Defined Protein Conjugates as Signaling Agents in Immunoassays

Abstract

Abstract Background: Conventional methods for conjugation of macromolecules, such as antibodies and reporter groups, typically yield a mixture ranging from unconjugated starting materials to large aggregates. We explored the use of a solid-phase process to allow improved control in conjugation of macromolecules for use in immunodiagnostic reagents. Methods: Activated components were sequentially delivered to an immobilized core protein, linking in concentric layers. For immunodiagnostic reagents, proteins with the desired signaling properties were added as interior layers and binding proteins were placed in the final surface layer. After assembly, the conjugates were released into solution by cleaving the linker holding the core protein to the support. Conjugates were prepared with use of three different reporter agents: R-phycoerythrin for microsphere fluorescence flow immunoassay, alkaline phosphatase for enzyme immunoassay, and acridinium for magnetic chemiluminescence immunoassay. For each reporter, six conjugates were prepared with various concentrations of both the reporter and an antibody directed against the α-subunit of thyroid-stimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. Results: Products ranged in mass from ∼1 to ∼20 MDa. HPLC analysis of the conjugates on a gel-permeation column showed sizes and chromophore contents highly consistent with the intended structures. In appropriate assay formats, the signal generated by a conjugate increased with incubation time, then plateaued at an intensity approximately proportional to the reporter content but relatively independent of the antibody con-tent of the conjugate. The time required to reach this maximum decreased with increasing antibody content. Conclusion: The high degree of structural control available with solid-phase assembly and the close correlation of structure with desired function of the resulting conjugates make this an attractive method for preparation of an important class of in vitro diagnostic reagents.