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QIA53 p53 ELISAPLUS (Autoantibody) Kit

p53 ELISAPLUS (Autoantibody) Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
QIA53
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      This product has been discontinued.



      We apologize for the inconvenience, but we do not currently have an alternative product.






      Kit is designed to measure circulating antibodies to p53 in human serum samples.
      Catalogue NumberQIA53
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Pipettors: 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips.
      • Precision repeating pipettor.
      • Wash bottle or multichannel dispenser for plate washing.
      • Microcentrifuge and tubes for sample preparation.
      • Vortex mixer.
      • Plate reader or spectrophotometer capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/595 nm or plate reader capable of measuring absorbance in 96-well plate at a single wavelength of 450 nm.
      • 500 or 1000 ml graduated cylinder.
      • Reagent reservoirs.
      • Distilled water of high quality.
      • Liquid household bleach for inactivating clinical specimens and decontamination of plate washer.
      • Disposable paper towels.
      References
      Product Information
      Detection methodColorimetric
      DeclarationManufactured by Dianova, GmbH, Germany. Not available for sale in Germany.
      Form96 Tests
      Format96-well plate
      Kit containsCoated 96-Well Plate, Calibrator, Negative Control, Detector Antibody Conjugate, TMB Substrate Solution, Dilution Buffer, Wash Buffer, Stop Solution, and a user protocol.
      Applications
      Biological Information
      Assay time2.5 h
      Sample TypeSerum
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/21/22-36/37/38-40

      Harmful by inhalation, in contact with skin and if swallowed.
      Irritating to eyes, respiratory system and skin.
      Limited evidence of a carcinogenic effect.
      S PhraseS: 22-24/25-26-36/37/39-45

      Do not breathe dust.
      Avoid contact with skin and eyes.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe Calbiochem® p53- ELISAPLUS (Autoantibody) Kit is designed to measure circulating antibodies to p53 in human serum samples.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C. Coated assay plates should be stored in the original foil bag sealed by the clip (provided).
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCoated 96-Well Plate, Calibrator, Negative Control, Detector Antibody Conjugate, TMB Substrate Solution, Dilution Buffer, Wash Buffer, Stop Solution, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA53 0

      Documentation

      Brochure

      Title
      Caspases and other Apoptosis Related Tools Brochure
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      p53 And Related Products
      User Protocol

      Revision23-November-2010 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C. Coated assay plates should be stored in the original foil bag sealed by the clip (provided).
      Intended useThe Calbiochem® p53- ELISAPLUS (Autoantibody) Kit is designed to measure circulating antibodies to p53 in human serum samples.
      BackgroundThe development of invasive tumors depends on discrete changes in specific genes controlling proliferation and tissue homeostasis. Tumor suppressor genes are vulnerable sites for critical DNA damage because they normally function as physiologic barriers against clonal expansion or genomic mutation and are able to hinder the growth and metastasis of cells driven to uncontrolled proliferation by oncogenes. Loss of tumor suppressor function can occur through damage to the genome by mutation, chromosomal rearrangement and nondisjunction, gene conversion, imprinting or mitotic recombination. Tumor suppressor activity can also be neutralized by interaction with other cellular proteins or viral oncoproteins. The p53 tumor suppressor gene is the most striking example because it is mutated in about 60-70% of most types of cancer arising from a wide spectrum of tissues. The p53 protein was discovered in the late 1970's as a cellular 53 kDa nuclear phosphoprotein. It has been implicated in the control of the cell cycle, DNA repair and synthesis, genomic plasticity and programmed cell death. Mutant forms can act as dominant oncogenes, whereas wild-type p53 has characteristics of a recessive tumor suppressor gene. Many studies have focused on the relevance of p53 mutations in human cancers. It was shown that 80% of the p53 mutations are missense mutations, usually altering protein confirmation and causing nuclear accumulation. For these studies it is necessary to prepare DNA, amplify the p53 exons and sequence the DNA, which is a complicated, time consuming and is an expensive technology. The relatively low levels of p53 protein in normal cells are generally undetectable when examined by immunohistochemical techniques, whereas in neoplastic cells carrying a missense mutation, the presence of p53 protein is easily demonstrated because of its prolonged half-life. For these studies it is necessary to have biopsies or other tumor material. Identification of p53 protein in serum, other body fluids or tumor extracts is variable and is dependent on the sensitivity of the assay used. One explanation for this is that the protein is being rapidly destroyed in serum or extracts by the action of proteases. Mutant p53 proteins, as tumor specific antigens, may be a target of the host immune systems. Studies have shown that about 25% of samples with different carcinomas have mounted a humoral immune response (antibodies) to abnormal levels of p53 protein. Thus, anti-p53 antibodies may be a future serological marker for malignancies and might be detected in samples negative for conventional tumor markers. In conclusion, these studies have shown that anti-p53 serum antibodies can be readily detected in a significant proportion of tumor samples.
      Principles of the assayThe Calbiochem® p53 ELISAPLUS (Autoantibody) Kit uses 96-well plates pre-coated with recombinant human wild-type p53 protein. The detector antibody is a horseradish peroxidase conjugated, purified goat anti-human polyclonal antibody which recognizes human IgG.

