title: Improved affinity coupling for antibody microarrays: engineering of double-(His)6-tagged single framework recombinant antibody fragments.
abstract: Antibody-based microarray is a novel technology with great promise in biomedicine that will provide unique means to perform global proteome analysis. In the process of designing the high-density antibody microarrays required, several critical key issues have been identified that remain to be resolved. In particular, there is a great need for specific and selective approaches enabling non-purified probes to be directly purified, orientated and coupled in a generic one-step procedure directly on the chip. In this study, we report on the successful design of affinity-tagged human recombinant single-chain fragment variable antibody fragments for improved affinity coupling in array applications. By replacing the standard single-histidine (His)(6)-tag with a consecutive double-(His)(6)-tag, the binding to Ni(2+)-nitrilotriacetic acid-coated substrates was significantly improved. Surface plasmon resonance analysis showed a significantly tighter binding with at least a threefold slower dissociation. The improved binding characteristics thus enabled non-purified probes even in the format of crude expression supernatants to be directly applied thereby eliminating the need for any time-consuming pre-purification step(s) prior to the immobilization. While the double-(His)(6)-tag probes were found to be expressed equally well as compared to the single-(His)(6)-tag probes, they displayed better long-term functional on-chip stability. Taken together, the results demonstrate the generic potential of double-(His)(6)-tag recombinant antibodies for the facile fabrication of high-density antibody microarrays.
authors.affiliation: Department of Immunotechnology, Lund University, Lund, Sweden.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Steinhauer
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
authors.forename: Farid
authors.initials: F
authors.lastname: Khan
authors.forename: Mingyue
authors.initials: M
authors.lastname: He
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Taussig
authors.forename: Carl A K
authors.initials: CA
authors.lastname: Borrebaeck
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D006639:Histidine