The identification of novel autoantigens by means of serological screening of a cDNA expression library constructed from multiple sclerosis brain tissues

• Main Author: Green, Melanie Leslie Dawn, 1973-
• Other Authors: Memorial University of Newfoundland. Faculty of Medicine
• Format: Thesis
• Language: English
• Published: 1999
• Subjects: Multiple sclerosis > Immunological aspects; Multiple sclerosis > Etiology; Antigen-antibody reactions; Multiple Sclerosis > immunology; Autoantigens; RNA; Lambda; Newfoundland studies; University of Newfoundland
• Online Access: http://collections.mun.ca/cdm/ref/collection/theses3/id/165875

Thesis (M.Sc.)--Memorial University of Newfoundland, 1999. Medicine Bibliography: leaves 156-206 Multiple sclerosis is the most common demyelinating disease of the central nervous system. It typically affects young adults, and as with many autoimmune diseases, MS affects more women than men. -- A large number of studies have concentrated on the identification of the antigen(s) responsible for inciting MS, but the "causative agent(s)" has yet to be found Some of these studies demonstrated that T and B lymphocytes isolated from MS patients are reactive with autoantigens, such as myelin basic protein, myelin oligodendrocyte protein, proteolipid protein, and myelin associated protein, and with viruses, such as Epstein-Barr virus, measles, and varicella-zoster. However, a role for any of these antigens in the initiation of multiple sclerosis has not yet been established -- This study was undertaken as an attempt to identify potential autoantigens in multiple sclerosis by using a modification of the SEREX (serological identification of antigens by recombinant expression cloning) technique developed by Sahin et al. (1995). This technique, unlike many of the techniques previously used by investigators to identify autoantigens in MS, makes no α priori assumptions as to the identity of the autoantigen. Messenger RNA was isolated from multiple sclerosis brain tissues and used to construct a cDNA library in a lambda phage vector. This vector was transfected into Escherichia colU protein expression was induced with isopropyl β-D-thiogalactopyranoside, and the proteins were transferred to nitrocellulose membranes. The membranes were screened with patients5 sera and positive clones were detected with a color reaction which recognizes IgG in patients' sera bound to the recombinant protein. Positive clones were subcloned to clonality and sequenced, and the sequences compared with DNA and RNA sequences in various databases. Three positive clones were used as probes in Northern blotting experiments to determine their relative expression levels in various tissues. One of these clones was identified as testican, the other two appear to be related gene products of the clone F4 transmembrane protein and KIAA0530.