Detection and coat protein gene characterization of an isolate of Grapevine virus B from corky bark-affected grapevines in Southern Brazil

An isolate of Grapevine virus B (GVB), obtained by indexing Vitis labrusca and V. vinifera grapevines on the indicator LN33, was transmitted mechanically to several Nicotiana species. The virus was partially purified from N. cavicola and the coat protein estimated at 23 kDa by SDS-PAGE. In negatively stained leaf extracts of experimentally inoculated N. cavicola and N. occidentalis, flexuous particles with cross banding were observed, predominantly measuring 750-770 x 12 nm, with a modal length of 760 nm. Decoration indicated a clear, positive reaction against AS-GVB. In DAS-ELISA, GVB was detected in N. cavicola and grapevine extracts, and Western blots showed homologous and cross reaction of GVB and GVA antisera with GVB coat protein. Using specific primers for GVB, a fragment of 594 bp, comprising the coat protein gene coding for 197 amino acids, was amplified by RT-PCR with viral RNA extracted from GVB-infected N. occidentalis. The nucleotide and the deduced amino acid sequences of the coat protein gene showed high identities with Italian and Japanese isolates of GVB.

Genetic diversity of begomoviruses infecting soybean, bean and associated weeds in Northwestern Argentina

The subtropical Northwestern region of Argentina (provinces of Tucumán, Salta, Jujuy, Santiago del Estero and Catamarca) suffers from a high incidence of the whitefly Bemisia tabaci, and the detection of begomoviruses is also common. The Northwest is the main bean-growing region of the country, and approximately 10% of Argentina's soybean crop is grown in this area. We have used a PCR-based assay to establish the identity and genetic diversity of begomoviruses associated with bean and soybean crops in Northwestern Argentina. Universal begomovirus primers were used to direct the amplification of a fragment encompassing the 5' portion of the capsid protein gene. Amplified fragments were cloned, sequenced and subjected to phylogenetic analysis to determine the sequence identity to known begomoviruses. The data indicated the presence of four distinct begomoviruses, all related to other New World begomoviruses. The prevalent virus, which was present in 94% of bean and soybean samples and also in two weed species, is closely related to Sida mottle virus (SiMoV). A virus with high sequence identity with Bean golden mosaic virus (BGMV) was found in beans. The two remaining viruses displayed less than 89% identity with other known begomoviruses, indicating that they may constitute novel species. One of these putative novel viruses was detected in bean, soybean and tomato samples.

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

Luminescence-based assay methods in drug discovery

The drug discovery process is facing new challenges in the evaluation process of the lead compounds as the number of new compounds synthesized is increasing. The potentiality of test compounds is most frequently assayed through the binding of the test compound to the target molecule or receptor, or measuring functional secondary effects caused by the test compound in the target model cells, tissues or organism. Modern homogeneous high-throughput-screening (HTS) assays for purified estrogen receptors (ER) utilize various luminescence based detection methods. Fluorescence polarization (FP) is a standard method for ER ligand binding assay. It was used to demonstrate the performance of two-photon excitation of fluorescence (TPFE) vs. the conventional one-photon excitation method. As result, the TPFE method showed improved dynamics and was found to be comparable with the conventional method. It also held potential for efficient miniaturization. Other luminescence based ER assays utilize energy transfer from a long-lifetime luminescent label e.g. lanthanide chelates (Eu, Tb) to a prompt luminescent label, the signal being read in a time-resolved mode. As an alternative to this method, a new single-label (Eu) time-resolved detection method was developed, based on the quenching of the label by a soluble quencher molecule when displaced from the receptor to the solution phase by an unlabeled competing ligand. The new method was paralleled with the standard FP method. It was shown to yield comparable results with the FP method and found to hold a significantly higher signal-tobackground ratio than FP. Cell-based functional assays for determining the extent of cell surface adhesion molecule (CAM) expression combined with microscopy analysis of the target molecules would provide improved information content, compared to an expression level assay alone. In this work, immune response was simulated by exposing endothelial cells to cytokine stimulation and the resulting increase in the level of adhesion molecule expression was analyzed on fixed cells by means of immunocytochemistry utilizing specific long-lifetime luminophore labeled antibodies against chosen adhesion molecules. Results showed that the method was capable of use in amulti-parametric assay for protein expression levels of several CAMs simultaneously, combined with analysis of the cellular localization of the chosen adhesion molecules through time-resolved luminescence microscopy inspection.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Leishmania chagasi using total antigen from lysed promastigotes. Fifty symptomatic mixed breed dogs from a region of high incidence of visceral leishmaniasis in Brazil were examined. The results showed that in the positive animals, diagnosed by cytological examination, the ELISA using protein A assay system (mean optical density ± SD / 2.078 ± 0.631) detected more antibodies than the anti-IgG assay (mean optical density ± SD / 1.008 ± 0.437), while in the negative animals, the results by both systems were similar. These results suggest that the ELISA assay using protein A peroxidase conjugated could be useful to detect early infected animals in endemic areas, and thus help to control the spread of the infection.

