Petri Saviranta

VTT Technical Research Centre of Finland, Esbo, Southern Finland Province, Finland

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Publications (35)130.86 Total impact

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    ABSTRACT: A quantitative multianalyte immunoassay utilizing luminescent upconverting single-crystal nanoparticles as reporters on an antibody array-in-well platform was demonstrated. Upconverting nanoparticles are inorganic rare earth doped materials that have the unique feature of converting low energy infrared radiation into higher energy visible light. Autofluorescence, commonly limiting the sensitivity of fluorescence-based assays, can be completely eliminated with photon upconversion technology because the phenomenon does not occur in biological materials. Biotinylated antibodies for three analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone) were printed in an array format onto the bottom of streptavidin-coated microtiter wells. Analyte dilutions were added to the wells, and the analytes were detected with antibody-coated upconverting nanoparticles. Binding of the upconverting nanoparticles was imaged with an anti-Stokes photoluminescence microwell imager, and the standard curves for each analyte were quantified from the selected spot areas of the images. Single analyte and reference assays were also carried out to compare with the results of the multianalyte assay. Multiplexing did not have an effect on the assay performance. This study demonstrates the feasibility of upconverting single-crystal nanoparticles for imaging-based detection of quantitative multianalyte assays.
    Analytical Chemistry 09/2012; 84(20):8628-34. · 5.70 Impact Factor
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    ABSTRACT: Clin Microbiol Infect ABSTRACT: 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.
    Clinical Microbiology and Infection 05/2012; · 4.58 Impact Factor
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    ABSTRACT: High-throughput RNAi screening is widely applied in biological research, but remains expensive, infrastructure-intensive and conversion of many assays to HTS applications in microplate format is not feasible. Here, we describe the optimization of a miniaturized cell spot microarray (CSMA) method, which facilitates utilization of the transfection microarray technique for disparate RNAi analyses. To promote rapid adaptation of the method, the concept has been tested with a panel of 92 adherent cell types, including primary human cells. We demonstrate the method in the systematic screening of 492 GPCR coding genes for impact on growth and survival of cultured human prostate cancer cells. The CSMA method facilitates reproducible preparation of highly parallel cell microarrays for large-scale gene knockdown analyses. This will be critical towards expanding the cell based functional genetic screens to include more RNAi constructs, allow combinatorial RNAi analyses, multi-parametric phenotypic readouts or comparative analysis of many different cell types.
    BMC Genomics 03/2011; 12:162. · 4.40 Impact Factor
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    ABSTRACT: We have developed a robust array-in-well test platform based on an oligonucleotide array, combining advantages of simple instrumentation and new upconverting phosphor reporter technology. Upconverting inorganic lanthanide phosphors have a unique property of photoluminescence emission at visible wavelengths under near-infrared excitation. No autofluorescence is produced from the sample or support material, enabling a highly sensitive assay. In this study, the assay is performed in standard 96-well microtiter plates, making the technique easily adaptable to high-throughput analysis. The oligonucleotide array-in-well assay is employed to detect a selection of ten common adenovirus genotypes causing human infections. The study provides a demonstration of the advantages and potential of the upconverting phosphor-based reporter technology in multianalyte assays and anti-Stokes photoluminescence detection with an anti-Stokes photoluminescence imaging device.
    Analytical Chemistry 02/2011; 83(4):1456-61. · 5.70 Impact Factor
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    ABSTRACT: Aneuploidy is among the most obvious differences between normal and cancer cells. However, mechanisms contributing to development and maintenance of aneuploid cell growth are diverse and incompletely understood. Functional genomics analyses have shown that aneuploidy in cancer cells is correlated with diffuse gene expression signatures and aneuploidy can arise by a variety of mechanisms, including cytokinesis failures, DNA endoreplication, and possibly through polyploid intermediate states. To identify molecular processes contributing to development of aneuploidy, we used a cell spot microarray technique to identify genes inducing polyploidy and/or allowing maintenance of polyploid cell growth in breast cancer cells. Of 5760 human genes screened, 177 were found to induce severe DNA content alterations on prolonged transient silencing. Association with response to DNA damage stimulus and DNA repair was found to be the most enriched cellular processes among the candidate genes. Functional validation analysis of these genes highlighted GINS2 as the highest ranking candidate inducing polyploidy, accumulation of endogenous DNA damage, and impairing cell proliferation on inhibition. The cell growth inhibition and induction of polyploidy by suppression of GINS2 was verified in a panel of breast cancer cell lines. Bioinformatic analysis of published gene expression and DNA copy number studies of clinical breast tumors suggested GINS2 to be associated with the aggressive characteristics of a subgroup of breast cancers in vivo. In addition, nuclear GINS2 protein levels distinguished actively proliferating cancer cells suggesting potential use of GINS2 staining as a biomarker of cell proliferation as well as a potential therapeutic target.
