Defined protein conjugates as signaling agents in immunoassays

Clinical Chemistry
John RussellStephen Stroupe

Abstract

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. 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 alpha-subunit of thyroid-stimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. Products...Continue Reading

References

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Sep 19, 2002·Bioconjugate Chemistry·John C RussellSie-Ting Wong

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Citations

Jan 21, 2016·Proceedings of the National Academy of Sciences of the United States of America·Gianluca VeggianiMark Howarth
Nov 3, 2010·Journal of Molecular Recognition : JMR·Paolo PengoGiorgio Fassina

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