Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

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.

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

BACKGROUND: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells. METHODS: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition. The ability of erythroid lineage cells to adhere to several surface modifications was examined. In situ hybridization methods were tested to find the best approach that is compatible with these cell preparations. RESULTS: The best glass slide coating for erythroid cells was found to be an antibody to glycophorin A, a red cell surface antigen. We were able to get excellent in situ hybridization signals in cells on flaskettes by modifying fixation and pretreatment parameters. CONCLUSIONS: The methods described here appear to be the best way of attaching a large number of erythroid lineage cells to slides and of detecting them by in situ hybridization.

Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target.

We have investigated whether maternal peripheral blood from the first trimester of pregnancy is a reliable source of identifiable trophoblast cells. The cells were enriched from 30 ml of venous blood, with multiple antibodies shown previously to enrich trophoblasts and a new cocktail based on known trophoblast surface features. Three different magnetic solid phases were tested to enrich trophoblasts, and both positive and negative cell enrichment strategies were examined. The cells were identified as trophoblast by morphology coupled with immunocytochemistry to co-localize cytokeratin with one of three IGF-II, PAI-1 or hPLH proteins or by in-situ hybridization with a mixture of 50 oligos directed to eight different expressed genes, alpha-HCG, IGF-II, PAI-1, HASH2, hPLH, p57(KIP2), PP5, H-19. While these tools worked beautifully in chorionic villi cell/sprout preparations and tissue sections, we could not detect and identify any trophoblasts in maternal peripheral blood even if the maternal peripheral blood was drawn 5-20 min following termination of pregnancy or from individuals maintaining the pregnancy. Based on our own experience and that of some reports in the literature, trophoblasts do not appear to be a viable candidate for fetal screening using maternal peripheral blood as the source. It is important to note that while trophoblast deportation is a biological phenomenon that has been described repeatable, they do not provide a means to perform prenatal genetic diagnosis.

Enrichment and identification of fetal trophoblast cells from first trimester maternal cervical lavage and uterine blood specimens.

First trimester prenatal diagnosis of fetal aneuploidies is an active area of research despite years of disappointing data employing maternal peripheral blood samples. To remedy this situation we have investigated other first trimester maternal specimens attempting to find a consistent fetal cell source. Using our previously established positive enrichment procedure along with a commercially available depletion method, fetal trophoblast cells were identified employing immunocytochemistry using an antibody cocktail or by using mRNA in-situ hybridization employing a cocktail of trophoblast specific probes. Fetal origin of positively identified cells was verified using interphase fluorescent in-situ hybridization (FISH) for X and Y-chromosomes. Artificial model systems were established that indicated yields of trophoblast cells and allowed the enrichment procedure to be optimized for minimal losses from maternal specimens. We demonstrate herein that blood drawn from maternal vessels near the placental implantation site to be the most consistent source of fetal cells from any first trimester maternal specimen described to date. In addition, a high yield of multinucleated syncytiotrophoblast cells was obtained using a cell depletion strategy to enrich the target cells. The safety of the procedure or even the clinical utility of blood drawn from maternal vessels near the placental implantation site is yet to be demonstrated.

Cloning and characterization of human erythroid membrane-associated protein, human ERMAP.

We describe here the cloning and characterization of the human gene ERMAP, identified by subtractive hybridization using early and late gestation human fetal liver. By in situ hybridization, we found human ERMAP to be expressed not only in erythoid cells in fetal liver and adult bone marrow, but also in reticulocytes and circulating erythroblasts in 8-12-week fetal cord blood. The human ERMAP protein is predicted to contain a transmembrane segment and one extracellular immunoglobulin fold (IgV). The cytoplasmic region contains a highly conserved B30.2 motif, multiple consensus sequences for kinases, and post-Golgi sorting signals. The protein was localized to the cell surface as shown by an antibody specific for a peptide predicted from the IgV fold. The amino acid sequence of human ERMAP is highly homologous with that of mouse ERMAP, but differs in the number of extracellular immunoglobulin folds. Human ERMAP represents a new unique member of the rapidly growing B30.2 domain proteins.

A growth signal with an artificially induced erythropoietin receptor-gp130 cytoplasmic domain heterodimer.

