QIA97 Cu/Zn Superoxide Dismutase ELISA Kit

QIA97
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      QIA97-1KIT
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          Description
          OverviewA colorimetric sandwich ELISA method for detecting Cu/Zn superoxide dismutase (SOD) in different types of biological samples.
          Catalogue NumberQIA97
          Brand Family Calbiochem®
          Application Data
          Recombinant Cu/Zn SOD was diluted in serial two-fold steps in PBS. The mean of three parallel titrations are plotted.



          Data represents the mean Cu/Zn SOD concentration and the coefficient of variation for each sample. The overall intra-assay coefficient of variation was determined to be 5.1%.

          Data represents the mean Cu/Zn SOD concentration and the coefficient of variation calculated on 18 determinations of each sample. The overall inter-assay coefficient of variation was determined to be 5.8%.

          Percent recoveries of expected values.
          Materials Required but Not Delivered 5 and 10 ml graduated pipettes
          5 to 1000 µl adjustable single channel micropipettes with disposable tips
          50 to 300 µl adjustable multichannel micropipette with disposable tips
          Multichannel micropipette reservoir
          Beakers, flasks, and cylinders necessary for preparation of reagents
          Multichannel wash bottle or automatic wash system for delivery of wash solution
          Plate reader capable of reading in 96-well plates at 450 nm with 620 nm as optional reference wavelength
          Glass-distilled or deionized water
          Calculator with program to perform linear regression analysis
          References
          ReferencesHerouart, D., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 3108.
          Niwa, Y., et al. 1993. Am. J. Pathol. 143, 312.
          Suzuki, H., et al. 1993. J. Clin. Invest. 91, 2727.
          Holzgreve, W., et al. 1991. Diagnose Labor. 41, 162.
          Porstmann, T., et al. 1991. Prenat. Diagn. 11, 195.
          Shull, S., et al. 1991. J. Biol. Chem. 266, 24398.
          Porstmann, T., et al. 1990. Hum. Genet. 85, 362.
          Porstmann, T., et al. 1988. Clin. Chim. Acta 171, 1.
          Jolly, S.R., et al. 1984. Circ. Res. 54, 277.
          Goebel, K.M. and Storck, U. 1983. Am. J. Med. 74, 124.
          Barra, D., et al. 1980. FEBS Lett. 120, 53.
          Sinet, P.M., et al. 1976. Exp. Cell Res. 97, 47.
          Harta, J.W. and Deutsch, H.F. 1972. Biol. Chem. 247, 7043.
          Product Information
          Detection methodColorimetric
          Form96 Tests
          Format96-well plate
          Kit containsPre-Coated 96-Well Plate, Anti-Cu/Zn SOD/HRP-Conjugate, Cu/Zn SOD Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Phosphate Buffered Saline, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
          Applications
          Key Applications Enzyme-Linked Immunosorbent Assay
          Biological Information
          Assay time1.5 h
          Sample TypeCell culture supernatant, serum, plasma, urine, amniotic fluid, and other biological fluids
          Species Reactivity
          • Human
          Physicochemical Information
          Sensitivity40 pg/ml
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 36/37/38

          Irritating to eyes, respiratory system and skin.
          S PhraseS: 26-36-45

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          Product Usage Statements
          Intended useThe Calbiochem® Cu/Zn Superoxide Dismutase ELISA Kit is designed to measure Cu/Zn superoxide dismutase (SOD) in cell culture supernatants and biological fluids like human serum, plasma, urine, and amniotic fluid.
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Multiple Toxicity Values, refer to MSDS
          Storage +2°C to +8°C
          Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
          Do not freeze Yes
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsPre-Coated 96-Well Plate, Anti-Cu/Zn SOD/HRP-Conjugate, Cu/Zn SOD Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Phosphate Buffered Saline, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
          Specifications

          Documentation

          Cu/Zn Superoxide Dismutase ELISA Kit SDS

          Title

          Safety Data Sheet (SDS) 

