title: Delineating monoclonal antibody specificity by mass spectrometry.
abstract: Generation of monoclonal antibody (mAb) libraries against antigens in complex matrices can prove a valuable analytical tool. However, delineating the specificity of newly generated antibodies is the limiting step of the procedure. Here, we propose a strategy for mAb production by injecting mice with complex biological fluid and mAb characterization by coupling immunoaffinity techniques with Mass spectrometry (immuno-MS). Mice were immunized against fractionated seminal plasma and mAbs were produced. Different immuno-MS protocols based on four types of solid support (i.e. polystyrene microtiter plates, NHS-activated agarose beads, tosyl-activated magnetic beads and MSIA™ pipette tips) were established. A well-characterized mouse monoclonal anti-KLK3 (PSA) Ab was used as a model to evaluate each protocol's robustness and reproducibility and to establish a set of criteria which would allow antigen characterization of newly developed Abs. Three of the newly generated Abs were analyzed using our optimized protocols. Analysis revealed that all assay configurations used were capable of antibody characterization. Furthermore, low-abundance antigens (e.g. ribonuclease T2) could be identified as efficiently as the high-abundance ones. Our data suggest that complex biological samples can be used for the production of mAbs, which will facilitate the analysis of their proteome, while the established immuno-MS protocols can offer efficient mAb characterization.
  
  
  CONCLUSIONS
  The inoculation of animals with complex biological samples is aiming at the discovery of novel disease biomarkers, present in the biological specimens, as well as the production of rare reagents that will facilitate the ultra-sensitive analysis of the biomolecules' native form. In the present study, we initially propose a general workflow concerning the handling of biological samples, as well as the monoclonal antibody production. Furthermore, we established protocols for the reliable and reproducible identification of antibody specificity using various immuno-affinity purification techniques coupled to mass spectrometry. Our data suggest that processed biological fluids can be used for the production of mAbs targeting proteins of varying abundance, and that various immuno-MS protocols can offer great capabilities for the mAb characterization procedure.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; Department of Clinical Biochemistry, University Health Network, Toronto, Canada.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Korbakis
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Prassas
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Davor
authors.initials: D
authors.lastname: Brinc
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.affiliation: Thermo Fisher Scientific BRIMS, Cambridge, MA, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.affiliation: Thermo Fisher Scientific BRIMS, Cambridge, MA, USA.
authors.forename: Mary F
authors.initials: MF
authors.lastname: Lopez
authors.affiliation: 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. Electronic address: ediamandis@mtsinai.ca.
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D054392:Adipokines; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology; D002853:Chromatography, Liquid; D000939:Epitopes / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D017430:Prostate-Specific Antigen / Q000276:immunology; D053719:Tandem Mass Spectrometry; D000090203:Zn-Alpha-2-Glycoprotein
chemical_list: D054392:Adipokines; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D006023:Glycoproteins; D017430:Prostate-Specific Antigen; C488891:AZGP1 protein, human; D000090203:Zn-Alpha-2-Glycoprotein
keywords: Immuno-MS; MSIA™ pipette tips; Magnetic beads; Microtiter plates; Monoclonal antibodies; NHS-activated beads