Purification of nuclear RNP for clinical and diagnostic application in a autoimmune experimental model

VALDEZ, J.C.; LEBLANC, J.G.; AJMAT, C.; ABBAS, L.; FIAD, L.; LORENTE, C.; VALVERDE, M.; HARO, M.I.

Tafi del Valle, Tucuman

Jornada; XXII Jornadas Científicas de la Asociación de Biología de Tucumán; 2005

Asociación de Biología de Tucumán

Mixed connective tissue disease (MCTD) is anautoimmune disorder associated with antiribonuclearprotein(U1 -snRNP) antibodies. The aim ofthis study was to obtain RNP to induce an experimentalmodel (in mice) and for use in ELISA assays. Materialsand methods: Purification of RNP: Blobel and Potter’smethods was used for the collection of the nuclei of ratliver. The RNP was purified by ultracentrifugation and byfractionated precipitation. The molecular weight of theisolated protein was determined by SDS -PAGE. ELISAassays: 96 well plates were sensitized with 1 mg RNP in100ml per well. We used MCTD patients´ sera as positivecontrols and normal individuals´ sera as negativecontrols. Sera of all induced animals were tested.Results and conclusions: the molecular weight of purifiedRNP was compatible with one fraction of U-1 snRNP (45kDa) and was reactive with the MCTD patients´ sera.Most animals inoculated with the RNP antigen werehighly reactive, others only somewhat. Non-inoculatedmice were all negative.