Human IL18BP / IL18BPa ELISA Pair Set

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Target Name (Antigen)
  • IL18BP
  • IL-18BP
  • Igifbp
  • MC54L
  • IL18BPa
  • interleukin 18 binding protein
  • IL18BP
  • Il18bp
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Purpose The ELISA Pair Set is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utlizes monoclonal antibody specific for IL18BP / IL18BPa coated on a 96-well plate. Standards and samples are added tothe wells, and any IL18BP / IL18BPa present binds to the immobilized antibody. The wells are washed and abiotinylated rabbit anti-IL18BPa polyclonal antibody is then added, producing an antibody-antigen-antibody "sandwich".To produces color in proportion to the amount of IL18BP / IL18BPa present in the sample strepavidin-HRP and TMBsubstrate solution are loaded. The absorbances of the microwell are read at 450 nm.
Sensitivity The minimum detectable dose of human IL18BP / IL18BPa was determined to be approximately 23.4 pg/mL. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard.
Characteristics Pair Set
Components Capture Antibody - 0.5 mg/mL of mouse anti-IL18BPa monoclonal antibody. Dilute to a workingconcentration of 2 μg/mL in CBS before coating.
Detection Antibody - Each vial contains 120μg biotinylated rabbit anti-IL18BPa polyclonal antibody.Reconstitute with sterile 1 mL distilled water. Dilute to a working concentration of 0.5μg/mL indetection antibody dilution buffer before use.
Standard - Each vial contains 42 ng of recombinant IL18BPa. Reconstitute standard powder with1mL detection antibody dilution buffer. A seven-point standard curve using 2-fold serial dilutions insample dilution buffer, and a high standard of 1500 pg/mL is recommended.
Streptavidin-HRP - 50 μL of streptavidin conjugated to horseradish-peroxidase. 1:2000 Dilution indetection antibody dilution buffer before use.
Material not included CBS - 0.05 M
Na2CO3 - 0.05 M
NaHCO3, pH 9.6, 0.2 μm filtered
TBS - 25 mM
Tris, adjust pH to 7.4 by HCl
Wash Buffer - 0.05 % Tween20 in TBS, pH 7.2 - 7.4
Blocking Buffer - 5 % BSA in Wash Buffer
Sample dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Detection antibody dilution buffer - 0.5 % BSA in wash buffer, pH 7.2 - 7.4, 0.2 μm filtered
Substrate Solution: To achieve best assay results, fresh substrate solution is recommended Substrate stock solution - 10 mg/mL TMB (Tetramethylbenzidine ) in DMSO Substrate dilution buffer - 0.05 M Na2HPO4 and 0.025 M citric acid , adjust pH to 5.5
Substrate working solution - For each plate dilute 250 μL substrate stock solution in 25 mL substrate dilutionbuffer and then add 80 μL 0.75 % H2O2 , mix it well
Stop Solution - 2 N H2SO4
Alternative Name IL18BP
Background Interleukin 18 binding protein (IL18BP) is a secreted glycoprotein which antagonizes the activity of theproinflammatory cytokine IL18 by binding to IL18 and blocking its biological activity. At least four isoformsdiffering in affinity for IL18 are produced from alternative splicing, and IL18 BP isoform a contains oneimmunoglobulin (Ig) -like C2-type domain. IL18BPa binds IL18 with high affinity, prevents the binding of IL18to its receptor, and inhibits the production of IFN- gamma, IL8, and activationof NF-kappa B, resulting inreduced T- helper type 1 (Th1) immune responses. IL18BPa is constitutively expressed and secreted inmononuclear cells, and its expression can be induced and enhanced through the IFN- gamma-mediatedpathway. The identified IFN-γ/IL18BPa negative feedback loop probably plays a key role in the modulation ofboth innate and adaptive immunity. A severe IL18/IL18BP imbalance is associated with hemophagocyticsyndrome (HPS) characterized by an uncontrolled activation of Th1 lymphocytes and macrophages. Inaddition, IL18 BPa is suggested to has a possible antimetastatic benefit, since IL18 plays a significant role intumor hepatic metastasis.
Application Notes Optimal working dilution should be determined by the investigator.

The human IL18BP / IL18BPa ELISA Pair Set is for the quantitative determination of human IL18BPa.This ELISA Pair Set contains the basic components required for the development of sandwich ELISAs.

Reagent Preparation
  1. Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100 μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4 °C.
    2. Aspirate each well and wash with at least 300 μL wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
    3. Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
    4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
Assay Procedure
  1. Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at roomtemperature.
    2. Repeat the aspiration/wash as in step 2 of plate preparation.
    3. Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1hour at room temperature.
    4. Repeat the aspiration/wash as in step 2 of plate preparation.
    5. Add 100 μL of Streptavidin-HRP to each well. Incubate for 1 hour at room temperature.
    6. Repeat the aspiration/wash as in step 2 of plate preparation.
    7. Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature (if substrate solutionis not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
    8. Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.9. Determine the optical density of each well immediately, using a microplate reader set to 450 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. 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. To determine the concentration of the unknowns, find the unknowns' 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 concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.

Restrictions For Research Use only
Format Lyophilized
Precaution of Use The Stop Solution suggested for use with this Pair Set is an acid solution. Wear eye, hand, face, andclothing protection when using this material.
Handling Advice Avoid repeated freeze-thaw cycles.
Storage 4 °C/-20 °C/-80 °C
Storage Comment Capture Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Detection Antibody: Aliquot and store at -20°C to -80°C for up to 6 months from date of receipt. Standard: Store lyophilized standard at -20°C to -80°C for up to 6 months from date of receipt. Aliquot and store the reconstituted Standard at -80°C for up to 1 month. Streptavidin-HRP: Store at 4°C and protect it from prolonged exposure to light. DO NOT FREEZE! It is stable for up to 6 months from date of receipt.
Expiry Date 6 months
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