      To perform the test, the sample or standard is pipetted into the wells and allowed to incubate for one hour at room temperature. This allows the circulating p53 antibodies to bind to the pre-coated p53 antigen. Following this incubation the plate is washed to rid of any unbound material. Next, a peroxidase-conjugated goat anti-human antibody is added to the wells which binds to any captured human p53 antibody. Following this incubation and a wash step, a chromogenic substrate is added to the wells. The horseradish peroxidase catalyses the conversion of the chromogenic substrate tetramethylbenzidine (TMB) from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of human p53 antibody in the test sample. The colored reaction product is quantified using a spectrophotometer.
      Quantitation is achieved by the construction of a calibrator curve using known titers of human p53 antibody. By comparing the absorbance obtained from a sample containing an unknown amount of human p53 antibody with that obtained from the calibrators, the titer of human p53 antibody in the test sample can be determined.
      Materials providedNote: Samples and calibrators should be assayed in duplicate. A calibrator curve must be included each time samples are analyzed. The following components are supplied and are sufficient for 96 determinations (including calibrators and negative controls).

      • 96-Well Plate (Kit Component No. JA1720): 1 microplate supplied ready to use, with 96 wells (12 strips of 8) in a tin-foil pouch with a desiccant pack. Wells are coated with recombinant human p53 protein.
      • Calibrator (Kit Component No. JA1710): 1 vial containing 1.5 ml of human serum with a defined amount of anti-human p53 antibody. The undiluted calibrator is always equivalent to the 1 I of P53 binding activity.
      • p53-A Positive Control (Kit Component No. JA1709): 1 vial containing 500 µl diluted human serum with defined p53 autoantibody concentration.
      • Negative Control (Kit Component No. JA1713): 1 vial containing 1 ml of human serum devoid of anti-human p53 antibody, read-to-use.
      • Sample Dilution Buffer (Kit Component No. JA1716): 6 vials containing a freeze-dried proteinmatrix with added sodium azide (0.05%). Reconstitute the vial by adding 12 ml of distilled water. Once reconstituted, this buffer is used to dilute samples and should be discarded after 14 days when stored at at 4°C.
      • Detector Antibody Conjugate (Kit Component No. JA1714): 1 bottle supplied ready to use, containing 12 ml of a peroxidase-conjugated goat anti-human polyclonal antibody.
      • TMB Substrate Solution (Kit Component No. JA1715): 1 bottle supplied ready to use, containing 12 ml of the chromogenic substrate, tetra-methylbenzidine (3,3,5,5-tetramethylbenzidine, (TMB)). This solution is light sensitive.
      • Stop Solution (Kit Component No. JA1718): 1 bottle supplied ready to use, containing 7.5 ml of 2 M HCl. Caution: corrosive.
      • Wash Buffer (Kit Component No. JA1719): 1 bottle containing 50 ml of a concentrated wash buffer (20X). To make 1 L of ready-to-use wash buffer, dilute 50 ml of the concentrate in 950 ml distilled water. Once reconstituted, this buffer should be discarded after 14 days when stored at at 4°C.
      Materials Required but not provided• Pipettors: 2-20 µl, 20-200 µl and 200-1000 µl precision pipettors with disposable tips.
      • Precision repeating pipettor.
      • Wash bottle or multichannel dispenser for plate washing.
      • Microcentrifuge and tubes for sample preparation.
      • Vortex mixer.
