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Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis.

Sørensen MD, Gonzalez Dosal R, Jensen KB, Christensen B, Kølvraa S, Jensen UB, Kristensen P - J. Biomed. Biotechnol. (2009)

Bottom Line: Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial.DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry.Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark.

ABSTRACT
Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising epsilon-Hb, one was chosen for further characterization, DAb1. DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry. Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express epsilon-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

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Flow cytometric analysis of (a) stained and nonstained K562. The lower circle shows the non-DAb1 stained cells while upper circle shows the stained cells (P1). All cells have been fixed, permeabilised, and PI stained before cytometry. (b) K562 cells spiked into paternal blood sample. Figure 3 shows 106 cell events. The P1 shows the population of stained K562 among the blood cells.
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fig3: Flow cytometric analysis of (a) stained and nonstained K562. The lower circle shows the non-DAb1 stained cells while upper circle shows the stained cells (P1). All cells have been fixed, permeabilised, and PI stained before cytometry. (b) K562 cells spiked into paternal blood sample. Figure 3 shows 106 cell events. The P1 shows the population of stained K562 among the blood cells.

Mentions: The lack of identified cells could be a result of the enrichment procedure; however, in order to avoid performing such enrichment, methods with a higher throughput such as FACS have to be applied. To explore the feasibility of using DAb1 to stain cells for flow cytometric analysis we mixed K562 cells stained with DAb1 with nonstained K562 cells, followed by FACS analysis. As can be seen in Figure 3(a) two pools appeared. Furthermore FACS was performed on blood sample spiked with K562 (Figure 3(b)). A population of cells is seen in the figure, consisting of K562 cells, confirmed by staining and morphology by microscopy (data not shown), thus providing proof of concept.


Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis.

Sørensen MD, Gonzalez Dosal R, Jensen KB, Christensen B, Kølvraa S, Jensen UB, Kristensen P - J. Biomed. Biotechnol. (2009)

Flow cytometric analysis of (a) stained and nonstained K562. The lower circle shows the non-DAb1 stained cells while upper circle shows the stained cells (P1). All cells have been fixed, permeabilised, and PI stained before cytometry. (b) K562 cells spiked into paternal blood sample. Figure 3 shows 106 cell events. The P1 shows the population of stained K562 among the blood cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712645&req=5

fig3: Flow cytometric analysis of (a) stained and nonstained K562. The lower circle shows the non-DAb1 stained cells while upper circle shows the stained cells (P1). All cells have been fixed, permeabilised, and PI stained before cytometry. (b) K562 cells spiked into paternal blood sample. Figure 3 shows 106 cell events. The P1 shows the population of stained K562 among the blood cells.
Mentions: The lack of identified cells could be a result of the enrichment procedure; however, in order to avoid performing such enrichment, methods with a higher throughput such as FACS have to be applied. To explore the feasibility of using DAb1 to stain cells for flow cytometric analysis we mixed K562 cells stained with DAb1 with nonstained K562 cells, followed by FACS analysis. As can be seen in Figure 3(a) two pools appeared. Furthermore FACS was performed on blood sample spiked with K562 (Figure 3(b)). A population of cells is seen in the figure, consisting of K562 cells, confirmed by staining and morphology by microscopy (data not shown), thus providing proof of concept.

Bottom Line: Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial.DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry.Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS).

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Aarhus, Denmark.

ABSTRACT
Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising epsilon-Hb, one was chosen for further characterization, DAb1. DAb1 binds to epsilon-Hb both in Western blots and immunocytochemistry. Several epsilon-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express epsilon-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

Show MeSH