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Timo Lövgren - One of the best experts on this subject based on the ideXlab platform.

  • Quantification of streptavidin adsorption in Microtitration wells.
    Analytical biochemistry, 2004
    Co-Authors: Lasse Välimaa, Kim Pettersson, Jaana Rosenberg, Matti Karp, Timo Lövgren
    Abstract:

    Streptavidin-coated Microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in Microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format Microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the Microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

  • A high-capacity streptavidin-coated Microtitration plate
    Bioconjugate chemistry, 2003
    Co-Authors: Lasse Välimaa, Kim Pettersson, Matti Karp, Markus Vehniäinen, Timo Lövgren
    Abstract:

    A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated Microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the Microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). ...

Lasse Välimaa - One of the best experts on this subject based on the ideXlab platform.

  • Comparison study of streptavidin-coated Microtitration plates.
    Journal of immunological methods, 2005
    Co-Authors: Lasse Välimaa, Katja Laurikainen
    Abstract:

    Streptavidin (SAv)-coated 96-well Microtitration plates from commercial suppliers (six plates) and our laboratory (two plates) were tested with respect to their binding capacity for small and large molecules, leaching, well-to-well variation and immunoassay performance. The binding capacities for europium-labelled biotin (Eu-biotin) varied from 4.4 to above 150 pmol/well depending on the plate, and for biotinylated monoclonal antibody from 1.2 to 6.4 pmol/well (190-1030 ng). Incubation for 1 h in regular immunoassay buffer resulted in leaching of 0.6-76 ng of SAv from unwashed wells and 0.5-60 ng from pre-washed wells. The desorbed quantity represented up to 3.4% of the maximal Eu-biotin binding capacity. Coating-related variation of 96 wells (CV% values) ranged from 1.2% to 8.0% when tested with respect to maximum binding capacity. Immunofluorometric assay for TSH as well as enzyme immunoassays for CA125 and PSA revealed immunoassay-related performance of the plates, regarding signal levels, variation and non-specific binding characteristics. Non-specific binding and variation tended to increase in the highest capacity plates, whereas some low capacity plates fulfilled the assay requirements optimally. Good performance with respect to capacity, coating homogeneity or leaching was not by definition reflected in the performance of two-site heterogeneous immunoassays.

  • Quantification of streptavidin adsorption in Microtitration wells.
    Analytical biochemistry, 2004
    Co-Authors: Lasse Välimaa, Kim Pettersson, Jaana Rosenberg, Matti Karp, Timo Lövgren
    Abstract:

    Streptavidin-coated Microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in Microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format Microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the Microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

  • A high-capacity streptavidin-coated Microtitration plate
    Bioconjugate chemistry, 2003
    Co-Authors: Lasse Välimaa, Kim Pettersson, Matti Karp, Markus Vehniäinen, Timo Lövgren
    Abstract:

    A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated Microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the Microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). ...

Victoria Wahl-jensen - One of the best experts on this subject based on the ideXlab platform.

  • A Semi-automated High-Throughput Microtitration Assay for Filoviruses.
    Methods in molecular biology (Clifton N.J.), 2017
    Co-Authors: Claire Marie Filone, David M. Miller, Victoria Wahl-jensen
    Abstract:

    The 50% tissue culture infectious dose (TCID50) endpoint dilution assay is one of the gold standard methods for measuring filovirus infectivity. We have increased virology Microtitration assay throughput at biosafety level (BSL)-4 by implementing automated liquid handling and semi-automated assay endpoint readout. Utilization of automated liquid handling for cell plating and virus dilution along with optimization of the assay endpoint readout, using a luminescent-based cell viability assay and an automated plate reader, has improved workflow efficiency, reduced operator burden and assay time, decreased assay variability, and increased data return.

  • Comparison of EBOV/Mak-C05 Recovery by qRT-PCR and Viability by Microtitration Assay.
    2016
    Co-Authors: Michael Schuit, David M. Miller, Mary S. Reddick-elick, Carly B. Wlazlowski, Claire Marie Filone, Artemas Herzog, Leremy A. Colf, Victoria Wahl-jensen, Michael Hevey, James W. Noah
    Abstract:

    The virus samples recovered from test surface coupons from Figs 2, 3 and 4 were analyzed by both qRT-PCR and virus Microtitration assay to determine whether virus material was efficiently recovered at low viability timepoints (n = 3). qRT-PCR analysis was performed on samples aged at (A) 22°C/41% RH, or (B) 28°C/90% RH. The ratio of virus genomic copies recovered from timepoints at the Microtitration limit of detection to recovery from timepoints immediately post-drying was determined and plotted as a percentage (black bars). This was compared to the virus viability ratio from the same timepoints, as determined by Microtitration assay (Log TCID50/mL) (gray bars). Actual mean percent recovery values are shown above each bar. Because no viable virus was detected at any timepoint in feces and % viable virus could not be calculated, % viable virus recovered in this matrix is indicated at

  • EBOV/Mak-C05 Surface Persistence in Simulated Vomit at Three Different Environments.
    2016
    Co-Authors: Michael Schuit, David M. Miller, Mary S. Reddick-elick, Carly B. Wlazlowski, Claire Marie Filone, Artemas Herzog, Leremy A. Colf, Victoria Wahl-jensen, Michael Hevey, James W. Noah
    Abstract:

    The surface-specific decrease in EBOV/Mak-C05 viability in simulated vomit was measured at (A) 22°C/17% RH (stainless steel and polypropylene only), (B) 22°C/41% RH, or (C) 28°C/90% RH. The Microtitration assay limit of detection (0.7 Log TCID50/mL) is indicated by a gray bar at the bottom of each graph.

