Browsing by Author "Leblebici, Pelin"
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- Monolith technology in isolation of human immunoglobulin G subclassesPublication . Leblebici, Pelin; Rodrigues, Alírio; Pais, Luís S.In the present study, a method was developed for the separation of hIgG into its subclasses with the advantages of monolithic technology. pH elution was applied using Protein A monolith column in a stepwise method and conditions were optimized. Since IgG3 has very low affinity to Protein A, elution was occurred immediately upon injection of the sample. IgG1 and IgG4 were eluted simultaneously under the applied conditions. Furthermore, IgG2 was separated with significant purity. According to the present results, monolithic Protein A column could be proposed as a time efficient separation media for the enrichment of IgG2 which is the most often directed antibody against bacterial pathogens.
- Separation of human immunoglobulin G subclasses on a protein A monolith columnPublication . Leblebici, Pelin; Leblebici, Mumin Enis; Ferreira-da-Silva, Frederico; Rodrigues, Alírio; Pais, Luís S.Monolithic columns have attracted significant attention for the purification of large biomolecules. In thepresent study, a step gradient elution method was evaluated for the separation of human immunoglobulinG (hIgG) into its subclasses on CIM (convective interaction media) r-protein A (recombinant protein A)monolithic column. hIgG was loaded onto the column and bound protein was eluted with a pH gra-dient. The subclass content of the eluted fractions was analyzed by enzyme-linked immunosorbentassay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be success-fully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealedthat enriched fractions of IgG1 and IgG2 could be obtained from purified hIgG in a 28 min long chro-matographic run. Three fractions with high IgG1 content (89.1%, 94.3% and 88.8%) were recovered.Furthermore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without anyadditives in buffers was proven to obtain enriched preparations of the two important subclasses withhigh throughput. The separation time can be reduced even more by increasing the flow rate without anyloss in selectivity, which will be beneficial in industrial scale applications.
- Separation of human immunoglobulin G subclasses using monolith technologyPublication . Leblebici, Pelin; Leblebici, Mumin Enis; Ferreira-da-Silva, Frederico; Ribeiro, António E.; Rodrigues, Alírio; Pais, Luís S.In the present study, a step gradient elution method was evaluated for the separation of human immunoglobulin G (hIgG) into its subclasses on CIM rwprotein A monolithic column. hIgG Seheme 1. Represantative structures of the human IgO subclasses [I). was loaded onto the column and bound protein was eluted with a pH gradient. The subclass content of the eluted fractions was analyzed by enzymewIinked immunosorbent assay (ELISA). Results showed that separation of IgG3 from the other three subclasses can be successfully achieved with high selectivity (100%) and throughput on monolithic media. It was also revealed that enriched fractions of IgG 1 and IgG2 could be obtained from purified hIgG in a 28 minutes long chromatographic run, Three fractions with high IgO 1 content (89.1%,94,3% and 88.8%) were recovered, Furthennore, IgG2 was enriched to 64% successfully. A rapid step gradient elution scheme without any additives in buffers was proven to obtain enriched prep~ions of the two important subclasses with high througbput.
- Separation of human immunoglobulin G subclasses using monolithic columnPublication . Leblebici, Pelin; Rodrigues, Alírio; Pais, Luís S.The present study aims to separate hIgG into its subclasses using monolithic stationary phase.