PET reagents usually use latex of approximately
200–300 nm for conjugating antibodies to facilitate
formation of larger immunecomplexes and thereby
generate detectable signals. This leads to decrease in
scattering of light at 90° and increase in forward scattering.
Turbidimetric assays, therefore, have better precision for
measuring larger immune complexes.
In the context of contemporary technology, turbidimetric
assays are gaining popularity over nephelometric
determination due to their simplicity and overall consistency.
Measures reduction in intensity
of transmitted light at 180° due
at an angle (usually 90°) away
nephelometric for small immune
Requires dedicated nephelometers
to slower reaction kinetics as
reaction can be monitored in a
Blanking has to be performed in
kinetics it is difficult to obtain a
Considerations for Measurements of Turbidimetric
As far back in the year 1929, Heidelberger and Kendall
have quantitatively described the formation of a
precipitate when reacting an antigen with an antibody.
They demonstrated that when an increasing amount of an
antigen is added to a constant amount of corresponding
antibody, the resulting degree of precipitate formed follows
a bell-shaped curve as shown in Figure 23.8. To obtain the
Heidelberger curve, the antigen concentration is plotted
against the absorbances obtained from measuring the AgAb reaction.
The Heidelberger-Kendall curve can be divided in three
In the first stage of the reaction, there is a large excess
of binding sites in the reaction mixture available for the
antigen to bind. First the antigen binding sites are quickly
saturated by antibody before cross-linking begins to occur.
This results in formation of small Ag-Ab complexes. In
this zone, the absorbances increase proportionally to the
In the second stage of reaction binding sites available for
the antigen are proportionate to the antigen concentration.
Here, the probability of cross-linking is more likely
resulting in formation of large immune complexes. As the
saturation point is reached, there is neither free antigen
nor free antibody in the reaction mixture. To this zone,
the absorbances increase with the increasing analyte
concentration, but does not increase proportionally.
In the third stage of the reaction, the relative concentration
of the antigen is so high that most of the binding sites are
overcrowded, hindering the formation of real precipitate
and favoring the formation of small immune complexes.
This is called the prozone effect or the hook effect. The term
“prozone” is inappropriately used to describe “postzone”
or “antigen excess” in day-to-day parlance. The existence of
prozone effect causes very high concentrations of antigen to
produce signals, which are similar to the signals generated, by
moderate concentrations of the same antigen. It is imperative
to know for the assay design as to what concentration
of analyte will cause a prozone effect in a turbidimetric
immunoassay for a given antibody reagent system.
When the Ag-Ab reaction takes place and the formation
of the immune complex is measured optically by
turbidimetry, then the absorbance and reaction kinetics in
the three zones will follow the following pattern:
Heidelberger-Kendall Absorbance curve
Antibody excess zone Increases towards maximum
Antigen excess zone Decreases below maximum
The Heidelberger-Kendall immunoprecipitin curve
forms the fundamental basis for all homogeneous Ag-Ab
assays including turbidimetry and is usually referred to as
provide a linear relationship between concentration
and turbidity. Estimating the concentrations of analytes
using a single standard, as in biochemical analysis
therefore, results in inaccurate results near the zone
of equivalence. The ∆A is directly proportional to the
concentration of analyte only in the initial region of
the antibody excess zone. Use of single standard for
calculating concentration of analyte may be acceptable
only for lower analyte concentrations. As the analyte
concentrations increase, the error in measurement
will start to magnify. Therefore, for having a larger
measuring range, the turbidimetric assays use that part
of the dose response curve, which covers the maximum
portion of the antibody excess region and demonstrates
a linear reaction as the standard curve.
The standard curve is plotted using a number of
standards containing different concentrations of analyte
being measured (usually 5–6). The highest concentration
of the standard is chosen in such a way that the analyte
absorbance at that concentration will lie on the linear
extreme of the standard curve. The lowest concentration of
the analyte is usually selected below the reference values
The linear range between the highest and lowest
standards used for the preparation of standard curve is
referred to as the measuring range of the assay (Fig. 23.9).
Optimization, Standardization and
Quality Control of Turbidimetric Assays
To measure the Ag-Ab reactions, reliably all the factors
that affect the reaction rate, other than the concentration
of the antibody, must be optimized and controlled. As
the reaction velocity is at its maximum under optimal
conditions, a larger analytical signal is obtained that can
be more accurately and precisely measured as compared
to a smaller signal obtained under suboptimal assay
Investigations, of the factors affecting Ag-Ab reactions
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