          Cu/Zn Superoxide Dismutase ELISA Kit Certificates of Analysis

          TitleLot Number
          QIA97

          References

          Reference overview
          Herouart, D., et al. 1993. Proc. Natl. Acad. Sci. USA 90, 3108.
          Niwa, Y., et al. 1993. Am. J. Pathol. 143, 312.
          Suzuki, H., et al. 1993. J. Clin. Invest. 91, 2727.
          Holzgreve, W., et al. 1991. Diagnose Labor. 41, 162.
          Porstmann, T., et al. 1991. Prenat. Diagn. 11, 195.
          Shull, S., et al. 1991. J. Biol. Chem. 266, 24398.
          Porstmann, T., et al. 1990. Hum. Genet. 85, 362.
          Porstmann, T., et al. 1988. Clin. Chim. Acta 171, 1.
          Jolly, S.R., et al. 1984. Circ. Res. 54, 277.
          Goebel, K.M. and Storck, U. 1983. Am. J. Med. 74, 124.
          Barra, D., et al. 1980. FEBS Lett. 120, 53.
          Sinet, P.M., et al. 1976. Exp. Cell Res. 97, 47.
          Harta, J.W. and Deutsch, H.F. 1972. Biol. Chem. 247, 7043.
          User Protocol

          Revision25-April-2016 RB
          Form96 Tests
          Format96-well plate
          Detection methodColorimetric
          Specieshuman
          StorageUpon arrival store the entire contents of the kit at 4°C.
          Intended useThe Calbiochem® Cu/Zn Superoxide Dismutase ELISA Kit is designed to measure Cu/Zn superoxide dismutase (SOD) in cell culture supernatants and biological fluids like human serum, plasma, urine, and amniotic fluid.
          BackgroundThe Superoxide Dismutase (SOD) family consists of metalloproteins that catalyze the dismutation of superoxide anion radicals to oxygen and hydrogen peroxide. It is ubiquitous in oxygen metabolizing cells, protecting these cells from direct and indirect oxygen-mediated free radical damage. Four types of SOD have been distinguished based upon their metal cofactors and distribution. Manganese (Mn SOD) is located mainly in the matrix of mitochondria of all aerobes, copper/zinc (Cu/Zn SOD) mainly in the cytoplasm of eukaryotic cells, iron (Fe SOD), in the cytosol, chloropasts or mitochondria of prokaryotes, and extracellular (EC SOD), which is found in the extracellular fluids or is membrane associated in mammals. While homology exists between Mn and Fe class enzymes, they display no homology with the Cu/Zn SOD. The human Cu/Zn SOD is a dimeric protein comprised of 2 subunits of 16 kDa each. Dissociation of these subunits is facilitated by the alkylation of the two sulfhydryl groups in the protein or by the removal of the copper and zinc ions. Cu/Zn SOD gene expression is induced by mediators of oxidative stress like sulfhydryl antioxidants, interleukin-1, and tumor necrosis factor.
          Principles of the assayThe Calbiochem® Cu/Zn Superoxide Dismutase ELISA Kit is a 96-well plate-based ELISA that utilizes a monoclonal antibody specific for Cu/Zn SOD to bind Cu/Zn SOD from unknown samples and standards. The captured enzyme is detected using a second anti-Cu/Zn SOD monoclonal antibody, conjugated to HRP. Substrate is added and the reaction stopped using an acid stop solution. The absorbance is read at 450 nm. The absorbance is directly proportional to the level of Cu/Zn SOD present in the samples. The concentration of Cu/Zn SOD in unknown samples is determined by comparison to a standard curve.
          Materials provided• Anti-Cu/Zn SOD mAb Coated 96-Well Plate (Kit Component No. JA6500): 1 plate, 96-wells coated with monoclonal antibody (mouse) to human Cu/Zn SOD, supplied as twelve 8-well strips
          • HRP-Conjugated Anti-Cu/Zn SOD Monoclonal Antibody (Kit Component No. JA6501): 2 vials, 20 µl each, with preservative
          • Cu/Zn SOD Standard (Kit Component No. JA6503): 2 vials, 500 µl each, supplied at 5 ng/ml, with preservative
          • Wash Buffer Concentrate (Kit Component No. JA6504): 1 bottle, 50 ml, supplied as 20X PBS with 1% Tween® 20 detergent and preservative
          • Assay Buffer Concentrate (Kit Component No. JA6505): 1 vial, 5 ml, supplied as 20X PBS with 1% Tween® 20 detergent, 10% BSA, and preservative
          • Phosphate Buffered Saline Concentrate (Kit Component No. JA6502): 1 vial, 5 ml, supplied as a 20X solution
          • Substrate Solution (Kit Component No. JA6513): 1 bottle, 15 ml, composed of TMB and stabilized hydrogen peroxide
          • Stop Solution (Kit Component No. JA6508): 1 bottle, 15 ml, 1 M Phosphoric Acid
          • Adhesive Plate Cover (Kit Component No. JA6512): 2 each
          Materials Required but not provided 5 and 10 ml graduated pipettes
          5 to 1000 µl adjustable single channel micropipettes with disposable tips
          50 to 300 µl adjustable multichannel micropipette with disposable tips
          Multichannel micropipette reservoir
          Beakers, flasks, and cylinders necessary for preparation of reagents
          Multichannel wash bottle or automatic wash system for delivery of wash solution
          Plate reader capable of reading in 96-well plates at 450 nm with 620 nm as optional reference wavelength
          Glass-distilled or deionized water
