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Mouse IL-12 p40 ELISA Set

Mouse IL-12 p40 ELISA Set

(RUO)
Mouse IL-12 p40 ELISA Set
This standard curve is for demonstration only. A standard curve must be run with each assay. \"Typical Standard Curve\" and 20-plate yield were obtained in the BD Biosciences Pharmingen laboratory, using the recommended procedure and manual plate washing.
This standard curve is for demonstration only. A standard curve must be run with each assay. \"Typical Standard Curve\" and 20-plate yield were obtained in the BD Biosciences Pharmingen laboratory, using the recommended procedure and manual plate washing.
Product Details
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BD OptEIA™
Mouse (QC Testing)
ELISA (Routinely Tested)
RUO
AB_2869032


Description

    Materials Provided  

  

    The OptEIA™ Set for mouse interleukin-12 (p40) (IL-12 (p40)) contains the components necessary to develop enzyme-linked immunosorbent assays (ELISA) for natural or recombinant mouse IL-12 (p40) in serum, plasma, and cell culture supernatants. Sufficient materials are provided to yield approximately 20 plates of 96-wells if the recommended storage, materials, buffer preparation, and assay procedure are followed as specified in this package.  

  

  

  

    Assay Optimization  

  

    BD OptEIA™ Sets allow flexible assay design to fit individual laboratory needs. To design an immunoassay with different sensitivity and dynamic range, the following parameters can be varied: Capture, Detection Antibody titers, Incubation time, Incubation temperature, Assay Diluent formulation, Buffer pH, ionic strength, protein concentration, Type of substrate, Washing technique (i.e., number of wash repetitions and soak times).  

  

  

  

    Standardization: This immunoassay is calibrated against purified Baculovirus-expressed recombinant mouse IL-12 (p40).  

Preparation And Storage

Store unopened reagents at 2-8°C. do not use reagents after expiration date, or if turbidity is evident. Before use, bring all reagents to room temperature (18-25°C). Immediately after use, return to proper storage conditions. Lyophilized standards are stable until expiration date. After reconstitution, immediately aliquot standard stock in polypropylene vials at 50 µl per vial anf freeze at -80°C for up to 6 months. If necessary, store at 2-8°C for up to 8 hours prior to aliquotting/freezing. Do not leave reconstituted standard at room temperature.

Recommended Assay Procedures

    Recommended buffers, solutions  

  

    Note: Do not use sodium azide in these preparations. Sodium azide inactivates the horseradish peroxidase enzyme.  

  

    The BD OptEIA™ Reagent Set A (Cat. No 550536) containing Coating Buffer, Assay Diluent, Substrate Reagents A and B, Stop Solution and 20X Wash Buffer Concentrate is recommended.  

  

    1. Coating Buffer - 0.2 M Sodium Phosphate, pH 6.5; 12.49 g Na2HPO4, 15.47 g NaH2PO4; q.s. to 1.0 L; pH to 6.5. Freshly prepare or use within 7 days of preparation, stored at 2-8°C.  

  

    2. Assay Diluent- PBS* with 10% FBS#, pH 7.0. The BD Pharmingen™ Assay Diluent (Cat. No. 555213) is recommended.   

  

    *Phosphate-Buffered Saline: 80.0 g NaCl, 11.6 g Na2HPO4, 2.0 g KH2PO4, 2.0 g KCL, q.s. to 10 L; pH to 7.0.   

  

    #Fetal Bovine Serum: Hyclone Cat. No. SH30088 (heatinactivated) recommended.   

  

    Freshly prepare or use within 3 days of preparation, with 2-8°C  storage.  

  

    3. Wash Buffer - PBS* with 0.05% Tween-20. Freshly prepare or use within 3 days of preparation, stored at 2-8°C.  

  

    4. Substrate Solution - Tetramethylbenzidine (TMB) and Hydrogen Peroxide. The BD Pharmingen™ TMB Substrate Reagent Set (Cat. No. 555214) is recommended.  

