title: Determination of binding specificities in highly multiplexed bead-based assays for antibody proteomics.
abstract: One of the major challenges of antibody-based proteomics is the quality assurance of the generated antibodies to ensure specificity to the target protein. Here we describe a single tube multiplex approach to simultaneously analyze the binding of antibodies to a large number of different antigens. This bead-based assay utilizes the full multiplexing capacity theoretically offered by the Luminex suspension array technology. A protocol for an increased coupling throughput for the immobilization of antigens was developed and used to set up complex and stabile 100-plex bead mixtures. The possibility of using a two-dimensional multiplexing, in terms of high numbers of both analytes and samples or as in this case antigens and antibodies, enables the specificity of 96 antibodies versus 100 different antigens to be determined in 2 h. This high throughput analysis will potentially have great impact on the possibility for the utilization of different antibody proteomics approaches where the quality assessment of antibodies is of the utmost importance.
authors.affiliation: Department of Proteomics, Royal Institute of Technology (KTH), SE-10691 Stockholm, Sweden.
authors.forename: Jochen M
authors.initials: JM
authors.lastname: Schwenk
authors.forename: Johan
authors.initials: J
authors.lastname: Lindberg
authors.forename: Mårten
authors.initials: M
authors.lastname: Sundberg
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Peter
authors.initials: P
authors.lastname: Nilsson
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D007118:Immunoassay / Q000379:methods*; D008863:Microspheres; D040081:Protein Array Analysis / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies