Additional file 4: Figure S4. Anti-exosome nanobodies bind EV-fractions separated by chromatography. Flow cytometry experiments show that both H1 and H6 strongly bind to exosomes present in the fraction 1 separated by IEX chromatography

Ario Marco (4764750)

Barbara Toffoletto (3564728)

Elisa Mazzega (692883)

Milica Popovic (252546)

Microbial Cell Factories

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MOESM4 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Background: The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. Methods: Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. Results: Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. Conclusions: Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3233

Popović, Milica

Mazzega, Elisa

Toffoletto, Barbara

de Marco, Ario

Cherry - Repository of the Faculty of Chemistry;  University of Belgrade

Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Popovic, Milica

De Marco, Ario

Repository of University of Nova Gorica

Isolation of anti‑extra‑cellular vesicle single‑domain antibodies by direct panning on vesicle‑enriched fractions

Milica Popovic

Elisa Mazzega

Barbara Toffoletto

Ario de Marco

Crossref

Additional file 3: Figure S3. Antibody differential binding to cell-derived EVs. EVs derived from HEK-293, SKBR3, and Jurkat cells were used to evaluate the binding preferences of the nanobodies H1 and B1 compared with the binding of the irrelevant clone nbVHH (AâC). The binding capacity of a commercial anti-CD9 antibody was tested with the same cell lines (D). Bars indicate median percentage of positively stained EV coated beads with anti-CD9âPE antibodies to H1-GFP, B1-GFP, and non-binding VHH coated beads with respect to autofluorescence of unstained EV coated beads. The error bars indicate standard deviations for triplicate measurements

MOESM3 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

Popović, Milica M.

Faculty of Chemistry Repository - Cherry

Abstract Background The thorough understanding of the physiological and pathological processes mediated by extracellular vesicles (EVs) is challenged by purification methods which are cumbersome, not reproducible, or insufficient to yield homogeneous material. Chromatography based on both ion-exchange and immune-capture can represent an effective method to improve EV purification and successive analysis. Methods Cell culture supernatant was used as a model sample for assessing the capacity of anion-exchange chromatography to separate distinct EV fractions and to isolate nanobodies by direct panning on whole EVs to recover binders specific for the native conformation of EV-surface epitopes and suitable to develop EV immune-capture reagents. Results Anion-exchange chromatography of cell culture supernatant separated distinct protein-containing fractions and all of them were positive for CD9, a biomarker associated to some EVs. This suggested the existence of several EV fractions but did not help in separating EVs from other contaminants. We further isolated several nanobodies instrumental for implementing immune-affinity protocols. These were able to immobilize EVs from both cell culture supernatant and biological samples, to be used in ELISA, flow-cytometry, and immune-purification. Conclusions Here we report the first successful isolation of anti-EV nanobodies for the use in immunoaffinity-based EV capture by panning a phage library directly on partially purified EVs. This achievement paves the way for the application of direct EV panning for the discovery of novel antibody-vesicle surface biomarker pairs and represents the preliminary requirement for the development of selective immune-capture that, in combination with anion-exchange chromatography, can simplify the systematic stratification of EV sub-populations and their individual characterization

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Additional file 1: Figure S1. Chromatographic separation of kit-purified EV-enriched fraction from culture media. Kit-precipitated EVs present in SKBR3 cell culture supernatant were separated using a large-pore anion-exchange monolith column. All the four separated fractions were analyzed by flow-cytometry and resulted positive for the EV marker CD9

MOESM1 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

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MOESM4 of Isolation of anti-extra-cellular vesicle single-domain antibodies by direct panning on vesicle-enriched fractions

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Cherry - Repository of the Faculty of Chemistry; University of Belgrade

Repository of University of Nova Gorica

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Faculty of Chemistry Repository - Cherry

Directory of Open Access Journals

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