Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications
-
Mohamed K. Al-Essa
,
Susanne Melzer
Abstract
This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4′,6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.
Acknowledgments
The authors would like to thank DAAD and The University of Jordan for their support (Bilateral Exchange of Academics program, 50015739). Also, special thanks are given to the Translational Center for Regenerative Medicine (TRM) at Leipzig University, Jordan University Hospital, and National Center for Diabetes, Endocrinology and Genetics for their generous support and use of their facilities. Further, the authors thank the German Federal Ministry of Education and Research’s Translational Center for Regenerative Medicine TRM Leipzig (BMBF, PtJ-Bio, 315883) and MaDaKos (BMBF, 16N10872, 990101-088) for grant support.
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©2018 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications
- Chemical profiling of Di-Wu-Yang-Gan Granules by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MSE technology
- Design, synthesis, and antibacterial activity of novel 8-methoxyquinoline-2-carboxamide compounds containing 1,3,4-thiadiazole moiety
- Pseudomonas and Burkholderia inhibit growth and asexual development of Phytophthora capsici
- Antiproliferative activity of synthesized some new benzimidazole carboxamidines against MCF-7 breast carcinoma cells
- Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection
- Kostchyienones A and B, new antiplasmodial and cytotoxicity of limonoids from the roots of Pseudocedrela kotschyi (Schweinf.) Harms
- Antimicrobial and antioxidant activities of two polyketides from lichen-endophytic fungus Preussia sp.
Articles in the same Issue
- Frontmatter
- Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications
- Chemical profiling of Di-Wu-Yang-Gan Granules by ultra performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MSE technology
- Design, synthesis, and antibacterial activity of novel 8-methoxyquinoline-2-carboxamide compounds containing 1,3,4-thiadiazole moiety
- Pseudomonas and Burkholderia inhibit growth and asexual development of Phytophthora capsici
- Antiproliferative activity of synthesized some new benzimidazole carboxamidines against MCF-7 breast carcinoma cells
- Overexpression of c-Myc enhances recombinant protein production in High Five cells after baculovirus infection
- Kostchyienones A and B, new antiplasmodial and cytotoxicity of limonoids from the roots of Pseudocedrela kotschyi (Schweinf.) Harms
- Antimicrobial and antioxidant activities of two polyketides from lichen-endophytic fungus Preussia sp.