We report a strategy for generating efficient signal transduction with unnatural heterologous receptor combinations. As previously described [Ueda, H., Kawahara, M. et al. (2000) J. Immunol. Methods 241, 159-170], chimeric receptors composed of the V(H)/V(L) domains of anti-hen egg lysozyme antibody HyHEL-10 and N-terminally truncated erythropoietin receptor (EpoR) can be activated by lysozyme. When the cytoplasmic domains of these receptors were substituted with one derived from gp130, IL-3 dependent Ba/F3 cells expressing both V(H)-gp130 and V(L)-gp130 grew dose-dependently when given lysozyme without IL-3. However, cells expressing the heterologous pair of V(H)-gp130 and V(L)-EpoR also showed more efficient and stricter lysozyme-dependent proliferation in the absence of IL-3, indicating this combination is as an efficient and strict signal transducer as wild-type EpoR. The immunoprecipitation data indicated the existence of a preformed V(H)-gp130 and V(L)-EpoR heterodimer in the absence of lysozyme, suggesting the crucial role of a receptor conformational change in signal triggering as well as wild-type EpoR and gp130. Phosphorylation of JAK2, STAT3, and STAT5 was observed upon the addition of lysozyme, suggesting the activation of both EpoR- and gp130-derived signals.

Replacing factor-dependency with that for lysozyme: affordable culture of IL-6-dependent hybridoma by transfecting artificial cell surface receptor.

Cytokines and growth factors are indispensable for the propagation and maintenance of factor-dependent mammalian cells. However, cytokines are often so expensive that the use of factor-dependent cells for industrial applications such as protein production is often not practical. Based on our previous design of a binary hen egg lysozyme (HEL)-specific receptor composed of portions of the anti-HEL antibody and the erythropoietin receptor, a new pair of chimeric receptors having the intracellular domain of gp130 were made and transfected to an interleukin-6 (IL-6)-dependent hybridoma, 7TD1. The clone expressing the two new receptors showed clear HEL dose-dependent cell growth and monoclonal antibody production in both serum-based and serum-free media without IL-6. These results establish the feasibility of applying receptor design to tailor cells for the inexpensive induction of cell growth for the purpose of producing therapeutic products.

Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer.

We describe a noncompetitive homogeneous bioluminescent immunoassay based on the antigen-dependent reassociation of antibody variable domains (open sandwich bioluminescent immunoassay, OS-BLIA). The reassociation of two chimeric proteins, an antibody heavy-chain fragment (V(H))-Renilla luciferase (Rluc) and an antibody light-chain fragment (V(L))-enhanced yellow fluorescent protein (EYFP), was monitored by a bioluminescence resonance energy transfer (BRET) between the two. Upon simple mixing of the reagents with the sample, an antigen-dependent increase in BRET was observed with a measurable concentration range of 0.1 to approximately 10 microg/ml antigen hen egg lysozyme. Compared with our comparable assays based on fluorescence resonance energy transfer (FRET), a 10-fold improvement in the sensitivity was attained, probably due to a reduction in reagent concentration.

Human homolog of the MutY repair protein (hMYH) physically interacts with proteins involved in long patch DNA base excision repair.

The human MutY homolog (hMYH) is a DNA glycosylase involved in the removal of adenines or 2-hydroxyadenines misincorporated with template guanines or 7,8-dihydro-8-oxodeoxyguanines. hMYH is associated in vivo with apurinic/apyrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and replication protein A (RPA) in HeLa nuclear extracts as shown by immunoprecipitation and Western blotting. However, binding of hMYH to DNA polymerases beta and delta was not detected. By using constructs containing different portions of hMYH fused to glutathione S-transferase, we have demonstrated that the APE1-binding site is at a region around amino acid residue 300, that the PCNA binding activity is located at the C terminus, and that RPA binds to the N terminus of hMYH. A peptide consisting of residues 505-527 of hMYH that contains a conserved PCNA-binding motif binds PCNA, and subsequent amino acid substitution identified Phe-518 and Phe-519 as essential residues required for PCNA binding. RPA binds to a peptide that consists of residues 6-32 of hMYH and contains a conserved RPA-binding motif. The PCNA- and RPA-binding sites of hMYH are further confirmed by peptide and antibody titration. These results suggest that hMYH repair is a long patch base excision repair pathway.