  

    5. Stop Solution - 1 M H3PO4 or 2 N H2SO4  

  

  

  

    Additional Materials Required  

  

    1. 96-well BD Falcon™ ELISA plates (Cat. No. 353279) are recommended  

  

    2. Microplate reader capable of measuring absorbance at 450 nm  

  

    3. Precision pipettes  

  

    4. Graduated cylinder, one liter  

  

    5. Deionized or distilled water  

  

    6. Wash bottle or automated washer  

  

    7. Log-log graph paper or automated data reduction  

  

    8. Tubes to prepare standard dilutions  

  

    9. Laboratory timer  

  

    10. Plate sealers or parafilm  

  

  

  

    Specimen Collection and Handling: Specimens should be clear, non-hemolyzed and non-lipemic.  

  

    Cell culture supernatants: Remove any particulate material by centrifugation and assay immediately or store samples at ≤-20°C. Avoid repeated freeze-thaw cycles.  

  

    Serum: Use a serum separator tube and allow samples to clot for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum and assay immediately or store samples at ≤-20° C. Avoid repeated freeze-thaw cycles.  

  

    Plasma: Collect plasma using citrate, EDTA, or heparin as anticoagulant. Centrifuge for 10 minutes at 1000 x g within 30 minutes of collection. Assay immediately or store samples at ≤-20° C. Avoid repeated freeze-thaw cycles.  

  

  

  

    Standards Preparation and Handling  

  

    1. Reconstitution: After warming lyophilized standard to room temperature, carefully open vial to avoid loss of material. Reconstitute lyophilized standard with 1.0 mL of deionized water to yield a stock standard. Allow the standard to equilibrate for at least 15 minutes before making dilutions. Vortex gently to mix.  

  

    2. Storage/ handling of reconstituted standard: After reconstitution, immediately aliquot standard stock in polypropylene vials at 50 μl per vial and freeze at -80°C  for up to 6 months. If necessary, store at 2-8° C for up to 8 hours prior to aliquotting/freezing. Do not leave reconstituted standard at room temperature.  

  

    3. Standards Preparation for Assay:  

  

    a. Prepare a 1000 pg/mL standard from the stock standard. Vortex to mix. (See dilution instructions on Instruction/Analysis Certificate.)  

  

    b. Add 300 μL Assay Diluent to 6 tubes. Label as 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.3 pg/mL, and 15.6 pg/mL.  

  

    c. Perform serial dilutions by adding 300 μL of each standard to the next tube and vortexing between each transfer. Assay Diluent serves as the zero standard (0 pg/mL).  

  

  

  

    Working Detector Preparation  

  

    (Note: One-step incubation of Biotin/Streptavidin reagents.) Add required volume of Detection Antibody to Assay Diluent. Within 15 minutes prior to use, add required quantity of Enzyme Reagent, vortex or mix well. For recommended dilutions, see lot-specific Instruction/Analysis Certificate. For a full 96-well plate, prepare 12 mL of Working Detector. Discard any remaining Working Detector after use.  

  

  

  

    Warnings and Precautions  

  

    1. Reagents which contain preservatives may be toxic if ingested, inhaled, or in contact with skin.  

  

    2. Handle all serum and plasma specimens in accordance with NCCLS guidelines for preventing transmission of blood-borne infections.  

  

    3. Capture Antibody contains < 0.1% sodium azide. Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.  

  

    4. Detection Antibody contains 1% BSA and ProClin™-150 as a preservative.  

  

    5. Enzyme Reagent contains 1% BSA and ProClin™-300 as preservative.  

  

    6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.  

  

  

  

    Recommended Assay Procedure  

  

    1. Coat microwells with 100 μL per well of Capture Antibody diluted in Coating Buffer. For recommended antibody coating dilution, see lot-specific Instruction/Analysis Certificate. Seal plate and incubate overnight at 4° C.  

