Typing of enteroviruses by use of microwell oligonucleotide arrays.

We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

Cloning, sequencing, expression and characterization of three anti-estradiol-17beta Fab fragments.

In order provide data for a basic understanding of the mechanisms of antibody specificity and for the design of antibodies with desired properties, we have sequence-analysed three high affinity anti-estradiol-17beta monoclonal antibodies. All three monoclonal antibodies to estradiol-17beta had been raised by conjugation of the 6-carboxymethyloxime derivative to protein carrier. The genes encoding heavy (Fd) and light (L) chains of these three antibodies were cloned and sequenced. The sequenced antibody chains were found to be from 46.0 to 89.7% sequence identical to a monoclonal antibody (DB3) binding a related steroid, progesterone. The Fd and L chains were paired with all possible Fd-L combinations and the corresponding proteins were expressed in Escherichia coli and characterized for their binding (immunoreactivity) to estradiol-17beta. Under the lac promoter and using the pelB signal sequences the production levels of the soluble (total) heavy and light chain Fab fragment combinations in periplasm and in supernatant varied from 115 to 2207 microg/l, while the immunoreactivity percentages (IR%) varied from < 1 to 45%. The production levels and IR% were dependent on the first constant domain subclasses of the heavy chain as well as the Fd-L chain combination expressed.

Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies.

The length of the heavy chain complementarity-determining region 2 (CDRH2) was extended beyond what is found in germline genes to improve the binding properties of an anti-estradiol antibody. The previous immunochemical characterization and the molecular modeling of the high affinity (Ka=3.9x10(8)) murine anti-estradiol antibody 57-2 suggested that a part of the antigen was loosely recognized by the antibody. The CDRH2, because of its close location but scarce contacts with the hapten, was considered as a conceivable target for mutagenesis. Libraries with either two, three or four random amino acid insertions in the tip of the CDRH2 loop were constructed and displayed on the M13 filamentous phage as Fab fragments. Mutations were introduced also into the rest of the VHdomain by error-prone polymerase chain reaction to allow the surrounding structures to adapt to the extended CDRH2. After the panning of the libraries with an antigen off-rate-based selection, a number of active clones, most of which showed significantly improved affinity and specificity, were isolated, characterized and sequenced. The results indicate that the structure of the antibody can tolerate a number of different insertions in the CDRH2 region. They also suggest that the repertoire of antibody libraries can be expanded by extending the length of the CDR loops beyond that naturally provided by the given set of germline genes. This kind of mutagenesis can be generally useful for the engineering of hapten-binding antibodies.

Structural analysis of an anti-estradiol antibody.

An anti-estradiol antibody with improved specificity is searched for by combining steroid analog binding studies, mutant antibodies obtained from a phage-display library and structural modeling. Three-dimensional models for the anti-estradiol antibody 57-2 were constructed by comparative model building. Estradiol and analogs were docked into the combining site and molecular dynamics simulation was used to further refine this area of the protein. Cross-reactivities measured against 36 steroid analogs were used to help in the docking process and to evaluate the models. The roles of a number of residues were assessed by characterization of cross-reactivity mutants obtained from a phage display library. The cross-reactivity data and the results observed for mutants are explained by the structural model, in which the estradiol D-ring inserts deeply into the binding site and interacts with the antibody through at least one specific hydrogen bond. The binding data strongly suggest that this hydrogen bond connects the estradiol 17-hydroxyl group with the side chain of Gln H35. As expected for the binding of a small aromatic molecule, the antibody binding site contains many aromatic residues, e.g. Trp H50, H95 and L96 and Tyr L32, L49 and Phe L91.

A sensitive model system for in vivo monitoring of baculovirus gene expression in single infected insect cells.

We have developed a fast and sensitive system for the in vivo analysis of gene expression in baculovirus infected lepidopteran insect cells. A recombinant baculovirus containing a luciferase gene from the click beetle, Pyrophorus plagiophthalamus, under transcriptional regulation of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) was used to infect a Spodoptera frugiperda cell line. Recombinant luciferase could be monitored by luminometry in real-time without disruption of the infected cells, allowing detection of synthesis as early as one hour after infection. The range of luminescence measurements was normally over four orders of magnitude, and the kinetics of luciferase synthesis and the levels of light produced in vivo closely correlated with the expression of polyhedrin in AcNPV infected cells when analyzed by SDS-PAGE. Additionally, single infected cells could be identified by CCD image analysis and flow cytometry.

