which future lots of calibrators can be assigned values. The secondary standards act as an intermediate between IRP primary standard and future lots of calibrators for assay

 

other conditions such as temperature, ionic strength

of the medium, characteristics of the antibody such as

avidity and affinity are important for formation of Ag-Ab

immune complex. These principles need to be applied to

the reagent system optimization for immunoturbidimetry.

FIG. 23.8: The quantitative immunoprecipitin curve

(Heidelberger-Kendall curve)

708 Concise Book of Medical Laboratory Technology: Methods and Interpretations Ionic Strength

It has been observed that the Ag-Ab reactions are strongly

influenced by the nature of ionic medium in which the

reaction is carried out. The ionic strength of the reaction

environment has a profound effect on the quantum

and the rate of the Ag-Ab reaction. As the ionic strength

increases, the depth of the electrical double layer that

forms around the charged molecules is compressed,

reducing the distance over which repulsive forces that

keep the molecules apart can act. This in effect leads to the

promotion of aggregation. The reduction in charge on the

other hand influences the electrostatic attraction between

oppositely charged species, thereby reducing the specific

binding between antigen and antibody.

pH

The reaction pH also influences the rate of aggregate

formation. The rate of reaction is found to be fairly

consistent at a pH of 6.0–8.0. Reduction in pH leads to some

proteins having net positive charge (those with a pH above

the reaction pH) leading to nonspecific agglutination with

negatively charged proteins or particles.

Temperature

As it is well known, temperature influences the rate

of formation of immune complexes and it should be

optimized to obtain accurate results. Assays are usually

designed with an incubation temperature of 37°C because

it is the most common temperature used in the routine of

clinical laboratories for clinical assays.

Enhancers

For enhancing the Ag-Ab reactions, polymeric compounds,

such as PEG and BSA, may be included in the buffer

system. These compounds facilitate the formation of

immune complex and help in amplification of signals and

improve the assay system sensitivity.

Interference

Many interfering factors, such as bilirubin and lipids, are

normally present in the samples apart from the analyte of

interest. High concentrations of some of these interfering

factors are frequently encountered in clinical samples. They

may influence signal generation and, therefore, can interfere

in the assay result. Minimizing the influence of these factors

help provide accurate and precise assay results.

Turbidimetric assays usually employ a suitable buffer

in the assay design to optimize assay conditions and the

desired ionic strength, pH and enhancement required for

the reaction medium. In addition, the buffer is also useful

in reducing the influence of interfering factors present

in the sample. The buffer used in turbidimetric assays is

generally referred to as activation buffer.

Characteristics of Antibody Used as a Reagent

Intrinsic characteristics of antibody employed as reagents

have a profound effect on the Ag-Ab reactions. The

specificity and affinity of the antibody to the antigenic sites

affect the sensitivity of the assay and signal generation

time. Usually, when high specificity and affinity antibodies

are used, a strong agglutination reaction will readily result.

In contrast, antibodies with low affinity, even if highly

specific, tend to react slowly and form a weak immune

complex, thereby lowering the detectable signal. The

avidity of antibody is also an important consideration in

the formation of immune complexes. This characteristic of

the antibody determines the degree of stability of the AgAb complexes at the antigen-binding sites. The tendency

of the complexes to dissociate and disperse decreases

substantially as the avidity of the antibody increases.

An antibody without cross-reactivity and with a good titer

is a prerequisite for a reliable turbidimetric assay utilizing

antibody as a principal reagent. In addition, the antibody

must be formulated as a clear solution to give a low reagent

blank and should be free from particulate matter.

Standardization and Calibration

During the course of treatment, individual patients are

likely to have tests carried out for the same analyte by

FIG. 23.9: Illustrating standard curve for IgA using lowest standard as

7 IU/mL and highest standard as 120 IU/mL

Diagnostic Immunology 709

different methods, and to have results checked against

reference intervals that were set elsewhere. To achieve

agreement between different methods, a single recognized

source of reference preparation is needed.

The reference preparation should:

¾ Have value assignment in meaningful units

¾ Be stable and identical to the analyte in the test samples

¾ Be free of interference from the test sample matrix

¾ Be standardized by a reference method

¾ Demonstrate intermethod agreements.

Most International Reference Preparations (IRPs) and

Certified Reference Materials (CRMs) such as CRM 470

for immunoassay analytes, can be obtained from the main

custodians of International Biological Standards such as

National Institute for Biological Standards and Controls

(NIBSC) or WHO and Community Bureau of Reference of

the Commission of European Communities (BCR) or the

IFCC.

As the availability of International Standards is limited,

it is a practice to prepare sets of secondary standards, from

which future lots of calibrators can be assigned values. The

secondary standards act as an intermediate between IRP

primary standard and future lots of calibrators for assay

runs. The calibrator sets are made in bulk and values are

assigned with reference to the secondary standards.

As discussed, the immunoturbidimetric assays require

a set of 5 or 6 calibrators to obtain a standard curve. The

quantitative values of unknown analyte obtained from

the standard curve will be highly dependent on correct

assignment of values to the calibrator used for preparing

the standard curve.