Defined Protein Conjugates as Signaling Agents in Immunoassays

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 ␣-subunit of thyroidstimulating hormone (TSH), and the complexes were tested in appropriate assay formats for measurement of TSH. Results: Products ranged in mass from ϳ1 to ϳ20 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-

Programmable polyproteams built using twin peptide superglues

“…Expressing modules individually and then linking the modules together would overcome these challenges as well as allow independent posttranslational modification of each module. Even the best noncovalent linkages (7)(8)(9) or reversible covalent linkages, including disulfide bonds (10,11), would allow rearrangement of polyproteams, so irreversible covalent linkage is required. There are a limited number of mutually unreactive (orthogonal) chemical reactions (12); therefore, it is impractical to link more than a few building blocks in a one-pot reaction.…”

Solid‐Phase Protein Modifications: Towards Precision Protein Hybrids for Biological Applications

“…Protein‐protein monoconjugation was reported as early as 1977 using a SP approach, but there were few advances over the following two decades [36] . Driven by increasing biomedical needs, this approach has regained attention in the last two decades and more elegant SPS strategies have since been devised to open access to a greater pool of functionalized proteins and higher‐order protein conjugates for immunodetection or as nanotransporters [5,10] . The preparation of protein conjugates by a SP approach offers several advantages in terms of ease of purification and quality control of the product due to formation of single species.…”

“…Several other protein‐protein monoconjugates were obtained in this fashion but are mainly limited to glycoproteins. Russell et al [5,73] . applied a modified thiol reactive SP to gain access to a series of defined protein conjugates (Figure 4a).…”

“…Moving onwards, the current third generation seeks to address new administration routes and the development of formulations that can give rise to drugs with higher efficacy, exemplified by monoclonal antibodies [3,4] . Besides therapeutics, proteins have also been employed in diagnostics such as antibodies in immunoassays [5] . Evidently, the advancement in recombinant technologies and synthetic capabilities has been a major driving force for the innovation of protein therapeutics and diagnostics.…”

Solid‐phase preparation of protein complexes

“…/jmr.1095 So far, all of these methods have displayed both advantages and limitations related to the specific chemical approach being used. Among the most interesting methods, Russell et al (2002;2004) proposed a batch-mode solid-phase protein conjugation procedure, exploiting the thiol-maleimide chemistry. However, this batch-wise method exploits the same chemistry for both conjugation and protein immobilization and does not allow the repeated use of the solid phase.…”