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

 

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.

Turbidimetry vs Nephelometry

Turbidimetry Nephelometry

Measures reduction in intensity

of transmitted light at 180° due

to the formation of immune

complexes

Measures scattering of light

at an angle (usually 90°) away

from the incident light, due

to the formation of immune

complexes

Can be performed on most

spectrophotometers

Sensitivity competitive with

nephelometric for small immune

complexes such as serum

proteins

More precise for measuring

large immune complexes

Requires dedicated nephelometers

Sensitive for measuring small

immune complexes such as

serum proteins

Less precise for measuring

large immune complexes due to

forward scattering of light

Blanking and reading reaction

can be performed in the same

measuring cuvette

Provides better precision due

to slower reaction kinetics as

blanking of immunochemical

reaction can be monitored in a

single cuvette

Blanking has to be performed in

separate measuring cuvette

Because of the fast reaction

kinetics it is difficult to obtain a

sample and a reagent, sample

and reagent blank in case of

nephelometry

Considerations for Measurements of Turbidimetric

Immunoassays (TIA)

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

zones as follows:

The Antibody Excess Zone

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

analyte concentration.

Zone of Equivalence

In the second stage of reaction binding sites available for

the antigen are proportionate to the antigen concentration.

Diagnostic Immunology 707

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.

The Antigen Excess Zone

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

Equilibrium Reaches 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

the dose response curve.

For many analytes of diagnostic importance, the AgAb reactions neither follow the Beer-Lambert law nor

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

of the analyte of interest.

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

conditions.

Investigations, of the factors affecting Ag-Ab reactions

were extensively studied by Heidelberger and Kendall. In

addition to the relative proportions of immune reactants,