          Calculator with program to perform linear regression analysis
          Precautions and recommendations As conditions may vary from assay to assay a standard curve must be established for every run.
          Bacterial, fungal, or cross contamination of samples or reagents may cause erroneous results.
          While disposable pipette tips, flasks or glassware are preferred, reusable glassware must be thoroughly washed and rinsed of all detergent prior to use.
          Insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty and fill wells as indicated for each wash cycle and do not allow wells to dry out at any time.
          The use of radioimmunotherapy has significally increased the number of patients with human anti-mouse IgG antibody (HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies resulting in false positive and false negatives. Serum samples containing antibodies to murine immunoglobulins can still be analyzed by adding murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) to the PBS.
          All chemicals should be considered as potentially hazardous. We recommend that this product be handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses, and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) for specific information.
          Do not mix or substitute reagents with those from other lots or kits from other sources.
          Do not expose kit reagents to strong light during storage or incubation.
          Do not pipette by mouth.
          Do not eat or smoke in areas where kit reagents or samples are handled.
          Avoid contact of skin or mucous membranes with kit reagents or specimens.
          Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
          Avoid contact of substrate solutions with oxidizing agents and metal.
          Avoid splashing or generation of aerosols.
          Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents.
          Exposure to acids will inactivate the conjugate.
          Glass-distilled water or deiionized water must be used for reagent preparation.
          Substrate solution must be a room temperature prior to use.
          Decontaminate and dispose specimens and all potentially contaminated materials as if they could contain infectious agents; the preferred method of decontamination is autoclaving for a minimum of 1 h at 121.5°C.
          Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1% sodium hypochlorite; allow 30 min for effective decontamination. Liquid waster containing acid must be neutralized prior to the addition of sodium hypochlorite.
          PreparationNote: Serum, EDTA plasma, citrate plasma, and heparin plasma are all suitable for use with this assay; it is recommended, however, for uniformity, to use the same type of samples in one assay. Serum and Plasma: Remove serum or plasma from the clot or red cells, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use. Do not use extensively hemolyzed or lipemic specimens. Store all samples at -20°C. Allow samples to warm to room temperature and dilute with PBS just prior to use. Avoid freeze/thaw cycles. Predilute serum or plasma samples 1:20 with PBS by adding 10 µl serum/plasma to 190 µl PBS. For fetal umbilical vein blood first adjust samples to ~2 x 107 erythrocytes/ml. Note on Stability of Samples: Aliquots of serum (unspiked and spiked with Cu/Zn SOD) were stored at -20°C, thawed several times, and assayed for the level of Cu/Zn SOD. There was no significant loss of Cu/Zn SOD following up to 5 freeze/thaw cycles. Aliquots of serum (unspiked and spiked with Cu/Zn SOD) were stored at -20°C, 4°C, room temperature, and 37°C and the levels measured after 24 h, 48 h, and 96 h. There was no significant loss of Cu/Zn SOD following storage under these conditions.
          Reagent preparation• Wash Buffer: If crystals have formed in the Wash Buffer Concentrate, warm it gently until the crystals are re-dissolved. To prepare enough Wash Buffer for the entire plate, add 50 ml Wash Buffer Concentrate to 950 ml deionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Store at 4°C. Wash Buffer is stable for up to 30 days. • Assay Buffer: Mix the Assay Buffer Concentrate well. To prepare enough Assay Buffer for the entire plate, add to 5 ml Assay Buffer Concentrate to 95 ml distilled or deionized water. Mix gently to avoid foaming. Store Assay Buffer at 4°C. Assay Buffer is stable for 30 days at 4°C. • PBS: Mix the contents of the PBS concentrate well. To prepare enough PBS for the entire plate, add 5 ml Phosphate buffered saline (PBS) concentrate to 95 ml distilled or deionized water. Mix gently to avoid foaming. PBS is stable for up to 30 days at 4°C. • HRP Conjugate: Dilute the HRP-Conjugated Anti-Cu/Zn SOD 1:5 just prior to use by adding 80 µl assay buffer to the tube containing HRP conjugate concentrate. Mix the contents of the tube well. Make an additional 1:100 dilution of the HRP-Conjugate with Assay Buffer in a clean plastic tube.
          Detailed protocolNote: Mix all reagents prior to use; mix gently to avoid foaming.