      • Plate reader or spectrophotometer capable of measuring absorbance in 96-well plates at dual wavelengths of 450 nm/595 nm or plate reader capable of measuring absorbance in 96-well plate at a single wavelength of 450 nm.
      • 500 or 1000 ml graduated cylinder.
      • Reagent reservoirs.
      • Distilled water of high quality.
      • Liquid household bleach for inactivating clinical specimens and decontamination of plate washer.
      • Disposable paper towels.
      Precautions and recommendations• Do not freeze any kit components - store at 4°C. Do not expose reagents to excessive light.
      • Use only the wells provided with the kit.
      • Use distilled water of the highest quality.
      • Do not mix reagents from different kits.
      • The buffers and reagents used in this kit contain preservatives. Care should be taken to avoid direct contact with these reagents.
      • The serum calibrator and negative control are HIV 1 and 2-negative, Hepatitis B and C-negative, but like all serum samples, should be treated as if infectious agents. Do not ingest, expose to open wounds or breathe aerosols. Wear protective gloves and dispose of biological samples properly.
      • Do not smoke, eat, or drink when performing the assay or in areas where samples or reagents are handled.
      • Do not permit contact of reagents with skin, metal or oxidizing agents. Dispose of tetramethylbenzidine (TMB) containing solutions in compliance with local regulations.
      • Wear disposable gloves and eye protection when handling Stop Solution (2 M hydrochloric acid).
      PreparationBlood should be collected in a tube without any additives. After coagulation at room temperature, separate serum from the clot by centrifugation. Samples may be stored for 24 h at 4°C. If the samples cannot be tested within 24 h, they should be aliquoted and stored at -20°C. Avoid repeated freezing and thawing.
      Reagent preparationBefore starting the assay, record the positions of the assay calibrators, negative control and samples. Remove reagents from refrigerator and allow them to reach room temperature before use. 1. Prepare wash solution by diluting the wash buffer to 1 liter with distilled water. 2. Reconstitute one vial of the Sample Dilution Buffer by adding 12 ml of distilled water. 3. Preparation of samples: Required volume is 100 µl per well. The samples must be diluted 1:100 in sample dilution buffer and should be assayed in duplicate. 4. Use 100 µl of the ready-to-use Negative Control.
      Detailed protocolThe Calbiochem® p53 ELISAPLUS (Autoantibody) Kit is provided with removable strips of wells so the assay can be carried out on separate occasions. Since conditions may vary, calibrators MUST be assayed each time the assay is performed. Calibrators, negative controls and samples should be assayed in duplicate. Disposable pipette tips and reagent troughs should be used for all transfers to avoid cross-contamination of reagents or samples.

      The assay should be done without interruption in order to prevent the drying of the plate wells. While pipetting, avoid bubble formation and make sure that no drops adhere to the sides of the wells, preventing the complete mixing of all reagents needed for the reaction to occur.

      The amount of p53 autoantibody in a test sample can be represented two ways using this kit:
      a) An antibody titer can be assigned to an unknown sample when a calibration curve is generated and assayed alongside the unknown sample.
      b) A p53 autoimmune index can be assigned to an unknown sample when the negative control and undiluted calibrator are assayed alongside the unknown sample.
      An explanation of how to generate both of these kinds of results is outlined in 'Evaluation of Results'.

