Purification of pituitary autoantigen by column liquid chromatography and chromatofocusing

Abstract

IntroductionPituitary autoantibodies can be determined both in patients with pituitary disease as well as patients with autoimmune endocrine diseases. The purpose of the study was to isolate and purify pituitary autoantigen using sera of patients and the microsomal fraction of the pituitary.Material and methodsTo isolate a pituitary autoantigen, patient sera were used, which showed a strong immune response to pituitary antigens. Pituitary microsomal fractions were prepared from pituitary tissue homogenates. In the study, sera of patients with pituitary disease, Addison and Graves’ disease were used. The initial stages were carried out by affinity chromatography on CN -Br sepharose column whereas purification was continued by column liquid chromatography on AcA54 Ultrogel. Chromatofocusing was performed by Polybuffer exchanger PBE 94.Results 125I-labeled pituitary antigens after isolation appeared in column chromatography in three peaks. The first peak contained 50-70 kDa proteins, the second peak – 17 to 22 kDa proteins and the third peak contains 125-iodides. Three fractions obtained from filtration on Ultrogel were separated in a polyacrylamide gel. In the first peak two bands 67 and 55 kDa appeared. The second peak contained low molecular weight substances, and the third peak contained 125I. The first peak from Ultrogel was isolated by chromatofocusing – the first peak with pH 5.9 and the second one with pH 4.9.ConclusionsIsolation and purification of pituitary autoantigen with the use of column liquid chromatography and chromatofocusing resulted in obtainment of two antigenic proteins of specific gravity of 67 and 55 kDa.

Incidence of pituitary autoantibodies in idiopathic diabetes insipidus

“…Finally, Sawicka et al . [31-33] observed that the incidence of pituitary antibodies in autoimmune endocrine diseases was 41% and that these antibodies react mostly with an autoantigen weighing 68 kDa. Despite the number of studies conducted so far, only a few pituitary autoantigens responsible for the autoimmune processes have been identified.…”