title: Regioselective covalent immobilization of recombinant antibody-binding proteins A, G, and L for construction of antibody arrays.
abstract: Immobilized antibodies are useful for the detection of antigens in highly sensitive microarray diagnostic applications. Arrays with the antibodies attached regioselectively in a uniform orientation are typically more sensitive than those with random orientations. Direct regioselective immobilization of antibodies on a solid support typically requires a modified form of the protein. We now report a general approach for the regioselective attachment of antibodies to a surface using truncated forms of antibody-binding proteins A, G, and L that retain the structural motifs required for antibody binding. The recombinant proteins have a C-terminal CVIX protein farnesyltransferase recognition motif that allows us to append a bioorthogonal azide or alkyne moiety and use the Cu(I)-catalyzed Huisgen cycloaddition to attach the binding proteins to a suitably modified glass surface. This approach offers several advantages. The recombinant antibody-binding proteins are produced in Escherichia coli, chemoselectively modified posttranslationally in the cell-free homogenate, and directly attached to the glass surface without the need for purification at any stage of the process. Complexes between immobilized recombinant proteins A, G, and L and their respective strongly bound antibodies were stable to repeated washing with PBST buffer at pH 7.2. However, the antibodies could be stripped from the slides by treatment with 0.1 M glycine·HCl buffer, pH 2.6, for 30 min and regenerated by shaking with PBS buffer, pH 7.2, at 4 °C overnight. The recombinant forms of proteins A, G, and L can be used separately or in combination to give glass surfaces capable of binding a wide variety of antibodies.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, USA.
authors.forename: Jin-soo
authors.initials: JS
authors.lastname: Seo
authors.forename: Sungwon
authors.initials: S
authors.lastname: Lee
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000906:Antibodies / Q000032:analysis*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics; D010462:Peptostreptococcus / Q000737:chemistry / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics; D013211:Staphylococcus aureus / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D013291:Streptococcus / Q000737:chemistry / Q000235:genetics
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C034284:IgG Fc-binding protein, Streptococcus; D055767:Immobilized Proteins; C116593:L-protein, Peptococcus magnus; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A