  

    2. Aspirate wells and wash 3 times with ≥ 300 μL/well Wash Buffer. After last wash, invert plate and blot on absorbent paper to remove any residual buffer.  

  

    3. Block plates with ≥ 200 μL/well Assay Diluent. Incubate at RT for 1 hour.  

  

    4. Aspirate/wash as in step 2.  

  

    5. Prepare standard and sample dilutions in Assay Diluent. See "Standards Preparation and Handling".  

  

    6. Pipette 100 μL of each standard, sample, and control into appropriate wells. Seal plate and incubate for 2 hours at RT.  

  

    7. Aspirate/ wash as in step 2, but with 5 total washes.  

  

    8. Add 100 μL of Working Detector (Detection Antibody + SAv-HRP reagent) to each well. Seal plate and incubate for 1 hour at RT.  

  

    9. Aspirate/ wash as in step 2, but with 7 total washes. Note: In this final wash step, soak wells in wash buffer for 30 seconds to 1 minute for each wash.  

  

    10. Add 100 μL of Substrate Solution to each well. Incubate plate (without plate sealer) for 30 minutes at room temperature in the dark.  

  

    11. Add 50 μL of Stop Solution to each well.  

  

    12. Read absorbance at 450 nm within 30 minutes of stopping reaction. If wavelength correction is available, subtract absorbance at 570 nm from absorbance 450 nm.  

  

  

  

    Assay Procedure Summary  

  

    1. Add 100 μL diluted Capture Ab to each well. Incubate overnight at 4°C.  

  

    2. Aspirate and wash 3 times.  

  

    3. Block plates: 200 μL Assay Diluent to each well. Incubate 1 hr RT  

  

    4. Aspirate and wash 3 times.  

  

    5. Add 100 μL standard or sample to each well. Incubate 2 hr RT.  

  

    6. Aspirate and wash 5 times.  

  

    7. Add 100 μL Working Detector (Detection Ab + SAv-HRP) to each well. Incubate 1 hr RT  

  

    8. Aspirate and wash 7 times (with 30 sec. to 1 min soaks)  

  

    9. Add 100 μL Substrate Solution to each well. Incubate 30 min RT in dark  

  

    10. Add 50 μL Stop Solution to each well. Read at 450 nm within 30 min with λ correction 570 nm.  

  

  

  

    Calculation of Results  

  

    Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from  

  

    each.  

  

    Plot the standard curve on log-log graph paper, with IL-12 (p40) concentration on the x-axis and absorbance on the y-axis. Draw the best fit  

  

    curve through the standard points. To determine the IL-12 (p40) concentration of the unknowns, find the unknown's mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the IL-12 (p40) concentration. If samples were diluted, multiply the IL-12 (p40) concentration by the dilution factor. Computer data reduction may also be employed, utilizing log-log regression analysis.  

  

  

  

    Specificity  

  

    Cross Reactivity: The following factors were tested in the BD OptEIA™ assay at ≥ 10 ng/mL and no cross-reactivity (value ≥ 4 pg/mL) was identified.  

  

    Recombinant Human: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 (p40), IL-12 (p70), IL-13, IL-15, G-CSF, GM-CSF, IFN-γ, CD23, Lymphotactin, MIP-1α, MIP-1β, MCP-1, MCP-2, NT-3, PDGF-AA, SCF, TNF, LT-α (TNF-β), VEGF  

  

    Recombinant Mouse: IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-15, IFN-γ, GM-CSF, MCP-1, TCA3, TNF  

  

    Recombinant Rat: IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF  

  

    Other: Viral IL-10 (1 ng/mL), Rabbit TNF  

  

    Note: This ELISA set will recognize mouse IL-12 (p70) [i.e it will recognize the (p40) subunit in the context of the (p70) complex]. To account for the complexed subunits, please utilize the BD OptEIA™ mouse IL-12 (p70) ELISA Set MN 555256.  

