Appendix XIV B. Immunochemical Methods

Immunochemical methods are based on the selective, reversible and non-covalent binding of antigens by antibodies. These methods are employed to detect or quantify either antigens or antibodies. The formation of an antigen-antibody complex may be detected, and the amount of complex formed may be measured by a variety of techniques. The provisions of this general method apply to immunochemical methods using labelled or unlabelled reagents, as appropriate.

The results of immunochemical methods depend on the experimental conditions and the nature and quality of the reagents used. It is essential to standardise the components of an immunoassay and to use, wherever available, international reference preparations for immunoassays.

The reagents necessary for many immunochemical methods are available as commercial assay kits, that is, a set including reagents (particularly the antigen or the antibody) and materials intended for the in vitro estimation of a specified substance as well as instructions for their proper use. The kits are used in accordance with the manufacturers' instructions; it is important to ascertain that the kits are suitable for the analysis of the substance to be examined, with particular reference to selectivity and sensitivity. Guidance concerning immunoassay kits is provided by the World Health Organisation, Technical Report Series 658 (1981).

Methods using labelled substances may employ suitable labels such as enzymes, fluorophores, luminophores and radioisotopes. Where the label is a radioisotope, the method is described as a "radio-immunoassay". The recommendations for the measurement of radioactivity given in the monograph on Radiopharmaceutical Preparations (0125) are applicable to immunoassays involving radioisotopes. All work with radioactive materials must be carried out in conformity with national legislation and internationally accepted codes of practice for protection against radiation hazards.

Immunoprecipitation methods include flocculation and precipitation reactions. When a solution of an antigen is mixed with its corresponding antibody under suitable conditions, the reactants form flocculating or precipitating aggregates. The ratio of the reactants which gives the shortest flocculation time or the most marked precipitation is called the optimal ratio, and is usually produced by equivalent amounts of antigen and antibody. Immunoprecipitation can be assessed visually or by light-scattering techniques (nephelometric or turbidimetric assay). An increase in sensitivity can be obtained by using antigen- or antibody-coated particles (e.g. latex) as reactants.

In flocculation methods, stepwise dilutions of one of the reactants is usually used whereas, in immunodiffusion (ID) methods, the dilution is obtained by diffusion in a gel medium: concentration gradients of one or both of the reactants are obtained, thus creating zones in the gel medium where the ratio of the reactants favours precipitation. While flocculation methods are performed in tubes, immunodiffusion methods may be performed using different supports such as tubes, plates, slides, cells or chambers.

Where the immunoprecipitating system consists of one antigen combining with its corresponding antibody, the system is referred to as simple; when it involves related but not serologically identical reactants, the system is complex and where several serologically unrelated reactants are involved, the system is multiple.

In simple diffusion methods, a concentration gradient is established for only one of the reactants diffusing from an external source into the gel medium containing the corresponding reactant at a comparatively low concentration.

Single radial immunodiffusion (SRID) is a simple quantitative immunodiffusion technique. When the equilibrium between the external and the internal reactant has been established, the circular precipitation area, originating from the site of the external reactant, is directly proportional to the amount of the antigen applied and inversely proportional to the concentration of the antibody in the gel.

In double diffusion methods, concentration gradients are established for both reactants. Both antigen and antibody diffuse from separate sites into an initially immunologically neutral gel.

Comparative double diffusion methods are used for qualitatively comparing various antigens versus a suitable antibody or vice versa. The comparison is based on the presence or absence of interaction between the precipitation patterns. Reactions of identity, non-identity or partial identity of antigens/antibodies can be distinguished.

Immunoelectrophoresis (IE) is a qualitative technique combining 2 methods: gel electrophoresis followed by immunodiffusion.

Crossed immunoelectrophoresis is a modification of the IE method. It is suitable both for qualitative and quantitative analysis. The first part of the procedure is an ordinary gel electrophoresis, after which a longitudinal gel strip, containing the separated fractions to be determined, is cut out and transferred to another plate. The electrophoresis in the second direction is carried out perpendicular to the previous electrophoretic run in a gel containing a comparatively low concentration of antibodies corresponding to the antigens. For a given antibody concentration and gel thickness, the relationship between the area of the respective precipitation peaks and the amount of the corresponding antigen is linear.

Electroimmunoassay, often referred to as rocket immuno-electrophoresis is a rapid quantitative method for determining antigens with a charge differing from that of the antibodies or vice versa. The electrophoresis of the antigen to be determined is carried out in a gel containing a comparatively lower concentration of the corresponding antibody. The test material and dilutions of a standard antigen used for calibration are introduced into different wells in the gel. During electrophoresis, migrating peak-shaped precipitation zones originating from the wells are developed. The front of the precipitate becomes stationary when the antigen is no longer in excess. For a given antibody concentration, the relationship between the distance travelled by the precipitate and the amount of antigen applied is linear.

Counter-immunoelectrophoresis is a rapid quantitative method allowing concentration gradients of external antigen and external antibody to be established in an electric field depending on the different charges. Dilutions of a standard for calibration and dilutions of the test material are introduced into a row of wells in a gel and a fixed amount of the corresponding reactant is introduced into an opposite row of wells. The titre of the test material may be determined as the highest dilution showing a precipitation line.

A number of modifications of crossed immunoelectrophoresis and electroimmunoassay methods exist.

Other techniques combine separation of antigens by molecular size and serological properties.

These may be performed by selective or non-selective stains, by fluorescence, by enzyme or isotope labelling or other relevant techniques. Selective staining methods are usually performed for characterisation of non-protein substances in the precipitates.

In translucent gels such as agar or agarose, the precipitation line becomes clearly visible in the gel, provided that the concentration of each of the reactants is appropriate.

A quantitative immunochemical method is not valid unless:

1)  The antibody or antigen does not significantly discriminate between the test and standard. For a labelled reactant, the corresponding reactant does not significantly discriminate between the labelled and unlabelled compound,

2)  The method is not affected by the assay matrix, that is, any component of the test sample or its excipients, which can vary between samples. These may include high concentrations of other proteins, salts, preservatives or contaminating proteolytic activity,

3)  The limit of quantitation is below the acceptance criteria stated in the individual monograph,

4)  The precision of the assay is such that the variance of the results meets the requirements stated in the individual monographs,

5)  The order in which the assay is performed does not give rise to systematic errors.

In order to verify these criteria, the validation design includes the following elements:

1)  The assay is performed at least in triplicate,

2)  The assay includes at least 3 different dilutions of the standard preparation and 3 dilutions of sample preparations of presumed activity similar to the standard preparation,

3)  The assay layout is randomised,

4)  If the test sample is presented in serum or formulated with other components, the standard is likewise prepared,

5)  The test includes the measurement of non-specific binding of the labelled reactant,

6)  For displacement immunoassay:

(a)  maximum binding (zero displacement) is determined,

(b)  dilutions cover the complete response range from values close to non-specific binding to maximum binding, preferably for both standard and test preparations.

To analyse the results, response curves for test and standard may be analysed by the methods described in 5.3. Statistical Analysis of Results of Biological Assays and Tests.

Significant non-parallelism indicates that the antibody or antigen discriminates between test and standard, and the results are not valid.

In displacement immunoassays, the values for non-specific binding and maximum displacement at high test or standard concentration must not be significantly different. Differences may indicate effects due to the matrix, either inhibition of binding or degradation of tracer.