title: Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study.
abstract: Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.
authors.affiliation: Department of Neurology, Mayo Clinic; Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic; watzlawik.jens@mayo.edu.
authors.forename: Jens O
authors.initials: JO
authors.lastname: Watzlawik
authors.affiliation: Department of Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic; Department of Pediatric and Adolescent Medicine, Mayo Clinic.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kahoud
authors.affiliation: Department of Neurology, Mayo Clinic; Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic.
authors.forename: Bharath
authors.initials: B
authors.lastname: Wootla
authors.affiliation: Department of Neurology, Mayo Clinic; Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic.
authors.forename: Meghan M
authors.initials: MM
authors.lastname: Painter
authors.affiliation: Department of Neurology, Mayo Clinic; Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic.
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Warrington
authors.affiliation: Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic; Division of Neonatal Medicine, Mayo Clinic.
authors.forename: William A
authors.initials: WA
authors.lastname: Carey
authors.affiliation: Department of Neurology, Mayo Clinic; Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic; Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic; rodriguez.moses@mayo.edu.
authors.forename: Moses
authors.initials: M
authors.lastname: Rodriguez
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D006801:Humans; D007075:Immunoglobulin M; D051379:Mice; D019006:Neural Cell Adhesion Molecules; D009474:Neurons; D015398:Signal Transduction
chemical_list: D007075:Immunoglobulin M; D019006:Neural Cell Adhesion Molecules