The stability and resolution of modern microprocessor driven spectrophotometers and automated clinical chemistry analyzers have greatly improved their ability to measure turbidity with dependable accuracy and

 

On the other hand, when the size of the immune

complex to be measured is more than 1/10th the size the

wavelength of the incident light of there is concentration

of scattered light in forward direction at an angle of 45°

or less, away from the axis of the incident light beam

(Fig. 23.7). This type of scattering is referred as RayleighDebye scattering.

Careful examination of both the figures (Figs 23.6 and

23.7) show that the intensity of scattered light for forward

and back scatter (0° and 180°) from small particles is equal

but less at 90° (Rayleigh scattering). As the size of the

FIG. 23.2: Forms of graphs showing change in reaction rate as a function of time. In A, the rate is constant in all the segments I, II and III. In B,

a measurement at segment II will be representative of constant rate, but at segment I (lag phase) and segment III it will be less. In C, the rate

falls of continuously

FIG. 23.3: Behavior of light in solution containing antigens where Io is

the intensity of incident light, lt is the intensity of transmitted light

and ‘b’ is the cuvette containing antigens in the reaction solution

A B C

Diagnostic Immunology 705

particle becomes larger, the angular dependence of light

scattering becomes dissymmetrical, increasing in forward

scattering and decreasing in backward scattering.

The Rayleigh and Rayleigh-Debye expressions provide

useful information about scattering of light by small and

intermediate size particles and are important for the

optimization of analytical instrumentation for measuring

light using turbidimetric and nephelometric assays.

The upper limit on size of immune complexes exhibiting

Rayleigh scattering is about 40 nm when a visible light

at 400 nm is used. Many of the plasma proteins such as

immunoglobulins, albumin, etc. fall below this limit. As

the immune complexes, become larger in size from 40 to

400 nm, the angular dependence of scattered light at 400

nm looses the symmetry around the 90° axis, and shows

an increase in forward scattering. Some plasma proteins

of the IgM class, aggregating immunoglobulin/antigen

FIG. 23.4: Behavior of light in solution containing immune-complexes

(Ag-Ab), where lo is the intensity of incident light, lt

 is the intensity

of transmitted light, ls is the intensity of scattered light and ‘b’ is the

cuvette containing immune-♥complexes in the reaction solution

FIG. 23.6: Illustrating Rayleigh scattering for a immune complex

with particle size < λ10 of the wavelength of incident light

FIG. 23.5: Detection principles in turbidimetry and nephelometry

where Io is the intensity of incident light, ‘b’ is the reaction cuvette, lt

is the intensity of transmitted light measured by detector at 180°, ls is

the intensity of scattered light measured by detectors placed at 90°

and 45°

FIG. 23.7: Illustrating Rayleigh-Debye scattering for a immune

complex with particle size > λ10 of the wavelength of incident light

706 Concise Book of Medical Laboratory Technology: Methods and Interpretations complexes fall into this size category. For measuring such

complexes a bigger wavelength of light depending on the

size of the immune complexes formed, should be used. For

latex based assays using a latex particle of approximately

200–300 nm, light of wavelength between 500–600 nm

would be ideal for measuring the immune complexes

formed.

Biochemical

measurements

Nephelometric

measurements

Turbidimetric

measurements

Measures absorbance

scattering at an by

colored complexes

formed as a result

of biochemical

reactions

Measures light

reduction in angle

away from the

incident light due

to formation of

immune complexes

Measures light at

180°, intensity of

light transmitted

(as absorbance)

at 180°due to the

formation of immune

complexes

Selection of Selection of Selection of

wavelength of light

is complementary

to the color of the

chemical complex to

be measured

wavelength of light

depends on the

size of the immune

complex formed

wavelength of light

depends on the size of

the immune complex

formed

The choice between turbidimetry and nephelometry will

depend on application and the available instrumentation.

Until recently, it was assumed that for relatively clear

solutions in which the transmission of light in the forward

direction is greater than 95% small changes in absorption

due to turbidity were difficult to measure with precision.

The stability and resolution of modern microprocessor

driven spectrophotometers and automated clinical

chemistry analyzers have greatly improved their ability

to measure turbidity with dependable accuracy and

precision. Turbidimetric methods have today become

competitive in sensitivity with nephelometric methods for

immunological quantitation of such solutions.

For some analytes, the signal amplification and assay

sensitivity requires the usage of conjugation chemistry

to attach antibodies to inert and uniform latex particles.

Such reagent systems are referred to as particle enhanced

turbidimetry (PET).