Genotyping of clinically relevant human adenoviruses by array-in-well hybridization assay.

A robust oligonucleotide array-in-well hybridization assay using novel up-converting phosphor reporter technology was applied for genotyping clinically relevant human adenovirus types. A total of 231 adenovirus-positive respiratory, ocular swab, stool and other specimens from 219 patients collected between April 2010 and April 2011 were included in the study. After a real-time PCR amplification targeting the adenovirus hexon gene, the array-in-well assay identified the presence of B03 (n = 122; 57.5% of patients), E04 (29; 13.7%), C02 (21; 9.9%), D37 (14; 6.6%), C01 (12; 5.7%), C05 (5; 2.4%), D19 (4; 1.9%), C06 (2; 0.9%), D08 (1; 0.5%), A31 (1; 0.5%) and F41 (1; 0.5%) genotypes among the clinical sample panel. The typing result was obtained for all specimens that could be amplified (n = 223; 97%), and specificity of the typing was confirmed by sequencing specimens representing each of the different genotypes. No hybridization signal was obtained in adenovirus-negative specimens or specimens with other viruses (n = 30). The array-in-well hybridization assay has great potential as a rapid and multiplex platform for the typing of clinically relevant human adenovirus genotypes in different specimen types.

Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.

Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.

Dual-label time-resolved immunofluorometric assay of free and total prostate-specific antigen based on recombinant Fab fragments.

BACKGROUND: Recombinant Fab fragments are attractive as reagents for novel sandwich immunoassays, but no such assays have been described. We developed a dual-label two-site immunoassay based entirely on recombinant Fab fragments and compared it to the same assay with intact monoclonal antibodies. METHODS: The capture Fab fragment, which binds free prostate-specific antigen (PSA) and PSA in complex with alpha(1)-antichymotrypsin on an equimolar basis, is site-specifically biotinylated and attached to the solid phase in streptavidin-coated microtitration wells. The Fab fragment that detects only free PSA is site-specifically labeled with a fluorescent europium chelate, and the Fab fragment that detects both free and serpin-complexed PSA in an equimolar fashion is labeled with a fluorescent terbium chelate. Time-resolved fluorescence is used to measure both europium and terbium signals in one well. RESULTS: The detection limits of the assay (mean + 3 SD of zero calibrator) were 0.043 and 0.28 microgram/L, respectively, for free and total PSA. The within-run and day-to-day CVs were 2-11% and 4-10%, respectively. Mean recoveries were 93% and 98% in female and male sera, respectively. Compared with the commercial ProStatus PSA Free/Total Assay, the intercepts of the regression equations (r >0. 99) were not significantly different from zero, and the slopes were 0.95-1.01. In one female serum sample, PSA was falsely increased with the monoclonal assay but was undetectable with the recombinant assay. CONCLUSIONS: The performance of this novel assay based on recombinant components is comparable to a conventional assay based on monoclonal antibodies. The more complete control of the essential characteristics of site-specifically derivatized recombinant Fab fragments will be valuable for the design of miniaturized and multianalyte assay concepts where correct antibody orientation, density, and capacity as well as uncompromised binding affinity are required.

Real-time measurement of cell permeabilization with low-molecular-weight membranolytic agents.

A new method for studying the action of membranolytic agents by simple measurement of light emitted from cells is described. It is based on the expression of the click beetle (Pyrophorus plagiophthalamus) luciferase gene (lucGR) in Escherichia coli, Bacillus subtilis and Spodoptera frugiperda cells in order to make them bioluminescent. The diffusion of the substrate for luciferase enzyme through the cell membranes is very low at physiological pH, and therefore a change in membrane permeability is seen as a change of in-vivo luminescence of cells. The cells used in this study represent different membrane structures, and thus allow a comparison of the reactions of the different membranes towards membranolytic agents in a real-time measurement. The dose-response data correlated well with target cell viable count. In addition, the time course of light emission as a consequence of permeabilizing compound is dose-dependent. The action of the compounds on prokaryotic and eukaryotic cells was found to be highly dependent on the permeabilizer used.

