title: Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids.
abstract: Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation. We produced mouse monoclonal antibodies, which detect in human serum or seminal plasma the native form of the human testis-expressed sequence 101 (TEX101) protein-a recently proposed biomarker of male infertility. Pairing of two monoclonal antibodies against unique TEX101 epitopes led to the development of an ELISA for the measurement of TEX101 in seminal plasma (limit of detection: 20 pg/ml) and serum (limit of detection: 40 pg/ml). Measurements of matched seminal plasma samples, obtained from men pre- and post-vasectomy, confirmed the absolute diagnostic specificity and sensitivity of TEX101 for noninvasive identification of physical obstructions in the male reproductive tract. Measurement of male and female serum samples revealed undetectable levels of TEX101 in the systemic circulation of healthy individuals. Immunocapture-SRM screening may facilitate development of monoclonal antibodies and immunoassays against native forms of challenging protein targets.
authors: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, Canada;
authors: Dimitrios
authors: D
authors: Korbakis
authors: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada;
authors: Davor
authors: D
authors: Brinc
authors: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada;
authors: Christina
authors: C
authors: Schiza
authors: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada;
authors: Antoninus
authors: A
authors: Soosaipillai
authors: ‖Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; **Department of Surgery, Division of Urology, Mount Sinai Hospital, University of Toronto, Canada.
authors: Keith
authors: K
authors: Jarvi
authors: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, Canada; ediamandis@mtsinai.on.ca andrei.drabovich@utoronto.ca.
authors: Andrei P
authors: AP
authors: Drabovich
authors: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; §Department of Clinical Biochemistry, University Health Network, Toronto, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada; ‖Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada; ediamandis@mtsinai.on.ca andrei.drabovich@utoronto.ca.
authors: Eleftherios P
authors: EP
authors: Diamandis
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D006825:Hybridomas; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008565:Membrane Proteins / Q000097:blood / Q000276:immunology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D012661:Semen / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008565:Membrane Proteins; C539319:TEX101 protein, human