  • EBOV/Mak-C05 Surface Persistence in Blood at Three Different Environments.
    2016
    Co-Authors: Michael Schuit, David M. Miller, Mary S. Reddick-elick, Carly B. Wlazlowski, Claire Marie Filone, Artemas Herzog, Leremy A. Colf, Victoria Wahl-jensen, Michael Hevey, James W. Noah
    Abstract:

    The surface-specific decrease in EBOV/Mak-C05 viability in human whole blood was measured at (A) 22°C/17% RH (stainless steel and polypropylene only), (B) 22°C/41% RH, or (C-D) 28°C/90% RH. The Microtitration assay limit of detection (0.7 Log TCID50/mL) is indicated by a gray bar at the bottom of each graph. The graphs in panels C-D are derived from two separate studies, as described in the methods section.

Kim Pettersson - One of the best experts on this subject based on the ideXlab platform.

  • Quantification of streptavidin adsorption in Microtitration wells.
    Analytical biochemistry, 2004
    Co-Authors: Lasse Välimaa, Kim Pettersson, Jaana Rosenberg, Matti Karp, Timo Lövgren
    Abstract:

    Streptavidin-coated Microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in Microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format Microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the Microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

  • A high-capacity streptavidin-coated Microtitration plate
    Bioconjugate chemistry, 2003
    Co-Authors: Lasse Välimaa, Kim Pettersson, Matti Karp, Markus Vehniäinen, Timo Lövgren
    Abstract:

    A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated Microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the Microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). ...

Matti Karp - One of the best experts on this subject based on the ideXlab platform.

  • Quantification of streptavidin adsorption in Microtitration wells.
    Analytical biochemistry, 2004
    Co-Authors: Lasse Välimaa, Kim Pettersson, Jaana Rosenberg, Matti Karp, Timo Lövgren
    Abstract:

    Streptavidin-coated Microtitration plates have an important role as a solid phase in clinical diagnostics. We have designed techniques for evaluating quantitative and functional aspects of streptavidin adsorbed in Microtitration wells. The theoretical monolayer adsorption capacity was modeled based on the molecular dimensions of the protein. Adsorbed streptavidin was quantified by direct labeling of protein with terbium chelate and with a sensitive bicinchoninic acid-based protein assay. A new small molecular weight (1037Da) reporter molecule, a europium-labeled biotin (Eu-biotin), was synthesized and used for monitoring adsorption and for determination of biotin-binding capacities of the streptavidin-coated wells. The theoretical monolayer adsorption of streptavidin yielded 6.20 pmol/cm(2) (370 ng) and consequently the theoretical adsorption capacity of a C12-format Microtitration well (200 microl liquid, coated area 1.54 cm(2)) was 9.55 pmol/well (570 ng). Adsorption properties of streptavidin from two suppliers were tested, one of which yielded 350-380 ng/well while the other yielded over 500 ng/well. The biotin binding capacities were about 11 and 14 pmol/well, respectively. We managed to quantify surface-adsorbed streptavidin with sensitive fluorescence and protein measurement methods in the Microtitration well. The new Eu-biotin reporter molecule enabled an exact and convenient determination of the biotin-binding capacities of streptavidin surfaces.

  • A high-capacity streptavidin-coated Microtitration plate
    Bioconjugate chemistry, 2003
    Co-Authors: Lasse Välimaa, Kim Pettersson, Matti Karp, Markus Vehniäinen, Timo Lövgren
    Abstract:

    A majority of current immunoassays rely on capturing a specific analyte on a solid phase to allow the separation of the bound analyte from nonbound components. Streptavidin-coated Microtitration plates are widely used for immobilization of capturing antibodies, since they provide a generic surface for immobilization of any biotinylated molecule and preserve biomolecule activity much better than direct passive adsorption. Our trials to further improve the properties of the plates resulted in a development of a modified plate, which has higher binding capacity than currently used control plate. The modified coat was prepared by cross-linking streptavidin chemically prior to adsorption onto the Microtitration well surfaces. The binding capacities of the plates were measured with biotinylated, europium-labeled molecules and labeled antigen. The immunoassay performance of the plates was studied with noncompetitive, sandwich-type assays of prostate specific antigen (PSA) and human chorionic gonadotropin (hCG). ...