The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle

An indirect enzyme-linked immunosorbent assay was developed to detect antigen-specific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm) was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

Development of a Real-time PCR test for porcine group A rotavirus diagnosis

Group A Rotavirus (RVA) is one of the most common causes of diarrhea in humans and several animal species. A SYBR-Green Real-Time polymerase chain reaction (PCR) was developed to diagnose RVA from porcine fecal samples, targeting amplification of a 137-bp fragment of nonstructural protein 5 (NSP5) gene using mRNA of bovine NADH-desidrogenase-5 as exogenous internal control. Sixty-five samples were tested (25 tested positive for conventional PCR and genetic sequencing). The overall agreement (kappa) was 0.843, indicating 'very good' concordance between tests, presenting 100% of relative sensitivity (25+ Real Time PCR/25+ Conventional PCR) and 87.5% of relative sensitivity (35- Real Time PCR/40- Conventional PCR). The results also demonstrated high intra- and inter-assay reproducibility (coefficient of variation ≤1.42%); thus, this method proved to be a fast and sensitive approach for the diagnosis of RVA in pigs.

Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing

Detection of adrenocortical autoantibodies in Addison's disease with a peroxidase-labelled protein A technique

Adrenocortical autoantibodies (ACA), present in 60-80% of patients with idiopathic Addison's disease, are conventionally detected by indirect immunofluorescence (IIF) on frozen sections of adrenal glands. The large-scale use of IIF is limited in part by the need for a fluorescence microscope and the fact that histological sections cannot be stored for long periods of time. To circumvent these restrictions we developed a novel peroxidase-labelled protein A (PLPA) technique for the detection of ACA in patients with Addison's disease and compared the results with those obtained with the classical IIF assay. We studied serum samples from 90 healthy control subjects and 22 patients with Addison's disease, who had been clinically classified into two groups: idiopathic (N = 13) and granulomatous (N = 9). ACA-PLPA were detected in 10/22 (45%) patients: 9/13 (69%) with the idiopathic form and 1/9 (11%) with the granulomatous form, whereas ACA-IIF were detected in 11/22 patients (50%): 10/13 (77%) with the idiopathic form and 1/9 (11%) with the granulomatous form. Twelve of the 13 idiopathic addisonians (92%) were positive for either ACA-PLPA or ACA-IIF, but only 7 were positive by both methods. In contrast, none of 90 healthy subjects was found to be positive for ACA. Thus, our study shows that the PLPA-based technique is useful, has technical advantages over the IIF method (by not requiring the use of a fluorescence microscope and by permitting section storage for long periods of time). However, since it is only 60% concordant with the ACA-IIF method, it should be considered complementary instead of an alternative method to IIF for the detection of ACA in human sera.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein a (C/EBPa) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBPa factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBPa factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBPa factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBPa factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.

Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

The carboxyl-terminal (CT) domain of connexin43 (Cx43) has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST)-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.