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. 2017 Oct 11;17(20):3462-3473.
doi: 10.1039/c7lc00402h.

Detection of Membrane-Bound and Soluble Antigens by Magnetic Levitation

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Free PMC article

Detection of Membrane-Bound and Soluble Antigens by Magnetic Levitation

Mikkel Schou Andersen et al. Lab Chip. .
Free PMC article

Abstract

Magnetic levitation is a technique for measuring the density and the magnetic properties of objects suspended in a paramagnetic field. We describe a novel magnetic levitation-based method that can specifically detect cell membrane-bound and soluble antigens by measurable changes in levitation height that result from the formation of antibody-coated bead and antigen complex. We demonstrate our method's ability to sensitively detect an array of membrane-bound and soluble antigens found in blood, including T-cell antigen CD3, eosinophil antigen Siglec-8, red blood cell antigens CD35 and RhD, red blood cell-bound Epstein-Barr viral particles, and soluble IL-6, and validate the results by flow cytometry and immunofluorescence microscopy performed in parallel. Additionally, employing an inexpensive, single lens, manual focus, wifi-enabled camera, we extend the portability of our method for its potential use as a point-of-care diagnostic assay.

Conflict of interest statement

Conflicts of interest

IG, AS and NS are co-inventors of the MELISSA technique, which is covered by a pending patent.

Figures

Figure 1
Figure 1. Magnetic levitation setup and the concept of average density
A. An Olympus Provis AX70 microscope was placed on its side to capture the images of the capillary tube. The microscope has three micro-manipulators, allowing for both horizontal and vertical search of the capillary tube B. Magnetic levitation setup showing the top and bottom magnet with the capillary tube mounted on an adjustable stage. C. Schematic of the capillary tube mounted between two magnets. D. An example of the detection of binding events with beads of different densities spiked with minimum number of cells. Upon cell-bead interaction, a bead-bead complex levitates between the high and low density beads. E. Schematic drawing of D.
Figure 2
Figure 2. Detection of Eosinophil and T cell-specific antigens by magnetic levitation
A. Schematic drawing of the formation of a bead-cell complex. B. Magnetic levitation of PBMCs with anti-CD3 antibody-coated beads showing formation of bead-CD3 positive T-cell complexes. The panel on the right shows the Gaussian fit of the whole image from the bitmap analysis (applies for all magnetic levitation images). C. Flow cytometry of the anti-CD3 antibody treated PBMCs identifying CD3 positive T-cells as approximately 27.4% of total cells. D. Magnetic levitation of PBMCs with IgG-control beads showing the absence of bead-cell complexes. E. Flow cytometry of PBMCs with IgG control F. Magnetic levitation of PMNs with anti-Siglec-8 antibody-coated identifying eosinophils. Inset showing magnified image of a bead-cell complex. G. Flow cytometry of the anti-Siglec-8-treated PMNs identifying eosinophils as approximately 3.0% of total cells. H. Magnetic levitation of PMNs with IgG-control beads showing the absence of bead-cell complexes. I. Flow cytometry of PMNs with IgG control. The experiments were performed twice.
Figure 3
Figure 3. Detection of red cell membrane antigens by magnetic levitation
A. Schematic drawing of formation of an antibody-coated bead-RBC complex. B–E. Magnetic levitation of RBCs with anti-CR1-coated beads from a low-CR1 expresser (B) compared to a high-CR1 expresser (D) with bitmap analyses differentiating the low and high CR1 expression levels. Flow cytometry of RBCs with anti-CR1 antibody performed in parallel confirms low (C) and high (E) CR1 expression levels. F. Magnetic levitation of Rh(−) RBCs spiked 1:100 with Rh(+) cells with anti-RhD antibody-coated beads showing the formation of bead-Rh(+) cell complexes. G. Magnetic levitation of Rh(+) RBCs with IgG control beads showing absence of bead-cell complexes H. Magnetic levitation of RBCs with anti-CD47-coated beads showing formation of bead cell complex. I. Magnetic levitation of RBCs with IgG control beads showing the absence of bead-cell complexes. The experiments were performed 3 times with two different sets of CR1 high and low expressers.
Figure 4
Figure 4. Detection of RBC-bound Epstein-Barr viral particles
A. Schematic drawing of detection of EBV bound to RBC antigen CR1 with anti-EBV antibody-coated beads. B. Magnetic levitation of RBCs with IgG control beads showing the absence of bead cell complex. C. Magnetic levitation of RBCs with anti-EBV antibody-coated beads showing the formation of bead cell complex. Inset showing magnified image of this interaction. D. The left frame shows bright field image of RBCs. The right frame shows magnified image of the square (upper left; DIC technique), TxRed-labeled CR1 (upper right), FITC-labeled EBV (lower left) and merged image (lower right). Arrows show co-localization (yellow) of CR1 and EBV. The white line represents 25 µM. E. Flow cytometry of EBV-negative (red) and EBV-positive (blue) RBCs performed in parallel. The experiment was performed four times.
Figure 5
Figure 5. Detection of soluble antigens by magnetic levitation
A. Schematic drawing of the interaction between a soluble antigen with detection antibody-coated 1.05 g/mL goat anti-mouse bead and capture antibody-coated 1.2 g/mL PMMA bead. B. Line graph showing the dose-response relationship between IL-6 concentration and the number of bead-bead complexes. C. Left panel: Control arm showing background binding of the capture and detection beads in the absence of IL-6. Right panel: Experimental arm showing the formation of bead-bead complexes in the presence of 10 pg/mL IL-6. The experiment was performed twice.
Figure 6
Figure 6. Detection of RBC CR1 antigen using Mini-MeLISSA
A. The compact Mini- MeLISSA B. Left panel: Deconstructed Mini-MeLISSA showing the two module pieces. The left is the light module that has a built-in 2.5 V LED light. The right is the camera shell module, housing the WiFi-enabled camera and the magnetic levitation setup with the two magnets and room for a squared 1×1 mm capillary tube, as described above. The specialized lens is placed behind the magnets with a fixed focus. Right: Upper-side-view of the two-piece module showing the light module to the left and the camera on the right. C. A cell phone image of anti-CR1 antibody-coated bead and RBC complexes from a high CR1 expresser taken with Mini-MeLISSA. Magnified image below shows bead cell complexes further enhanced to the right. Magnetic levitation of RBCs from the same donor with IgG control beads shows the absence of bead-cell complexes.

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