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Isolation of HLA-G+ cells using MEM-G/9 antibody-conjugated magnetic nanoparticles for prenatal screening: a reliable, fast and efficient method - PubMed

  • ️Fri Jan 01 2021

. 2021 Sep 20;11(49):30990-31001.

doi: 10.1039/d1ra05988b. eCollection 2021 Sep 14.

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Isolation of HLA-G+ cells using MEM-G/9 antibody-conjugated magnetic nanoparticles for prenatal screening: a reliable, fast and efficient method

Elaheh Emadi et al. RSC Adv. 2021.

Abstract

The development of an effective and noninvasive early method for obtaining fetal cells is crucial to prenatal screening. Despite proving the presence of fetal cells in the reproductive tract, their use is limited due to their inability to properly isolate them from maternal cells. Magnetic-activated cell sorting (MACS) is a simple technique to separate cells. The present study aimed to develop a MACS-based platform for the isolation of the HLA-G expressing trophoblast cells. For this purpose, first, the triazine functionalized MNPs were synthesized and characterized. Then, MNPs were directly and indirectly conjugated by the MEM-G/9 antibodies targeting HLA-G+ cells. The antibody amount on the surface of the nanoparticles was determined with the Bradford assay. The cell capture efficiency was also investigated. Various characterization methods confirmed the successful nanoparticle synthesis and antibody conjugation. The optimal initial antibody amount for the immobilization was about 20 μg and the optimal time was 3 h. The antibody-nanoparticles by the indirect method had better targeting and capture efficiency than the direct method. The MNPs indirectly conjugated with antibodies are an efficient tool for cell isolation and present considerable potential to be applied in biomedical fields.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Schematic diagram of the synthesis route of the antibody immobilization on triazine-functionalized magnetic nanoparticles. Abs: antibodies, APTES: 3-aminopropyl triethoxysilane, TCT: trichlorotriazine, TEOS: tetraethyl orthosilicate, THF: tetrahydrofuran.
Fig. 2
Fig. 2. (A) Effect of the initial amount of HLA-G antibody on immobilization percentage (the red graph) and amount of immobilized antibody on nanoparticles (the blue graph). (B) Effect of the initial amount of anti-mouse IgG antibody on immobilization percentage (the red graph) and amount of immobilized antibody on nanoparticles (the blue graph). (C) Effect of reaction time on the immobilization percentage (the red graph) and amount of immobilized antibody on nanoparticles (the blue graph).
Fig. 3
Fig. 3. Characterization of MNPs and Ab-MNPs. (A) The FTIR spectra of (1) Fe3O4, (2) Fe3O4@SiO2, (3) Fe3O4@SiO2-APTES, (4) Fe3O4@SiO2-APTES-TCT and (5) Fe3O4@SiO2-APTES-TCT-Ab nanoparticles. (B) The TEM image (1) and size distribution histogram (2) of Fe3O4@SiO2-APTES-TCT nanoparticles. (C) The EDX spectrum of (1) Fe3O4, (2) Fe3O4@SiO2, (3) Fe3O4@SiO2-APTES and (4) Fe3O4@SiO2-APTES-TCT. (D) The Zeta potential analysis of Fe3O4, Fe3O4@SiO2, Fe3O4@SiO2-APTES and Fe3O4@SiO2-APTES-TCT. (E) Room temperature magnetization curves of Fe3O4@SiO2 (●), Fe3O4@SiO2-APTES (▲) and Fe3O4@SiO2-APTES-TCT (♦). (F) The TGA spectra of (a) Fe3O4@SiO2-APTES-TCT and (b) Ab-MNPs.
Fig. 4
Fig. 4. The fluorescent microscopy images of the reactivity of immobilized MEM-G/9 antibodies on Fe3O4@SiO2-APTES-TCT nanoparticles with the FITC-labeled IgG secondary antibodies (A) and the reactivity of BSA-blocked nanoparticles without MEM-G/9 antibody conjugation (control) with the FITC-labeled IgG secondary antibody (B). Scale bars = 5 μm, 1000× magnification.
Fig. 5
Fig. 5. The light microscopy images of Ab-MNPs tendency to be HLA-G-positive JEG-3 cells. Unlabeled cells with MEM-G/9 Ab incubated with IgG-MNPs (A) and JEG-3 cells incubated with the BSA-blocked MNPs without MEM-G/9 antibody conjugation (C) (as controls). Tendency of the MNPs conjugated indirectly to MEM-G/9 to target JEG-3 cells (B) and tendency of the MNPs conjugated directly to MEM-G/9 to target JEG-3 cells (D). Scale bars = 20 μm, 400× magnification.
Fig. 6
Fig. 6. Isolated JEG-3 cells by indirect immunomagnetic method (A) and by direct immunomagnetic method (B). Scale bars = 20 μm, 400× magnification. The cell capture efficiency in indirect vs. direct immunomagnetic method. Error bars display standard error of mean (SEM) and the asterisk displays a statistically significant difference (p ≤ 0.05).
Fig. 7
Fig. 7. The fluorescent images of cells in the precipitates after incubation with Ab-MNPs and separation under magnetic field. (A) The target JEG-3 cells isolated with the MNPs conjugated indirectly to MEM-G/9 from the non-target SKBR-3 cells (1–4). (B) The target JEG-3 cells isolated with the MNPs conjugated directly to MEM-G/9 (1–4). JEG-3 (DAPI = blue), SKBR-3 (PI = red) and MNPs (grey dots). Scale bars = 50 μm, 100× magnification. (C) The fluorescent image of cells in the supernatant after incubation with Ab-MNPs and separation under magnetic field. The HLA-G-positive JEG-3 cells not isolated by the Ab-MNPs (DAPI = blue) (1). The non-target SKBR-3 cells not isolated by the Ab-MNPs (PI = red) (2). Section (3) is a merging of (1) and (2) images. Scale bars = 50 μm, 100× magnification.

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