Open sandwich enzyme-linked immunosorbent assay for the quantitation of small haptens.

The quantitation of low-molecular-weight haptens has been difficult with conventional sandwich immunoassays due to their small size. Many researchers have attempted to develop sandwich assays for haptens due to the significant advantages of the sandwich format over competitive assays including greater dynamic range, ease of automation, and sensitivity. Here we apply the open-sandwich ELISA (OS-ELISA), an immunoassay based on antigen-dependent stabilization of antibody variable regions (V(H) and V(L) domains), to hapten quantitation. Two fusion proteins, the high-affinity mutant V(H) domain from anti-4-hydroxy-3-nitrophenacetyl (NP) antibody B1-8 tethered with Escherichia coli alkaline phosphatase (V(H)(W33L)-PhoA) and the V(L) domain from the same antibody tethered with Streptococcus sp. protein G, were made. These fusion proteins when added together achieved Fv reassociation consequent to the addition of NP. Signal was generated in a direct relationship to the NP concentration with better sensitivity compared with competitive immunoassay, demonstrating this assay to be a quick noncompetitive alternative to the conventional assays for small compounds, such as environmental pollutants, drugs of abuse, and therapeutic drugs. With our previous demonstration that the OS-ELISA works well with large proteins, the OS-ELISA becomes the first practical immunoassay approach capable of quantifying any molecule regardless of their size.

The familial aggregation of the lesser variant in biological and nonbiological relatives of PDD probands: a family history study.

OBJECTIVE: To determine the risk of the lesser variant (or PDD-like traits) in the biological and nonbiological second- and third-degree relatives of PDD probands using a screening questionnaire and to investigate the extent to which the risk of the lesser variant differs according to various characteristics of the proband. METHOD: The sample consists of a series of 34 nuclear families with 2 affected PDD children (multiplex, MPX), 44 families with a single PDD child (simplex, SPX), and 14 families who adopted a PDD child. Data on characteristics of the lesser variant in 1362 biological and 337 nonbiological second- and third-degree relatives were collected from parents by telephone interview and from several maternal and paternal relatives by questionnaire. RESULTS: All components of the lesser variant were more common in biological relatives (BR) than nonbiological relatives (NBR), confirming the familial aggregation of the traits. Proband characteristics associated with an increased risk of the lesser variant in relatives were a higher level of functioning and coming from a MPX family. CONCLUSIONS: These findings on the familial aggregation of the lesser variant suggest that the genes for PDD also confer susceptibility to the lesser variant and that PDD may be a genetically heterogeneous disorder.

Cell-growth control by monomeric antigen: the cell surface expression of lysozyme-specific Ig V-domains fused to truncated Epo receptor.

Previously we have shown that the V(H) and V(L) fragments of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 are weakly associated but can be driven together by antigen. By joining these antibody variable domains to the cytoplasmic portion of the murine erythropoietin receptor, we created a chimeric growth factor receptor that could be activated by HEL. After co-transfection with two plasmids encoding the respective chimeric receptors in IL-3 dependent murine pro-B Ba/F3 cells, a portion of the cells survived under antigen dependent stimulation without IL-3. These surviving cells all showed coexpression of the two chimeric receptor chains and demonstrated HEL dose-dependent growth stimulation without IL-3. When another IL-3 dependent cell line 32D was transfected with a variant of such chimeric receptor with a linker peptide (Gly-Ser-Gly) inserted between V(H)/V(L) and EpoR domains, an improved growth response was attained. These observations suggest the utility of heterodimeric Fv chimeric receptors in creating cells that respond to monomeric antigen.

High functioning autism and Childhood Disintegrative Disorder in half brothers.

Childhood Disintegrative Disorder (CDD) is grouped with autism as a subtype of Pervasive Developmental Disorder (PDD) in ICD-10 and DSM-IV. This is the first report of autism and CDD cosegregating within a sibship. J. P. and M. P. are half-brothers with the same mother. J. P. is an 18-year-old with impairments in communication, social reciprocity, and stereotypies and was diagnosed with autism. M. P. is a 7-year-old who developed normally to 2 years 4 months. He then underwent a profound regression, becoming nonverbal and socially withdrawn, and lost adaptive skills. Investigations did not reveal any neurodegenerative process. M. P. was diagnosed with CDD. The rarity of the two conditions suggests a shared transmissible mechanism. The implications for autism/PDD genetic studies are discussed.

Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay.

A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted. Two chimeric proteins, Trx-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB - mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL. A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the V(H) and V(L) domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1-100 microg/ml could be non-competitively determined. The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples. This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Detecting Attention Deficit Hyperactivity Disorder in a communications clinic: diagnostic utility of the Gordon Diagnostic System.

This study examines the sensitivity, specificity, predictive values, and likelihood ratios of laboratory measures of attention and impulsivity (the Gordon Diagnostic System; GDS) in 99 school-aged boys with a history of suspected language disorders. Classification analyses comparing scores from these tests with parent and teacher ratings of attention deficit hyperactivity disorder (ADHD) symptoms revealed low positive predictive values (20.0% to 36.8%) and high negative predictive values (71.9% to 87.9%). Likelihood ratios for abnormal test scores were low to moderate (0.74 to 1.73), suggesting that these tests may not accurately identify children with ADHD. Likelihood ratios for normal scores were also low to moderate (0.41 to 1.16). These findings suggest that GDS scores have clinical utility in ruling out a diagnosis of ADHD, but not in confirming the diagnosis in a clinic population of boys with communicative disorders who are at risk for developing ADHD.

Homogeneous noncompetitive immunoassay based on the energy transfer between fluorolabeled antibody variable domains (open sandwich fluoroimmunoassay).

The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (VH) and light chain (VL) fragments. The VH and VL fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1-100 micrograms/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.

Open sandwich ELISA with V(H)-/V(L)-alkaline phosphatase fusion proteins.

The Sandwich ELISA is a widely used technique to measure antigen concentration. Recently, a novel ELISA based on the interchain interaction of separated V(H) and V(L) chains from a single antibody variable region (Fv) was proposed (Open Sandwich ELISA). Since it employs a single antibody recognizing one epitope, the assay requires, in essence, only one cycle of incubation and washing steps. To demonstrate this directly, we have constructed a recombinant gene fusion encoding the V(H) chain of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 and Escherichia coli alkaline phosphatase (V(H)-PhoA). The same type of gene fusion using V(L) chain instead of V(H) chain (V(L)-PhoA) was also constructed and the proteins were obtained with an E. coli expression/secretion system. Open Sandwich ELISAs were performed using microtiter plates with immobilized V(L) or V(H) fragment, and V(H)-PhoA or V(L)-PhoA, respectively, as the detection reagent which was simultaneously added to each well with samples. As a result, HEL concentrations in the samples were determined after one round of incubation and washing steps, with a signal generated in a direct relationship to the concentration of HEL added to the reaction mixture. The minimum detectable HEL concentration was approximately 10 ng/ml, which was almost equal to the value previously obtained with plate-immobilized V(L) and V(H) fragment displayed on M13 phage. When the active-site mutant V(H)-PhoA(D101S) was employed instead of V(H)-PhoA and reacted at an optimum pH of 10, a significant enhancement in signal was attained.

Familial factors influence level of functioning in pervasive developmental disorder.

OBJECTIVE: To determine whether siblings with pervasive developmental disorders (PDD) tend to have the same type and number of PDD symptoms or a similar level of functioning. METHOD: The familial correlations for PDD subtype, symptom totals, adaptive behaviors, and nonverbal IQ were calculated for 94 children with PDD from 46 families. RESULTS: On variables measuring PDD symptoms, only impairments in nonverbal communication and verbal/nonverbal status tended to run true within families. There was no familial aggregation of PDD subtype. In contrast, measures of nonverbal IQ and adaptive behaviors in socialization and communication showed a moderate degree of familial resemblance. The degree of familial resemblance did not change if the analysis was restricted only to those families in which both affected children met criteria for autism. CONCLUSION: Insofar as the familial resemblance seen in PDD is due to genetic factors, these data provide some evidence that higher- and lower-functioning PDD children may arise from separate genetic mechanisms. Current gene-mapping studies of PDD may need to take this evidence of genetic heterogeneity into account.