CD34+ stem cell counting using labeled immobilized anti-CD34 antibody onto magnetic nanoparticles and EasyCounter BC image cytometer

• Published in: Analytical biochemistry 2020-12, Vol.610, p.113929-113929, Article 113929
• Main Authors: Krasteva, Dimitrina R., Ivanov, Yavor L., Chervenkov, Triphon G., Gabrovska, Katya I., Godjevargova, Tzonka I.
• Format: Article
• Language: English
• Subjects: Antibodies - chemistry; Antibodies - immunology; Antibodies, Immobilized - chemistry; Antibodies, Immobilized - immunology; Antigens, CD34 - immunology; Antigens, CD34 - metabolism; CD34; CD45; Flow Cytometry - methods; Fluorescent Dyes - chemistry; Humans; Immunoassay; Leukocyte Common Antigens - immunology; Leukocyte Common Antigens - metabolism; Magnetic nanoparticles; Magnetite Nanoparticles - chemistry; Stem cells; Stem Cells - cytology; Stem Cells - metabolism
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113929

The ability of immobilized conjugate anti-CD34+ monoclonal antibody-dR110 and free conjugate anti-CD45+ monoclonal antibody-ATTO620 to precisely enumerate CD34+ stem cells and CD45+ cells in apheresis samples were evaluated. The conjugates anti-CD34+ antibody–dR110 and anti-CD45+– antibody-ATTO620 were prepared. Functionalized magnetic nanoparticles (MNPs) were synthesized. The anti-CD34+ antibody–dR110 conjugate was immobilized on the modified MNPs using a carbodiimide method. The stem cell count in thawed apheresis samples was determined using the free and the immobilized conjugate anti-CD34+ antibody–dR110 on MNPs and an image cell counter EasyCounter BC. A higher stem cell count and more accurate results were obtained with the immobilized conjugate, because a separation and concentration of the stem cells bound to antibody–dR110 on MNPs by external magnet were performed. Coefficients of variation of CD34+ cell count in apheresis samples, determined by EasyCounter BC, were ranged from 5.5 to 6.9% and those of CD45+ cell count from 3.8 to 4.7%. The viability of CD34+ cells was high from 98.5 to 99.6%. It was found that correlation coefficient between the flow cytometer and automatic cell counter, using free anti-CD34+ antibody–dR110 was 0.94, and when using immobilized anti-CD34+antibody–dR110 on MNPs, the correlation coefficient was 0.97. [Display omitted] •The obtained immobilized conjugate anti-CD34+ monoclonal antibody-dR110 were enumerated CD34+ stem cells in apheresis samples.•The separation and the concentration of the stem cells bound to antibody–dR110 on MNPs by external magnet were performed.•Accurate results were obtained with the immobilized conjugate anti-CD34+ monoclonal antibody-dR110 and automatic EasyCounter BC.