Coordination complexes as molecular glue for immobilization of antibodies on cyclic olefin copolymer surfaces

A novel metal-based chelating method has been used to provide an order of magnitude increase in immunoassay performance on cyclic olefin copolymer (COC) plastics compared with passive binding. COCs are hydrophobic, and without surface modification they are often unsuitable for applications where protein adhesion is desired. When interacting with the bare plastic, the majority of the bound proteins will be denatured and become nonfunctional. Many of the surface modification techniques reported to date require costly equipment setup or the use of harsh reaction conditions. Here, we have successfully demonstrated the use of a simple and quick metal chelation method to increase the sensitivity, activity, and efficiency of protein binding to COC surfaces. A detailed analysis of the COC surfaces after activation with the metal complexes is presented, and the immunoassay performance was studied using three different antibody pairs

Challenges for protein chemical synthesis in the 21st century: Bridging genomics and proteomics

The Human Genome Project and other major sequencing projects have rapidly provided a vast array of new protein sequences. In the postgenomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. In the epoch, the advantages of protein chemical synthesis will complement recombinant-DNA methods, and will be used to provide rapid access to small proteins or functional receptor domains. In this review the key methodological advances that have made the synthesis of long peptides and small proteins more effective will be presented. Focus is given to the issues and goals of contemporary chemical protein synthesis, including (1) the rapid chain assembly of tailored peptide segments for use in ligation strategies, and (2) development of highly efficient and universal chemoselective ligation strategies. (C) 2000 John Wiley & Sons, Inc

High-throughput optimization of surfaces for antibody immobilization using metal complexes

Using a high-throughput surface discovery approach, we have generated a 1600-member library of metal-containing surfaces and screened them for antibody binding potential. The surface library assembly involved graft modification of argon plasma-treated polyvinylidenedifluoride (PVDF) membranes with alternating maleic anhydride-styrene copolymer followed by anhydride ring opening with a range of secondary amines and microarray contact printing of transition metal complexes. The microarrays of metal-containing surfaces were then tested for their antibody binding capacity by incubation with a biotinylated mouse antibody in a chemiluminescence assay. A total of 11 leads were identified from the first screen, constituting a "hit" rate of 0.7%. A smaller 135-member surface library was then synthesized and screened to optimize existing hits and generate additional leads. To demonstrate the applicability of these surfaces to other formats, high-binding surface leads were then transferred onto Luminex beads for use in a bead flow cytometric immunoassay. The novel one-step antibody coupling process increased assay sensitivity of a Luminex tumor necrosis factor immunoassay. These high-binding surfaces do not require prior incorporation of polyhistidine tags or posttreatments such as oxidation to achieve essentially irreversible binding of immunoglobulin G