AccuSignal™ Nuclease ELISA Kit for Impurity Detection

• 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.

• 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

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.
Note: This is the “reference stock solution” that will be used below to make the standards.
2. Prepare dilutions of standard.
3. Test samples should be diluted in Nuclease Kit Sample Buffer based on empirically determined criteria for each sample.

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.

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.

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.

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.

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.