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IgG2b ELISA Kit

Reactivity: Mouse Colorimetric Sandwich ELISA
Catalog No. ABIN956085
  • Target See all IgG2b products
    IgG2b
    Reactivity
    • 2
    • 1
    Mouse
    Detection Method
    Colorimetric
    Method Type
    Sandwich ELISA
    Application
    ELISA
    Purpose
    The Mouse IgG2B ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative determination of IgG2B in mouse biological samples.
    Analytical Method
    Quantitative
    Characteristics
    In this assay the IgG2B present in the sample reacts with the anti-IgG2B antibody which has been adsorbed to the surface of polystyrene microtiter wells. After the removal of unbound proteins by washing, anti-IgG2B antibody conjugated with horseradish peroxidase (HRP) is added. This HRP-conjugated antibody forms a complex with the previously bound IgG2B. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3',5,5'- tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of IgG2B in the sample tested, thus, the absorbance, at 450 nm, is a measure of the concentration of IgG2B in the test sample. The quantity of IgG2B in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected for sample dilution.
    Components
    1. Diluent Concentrate: One bottle containing 50 mL of a 5X concentrated diluent running buffer.
    2. Wash Solution Concentrate: One bottle containing 50 mL of a 20X concentrated wash solution.
    3. Enzyme-Antibody Conjugate Concentrate: One vial containing 150 µL of a 100X concentrated affinity-purified anti-mouse IgG2B antibody conjugated with HRP in a stabilizing buffer.
    4. TMB Substrate Solution: One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
    5. Stop Solution: One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
    6. Microtiter Plate: Twelve removable eight-well strips in well holder frame. Wells are coated with affinity-purified anti-mouse IgG2B.
    7. Mouse IgG2B Calibrator: One vial containing 1.4 mL with 660 ng/mL of Mouse IgG2B Calibrator.
    8. Positive Control: One vial containing 50 µL of serum with 0.1% sodium azide. See the Control Certificate for the concentration.
    Material not included
    Precision pipettes (2 µL to 200 µL) for making and dispensing diluions
    Test tubes
    Microplate washer/aspirator
    Distilled or de-ionized H2O Microplate reader
    Assorted glassware for the preparation of reagents and buffer solutions
    Timer
    Vortex mixer
  • Plate
    Pre-coated
    Reagent Preparation
    1. Diluent Concentrate: The Diluent solution supplied is a 5X concentrate and must be diluted 1:5 with distilled or de-ionized water.
      2. Wash Solution Concentrate: The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to 30-35°C before dilution can dissolve crystals.
      3. Enzyme-Antibody Conjugate Concentrate: Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL of Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently. Avoid foaming.
      4. TMB Substrate Solution: Ready to use as supplied.
      5. Stop Solution: Ready to use as supplied.
      6. Microtiter Plate: Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will not be used in the assay and place back in pouch and re-seal along with desiccant.
      7. Mouse IgG2B Calibrator: The calibrator is now at a concentration of 660 ng/mL. Prepare the Mouse IgG2B Calibrators immediately prior to use according to the table below. Mix well between each step. Avoid foaming.
      8. Positive Control: The concentration and recommended dilution are provided on the Control Certificate. Before use, briefly centrifuge the Positive Control to allow all of the liquid to collect in the bottom of the vial.
    Calculation of Results
    1. Subtract the average background value from the test values for each sample.
      2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a four- parameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
      3. Interpolate test sample values from the calibration curve. Correct for sample dilution factor to arrive at IgG2B concentration in original sample.

      1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the information contained in the package insert instructions and with adherence to good laboratory practice.
      2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware, quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation time or temperature.
    Restrictions
    For Research Use only
  • Storage
    4 °C
    Storage Comment
    1. Complete Kit: The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: See long term storage recommendations below for the Mouse IgG2B Calibrator and Positive Control.
    2. Diluent: The 5X Diluent Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions should be stored at 4°C.
    3. Wash Solution: The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
    4. Enzyme-Antibody Conjugate: Undiluted anti-IgG2B-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The working conjugate solution is stable for up to 8 hours.
    5. TMB Substrate Solution: The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
    6. Stop Solution: The Stop Solution should be stored at 4°C and is stable until the expiration date.
    7. Microtiter Plate: Anti-mouse IgG2B coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouch with a desiccant pack.
    8. Mouse IgG2B Calibrator: Long Term Storage : Upon receipt, aliquot the calibrator and store them frozen. They will be stable until the expiration date.
    Expiry Date
    The expiry date is stated on the label.
  • Target See all IgG2b products
    IgG2b
    Abstract
    IgG2b Products
    Synonyms
    IgG2b ELISA Kit, Igh-3 ELISA Kit, gamma2b ELISA Kit, immunoglobulin heavy constant gamma 2B ELISA Kit, Ighg2b ELISA Kit
    Target Type
    Antibody
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