Immunogenicity assays formats

Immunogenicity assays for investigating the frequency and consequences of antibody development against a protein therapeutic agent are typically based on an immunoassay technique (mostly ELIS As of various types). However, other assay formats are available such as radioimmunoprecipitation assay, surface plasmon resonance, and electrochemiluminescence [3]. Assays for measuring antibody response should be established in the early preclinical stage of development to estimate the value of the applied animal models (see Chapters 16 and 20). 

Of probable interest to the reader of this volume are the many applications of SPR to assays for the detection and characterization of antibodies elicited by therapeutic treatment with biologic macromolecules [166 169]. Given the current intense interest in the development of protein and ODN macromolecules as potential therapeutics, as well as the developing interest in biosimilar protein products, immunogenicity of macromolecules will continue to be investigated for the foreseeable future. Further discussion of assay formats for the detection and characterization of antibodies elicited by treatment with macromolecular therapeutics is presented elsewhere in this volume. 

To develop assays for d and 1-methadone, the hemisuccinates of a-l-methadol (Fig, 4 [4a]) were conjugated to bovine thyroglobulin (54). The resulting immunogens (Fig. 4 [4b]) caused formation of antisera that were quite selective when used with enantiomerically pure tritium-labeled d- or /-methadone. The cross-reaction of the I isomer with the d antiserum was less than 1%, and cross-reaction of /-methadone with the d antiserum was about 3%. The racemate exhibited cross-reactions of 56-57% (55). 

The competition assay was designed which followed the standard indirect ELISA format (17-18 . The methoprene conjugate was bound to a solid support in the form of a microtiter plate. Free methoprene in methanol (5 fib) was added to the pre-coated wells followed by methoprene-specific antiserum. The antibodies were allowed to compete for both immunogen-bound and free methoprene. Enzyme-conjugated, goat-antirabbit antibody was added, followed by substrate, and the color was allowed to develop. The absorbance of substrate over a range of methoprene concentrations can be drawn as a standard curve, which is presented as percent inhibition of the assay (Figure 6). The 50% inhibition (I5g) of methoprene was at a concentration of approximately 50 ng/mL. 

A number of experiments performed thereafter were supportive for the immune-based etiology of zimeldine-induced adverse effects (Kristofferson Nilsson, 1989). Three individuals occupationally exposed to zimeldine developed allergy to the compound and showed positive patch and skin prick tests and positive response to zimeldine in the lymphocyte transformation test. Patients with a history of zimeldine-induced disease showed marked lymphocyte transformation test responses to zimeldine as well as two metabolites (norzimeldine and CPP200). These findings indicate that zimeldine may be immunogenic indeed, zimeldine has been shown to be positive in the popliteal lymph node assay, based on cell numbers and including germinal centre formation and production of IgM and IgG antibodies (Thomas et al., 1989). 

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