    Neoplasia (New York, N.Y.) 11/2010; 12(11):877-88. · 5.48 Impact Factor
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    ABSTRACT: Cell division is orchestrated by a complex protein network that aims to maintenance of genomic stability. Visualisation of mitotic protein-protein associations in space and time has been limited due to the lack of proper biochemical and easy-to-use imaging tools. Here we report adaptation of the in situ proximity ligation assay (is-PLA) to study mitotic protein interactions with spatio-temporal resolution. We examined the composition of the Chromosomal Passenger Complex (CPC) at various mitotic phases and after chemical treatments using is-PLA with antibodies against the core CPC subunits Aurora B, INCENP, Survivin and Borealin. Our results support the notion that the core CPC functions as a single structural unit at centromeres in early mitosis and at central spindle after the onset of anaphase. Treatment of cells with the Aurora B inhibitor ZM447439 diminished the is-PLA signals at centromeres suggesting that Aurora B activity contributes to structural maintenance and/or proper subcellular localization of the core CPC. Is-PLA-based analysis of interaction between INCENP and Polo-like kinase 1 (Plk1) proposes that the kinase co-travels with CPC during late mitosis. The data illustrates both the strengths and limitations of the is-PLA in the analysis of mitotic macromolecule associations at sub-organelle level.
    Molecular oncology 10/2010; 5(1):105-11. · 6.70 Impact Factor
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    ABSTRACT: Oncogene Vol.28, 3926 - 3936
    Oncogene 09/2009; · 8.56 Impact Factor
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    ABSTRACT: 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.
    Journal of clinical microbiology 05/2009; 47(6):1863-70. · 4.16 Impact Factor
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    ABSTRACT: Journal of Clinical Microbiology Vol.47 Nr.6, 1863 - 1870
    01/2009;
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    ABSTRACT: ProteomeBinders is a new European consortium aiming to establish a comprehensive resource of well-characterized affinity reagents, including but not limited to antibodies, for analysis of the human proteome. Given the huge diversity of the proteome, the scale of the project is potentially immense but nevertheless feasible in the context of a pan-European or even worldwide coordination.
    Nature Methods 02/2007; 4(1):13-7. · 23.57 Impact Factor
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    ABSTRACT: A method for evaluation of toxicity of aqueous solutions is described based on the use of genetically engineered Escherichia coli. The genes encoding bacterial luciferase have been cloned from Vibrio harveyi to a deep rough mutant of E. coli under the control of the lac promoter. Light production by this strain has been stabilized by optimizing several parameters having an effect on the gene expression. Toxicity measurements were performed for selected metals and organic solvents to determine the sensitivity of the test strain. Effective concentrations calculated from these measurements show that this method has a sensitivity equal to other normally used methods. The test can be performed using buffers with low ionic strength without any significant change in the stability of the light emitted. Moreover, the method does not necessitate the use of special equipment or skills.
    Environmental Toxicology and Water Quality 06/2006; 5(4):337 - 350.
  • Petri Saviranta, Olli Kallioniemi
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    ABSTRACT: A complete set of high-quality antibodies against the human proteome would constitute a remarkable resource for the analysis of protein abundances in tissues, facilitating systematic protein expression profiling in health and disease. In two recent papers, Swedish researchers describe a concerted effort towards producing a 'Human Protein Atlas' by combining high-throughput antibody generation with immunohistochemical profiling of tissue microarrays.
    Trends in Biotechnology 06/2006; 24(5):195-7. · 9.66 Impact Factor
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    ABSTRACT: Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.
    Biochimica et Biophysica Acta 07/2005; 1724(1-2):146-54. · 4.66 Impact Factor
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    ABSTRACT: Stability of single-chain Fvs (scFvs) can be improved by mutagenesis followed by phage display selection where the unstable variants are first inactivated by, for example, denaturing treatment. Here we describe a modified strategy for the selection of stabilized antibody fragments by phage display, based on denaturation under reducing conditions. This strategy was applied to an anti-thyroid-stimulating hormone (TSH) scFv fragment which refolded remarkably during the selection if denaturation was carried out in conventionally used non-reducing conditions. Refolding was, however, efficiently prevented by combining denaturation with reduction of the intra-domain disulfide bridges, which created favourable conditions for selection of clones with improved stability. Using this strategy, scFv mutants with 8-9 degrees C improved thermal stability and 0.8-0.9 M improved stability for guanidinium chloride were found after 4-5 enrichment cycles. The most stable mutants selected contained either Lys(H)66Arg or Asn(H)52aSer mutations, which are known to stabilize other scFvs. Periplasmic expression level of the mutants was also improved.
