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Standardization of immunoassays
The aim of standardization is to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, can be readily compared. Standardization is especially desirable for immunoassays because all external quality assessment surveys have shown that this type of method involves a much greater variability than traditional assays. A major problem in immunoassays is that the recognition of the analyte is determined by the reagent (i.e. an antibody or conversely, if antibodies are to be determined, an antigen) using a limited portion of the molecule, the epitope or the paratope, respectively. Therefore, the specificity of whole process is questionable, even if the specificity of the epitope/paratope reaction is fair. Biological variability is such that many molecules possessing the same recognition site coexist in biological fluids. A first example is the different secretion forms of hormones, their fragments, and other related hormones which occur simultaneously in the blood. A second example is the high variety of antibodies possessing the same paratope. Many other examples can be cited. Thus, a precise definition of the analyte should be given, dependent on the aim of the assay. This is a major conceptual difficulty because several different molecules having the same recognition site may cooperate in a physiological function. In this context, it may be useful to assay them simultaneously, but it is impossible to express their concentrations in common mass units. Another major difficulty in immunoassay standardization is due to the fact that such methods operate at very low concentrations and are sensitive to the environmental conditions created by ambient molecules existing in much higher concentrations. These "non-specific" effects create problems of non-comparability due to the prevalent conditions in different samples. From the different causes mentioned above, it follows that the major difficulty is to ensure perfect identity of the conditions in the calibrator and in the sample (analyte and matrix). The current procedures of assay standardization are reviewed and their deficiencies are stressed. Recent proposals for rationalization of standardization of immunoassays are described.
The aim of standardization is to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, can be readily compared. Standardization is especially desirable for immunoassays because all external quality assessment surveys have shown that this type of method involves a much greater variability than traditional assays. A major problem in immunoassays is that the recognition of the analyte is determined by the reagent (i.e. an antibody or conversely, if antibodies are to be determined, an antigen) using a limited portion of the molecule, the epitope or the paratope, respectively. Therefore, the specificity of whole process is questionable, even if the specificity of the epitope/paratope reaction is fair. Biological variability is such that many molecules possessing the same recognition site coexist in biological fluids. A first example is the different secretion forms of hormones, their fragments, and other related hormones which occur simultaneously in the blood. A second example is the high variety of antibodies possessing the same paratope. Many other examples can be cited. Thus, a precise definition of the analyte should be given, dependent on the aim of the assay. This is a major conceptual difficulty because several different molecules having the same recognition site may cooperate in a physiological function. In this context, it may be useful to assay them simultaneously, but it is impossible to express their concentrations in common mass units. Another major difficulty in immunoassay standardization is due to the fact that such methods operate at very low concentrations and are sensitive to the environmental conditions created by ambient molecules existing in much higher concentrations. These "non-specific" effects create problems of non-comparability due to the prevalent conditions in different samples. From the different causes mentioned above, it follows that the major difficulty is to ensure perfect identity of the conditions in the calibrator and in the sample (analyte and matrix). The current procedures of assay standardization are reviewed and their deficiencies are stressed. Recent proposals for rationalization of standardization of immunoassays are described.