  

    Note: Although not directly tested by BD Biosciences, the IL-12 (p40) subunit has been reported to be shared with IL-23.  

  

  

  

    Limitations of the Procedure  

  

    · Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-assayed.  

  

    · Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.  

  

    · BD OptEIA™ Sets are intended for use as an integral unit. Do not mix reagents from different Set batches. Reagents from other manufacturers are not recommended for use in this Set.  

Product Notices

  1. For online training for BD OptEIA™ Set ELISA Techniques, please refer to http://www.bdbiosciences.com/OptEIA/downloads.shtml
  2. Samples that generate absorbance values higher than the standard curve should be diluted with Standard Diluent and re-assayed.
  3. Interference by drug metabolites, soluble receptors, or other binding proteins in specimens has not been thoroughly investigated. The possibility of interference cannot be excluded.
  4. BD OptEIA™ Sets are intended for use as an integral unit. Do not mix reagents from different Set batches. Reagents from other manufacturers are not recommended for use in this Set.
  5. Reagents which contain preservatives may be toxic if ingested, inhaled, or in contact with skin.
  6. Handle all serum and plasma specimens in accordance with NCCLS guidelines for preventing transmission of blood-borne infections.
  7. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. ProClin is a trademark of Rohm and Haas Company.
555165 Rev. 1
Components
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Description Quantity/Size Part Number EntrezGene ID
Capture Antibody Purified Anti-Mouse IL-12 P40/P70 N/A 51-26191E 16160
Detection Antibody Biotin Anti-Mouse IL-12 P40 N/A 51-26192E 16160
Recombinant Mouse IL-12 P40 Lyophilized Standard N/A 51-26196E 16160
Enzyme Reagent Streptavidin-horseradish peroxidase conjugate (SAv-HRP) N/A 51-9002813 N/A
555165 Rev. 1
Citations & References
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Development References (12)

  1. Ashkar S, Weber GF, Panoutsakopoulou V, et al. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science. 2000; 287(5454):860-864. (Biology). View Reference
  2. Chougnet, et al. J Immunol. 2001; 3210-3217. (Biology).
  3. D'Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993; 178(3):1041-1048. (Biology). View Reference
  4. D'Andrea A, Rengaraju M, Valiante NM, et al. Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells. J Exp Med. 1992; 176(5):1387-1398. (Biology). View Reference
  5. He J, Gurunathan S, Iwasaki A, Ash-Shaheed B, Kelsall BL. Primary role for Gi protein signaling in the regulation of interleukin 12 production and the induction of T helper cell type 1 responses . J Exp Med. 2000; 191(9):1605-1610. (Biology). View Reference
  6. Iwasaki A, Kelsall BL. Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer's patch dendritic cells. J Immunol. 2001; 166(8):4884-4890. (Biology). View Reference
  7. Iwasaki A, et al. J Exp Med. 2000; 229-239. (Biology).
  8. Ling P, Gately MK, Gubler U, et al. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J Immunol. 1995; 154(1):116-127. (Biology). View Reference
  9. Morelli AE, Larregina AT, Ganster RW, et al. Recombinant adenovirus induces maturation of dendritic cells via an NF-kappaB-dependent pathway. J Virol. 2000; 74(20):9617-9628. (Biology). View Reference
  10. Oppmann B, Lesley R, Blom B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.. Immunity. 2000; 13(5):715-25. (Biology). View Reference
  11. Schoenhaut DS, Chua AO, Wolitzky AG, et al. Cloning and expression of murine IL-12. J Immunol. 1992; 148(11):3433-3440. (Biology). View Reference
  12. Zeh HJ III, Tahara H, Lotze MT. Interleukin-12. In: Thomson AW, ed. The Cytokine Handbook. London: Academic Press Limited; 1994:239-256.
View All (12) View Less
555165 Rev. 1

Please refer to Support Documents for Quality Certificates

Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described

Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.

Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.