      1. To generate the samples for the calibration curve, dilute the Calibrator 1:1.5, 1:2, 1:3, 1:4, and 1:6 in sample dilution buffer. Including the undiluted calibrator, this results in a curve with 1, 0.67, 0.5, 0.33, 0.25 and 0.16 Units. 1 Unit [U] is defined as the p53 binding activity corresponding to the binding activity of the calibrator (human serum sample with controlled and defined p53 autoantibody titer). Make enough to provide 100 µl per well, i.e. 200 µl for duplicates.

      2. Secure the required strips to the plate. (Unused strips should be stored in the tin-foil pouch with the desiccant, clipped and stored at 4°C.) Add 200 µl of 1X wash buffer to each well and incubate for three minutes at room temperature. Invert the plate over sink and shake vigorously. Remove any residual wash buffer by tapping plate on paper towels.

      3. Add 100 µl per well of calibrators, negative control (undiluted), positive control (diluted (1:3), and diluted samples immediately after washing. Incubate for 1 h at room temperature.

      4. Wash wells 5 times with 1X Wash Buffer making sure each well is filled completely. Each well is washed by filling with 200 µl of 1X Wash Buffer (using a multichannel pipette, automatic plate washer or wash bottle). It is essential to completely remove the Wash Buffer after each step and to ensure that the wells do not contain any buffer after the last wash. This can be achieved by inverting the plate and tapping it on paper towels.

      5. Add 100 µl of the Detector Antibody Conjugate solution (ready to use) to each well. Incubate for 1 h at room temperature. Wash plate as described in step 4.

      6. Add 100 µl of the Substrate Solution (ready to use) to each well. Incubate for 30 min at room temperature in the dark.

      7. Add 50 µl of Stop Solution (ready to use) to each well to stop the enzymatic reaction. It is important that the stop solution is added in the same order as the substrate solution.

      8. Read absorbance at 450 nm. (We recommend using the 620 or 750 nm reference filter in order to compensate for possible differences in the material of the plate. Readings may be done at a single wavelength of 450 nm; however, backgrounds and readings may be higher due to plate contributions).
      CalculationsAnalysis
      Read absorption at 450 nm (reference wavelength 620 nm or 750 nm). Subtract the E450 value of the blank from all other E450 values.

      Allowed absorption rang of controls
      After correct carry out, the absorption of the controls should be in the range as detailed below:

      Internal Quality Assurance
      Part of the kit is a positive control for enabling an internal Quality Assurance. The positive control contains p53 autoantibodies in a concentration of 14 U/ml. As an internal control a 1:3 dilution of the positive control should be included in each measurement. For the assay run to be valid calculation of positive control titer should result in 1,4 U/test (±25%)

      Interpretation of results
      Construct a calibration curve using different dilutions of calibrator. This calibration curve is a linear regression curve that cuts the x-axis at 0. Cut-off is 60 U/ml, samples should be defined as negative if their titer is ≤60 U/ml.

      negative samples: Titer <60 U/ml
      positive samples: Titer >120 U/ml
      critical: Titer 60-120 U/ml
      Serum samples having a titer of 60-120 U/ml are defined "critical" meaning that there is a probable presence of p53 autoantibodies.

      1 Unit is defined as p53 binding activity that corresponds to the binding activity of 100 µl undiluted calibrator.

      Example:

      Dilution of calibrator
      Dilute the calibrator (10 U/ml) with sample dilution buffer. Use undiluted and diluted calibrator for calculating the calibration curve. We recommend 4-6 measurements. The absorption (E450) of undiluted calibrator corresponds to exactly 1 Unit (1U/test).




      Serum sample
      Diluted your positive serum samples in sample dilution buffer that the absorption is in the range of your calibration curve. It is possible to dilute until 1:5000. Abosrptions of samples which are out of calibration range must be tested again in higher di;ution. On calculation of antibody titer of serum considered the corespondent dilution.

      antibody titer calibration curve [U/test] x dilutionserum = antibody titer serum undiluted [U/test]
      antibody titer calibration curve [U/test] x 10 = antibody titer serum undiluted [U/test]
      Protocol Summary
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.