          1. Remove the desired number of strips and return the unused portion to the foil pouch and store at 4°C. Each sample, standard, blank, and optional control sample should be assayed in duplicate.
          2. Wash the wells with approximately 300 µl Wash buffer; thoroughly aspirate the contents of the wells between washes. Please note that insufficient washing at any step in the assay will result in false results. Completely empty wells before each wash. Take care not to scratch the surface of the wells. After the last wash, empty wells and tap on absorbent pad or paper towel to remove excess buffer. Use the strips immediately after washing or place upside down on a wet absorbent paper for not longer than 15 min. Do not allow wells to dry.
          3. Add 100 µl PBS to all (duplicate) standard wells leaving the first wells empty. Prepare standard dilutions by adding 200 µl Cu/Zn SOD standard to wells A1 and A2. Mix the contents of wells A1 and A2 by repeated aspiration and carefully transfer 100 µl to wells B1 and B2, respectively. Take care not to scratch the inner surface of the wells. Continue this procedure five times, creating two rows of Cu/Zn SOD standard dilutions ranging from 5 ng/ml to 80 pg/ml. Discard 100 µl of the contents from the last wells.
          4. Add 100 µl PBS to designated (duplicate) blank wells.
          5. Add 90 µl PBS to designated (duplicate) sample wells.
          6. Add 10 µl of each pre-diluted sample, in duplicate, to designated wells.
          7. Add 50 µl HRP Conjugate to all wells, including the blank wells.
          8. Cover the plate with a plate cover and incubate at room temperature for 1 h on a rotator platform.
          9. Remove plate cover and empty wells. Wash the wells 3 times as outlined in step 2 above.
          10. Add 100 µl Substrate Solution to all wells, including the blank wells.
          11. Incubate the plate at room temperature for 10 min. Avoid direct exposure to intense light. It is recommended to add the Stop solution when the highest standard has developed a dark blue color. Alternatively, the point at which the substrate reaction should be stopped may be determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2.0 absorbance points, therefore the color development and the substrate reaction must be stopped before positive wells are no longer properly recordable.
          12. Stop the reaction by quickly adding 100 µl Stop solution to each well, including the blank wells. It is important that the Stop solution be spread quickly and uniformly throughout the wells to completely stop the reaction. Results must be read immediately after the Stop solution is added or within 1 h if the strips are stored at 4°C in the dark.
          13. Read the absorbance of each well using an ELISA plate reader set at 450 nm as the primary wave length and 620 nm as the reference wave length (optional); 610 nm to 650 nm is acceptable. Blank the plate reader using the blank wells. Determine the absorbance of the samples and the Cu/Zn SOD standards. Note: incubation without shaking may result in lower than expected absorbance values.
          Calculations1. Calculate the average absorbance values for each set of duplicate standards and samples. 2. Create a standard curve by plotting the mean absorbance for each standard concentration on the y-axis against the Cu/Zn SOD concentration on the x-axis. Draw a best-fite curve through the points on the graph. 3. Determine the concentration of Cu/Zn SOD for each sample by finding the mean absorbance value on the y-axis and extending a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the x-axis and read the corresponding Cu/Zn SOD concentration. For samples that have been diluted according to the instructions outlined in this user protocol (1:200 dilution), the concentration read from the standard curve must be multiplied by a dilution factor of 200. Calculation of samples with an absorbance exceeding the range of the standard curve may result in incorrect/low Cu/Zn SOD levels. Such samples should be re-analyzed at higher dilution (1:400 to 1:800) in PBS to accurately quantitate the Cu/Zn SOD level. Values obtained should be within the expected range of known Cu/Zn SOD controls. 4. It is suggested that each end-user establish a control sample of known Cu/Zn SOD concentration to be included as an addtional postivie control with each assay. If values obtained are not within the expected range of this control, the assay results may be invalid.
          Standard curve

