Particle size
2. Concentration of particles
3. Molecular weight of particles
4. Wavelength dependence
5. Effect of polarization of incident light
6. Distance of observation

5. LIGHT SCATTERING
3 types
1. Wavelength of light > particle size - RAYLEIGH
 light symmetrically scattered around the particle –
RAYLEIGH
 eg – Ig, Albumin

6. LIGHT SCATTERING
2. Wavelength of light < particle size - MIE THEORY
 light appears scattered forward due to destruction out of
phase background scatter- MIE THEORY
 Particle size – 7000- 40,000nm like in RBC and bacteria .

7. LIGHT SCATTERING
 3. wavelength of light = particle size – RAYLEIGH DEBYE
SCATTER
 light scattered is more in forward than in backward direction –
RAYLEIGH DEBYE SCATTER
 Application
 Light scatter analysis is used for Ag- Ab reactions with size 250 –
1500nm its Rayleigh Debye scatter and blank scatter by Rayleigh.
RAYLAIGH DEBYE

8.  Wavelength dependence of light scattering:
 Intensity of light scattered is inversely proportional to the
wavelength of incident light.
 Scattered light intensity is inversely related to distance from
the particle to detector.
 Concentration and molecular weight:
 From equation, it direct relationship of light scattering to the
conc & molecular weight of particle.

9. INSTRUMENTATION OF
NEPHELOMETER
1. Light source- quartz halogen lamp,mercury arc lamps, xenon
lamps,lasers.
Lasers:
stable, collimated intense light beams,
Reduces stray light, background scatter
2. Collimating optics
3. Sample cell
4. Collection optics –light scattering optics
- detector filter
- detector(PMD)

10. INSTRUMENTATION OF
NEPHELOMETER

11. INSTRUMENTATION OF TURBIDIMETER:

12. LIMITATIONS
 Antigen excess
 Ag -Ab reactions are complex and appear to result in a
mixture of aggregate sizes .
 Turbidity ↑→ adding Ag to Ab & then ↓ →marking the
beginning of antigen excess.

13. LIMITATIONS
 Matrix effects
 Particles, solvent and serum macromolecules scatter light.
 Lipoprotein and chylomicrons in lipemic samples→
background interference
 This is avoided by rate measurements with elimination of
initial sample blank
 Large particles: suspended dust → background interference
 Filtering all buffers, diluted antisera before analysis.

14. APPLICATIONS
 Quantify AA , proteins , vitamins , glycogen , and antibiotics
in blood.
 Quantification of urine, csf protein( conc is less) by
immunonephalometry
 Quantification of urine ALB, ASO, CRP, U.MAU →
Immunoturbidimetry

15. DIFFERENCE BETWEEN
NEPHELOMETRY AND TURBIDIMETRY
1. Mercury arc lamp
2. Rectangular cuvette used
3. Scattered light is measured
4. Measured at 90 deg
5. PMT is detector
1. Tu / Du lamp is used
2. Semi octagonal cuvette
3. Light transmitted is
measured
4. Measured in straight line
5. Photocell is detector
Nephalometry Turbidimetry

16. REFLECTANCE SPECTROPHOTOMETRY
 Beam of light is directed at a flat reaction surface & the
reflected light is quantified.
 Reaction mixture in a carrier is illuminated with diffuse
light, & the intensity of the reflected light from the
chromogen is compared with the intensity of the light
reflected from a reference surface.

17.  The reflected light intensity is non linear in relation to
conc of analyte.
DR = log ( Ro/Rtest)
 Kubelka-Munk or Clapper-Williams transformation
equation used to convert the data into linear format.

18. INSTRUMENTATION :
 Components are same as
Absorbance photometry.
except that the
geometry of the system is
modified so that the light
source & the detector are
on one side of the sample.

19. USES:
 Used as quantitative measurement of surface
reactions such as dipstick or Dry film chemistry
system.

20. REFERENCE
 Clinical chemistry: Kaplan
 Clinical chemistry: TIETZ

23. IMMUNONEPHELOMETRY
 Principle
o Ag +Ab form small aggregates that scatter light – turbid
appearance
o These agg to form large matrix as seen in immunoppt assays
like double diff or radial immunodiff .
o Light scatter intensity α amt of ppt in Ab excess
o Agg from primary reaction –seconds to minutes
o Secondary reaction- takes hours
o Light scatter assay measure early 2nd order reaction bet Ag
and Ab
Agg formation enhanced by addition of solu polyethylene glycol
of conc 2% to 4%

24. IMMUNONEPHELOMETRY
Monoclonal Ab reagents
 Polyclonal Ab need monitoring of titre specificity
and affinity .
 This is overcome by use of monoclonal Ab

25. IMMUNONEPHELOMETRY
 Sample req and preparation – serum urine CSF
 Reagents
 Instrumentation
 Common pitfalls
 Ab excess
 high background scatter
 interference by coloured solu
 Mixing insufficient
 Limitations
 Diff to determine if ppt is in Ag or Ab excess

26. LIGHT SCATTER INHIBITION
IMMUNOASSAY
 NINIA
 1ST described for progesterone by CAMBIASO ET AL 1974
 Principle – ppt from antihapten is inhibited by adding free
hapten
 Used for rapid analysis of drugs in mg/l like phenytoin ,
phenobarbital , theophylline .

27. LIGHT SCATTER INHIBITION
IMMUNOASSAY
 Sample req and prep – serum
 Reagents
 Instrumentation
 Common pitfalls
 Reaction should be in antigen excess.

28. ADDITIONAL ASSAY
MODIFICATION
1. Particle enhanced light scatter
 Type of agglutination procedure
 Ag or Ab coupled with inert carrier particles like
polystyrene latex beads
 Fast signal transmission and economy of reagents
 Eg latex fixation test for detection of RF
2. Monoclonal Ab reagents
 Polyclonal Ab need monitoring of titre specificity and
affinity .
 This is overcome by use of monoclonal Ab .