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Optimization of unnicked ?2-glycoprotein I and high avidity anti-?2-glycoprotein I antibodies isolation.

ABSTRACT: Patient biological material for isolation of ?2-glycoprotein I (?2GPI) and high avidity IgG anti-?2-glycoprotein I antibodies (HAv anti-?2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked ?2GPI and HAv a?2GPI to gain unmodified proteins in higher yields/purity. Isolation of ?2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv a?2GPI affinity chromatographies with immobilized protein G and human ?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 ?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 ?2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-?2GPI from different matrixes was 13.8%, as determined by our in-house anti-?2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked ?2GPI and HAv anti-?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%.

SUBMITTER: Artenjak A

PROVIDER: S-EPMC3987788 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Optimization of unnicked β2-glycoprotein I and high avidity anti-β2-glycoprotein I antibodies isolation.

Artenjak Andrej A Leonardi Adrijana A Križaj Igor I Ambrožič Aleš A Sodin-Semrl Snezna S Božič Borut B Cučnik Saša S

Journal of immunology research 20140123

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 ...[more]

PMID: 24741579

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Project description:BackgroundMore than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis.MethodsTo isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing.ResultsThe CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2-40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum.ConclusionsThe newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

Dataset Information

Optimization of unnicked ?2-glycoprotein I and high avidity anti-?2-glycoprotein I antibodies isolation.

Publications

Optimization of unnicked β2-glycoprotein I and high avidity anti-β2-glycoprotein I antibodies isolation.

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