          Figure 1: Standard Curve

          Recombinant Cu/Zn SOD was diluted in serial two-fold steps in PBS. The mean of three parallel titrations are plotted.

          Table 1: Typical Data Obtained With the Cu/Zn Standard

          Sensitivity40 pg/ml
          Sensitivity NotesThe limit of detection for Cu/ZnSOD, defined as the analyte concentration resulting in an absorption significantly higher than the absorption of the dilution medium (mean plus three standard deviations) was determined to be 40 pg/ml (mean of 10 independent assays).
          Assay Range NotesA panel fo 22 serum samples from apparently health blood donors (male and female) was tested for Cu/Zn SOD. The detected levels ranged between 22.5 and 102.9 ng/ml with a mean level of 56.5 ng/ml and a standard deviation of 20 ng/ml. Normal Cu/Zn SOD levels may vary depending on the serum used. Measurement of Cu/Zn SOD from erythrocytes of fetal umbilical vein blood resulted in levels ranging from 11-16 ng SOD per 1 X 10⁶ fetal erythrocytes for normals and >20 ng SOD per 1 X 10⁶ fetal erythrocytes from fetuses with Down's Syndrome.
          RecoverySpiked samples were prepared by adding four different levels of Cu/Zn SOD to 2 human serum samples with different base levels of Cu/Zn SOD. Recoveries were determined in two independent experiments.

          Table 2: Recovery

          ReproducibilityIntra-Assay Reproducibility within the assay was evaluated in three independent experiments. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of Cu/Zn SOD. Two standard curves were run on each plate.

          Table 3: Intra-Assay Reproducibility

          Data represents the mean Cu/Zn SOD concentration and the coefficient of variation for each sample. The overall intra-assay coefficient of variation was determined to be 5.1%.

          Inter-Assay Assay to assay reproducibility within one laboratory was evaluated in three independent experiments by three technicians. Each assay was carried out with 6 replicates of 8 serum samples containing different concentrations of Cu/Zn SOD. Two standard curves were run on each plate.

          Table 4: Inter-Assay Reproducibility

          Data represents the mean Cu/Zn SOD concentration and the coefficient of variation calculated on 18 determinations of each sample. The overall inter-assay coefficient of variation was determined to be 5.8%.

          ParallelismFour serum samples with different levels of Cu/Zn SOD were assayed at four serial two-fold dilutions (1:200-1:1600) covering the working range of the standard curve. Recoveries ranged from 80% to 107% with an overall mean of 90%.

          Table 5: Parallelism

          Percent recoveries of expected values.

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          Tween® is a registered trademark of ICI Americas, Inc.