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Use of immuno-magnetic beads for direct capture of nanosized microparticles from plasma
Yuana Yuana1, Susanne Osanto, Rogier M Bertina
1Department of Clinical Oncology, Leiden University Medical Centre, The Netherlands.
Abstract:
Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 μl platelet-poor plasma (PPP) was needed in combination with 135 μl (27 μg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 μl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.
Insights
A new immuno-magnetic bead method isolates microparticles carrying tissue factor (TF) activity from plasma. This technique identifies the cellular source of TF-carrying microparticles, crucial for understanding diseases like cancer and cardiovascular conditions.
Area of Science:
- Biochemistry
- Immunology
- Hematology
Background:
- Increased microparticle tissue factor (TF) activity is linked to cancer, cardiovascular, and inflammatory diseases.
- Current methods like flow cytometry can analyze microparticle subsets but cannot identify which carry active TF or their cellular origin.
Purpose of the Study:
- To develop and optimize an immuno-magnetic bead method for capturing microparticle subsets directly from plasma.
- To determine the cellular origin of microparticles exhibiting active TF.
Main Methods:
- An immuno-magnetic bead assay was developed using biotinylated anti-human CD41 monoclonal antibody (MoAb) and streptavidin beads for platelet-derived microparticle (PMP) capture from plasma.
- The method was validated using anti-CD14 MoAb to capture CD14-positive microparticles from monocyte-derived microparticles (MoMPs).
Main Results:
- The optimized method achieved complete separation of PMPs from 100 μl of platelet-poor plasma (PPP).
- TF activity was detected in both CD14-positive and CD14-negative fractions of MoMPs, indicating diverse cellular origins.
Conclusions:
- The developed immuno-magnetic bead method effectively captures microparticle subsets from plasma.
- This technique can identify the cellular source of TF-carrying microparticles, offering new insights for disease research.