    Journal of Immunological Methods 02/2005; 296(1-2):159-70. · 2.23 Impact Factor
  • Ville Santala, Petri Saviranta
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    ABSTRACT: The search for high-affinity antibodies from phage libraries by panning is often complicated by inefficient detachment of the captured phage particles from the antigen-coated solid phase. General elution methods, independent on the bioaffinity interaction, would be desirable for high-throughput antibody screening. We have developed a bioaffinity-independent elution method based on the use of commercially available streptavidin paramagnetic particles.
    Journal of Immunological Methods 02/2004; 284(1-2):159-63. · 2.23 Impact Factor
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    ABSTRACT: The anti-17beta-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The double-mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.
    Protein Science 12/2003; 12(11):2549-58. · 2.74 Impact Factor
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    ABSTRACT: Sulfa-antibiotics (sulfonamides) are widely used in veterinary medicine. Meat and milk from treated animals can be contaminated with sulfa residues. Current sulfonamide assays are unfit for screening of food, because they are either too laborious, insensitive or specific for a few sulfa compounds only. An immunoassay for detection of all sulfas in a single reaction would be useful for screening. Previously we have improved the broad specificity sulfa binding of antibody 27G3 with random mutagenesis and phage display. In order to improve the properties of this antibody further, mutants from the previous study were recombined and more mutations introduced. These new libraries were enriched with phage display and several different mutant antibodies were isolated. The cross-reaction profile of the best mutant was better than that of the wild-type antibody and the mutants of the previous study: it was capable of binding 10 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas 5- to 11-fold better than the mutants of the previous study.
    Protein engineering 02/2003; 16(1):37-46.
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    ABSTRACT: Bacterial luciferases and the genes encoding these light-emitting enzymes have an increasing number of applications in biological sciences. Temperature lability and the heterodimeric nature of these luciferases have been the major obstacles for their widespread use, for instance, as genetic reporters. Escherichia coli expressing wild-type Photorhabdus luminescens luciferase was found to produce eight times more light than the corresponding Vibrio harveyi luciferase clone in vivo at 37 degrees C. Three monomeric luciferases were created by translationally fusing the two genes encoding luxA and luxB proteins of P. luminescens. These clones were equally active in producing light in vivo when cultivated at 37 degrees C compared to cultivation at 30 degrees C. The fusion containing the longest linker showed the highest activity. In vitro, the monomeric luciferases were less active having at best 20% of activity of the wild-type enzyme due to the partial formation of insoluble aggregates. The results suggest that P. luminescens luciferase and monomeric derivatives thereof should be more suitable than the corresponding V. harveyi enzyme to be used as reporters in cell types which need cultivation at elevated temperatures.
    Biochemical and Biophysical Research Communications 10/2002; 296(5):1072-6. · 2.28 Impact Factor
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    ABSTRACT: Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range.
    Journal of Agricultural and Food Chemistry 08/2002; 50(15):4194-201. · 3.11 Impact Factor
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    ABSTRACT: 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. 1999 Academic Press Keywords: antibody engineering; CDRH2; hapten; insertion; phage display Introduction The structure, chemical nature and the relative orientations of the six complementarity-determining region (CDR) loops (L1, L2, L3 in the light chain and H1, H2 and H3 in the heavy chain) attached to a conserved [-sheet framework primarily determine the antigen binding properties of an antibody. As first shown by Chothia & Lesk (1987), five of the CDR loops, e.g. the CDR1 and CDR2 loops of both chains and the CDRL3, typically Present address: S. Pauprio, Centro de Eshados de Paramiloidose, Instihato Nacional de Sadde Dr. Ricardo Jorge, 4050 Porto, Porhagal. Abbreviations used: 2aa, two amino acid insertion library; 3aa, three amino acid insertion library; 4aa, fo
    03/2001;

Publication Stats

490 Citations
130.86 Total Impact Points

Institutions

  • 2011
    • VTT Technical Research Centre of Finland
      Esbo, Southern Finland Province, Finland
  • 1997–2009
    • University of Turku
      • • Department of Virology
      • • Turku Centre for Biotechnology
      • • Department of Biochemistry and Food Chemistry
      Turku, Western Finland, Finland