IL-18 ELISA Kit

Catalog No. ABIN7014027

Product Details IL-18 ELISA Kit

Target: IL-18 (IL18) (Interleukin 18 (IL18))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.563 pg/mL - 100 pg/mL

Minimum Detection Limit: 1.563 pg/mL

Application: ELISA

Purpose: For quantitative detection of IL-18 in serum, plasma, tissue homogenates and other biological fluids.

Sample Type: Plasma, Serum, Tissue Homogenate

Analytical Method: Quantitative

Specificity: This assay has high sensitivity and excellent specificity for detection of IL -18. No significant cross-reactivity or interference between IL-18 and analogues were observed.

Sensitivity: 0.983 pg/mL

Grade: High Sensitivity

Components:

• ELISA Microplate (Dismountable)
• Lyophilized Standard
• Sample Dilution Buffer
• Biotin-labeled Antibody (Concentrated)
• Antibody Dilution Buffer
• HRP-Streptavidin Conjugate (SABC)
• SABC Dilution Buffer
• TMB Substrate
• Stop Solution
• TMB Substrate
• Stop Solution
• Wash Buffer (25x)
• Plate Sealer
• Instruction manual

Application Details

Comment:

Do not mix reagents of different batches and different product numbers in the kit, otherwise the kit will not work properly

Sample Volume: 50 μL

Plate: Pre-coated

Protocol:

1. Set standard, test samples, control (blank) wells on the pre-coated plate respectively, and then, records their positions.
2. Add 100ul standard or sample to each well and incubate for 90 minutes at 37°C.
3. Aspirate and wash plates 2 times.
4. Add 100ul Biotin-labeled antibody working solution to each well and incubate for 60 minutes at 37°C.
5. Aspirate and wash plates 3 times.
6. Add 100ul SABC Working Solution into each well and incubate for 30 minutes at 37°C.
7. Aspirate and wash plates 5 times.
8. Add 90ul TMB Substrate Solution. Incubate 10-20 minutes at 37°C.
9. Add 50ul Stop Solution. Read at 450nm immediately and calculation.

Reagent Preparation:

Bring all reagents and samples to room temperature for 20 minutes before use.

1. Wash Buffer: Dilute 30 mL Concentrated Wash Buffer to 750 mL Wash Buffer with deionized or distilled water. Put unused solution back at 2-8 °C.
   Note: If crystals have formed in the concentrate, you can warm it with 40 °C water bath (Heating temperature should not exceed 50 °C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
2. Standards:
   a) Add 1 mL Sample Dilution Buffer into one Standard tube (labeled as zero tube), keep the tube at room temperature for 10 minutes and mix them thoroughly.
   Note: If the standard tube concentration higher than the range of the kit, please dilute it and label as zero tube.
   b) Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3 mL of the Sample Dilution Buffer into each tube. Add 0.3 mL of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3 mL from 1st tube to 2nd tube and mix them thoroughly, and so on. Sample Dilution Buffer is used for the blank control.
   Note: It is best to use Standard Solutions within 2 hours.
3. BS Working Solution: Prepare it within 15 minutes before experiment.
   a) Calculate required total volume of the working solution: 0.1 mL/well x quantity of wells. (Allow 0.1-0.2 mL more than the total volume.)
   b)Dilute the BS with BS Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1 μL of BS into 99 μL of BS Dilution Buffer.)
   Note: If crystals have formed in the BS, you can warm it with water (temperature should not exceed 30 °C) and mix it gently until the crystals have completely been dissolved.
4. Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution: Prepare it within 30 minutes before experiment. a）Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total volume.) b）Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1ul of SABC into 99ul of SABC Dilution Buffer.)

Sample Preparation:

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturation may occur in these samples, leading to false results. Samples should therefore be stored for a short period at 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thaw cycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged to remove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determine compatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibility of causing a deviation due to the introduced chemical substance. The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in the range of the standard curve, the optimal sample dilution for the particular experiment has to be determined.

Assay Procedure:

When diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB Substrate for 30 minutes at 37°C. It is recommended to plot a standard curve for each test.

