Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications

Author:

Al-Essa Mohamed K.1ORCID,Melzer Susanne2,Tarnok Attila34,Hadidi Kamal A.56,El-Khateeb Mohammed57

Affiliation:

1. Department of Physiology and Biochemistry, Faculty of Medicine , The University of Jordan , Amman 11942 , Jordan , Phone: +962 6 5355000-23477, Fax: +962 6 5300820

2. Clinical Trial Centre Leipzig , University of Leipzig , Leipzig , Germany

3. Translational Center for Regenerative Medicine (TRM)/Sächsischer Inkubator für Klinische Translation (SIKT) , Leipzig , Germany

4. Department of Therapy Validation, Fraunhofer-Institut für Zelltherapie und Immunologie IZI , Leipzig , Germany

5. Department of Pathology, Faculty of Medicine , The University of Jordan , Amman , Jordan

6. Deanship of Higher Studies , The University of Jordan , Amman , Jordan

7. National Center for Diabetes, Endocrinology and Genetics (NCDEG) , Amman , Jordan

Abstract

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.

Publisher

Walter de Gruyter GmbH

Subject

General Biochemistry, Genetics and Molecular Biology

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