open access

Vol 60, No 6 (2009)
Original papers
Published online: 2009-12-30
Submitted: 2013-02-15
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Isolation of pituitary autoantigen by affinity chromatography

Paweł Gut, Jerzy Kosowicz, Katarzyna Ziemnicka, Maciej Bączyk, Jerzy Sowiński
Endokrynologia Polska 2009;60(6):455-460.

open access

Vol 60, No 6 (2009)
Original papers
Published online: 2009-12-30
Submitted: 2013-02-15

Abstract


Introduction: In sera of pituitary disease patients and other autoimmune endocrine disease are detectable pituitary autoantibodies. Until now characterization of pituitary antigen is still unknown. The aim of our study was isolation and characterization of pituitary autoantigen by affinity chromatography. For isolation we have used microsomal fraction of human pituitary.
Material and methods: For immunoglobulins isolation have been used sera of pituitary disease, Addison disease and Graves-Basedow disease patients with detectable pituitary autoantibodies. For pituitary antigen isolation have been used microsomal fraction of human pituitary obtained by ultracentrifugation and solubilisation. Immunoglobulins isolation was performed on Sepharose 4B–Protein A. Immunosorbent was performed on CNBr-activated Cl-4B Sepharose. Desorbtion was conducting by 0.2 mol/L glicine and 1 mol/L, 3 mol/L guanidine. The estimation of isolated proteins was performed by immunoblotting.
Results: Isolation of immunoglobulins from patients sera was done 12 times receiving from 8.5 up to 13.5 mg of IgG from 1-1.5 ml of sera. In desorbtion we have received from 0.026 up to 0.150 mg of antigen proteins. For molecular weight estimation isolated proteins have been labeled by 125I and run on SDS/PAGE with autoradiography. Autoradiography shown us two lines with 67 kDa and 55 kDa and low weight protein line.
Conclusions: Isolation of pituitary autoantigen by affinity chromatography shown two different antigen proteins with 67 kDa and 55 kDa.

Abstract


Introduction: In sera of pituitary disease patients and other autoimmune endocrine disease are detectable pituitary autoantibodies. Until now characterization of pituitary antigen is still unknown. The aim of our study was isolation and characterization of pituitary autoantigen by affinity chromatography. For isolation we have used microsomal fraction of human pituitary.
Material and methods: For immunoglobulins isolation have been used sera of pituitary disease, Addison disease and Graves-Basedow disease patients with detectable pituitary autoantibodies. For pituitary antigen isolation have been used microsomal fraction of human pituitary obtained by ultracentrifugation and solubilisation. Immunoglobulins isolation was performed on Sepharose 4B–Protein A. Immunosorbent was performed on CNBr-activated Cl-4B Sepharose. Desorbtion was conducting by 0.2 mol/L glicine and 1 mol/L, 3 mol/L guanidine. The estimation of isolated proteins was performed by immunoblotting.
Results: Isolation of immunoglobulins from patients sera was done 12 times receiving from 8.5 up to 13.5 mg of IgG from 1-1.5 ml of sera. In desorbtion we have received from 0.026 up to 0.150 mg of antigen proteins. For molecular weight estimation isolated proteins have been labeled by 125I and run on SDS/PAGE with autoradiography. Autoradiography shown us two lines with 67 kDa and 55 kDa and low weight protein line.
Conclusions: Isolation of pituitary autoantigen by affinity chromatography shown two different antigen proteins with 67 kDa and 55 kDa.
Get Citation

Keywords

pituitary autoantigen; isolation; pituitary autoantibodies

About this article
Title

Isolation of pituitary autoantigen by affinity chromatography

Journal

Endokrynologia Polska

Issue

Vol 60, No 6 (2009)

Pages

455-460

Published online

2009-12-30

Bibliographic record

Endokrynologia Polska 2009;60(6):455-460.

Keywords

pituitary autoantigen
isolation
pituitary autoantibodies

Authors

Paweł Gut
Jerzy Kosowicz
Katarzyna Ziemnicka
Maciej Bączyk
Jerzy Sowiński

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