Cargando…

Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation

Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified prot...

Descripción completa

Detalles Bibliográficos
Autores principales: Artenjak, Andrej, Leonardi, Adrijana, Križaj, Igor, Ambrožič, Aleš, Sodin-Semrl, Snezna, Božič, Borut, Čučnik, Saša
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987788/
https://www.ncbi.nlm.nih.gov/pubmed/24741579
http://dx.doi.org/10.1155/2014/195687
_version_ 1782311934172856320
author Artenjak, Andrej
Leonardi, Adrijana
Križaj, Igor
Ambrožič, Aleš
Sodin-Semrl, Snezna
Božič, Borut
Čučnik, Saša
author_facet Artenjak, Andrej
Leonardi, Adrijana
Križaj, Igor
Ambrožič, Aleš
Sodin-Semrl, Snezna
Božič, Borut
Čučnik, Saša
author_sort Artenjak, Andrej
collection PubMed
description Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified proteins in higher yields/purity. Isolation of β2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aβ2GPI affinity chromatographies with immobilized protein G and human β2GPI 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 β2GPI 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 β2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-β2GPI from different matrixes was 13.8%, as determined by our in-house anti-β2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β2GPI and HAv anti-β2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%.
format Online
Article
Text
id pubmed-3987788
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Hindawi Publishing Corporation
record_format MEDLINE/PubMed
spelling pubmed-39877882014-04-16 Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation Artenjak, Andrej Leonardi, Adrijana Križaj, Igor Ambrožič, Aleš Sodin-Semrl, Snezna Božič, Borut Čučnik, Saša J Immunol Res Research Article Patient biological material for isolation of β2-glycoprotein I (β2GPI) and high avidity IgG anti-β2-glycoprotein I antibodies (HAv anti-β2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked β2GPI and HAv aβ2GPI to gain unmodified proteins in higher yields/purity. Isolation of β2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aβ2GPI affinity chromatographies with immobilized protein G and human β2GPI 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 β2GPI 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 β2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-β2GPI from different matrixes was 13.8%, as determined by our in-house anti-β2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked β2GPI and HAv anti-β2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%. Hindawi Publishing Corporation 2014 2014-01-23 /pmc/articles/PMC3987788/ /pubmed/24741579 http://dx.doi.org/10.1155/2014/195687 Text en Copyright © 2014 Andrej Artenjak et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Artenjak, Andrej
Leonardi, Adrijana
Križaj, Igor
Ambrožič, Aleš
Sodin-Semrl, Snezna
Božič, Borut
Čučnik, Saša
Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title_full Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title_fullStr Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title_full_unstemmed Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title_short Optimization of Unnicked β2-Glycoprotein I and High Avidity Anti-β2-Glycoprotein I Antibodies Isolation
title_sort optimization of unnicked β2-glycoprotein i and high avidity anti-β2-glycoprotein i antibodies isolation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987788/
https://www.ncbi.nlm.nih.gov/pubmed/24741579
http://dx.doi.org/10.1155/2014/195687
work_keys_str_mv AT artenjakandrej optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT leonardiadrijana optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT krizajigor optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT ambrozicales optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT sodinsemrlsnezna optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT bozicborut optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation
AT cucniksasa optimizationofunnickedb2glycoproteiniandhighavidityantib2glycoproteiniantibodiesisolation