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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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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.
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fig1: 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.

Mentions: Sequencing of these 12 potentially specific antibodies revealed four different clones; one was represented 8 times (DAb1), one was represented twice (1.9A), and two were represented once (3.9G and 4.8E). The repeated representation of clones is normal, when performing more than one round of selection. Clone DAb1, carried an Amber stop codon within the reading frame. This was restored using site directed mutagenesis. The four different phage antibodies, were tested in Western blot against a control protein carrying a His-tag, extract from RBC, K562 cell extract, and the recombinant ε-Hb (Figure 1). The four antibodies all recognised purified ε-Hb together with one or more proteins in the K562 extract. Several bands were present in the lane containing the recombinant ε-Hb, representing monomers, dimmers, and higher complexes of the ε-Hb chain. None of the antibodies recognised the His-tag protein; however, clone 3.9G showed a very weak recognition of one protein in the RBC extract.


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: 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.
Mentions: Sequencing of these 12 potentially specific antibodies revealed four different clones; one was represented 8 times (DAb1), one was represented twice (1.9A), and two were represented once (3.9G and 4.8E). The repeated representation of clones is normal, when performing more than one round of selection. Clone DAb1, carried an Amber stop codon within the reading frame. This was restored using site directed mutagenesis. The four different phage antibodies, were tested in Western blot against a control protein carrying a His-tag, extract from RBC, K562 cell extract, and the recombinant ε-Hb (Figure 1). The four antibodies all recognised purified ε-Hb together with one or more proteins in the K562 extract. Several bands were present in the lane containing the recombinant ε-Hb, representing monomers, dimmers, and higher complexes of the ε-Hb chain. None of the antibodies recognised the His-tag protein; however, clone 3.9G showed a very weak recognition of one protein in the RBC extract.

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