Phone:
+1 877 302 8632
Fax:
+1 888 205 9894 (Toll-free)
E-Mail:
orders@antibodies-online.com

AccuSignal™ Nuclease ELISA Kit for Impurity Detection

ELISA, Imp De Colorimetric Quantitative
Catalog No. ABIN7505815
  • Key Features
    • Impurity detection in the manufacturing of biological drugs.
    • The AccuSignal™ Nuclease ELISA Kit is designed for sensitive and reliable quantification of nucleases in therapeutic products, including DENARASE®, Benzonase®, and Turbonuclease.
    • Offers a broad quantification spectrum, maintaining outstanding dilution linearity, instilling trust in the accuracy of results over a wide array of nuclease concentrations.
    • Delivers consistent and repeatable outcomes, characterized by minimal intra- and inter-assay variability.
    • Tailored antibody specificity allows for application across diverse materials, accommodating various nuclease products from multiple suppliers.
    • Components: 96-well strip plate, plate sealer, nuclease standard, biotinylated detection antibody, Streptavidin-HRO (100X), buffers.
    Target See all Nuclease products
    Nuclease
    Detection Method
    Colorimetric
    Host
    Rabbit
    Clonality
    Polyclonal
    Method Type
    Sandwich ELISA
    Detection Range
    0.03 ng/mL - 20 ng/mL
    Minimum Detection Limit
    0.03 ng/mL
    Application
    ELISA, Impurity Detection (Imp De)
    Purpose
    Nuclease ELISA kit is designed for the quantitative detection of Nuclease/NucA in serum, plasma, and hybridoma cell supernatants.
    Brand
    AccuSignal™
    Analytical Method
    Quantitative
    Specificity
    Benzonase, Nuclease, Endonuclease, Serratia marcescens endonuclease, NucA, Denarase
    Sensitivity
    3 ng/mL
    Components
    This kit contains: Nuclease antibody coated 96-well strip plate, Plate Sealer, Nuclease standard, biotinylated Detection Antibody, streptavidin-peroxidase conjugate, along with buffers and protocol.
    Material not included
    • Microplate shaker (up tp 450 rpm)
    • Interval timer
    • Multichannel pipettor (50-300 µL)
    • Precision single pipettes (10 µL, 35 µL, 100 µL, 1000 µL, etc.)
    • Disposable pipette tips
    • Deionized water
    • Disposable microcentrifuge Tube(s) or microplate
    • Polypropylene centrifuge tubes (15 mL)
    • Spectrophotometer microplate reader (450 nm absorbance, 630–650 nm reference filter)
    • Disposable gloves
    • Graduated cylinder
    • Reagent reservoirs
    • Vortex mixer
    • Stir plate & magnetic stir bar
    • Absorbent paper
    Product Specific Information

    The Nuclease ELISA Kit is an essential tool specifically designed to sensitively and robustly quantify the amount of nucleases in therapeutic products, including DENARASE®, Benzonase®, and Turbonuclease. This kit utilizes a sandwich ELISA format, leveraging the specificity and affinity of pre-immobilized anti-nuclease antibodies. The biotin-conjugated detection combined with streptavidin-HRP guarantees unparalleled sensitivity, enabling accurate measurement of nucleases across a variety of therapeutic materials. It facilitates the assessment of nuclease content in biologically derived therapeutics, ensuring their stability and quality throughout the manufacturing process. Additionally, it aids in evaluating nucleases in gene therapy vectors, a critical factor in maintaining the integrity of these vectors for effective therapeutic gene delivery. It also helps in validating the nuclease levels in pharmaceutical formulations, protecting their potency and preventing potential degradation. By providing a reliable and standardized method for the quantification of nucleases, the Nuclease ELISA Kit not only ensures compliance with strict regulatory requirements but also accelerates research and development processes.

    Main application areas for the AccuSignal™ Nuclease ELISA Kit

    Biological Therapeutics: Evaluate nuclease levels in biologically-sourced therapeutics to guarantee their stability and quality during production.

    Gene Therapy Vectors: Aid in assessing nuclease presence in gene therapy vectors, essential for preserving their integrity for successful gene delivery treatments.

    Vaccine Production: Confirm nuclease concentrations in pharmaceutical formulations to ensure their effectiveness and prevent potential degradation.

    The user-friendly approach and robust methodology streamline workflows, enabling scientists to focus on advancing therapeutic innovations with confidence. As an indispensable instrument, it promotes precision, reliability, and efficiency in the evaluation of nucleases in therapeutic products, while ensuring safety and efficacy.

  • Application Notes
    This kit was shown to positively detect commercially available nuclease variants, such as Benzonase, Denarase and other analogs.
    Assay Time
    3 h
    Reagent Preparation

    Preparation of Standards & Test Samples

    1. Reconstitute the standard vial with 1.0 mL deionized water to obtain a final concentration of 200 ng/mL.
    2. Note: This is the “reference stock solution” that will be used below to make the standards.
    3. Prepare dilutions of standard.
    4. Test samples should be diluted in Nuclease Kit Sample Buffer based on empirically determined criteria for each sample.

    Detection antibody working solution preparation
    1. Add 110 µL of conjugated antibody to 11 mL of Sample Buffer for use in a full 96-well assay.
    2. Mix well by pipette or inversion. Do not vortex.
    3. Distribute antibody working solution as described in the assay procedure.
    Note: Volumes may be adjusted so long as final working concentration remains as specified.

