Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation. (23rd January 2014)

• Record Type: Journal Article
• Authors: Artenjak, Andrej; Leonardi, Adrijana; Križaj, Igor; Ambrožič, Aleš; Sodin-Semrl, Snezna; Božič, Borut; Čučnik, Saša
• Other Names: De Carvalho Jozélio Freire Academic Editor.
• Is Part Of: Journal of immunology research. Volume 2014(2014)
• Publication Date: 2014-01-23
• Subjects: Immunology -- Periodicals; Immunology -- Research -- Periodicals; 616.07905
• DOI: 10.1155/2014/195687
• Languages: English
• ISSNs: 2314-8861

Abstract

Abstract : Patient biological material for isolation of β 2-glycoprotein I ( β 2GPI) and high avidity IgG anti- β 2-glycoprotein I antibodies (HAv anti- β 2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β 2GPI and HAv a β 2GPI to gain unmodified proteins in higher yields/purity. Isolation of β 2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv a β 2GPI affinity chromatographies with immobilized protein G and human β 2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified β 2GPI increased from 6.56 mg to 9.94 mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from β 2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti- β 2GPI from different matrixes was 13.8%, as determined by our in-house anti- β 2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β 2GPI and HAv anti- β 2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%.