สารบัญ

14.5 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 standardize 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.

METHODS IN WHICH A LABELLED ANTIGEN OR A LABELLED ANTIBODY IS USED

      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 “radioimmunoassay”. The recommendations for the measurement of radio activity given under radiopharmaceutical preparations 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.

      Radioimmunoassay methods (RIA) The principle of the assay method is that radioactive labelled antigen competes with the non-labelled antigen of a sample under test for the antibody with which the labelled and non-labelled antigens are mixed. The more of the antigen in the test sample the less chance the labelled antigen has of combining with a limited number of antibody molecules that are available in the antibody serum. The amount of labelled antigen bound to antibody is detemined by the amount of unlabelled antigen that competes in test sample. Thus, by measuring the quantity of labelled antigen combined with antibody (using isotope counting equipment) a measure of the antigen in the test sample can be obtained.

      Enzyme-linked immunosorbent assay methods (ELISA) Where the label is enzyme, the method is described as Enzyme-linked immunosorbent assay. The method involves coupling to enzymes which give a coloured soluble reaction products after enzyme substrate, a colourless substrate, is added. The enzymes such as horseradish peroxidase or alkaline phosphatase are linked to either antibody or antigen molecules. The presence of the enzyme-linked molecule is detected by means of the enzyme substrate and can be measured by a spectrophotometer. Either the labelled antigen or the antibody can be attached to an insoluble support, such as plastic beads or plastic agglutination plates.

METHODS IN WHICH AN UNLABELLED ANTIGEN OR ANTIBODY IS USED

      Immunoprecipitation methods 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 that 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 lightscattering techniques (nephelometric or turbidimetric assay). An increase insensitivity can be obtained by using antigen- or antibody-coated particles (such as latex) as reactants.

      In flocculation methods, stepwise dilutions of one of the reactants are 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 reactants 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 in a neutral (inert) gel by allowing both reactants to diffuse into the gel from separate sites.

      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, nonidentity or partial identity of antigens or antibodies can be distinguished.

      Agglutination methods In this reaction the antigen is part of the surface of some particulate material such as a red cell, bacterium or perhaps an inorganic particle (e.g. polystyrene latex) which has been coated with antigen. Antibody added to a suspension of such particles combines with the surface antigens and links them together to form clearly visible aggregates or agglutinates. This clearly visible agglutinates is the end-point of the test. The reciprocal of the antiserum dilution made at the end-point is known as the titre of the antiserum and is a measure of the number of antibody unit per volume of serum.

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

      Crossed immunoelectrophoresis is a modification of the IE method. It is suitable for both 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 at an angle of 90º 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 immunoelectrophoresis, 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.

      Visualization and characterization of immunoprecipitation line These may be performed by selective or non-selective stains, by fluorescence, by enzyme or isotope labelling, or by other relevant techniques. Selective staining methods are usually performed for characterization 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.