    Streptavidin-HRP Working Solution Preparation
    1. Add 110 µL of Streptavidin-HRP to 11 mL of Sample Buffer, respectively for use in a full 96-well assay.
    2. Mix well by pipette or inversion. Do not vortex.
    3. Distribute Streptavidin-HRP working solution as described in the assay procedure.
    Note: Volumes may be adjusted so long as final working concentration remains as specified

    Wash Buffer (1X) Preparation
    1. Add 50 mL of Nuclease Kit Wash Buffer (10X) to 450 mL of deionized water.
    2. Mix for at least 10-minutes using a magnetic stir bar.
    Note: The wash buffer (1X) can be stored at room temperature (15°C to 25°C) for up to 2-weeks, after which it should be discarded.

    Assay Procedure
    1. To each well add 100 µL of unknown or standard sample per well and incubate at room temperature for 60-minutes with shaking at 450 revolutions per minutes (rpm) on a shaker.
    2. Wash the wells with Wash Buffer as follows:
      • Decant the contents of the wells manually with a hard, rapid downward motion. Fluid should be captured in a receptable designed to collect the waste. Remove all residual reagent from the microplate by tapping it on absorbent paper with the opening facing downwards.
      • Fill each well with 300 µL of Washing Buffer with a multichannel pipettor.
      • Decant the Washing Buffer from the wells with a hard, rapid downward motion. Remove all residual solution from the microplate by tapping it on absorbent paper with the opening facing downwards.
      • Repeat steps ii and iii two more times (total of 3 washings). Do not leave any residual moisture in the wells on each washing step.
    3. To each well add 100 µL of detection antibody working solution
    4. Incubate at room temperature for 60-minutes, covered to protect from light, with shaking at 450 rpm.
    5. Following 60-minute incubation, wash with Wash Buffer as follows:
      • Decant the contents of the wells manually with a hard, rapid downward motion. Fluid should be captured in a receptable designed to collect the waste. Remove all residual reagent from the microplate by tapping it on absorbent paper with the opening facing downwards.
      • Fill each well with 300 µL of Washing Buffer with a multichannel pipettor.
      • Decant the Washing Buffer from the wells with a hard, rapid downward motion. Remove all residual solution from the microplate by tapping it on absorbent paper with the opening facing downwards.
      • Repeat steps ii and iii two more times (total of 3 washings). Do not leave any residual moisture in the wells on each washing step.
    6. To each well add 100 µL of Streptavidin-HRP working solution
    7. Incubate at room temperature for 20-minutes, covered to protect from light, with shaking at 450 rpm.
    8. Following 20-minute incubation, wash with Wash Buffer as follows:
      • Decant the contents of the wells manually with a hard, rapid downward motion. Fluid should be captured in a receptable designed to collect the waste. Remove all residual reagent from the microplate by tapping it on absorbent paper with the opening facing downwards.
      • Fill each well with 300 µL of Washing Buffer with a multichannel pipettor.
      • Decant the Washing Buffer from the wells with a hard, rapid downward motion. Remove all residual solution from the microplate by tapping it on absorbent paper with the opening facing downwards
      • Repeat steps ii and iii two more times (total of 3 washings). Do not leave any residual moisture in the wells on each washing step.
    9. Next add 100 µL per well of room temperature TMB solution and incubate the plate with TMB solution at room temperature for 20 minutes (covered and protected from light).
    10. Then add 100 µL of stop solution per well. Gently tap the plate to mix, ensuring no bubbles are formed, and read plate within 5 minutes after stopping the reaction.
    11. On plate reader, measure absorbance at 450 nm with the reference wavelength set at 630–650 nm.
    Calculation of Results

    Follow the steps below to estimate the nuclease concentration of the test samples.

    1. Calculate the relative OD 450 using the following formula: Relative OD 450 = (OD 450 of well) – (OD 630-650 nm of the well)
    2. Calculate the mean relative OD 450 of the replicates for each standard solution.
    3. Plot the standard solutions data as mean relative OD 450 for each standard solution (Y) vs the respective concentration of the standard solutions (X).
    4. Fit the standard solution data with a 4-parameter logistic (4-PL) curve. Weight by 1/Y^2 is intended to be used during generation of 4-PL curve
    5. Estimate the Nuclease concentration of each test sample well using interpolation from the standard curve. Calculate the average of each respective sample solution concentration.
    Note: If the assay samples are from dilutions, multiply the concentrations obtained from interpolations by the dilution factor.
    Note: If the spectrometer used for the assay does not automatically subtract the reference wavelength, do this manually.

    Assay Precision
    Intra-and inter-assay CV% <20%
    Restrictions
    For Research Use only
  • Storage
    4 °C
    Storage Comment
    The kit and reagents should be stored at 2-8°C. Allow reagents to reach room temperature (18-26°C) before use and may be used until the expiration date. It is recommended to aliquot the reconstituted standard solution and store it at -20°C to avoid freeze/thaw cycles.
  • Target
    Nuclease
    Alternative Name
    nucA (Nuclease Products)
    Background
    Nucleases are secreted by Serratia marcescens into the medium it surrounds. The enzyme is a sugar-nonspecific hydrolase, capable of cleaving both RNA and DNA in either double or single stranded form. It requires divalent cations, preferably Mg2+, and is functional across a broad pH range from 6 to 10 (optimal at 8-8.5) and wide temperature ranges between 35°C and 44°C. The ability of Serratia to secrete nuclease appears to be regulated. Bacterial cultures at differing cell densities display different kinetics and efficiencies of nuclease secretion [i.e. growth medium, growth conditions, and host cell mutations]. Anti-Nuclease/NucA Antibody is useful for researcher interested in identifying nucleic acid contamination.
You are here: