Preparation of a monoclonal antibody to citrullinated epitopes: Its characterization and some applications to immunohistochemistry in human brain

• Published in: Glia 2002-03, Vol.37 (4), p.328-336
• Main Authors: Nicholas, Anthony P., Whitaker, John N.
• Format: Article
• Language: English
• Subjects: Actin Cytoskeleton - immunology; Actin Cytoskeleton - metabolism; Actin Cytoskeleton - ultrastructure; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibody Specificity - immunology; astrocyte; Astrocytes - cytology; Astrocytes - immunology; Astrocytes - metabolism; Biological and medical sciences; Brain - cytology; Brain - immunology; Brain - metabolism; citrulline; Citrulline - immunology; Citrulline - metabolism; Epitope Mapping - methods; Epitopes - immunology; Fundamental and applied biological sciences. Psychology; glial fibrillary acidic protein; Glial Fibrillary Acidic Protein - metabolism; Guinea Pigs; Humans; Immunoglobulin M - immunology; Immunoglobulin M - isolation & purification; Immunohistochemistry - methods; Isolated neuron and nerve. Neuroglia; multiple sclerosis; myelin basic protein; Nerve Fibers, Myelinated - immunology; Nerve Fibers, Myelinated - metabolism; Nerve Fibers, Myelinated - ultrastructure; Vertebrates: nervous system and sense organs
• ISSN: 0894-1491; 1098-1136
• DOI: 10.1002/glia.10039

Using hybridoma technology, an IgM monoclonal antibody (mAb), designated as F95, was developed against a deca‐citrullinated peptide (DCP) consisting of 10 citrulline residues and a carboxyl Gly‐Gly‐Cys through which DCP was covalently linked to an activated carrier protein, keyhole limpet hemocyanin (KLH). Clones were selected on the basis of not reacting with human unmodified and noncitrullinated myelin basic protein (MBP), MBP‐C1, but reacting well with human citrullinated MBP (MBP‐C8). When tested by ELISA, this mAb demonstrated minimal reactivity with human MBP‐C1, varying reactivity with the C2–C5 isomers of human MBP, moderate binding with guinea pig MBP‐C8, and strong reactivity with human MBP‐C8. By ELISA, mAb F95 was directed predominantly against citrulline, not MBP, as revealed by its binding to DCP linked with activated KLH, bovine serum albumin (BSA), or ovalbumin (OA), but not with KLH, BSA, or OA alone. Immunohistochemistry of normal human brain demonstrated that F95 stained central nervous system myelin and a subset of astrocytes. Given the citrulline‐directed features of mAb F95, this immunohistochemical pattern suggests that certain astroglial filaments expressing glial fibrillary acidic protein also contain citrulline‐bearing components. These potentially implicate citrullinated proteins, notably in astroglial filaments, in a variety of normal and pathological neurobiological processes. GLIA 37:328–336, 2002. © 2002 Wiley‐Liss, Inc.