Abstract
A method for affinity purification of autoantibodies from a membrane strip using small volumes of human sera is described. The membrane strip is excised from a western blot containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance in a HeLa cell extract. The protein mixture is resolved on a preparative SDS polyacrylamide gel and transferred to nitrocellulose membrane. A couple of strips are excised from either side of the blotted membrane and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification. We have used this procedure to affinity purify antibodies to a 70,000 molecular weight protein derived from HeLa cell extract. A sham band, excised away from the target antigen, was used as a control for sham purification of autoantibodies. The autoantibodies purified in this manner reproduced the multiple nuclear dot anti-nuclear antibody pattern obtained using 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
Harlow E, Lane D (1988) Electrophoresis. Appendix I. In: Harlow E, Lane D (eds) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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
Acknowledgement
This work was supported by NIH grant ARO1844 and Oklahoma Center for the Advancement of Science and Technology to RHS.
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Kurien, B.T. (2015). Membrane Strip Affinity Purification of Autoantibodies. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_27
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DOI: https://doi.org/10.1007/978-1-4939-2694-7_27
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2693-0
Online ISBN: 978-1-4939-2694-7
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