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Background Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood. Methods Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor. These methods were compared with antibody selection directly from whole blood. The total yield of erythroblasts was determined for each method. Results Red cell lysis is not recommended because the lysis step cannot be well controlled. Density separation followed by antibody selection works well. However, a faster and simpler method, antibody selection directly from whole blood using Immunicon Ferrofluid and magnetic separators, works as well and has the potential to yield even more cells. Conclusions Considering the need for a simple and quick method for selecting fetal cells from maternal blood, we suggest selection directly from whole blood. Cytometry 45:267–276, 2001. © 2001 Wiley-Liss, Inc.

The ligand specificities and binding affinities of three recombinant pheromone binding proteins (PBPs) of two saturniid moths (genus Antheraea) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [3H]-6E,11Z-hexadecadienyl acetate and [3H]-4E,9Z-tetradecadienyl acetate. The new binding assay, in which nonspecific adsorption to a plastic vessel is suppressed by presaturation of the surface with a 1-alkanol, allows measurement of dissociation constants (KD) for lipophilic ligands for their carrier proteins. The three PBPs showed KD values for [3H]-6E,11Z-16:Ac and [3H]-4E,9Z-14:Ac between 0.6 and 30 microM, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligands. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:Ac than for 4E,9Z-14:Ac, while Aper-2 showed a 3.5-fold preference for binding the shorter chain compound. In addition, for the Apol-3 PBP, displacement of [3H]-6E,11Z-16:Ac binding by other pheromone components or analogs showed a clear trend in relative binding affinity: 6E,11Z-16:Ac > 4E,9Z-14:Ac > 6E,11Z-16:Al approximately 16:Ac > 6E,11Z-16:OH > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguously demonstrate the specificity of the PBP-pheromone interaction. Moreover, this assay offers the potential for determining ligand specificities for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.

The bacterially expressed recombinant pheromone binding protein (PBP) of Antheraea polyphemus was photoaffinity labeled with (6E,11Z)-[3H]hexadecadienyl diazoacetate, a photoactivatable analog of the naturally occurring acetate pheromone. Radiolabeled peptides were separated from an endoproteinase Lys-C digestion by HPLC and characterized by Edman degradation. The label was exclusively found in the Asp39-Lys58 fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be alpha-helical. The adjoining LAGCAINCLATK fragment, which is highly conserved in PBP sequences, was predicted to be a hydrophobic beta-strand and has been proposed to be important in recognition of the alkadienyl chain. Edman degradation confirmed the location of the covalently attached ligand at Thr44 of the smaller hydrophilic peptide. In addition, the synthesis of the newly identified pheromone component (4E,9Z)-tetradecadienyl acetate and its photoaffinity analog, (4E,9Z)-[3H]tetradecadienyl diazoacetate, is also described. Mapping of PBP photoaffinity labeled by (4E,9Z)-[3H]14:Dza revealed that the hydrophobic region Asp21-Lys38 adjacent to the primary binding domain Asp39-Lys58 contained a second modification site. The 14-carbon odorant molecule thus had two binding positions within the recognition site, while only a single binding position was available to the 16-carbon pheromone.

Pheromone specificity in the Lepidoptera is encoded in protein components of the antennal sensillum lymph and dendritic membrane. In this paper, we highlight recent work on the molecular determinants of pheromone binding affinity of pheromone binding proteins (PBPs) of three genera. First, we describe new cDNA sequences for Lymantria dispar (Lymantriidae) and Agrotis segetum (Noctuidae). These data enrich the conclusions derived from our functional studies. Secondly, we indicate how preparation of multimilligram quantities of the recombinant PBP 'Apol-3' (originally from Antheraea polyphemus) has provided a platform (i) to determine the ligand binding sites using photoaffinity labeling, (ii) to conduct structural analysis by CD and NMR, and (iii) to measure binding affinities using a new binding assay. Thirdly, we describe the use of expression-cassette PCR technology to prepare two related PBPs from Antheraea perneyi to test binding affinities of naturally-occurring homologous PBPs. Our results support a model in which ligand specificity for chain length, double bond position, and terminal functionality is partially encoded in the PBPs. We propose that the final decoding is accomplished when the PBP-pheromone complex activates a G-protein coupled seven-transmembrane domain receptor that contains recognition sites for both the presented pheromone and the presenting PBP.