Abstract
This chapter describes a technique for purifying autoantibodies off a membrane strip using small human sera volumes. This method used a membrane strip cut out from a western blot having a target antigen transferred electrophoretically from a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). This process is a valuable substitute for affinity column chromatography, especially when the protein of interest is of low abundance in a HeLa cell extract. The protein sample is separated on a preparative SDS polyacrylamide gel and transferred to a nitrocellulose membrane. A couple of strips are cut out from either side of the blotted membrane and immunoblotted with particular antisera to detect the protein of interest. Then, the targeted protein band is cut horizontally and used for affinity purification. We affinity-purified antibodies binding to a 70,000-molecular-weight protein obtained from HeLa cell extract using this procedure. A mock band, cut from an area away from the target antigen, served as a control band for mock purification of autoantibodies. The autoantibodies obtained in this way duplicated the multiple nuclear dot (MND) antinuclear antibody (ANA) pattern got with crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody.
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References
Kurien BT, Scofield RH (2006) Autoantibody determination in the diagnosis of systemic lupus erythematosus. Scand J Immunol 64:227–235. Review
Hansen BU, Eriksson S, Lindgren S (1991) High prevalence of autoimmune liver disease in patients with multiple nuclear dot, anti-centromere, and mitotic spindle antibodies. Scand J Gastroenterol 26:707–713
Bernstein RM, Neuberger JM, Bunn CC, Callender ME, Hughes GR, Williams R (1984) Diversity of autoantibodies in primary biliary cirrhosis and chronic active hepatitis. Clin Exp Immunol 55:553–560
Electrophoresis (1988) Appendix I. In: Harlow E, Lane D (eds) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 283–318
Kurien BT, Scofield RH (1998) Multiple nuclear dot antinuclear antibody in patients without primary biliary cirrhosis. Indian J Biochem Biophys 35:41–47
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Proc Natl Acad Sci U S A 76:4350–4354
Olmsted JB (1981) Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem 256:11955–11957
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Kurien, B.T. (2021). Purification of Autoantibodies Bound to an Autoantigen Immobilized on a Membrane Strip. In: Western Blotting for the Non-Expert. Techniques in Life Science and Biomedicine for the Non-Expert. Springer, Cham. https://doi.org/10.1007/978-3-030-70684-5_28
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DOI: https://doi.org/10.1007/978-3-030-70684-5_28
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