Immunoassay for macromolecules

Findlay JWA, Das I Validation of immunoassays for macromolecules from biotechnology. J. Clin. Ligand Assay (1998)... 

The primary focus in the analytical considerations of immunoassays for macromolecules should be in the development phase. By thoroughly defining each component of the immunoassay in the first phase of the assay life cycle, the resulting validation plan should be concise and the validation process should be straightforward. Several facets of the assay defined in the late development stage, such as specificity and dilutional linearity, can appropriately be included in the final validation report. 

Binodh DeSilva, Amgen, Inc., Thousand Oaks, California, Analytical Considerations for Immunoassays for Macromolecules... 

Cellhouse operating parameters, 26 572t Cell interconnects, SOFC, 72 226 Cell macromolecules, enzyme immunoassay for, 74 143 Cell-mediated immune response (CMI),... 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

VALIDATION CONSIDERATIONS FOR IMMUNOASSAYS FOR LOW-MOLECULAR-WEIGHT XENOBIOTICS AND MACROMOLECULES... 

Although most immunoassays have used polyclonal antibodies as the critical binding reagents, development of monoclonal antibodies by Kohler and Milstein in 1975,has resulted in their widespread use, particularly in assays for macromolecules. Their unique epitope specificity conveys advantages in double antibody immunoassays for proteins, where one monoclonal antibody may be used to capture the protein by a specific subunit or epitope, and another, directed against a... 

A broad spectrum of immunoassays for low-molecular weight haptens, macromolecules, and microorganisms have been made available in recent years through the enormous progress in immunological research, especially in the preparation of monoclonal antibodies. About one billion immunoassays are sold per year. 

The optimization and validation of immunoassays for immunogenicity (ADA) testing has been described in detail in several publications [9,14,33,34]. In this section, we will describe the evaluation of relevant performance characteristics (validation parameters) that require the most effort. Some of these are different from the validation of traditional bioanalytical pharmacokinetic (PK) methods for macromolecules [35 37]. Precision, specificity, robustness, and ruggedness are determined similarly between ADA and PK methods. However, recovery/accuracy, sensitivity, stability, linearity, system suitability controls, and selectivity are treated differently between these two types of assays. 

The ideal PK immunoassay standard curve, which is nonlinear and heteroscedastic, is derived from solutions of well-characterized macromolecules added to a relatively nonreactive sample matrix. However, rarely does one encounter an ideal situation when describing bioanalytical methodology, thus developing and validating analytical methods for macromolecules, and analyzing samples from preclinical or clinical trials must include an evaluation of these variables and possibly many others. Careful consideration must be given to the topics described in this chapter to achieve the goal of accurate and reproducible quantification of biotherapeutics necessary for pharmacokinetic analysis. 

The fluorescence polarization immunoassay is used for routine, automated immunoassay of small molecules, such as drugs. It depends on the principle that a fluorophore attached to a macromolecule such as an antibody is not free to rotate in solution. If polarized light is used to stimulate the fluorophore to fluoresce, emission from the bound fluorophore (attached to the antibody, which is bound to a surface) will continue to be polarized, but polarization will be lost from free fluorophore (111). 

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