Ultra Nuclease Assay Kit (ELISA)
- Wide Application: It can degrade all forms of DNA and RNA and digest them into a length of 3-5 bases.
- High purity & Activity: Purity ≥99%, specific activity ≥1.1×106U/mg.
- Strong Adaptability: Able to maintain high stability and digestive activity under conditions including 6M Urea, 0.1M Guanidine HCl, 0.4%Triton X-100, 0.1% SDS, 1mM EDTA or 1mM PMSF.
- Compliance with Pharmacopoeia: Animal-origin free, β -amide antibiotics free. High Production Standard.
- High Production Standard: GMP-standard production, meeting requirements for large-scale use from R&D to production.
UltraNuclease is a genetically engineered broad-spectrum nuclease originating from Serratia Marcescens. It can degrade all forms of DNA and RNA, including single-stranded, double-stranded, linear, natural and denatured nucleic acids, and digest them into 5′-oligonucleotide monophosphate of 3-5 base in length with no base-specific recognition. Therefore, it is the most ideal enzyme preparation for removing nucleic acid.
Principle of the Procedure
This UltraNuclease ELISA kit is a sandwich ELISA to be performed in a microplate format. Sample potentially containing endonuclease are incubated in microtiter plate wells which have been pre-coated with an affinity purified anti-endonuclease capture antibody. After incubation and a washing step in which unbound components are removed, an enzyme-conjugated, anti-endonuclease detector antibody is added. The immunological reactions result in the formation of a sandwich complex of solid phase antibody-endonuclease-enzyme labeled antibody. After a final washing step, a substrate solution is added to the wells and reacted, resulting in color development. The optical density is measured photometrically and is proportional to the analyte concentration present in the wells. The endonuclease concentration in unknown samples can be calculated based on the corresponding standard curve.
Kit Component
- ELISA microplate….……….………………….8×12 wells
- Enzyme conjugate (100×)…………………0.15mL×1
- Dilution buffer…..………..……………………30mL×1
- Substrate solution……………..….…………6mL×2
- Stop solution…….……….……..……………..6mL×1
- Wash concentrate (10×)…………………..50mL×1
- Standard concentrate……………………..0.5mL×1
- Sealing film………….…….….………………..4 films
- Manual……………………..……………………..1 copy
Storage and Stability
- Unopened kit should be stored at 2℃ to 8℃ for stability until the expiration date printed on the kit.
- Opened reagents should be stored at 2℃ to 8℃ and are stable for 6 weeks.
Equipment required
- Microplate reader spectrophotometer with dual wavelength capability at 450 and 650nm. (If your plate reader does not provide dual wavelength analysis you may read at just the 450nm wavelength.)
- Microtiter plate rotator (400-600 rpm).
Reagent Preparation
- Bring all reagents to room temperature (23℃±2℃) before use.
- Prepare washing buffer (1×) by diluting 50mL of the Wash concentrate (10×) with 450mL of deionized or distilled water.
- Enzyme conjugate (1×) : Dilute to work concentrate at 1:99 by dilution buffer before use.
- Dilute the Standard concentrate (1000ng/mL) with dilution buffer to 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.63ng/mL, 0.31ng/mL, 0ng/mL. Standards (20ng/mL to 0ng/mL) are prepared according to the following dilution scheme:
Assay Procedure
- Take out the required strips form the foil of the ELISA microplate after reaching room temperature. Remaining plate strips not used in the assay should be repacked in the bag with desiccant. Close the bag tightly for refrigerated storage.
- Pipette 100μL of standards (20ng/mL to 0ng/mL) or samples into each well.
- Cover and incubate on shaker at 500rpm for 60 min at room temperature.
- Dump contents of the wells. Blot and gently but firmly tap over absorbent paper to remove most of the residual liquid. Fill wells generously with 300μL/well of washing buffer (1×). Dump and tap again. Repeat for a total of 5 washes.
- Pipette 100μL of the enzyme conjugate (1×) into each well. Cover and incubate on shaker at 500rpm for 30 min at room temperature.
- Dump contents of the wells. Blot and gently but firmly tap over absorbent paper to remove most of the residual liquid. Fill wells generously with 300μL/well of washing buffer (1×). Dump and tap again. Repeat for a total of 5 washes.
- Pipette 100μL of the Substrate solution into each well. Cover and incubate for 30 min at room temperature. Protect from light. (DO NOT SHAKE.)
- Pipette 50μL of the Stop solution into each well. Read absorbance at 450/650nm within 15 min.
Assay Flowchart
Calculation of Result
- Calculate the mean absorbance for each standard, control and sample and subtract average zero standard optical density. It is possible not to subtract the optical density of zero standard. Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. Absorbances of samples are then interpolated from this standard curve.
- Most graphing software can help make the curve and cubic usually provide the best fit, though other equations (e.g. 4 parameter logistic fit) can also be tried to see which provides the most accurate.
Performance Characteristics
- Lower limit of detection: 0.06 ng/mL
- Lower limit of quantitation: 0.2 ng/mL
- Linearity: 0.2~20 ng/mL
- Precision: Intra assay CV ≤10%, Inter assay CV ≤15%
- Recovery: 80%~120%
- Specificity: This immunoassay is calibrated against UltraNuclease, available from Wuhan
Hzymes Biotechnology Co., Ltd. Endonuclease from other suppliers may show a different reactivity with regard to sensitivity and accuracy, therefore the compatibility of the kit calibration to the individual endonuclease product must be verified.
Precautions
- TMB reaction temperature and time is critical, please control them according to the instruction strictly.
- Thorough washing is essential to proper performance of this assay. The manual method described in the assay procedure is preferred for best precision, sensitivity and accuracy.
- All reagents should be shaken well before use. When adding samples, add the sample to the bottom of the well of the ELISA plate, avoid adding to the upper part of the well wall, and be careful not to splash or generate bubbles when adding samples.
- If crystals have formed in buffer solution, warm to room temperature and mix gently until the crystals have completely dissolved.
- Avoid the assay of samples containing sodium azide (NaN3) which will destroy the HRP activity of the conjugate and could result in the underestimation of UltraNuclease levels.
- Remove nucleic acids wrapped around the surface of virus particles, to promote virus release and purification.
- Remove the exogenous nucleic acids in vaccine products, to lower the risk of residual toxicity of nucleic acid and improve product safety.
- Reduce feed viscosity caused by nucleic acid during drug purification, accelerate the process of purification treatment, and increase protein recovery yield.
- Remove nucleic acids in gene therapy, to obtain purified adeno-associated virus.
- Used in sample preparation during column chromatography, electrophoresis and western blotting, to improve resolution and recovery rate.
- Prevent cell clustering
The Certificate Of Analysis (COA) & Material Safety Data Sheet (MSDS) Is A Signed Document That Includes The Storage Temperature,
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