Expression of prostate specific antigen on the surface of a filamentous phage.

We have constructed two phagemid vectors containing the gene coding for human Prostate Specific Antigen (PSA) translationally fused to a pelB signal sequence and minor coat protein III of phage fd leading to phage particles that carry PSA on their tips. Phages were characterized by Western blotting and by panning, using biotinylated monoclonal anti-PSA antibodies immobilized onto streptavidin-coated microtitration wells. Binding of PSA-phages was 10(2)- to 5 x 10(3)-fold more effective than that of wild-type phages. Trypsin treatment of the phage abolished the specific binding. Competitive panning with different concentrations of added purified PSA demonstrated that binding was PSA specific. Finally, Western-blot analysis confirmed that full-length and shorter degradation products of the fusion between PSA and protein III were visible as probed with anti-PSA antibody.

In vitro enzymatic biotinylation of recombinant fab fragments through a peptide acceptor tail.

We describe the site-specific enzymatic biotinylation of recombinant anti-estradiol Fab fragments through a 13 amino acid acceptor peptide translationally fused to the C-terminus of the Fd chain. The Fab-peptide fusion proteins were secreted to the periplasm of Escherichia coli, purified, and biotinylated in vitro using biotin ligase, biotin, and ATP. The E. coli biotin ligase (the BirA protein) was produced as a novel N-terminal fusion protein with glutathione S-transferase (GST) and purified in one step from bacterial cell lysate using a Glutathione Sepharose affinity column. The purified fusion protein worked as such (without cleavage of the GST part) for the in vitro biotinylation of the Fab fragments. After the removal of nonbiotinylated Fab fragments by monomeric avidin chromatography, the overall yield of biotinylated Fab was 40%. The site-specifically biotinylated Fab fragments (BioFab) were tested in streptavidin-coated microtitration wells, to which they were shown to bind linearly with respect to the amount of BioFab added, specifically as indicated by biotin inhibition, and tightly with a half-life of several days. Moreover, the enzymatic BioFab exhibited uniform antigen binding affinity unlike the same recombinant Fab fragments biotinylated through random chemical conjugation to surface lysines. Finally, the BioFab demonstrated its potential as a well-behaving immunoassay reagent in a model competitive assay for estradiol.

Engineering the steroid-specificity of an anti-17beta-estradiol Fab by random mutagenesis and competitive phage panning.

We have employed random mutagenesis and phage display to improve the steroid-specificity of an anti-17beta-estradiol Fab fragment. The VH domain was mutated using error-prone PCR; the mutation rate was controlled by adjusting the number of effective duplications. A phage library of 2 x 10(6) independent mutants was generated, each mutant containing on average 24 amino acid changes. We selected for decreased testosterone (TES) cross-reactivity by adding a large excess TES as a competitor to the panning reactions. After four panning rounds, the cross-reactivities of the individual mutant clones ranged from 19 to 4%, showing up to 20-fold improvement over the original value (78%). Estradiol affinities were mainly unchanged. Sequencing of the VH regions revealed two hot spots, one located around Ser32 in CDR1 and the other around Thr52A in CDR2, while no mutations were found in CDR3. Although most clones had multiple mutations, it was possible to deduce the residues relevant to the improved specificity by comparing the sequences and binding data of the mutants. We demonstrated that controlled error-prone PCR mutagenesis is a rapid method to identify such key residues, lending itself to the scanning of 'lead' positions for further mutagenesis by other methods.

Linkage studies in a new X-linked myopathy, suggesting exclusion of DMD locus and tentative assignment to distal Xq.

We here report linkage studies in a family suffering from a recently described hereditary muscle disease named X-linked myopathy with excessive autophagy (XMEA). Significant lod scores excluding linkage to the Duchenne-Becker muscular dystrophy locus were found. Several other loci on the short and long arms of the X chromosome produced negative lod scores, whereas probe DX13-7 defining locus DXS15 showed no recombinants and a lod score of z = 0.903 at theta = .0. Further studies should be done to determine whether the gene for XMEA is (1) located at Xq and (2) caused by a mutation of the Emery-Dreifuss muscular dystrophy gene, which has been assigned to the same region.