Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations
High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates....
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| Format: | Article |
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MDPI AG
2021-02-01
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| Series: | Biomedicines |
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| Online Access: | https://www.mdpi.com/2227-9059/9/2/206 |
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| author | Rikke W. Rasmussen Jaco Botha Frederik Prip Mathilde Sanden Morten H. Nielsen Aase Handberg |
| author_facet | Rikke W. Rasmussen Jaco Botha Frederik Prip Mathilde Sanden Morten H. Nielsen Aase Handberg |
| author_sort | Rikke W. Rasmussen |
| collection | DOAJ |
| description | High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs. |
| first_indexed | 2024-03-09T00:45:24Z |
| format | Article |
| id | doaj.art-236dbb89354942c09aedefa361700a70 |
| institution | Directory Open Access Journal |
| issn | 2227-9059 |
| language | English |
| last_indexed | 2024-03-09T00:45:24Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomedicines |
| spelling | doaj.art-236dbb89354942c09aedefa361700a702023-12-11T17:30:31ZengMDPI AGBiomedicines2227-90592021-02-019220610.3390/biomedicines9020206Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV PopulationsRikke W. Rasmussen0Jaco Botha1Frederik Prip2Mathilde Sanden3Morten H. Nielsen4Aase Handberg5Department of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkDepartment of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkDepartment of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkDepartment of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkDepartment of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkDepartment of Clinical Biochemistry, Aalborg University Hospital, 9000 Aalborg, DenmarkHigh-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.https://www.mdpi.com/2227-9059/9/2/206high-resolution flow cytometryextracellular vesiclesprotein aggregates |
| spellingShingle | Rikke W. Rasmussen Jaco Botha Frederik Prip Mathilde Sanden Morten H. Nielsen Aase Handberg Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations Biomedicines high-resolution flow cytometry extracellular vesicles protein aggregates |
| title | Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations |
| title_full | Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations |
| title_fullStr | Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations |
| title_full_unstemmed | Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations |
| title_short | Zoom in on Antibody Aggregates: A Potential Pitfall in the Search of Rare EV Populations |
| title_sort | zoom in on antibody aggregates a potential pitfall in the search of rare ev populations |
| topic | high-resolution flow cytometry extracellular vesicles protein aggregates |
| url | https://www.mdpi.com/2227-9059/9/2/206 |
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