Epsilon haemoglobin specific antibodies with applications in noninvasive prenatal diagnosis

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.

Figure 1

Western blots performed with antibodies, DAb1(a), 1.9A(b), 4.8E(c), and 3.9G(d). The four different clones each recognises the recombinant ε-Hb, giving a correct band of 17 kDa (ε-Hb protein fused with His-Tag; adding 8 amino acids). Furthermore they all recognise a slightly smaller band of 146 amino acid residues in the K562 extract, containing the ε-Hb. A difference is observed for the recognised band in the lane containing the recombinant ε-Hb and the ε-Hb in the K562 extract. Lane H: a control his-tagged protein, R: adult red blood cell extract, K: K562 cell extract, and E: recombinant ε-Hb chain (2,5 μg). (e) Representative ponceau staining of the blotted proteins. M: the marker.

Figure 2

Staining of enriched maternal blood sample taken as post-CVS. First enrichment performed by CD-71 directed MACS. Subsequently cells were mounted on glass slides and permeabilized with methanol followed by immunostaining with FITC conjugated DAb1 (30 μg/mL). Subsequently FISH was performed to karyotype the cells. Embryonic nucleated red blood cells (primitive erythroblasts) identified with DAb1, directly conjugated with FITC (green) is shown. Subsequently, FISH was performed against both the X- (blue) and Y-chromosomes (red), confirming the origin of the immunostained cells. X-chromosomes of nonimmunostained nucleated maternal cells (seen as blue dots), are located around the identified foetal erythroblasts.

Figure 3

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.