1. Set standard, test samples (diluted at least 1/2 with Sample Dilution Buffer), control (blank) wells on the pre-coated plate respectively, and then, records their positions. It is recommended to measure each standard and sample in duplicate.
2. Prepare Standards: Aliquot 100ul of zero tube, 1sttube, 2ndtube, 3rdtube, 4thtube, 5thtube, 6thtube and Sample Dilution Buffer (blank) into the standard wells.
3. Add Samples: Add 100ul of properly diluted sample into test sample wells.
4. Incubate: Seal the plate with a cover and incubate at 37°C for 90 minutes.
5. Wash: Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells dry completely at any time.
6. Biotin-labeled Antibody: Add 100ul Biotin-labeled antibody working solution into above wells (standard, test sample and blank wells). Add the solution at the bottom of each well without touching the sidewall, cover the plate and incubate at 37°C for 60 minutes.
7. Wash: Remove the cover, and wash plate 3 times with Wash Buffer, and let the Wash Buffer stay in the wells for 1-2 minutes each time.
8. HRP-Streptavidin Conjugate (SABC): Add 100ul of SABC Working Solution into each well, cover the plate and incubate at 37°C for 30 minutes.
9. Wash: Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minutes each time.
10. TMB Substrate: Add 90ul TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 minutes. (Before adding TMB into wells, equilibrate TMB Substrate for 30 minutes at 37°C.). (Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. You can terminate the reaction when apparent gradient appeared in standard wells.)
11. Stop: Add 50ul Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solution should be as the same as the TMB Substrate Solution.
12. OD Measurement: Read the O.D. absorbance at 450nm in Microplate Reader immediately after adding the stop solution.

Assay Precision: Intra-Assay: CV<8% Inter-Assay: CV<10%

Restrictions: For Research Use only

Handling

Storage: 4 °C,-20 °C

Storage Comment:

1. For unopened kit: All reagents should be stored according to the labels on the vials. The Standard and 96-well Strip Plate should be stored at -20 °C upon receipt, while the other reagents should be stored at 4 °C.
2. For opened kits: the remaining reagents must be stored according to the above storage conditions. In addition, please return the unused wells to the foil pouch containing the desiccant and seal the foil pouch with the zipper.

Expiry Date: 6 months

Target Details for IL-18

Target: IL-18 (IL18) (Interleukin 18 (IL18))

Alternative Name: Interleukin 18 (IL18 Products)

Background: Interleukin-18 (IL-18) is a proinflammatory cytokine in the IL-1 family that exerts distinct immune effects depending on the local cytokine environment. It is expressed as a 24 kDa precursor by endothelial and epithelial cells, keratinocytes, gamma δT cells, and phagocytes. The precursor is activated intracellularly by Caspase-1 mediated proteolysis to release the 17 kDa mature cytokine. The precursor can also be released by necrotic cells for extracellular cleavage by multiple proteases. IL -18 activation is induced by infection or tissue damage and contributes to disease pathology in chronic inflammation. IL -18 binds to the widely expressed IL-18 R alpha which recruits IL-18 R beta to form the signaling receptor complex. Its bioactivity is negatively regulated by interactions with IL-18 binding proteins and virally encoded IL-18BP homologs. In the presence of IL-12 or IL-15, IL-18 enhances anti-viral Th1 immune responses by inducing IFN-gamma production and the cytolytic activity of CD8+ T cells and NK cells. In the absence of IL-12 or IL-15, however, IL-18 promotes production of the Th2 cytokines IL-4 and IL-13 by CD4+ T cells and basophils. In the presence of IL-1 beta or IL-23, IL-18 induces the antigenindependent production of IL-17 by gamma δ T cells and CD4+ T cells. IL-18 also promotes myeloid dendritic cell maturation and triggers neutrophil respiratory burst. In cancer, IL-18 exhibits diverse activities including enhancing anti-tumor immunity, inhibiting or promoting angiogenesis, and promoting tumor cell metastasis. Alternative splicing in human ovarian cancer generates an isoform that is resistant to Caspase-1 activation. A cell surface form can be expressed on M-CSF induced macrophages and released in response to bacterial endotoxin.

Pathways: Cellular Response to Molecule of Bacterial Origin, Activated T Cell Proliferation, Cancer Immune Checkpoints, Inflammasome