Contents: Mouse IL-23 ELISA Ready-SET-Go! 'IS' ELISA Kit with Pre-Coated Plates (Improved sensitivity assay with 5B2 capture antibody)
Catalog Number: 88-7923
Sensitivity: 8 pg/ml
Standard Curve Range: 1000 pg/ml - 8 pg/ml
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*International customers: Please contact your distributor for region specific pricing.
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Questions? Please consult our answers to frequently asked questions at
http://www.ebioscience.com/faq.
Description
This Mouse IL-23 ELISA Ready-SET-Go! 'IS' ELISA Kit with Pre-Coated Plates (Improved sensitivity assay with 5B2 capture antibody) contains the necessary reagents, buffers and diluents for performing quantitative, enzyme-linked immunosorbent assays (ELISA). The 5B2 antibody in combination with the C17.8 antibody yields an improved IL-23 sandwich ELISA with greater sensitivity than was achievable with previous assays. This ELISAkit is specifically engineered for accurate and precise measurement of mouse IL-23 protein levels from samples including serum, plasma, and supernatants from cell cultures. The assay demonstrates parallelism in measuring recombinant and native mouse IL-23 proteins with a standard curve range of 4 pg/ml to 500 pg/ml and assay sensitivity below 4 pg/ml. Native mouse IL-23 was detected in supernatants from bone marrow-derived, LPS-activated dendritic cells. The use of a p19-specific capture antibody and a p40-specific detection antibody renders this assay exquisitely specific for mouse IL-23. IL-12 p40 homodimer and IL-12 p70 were each run in the assay at 500 ng/ml with no interference or cross-reactivity observed. A panel of 20 unrelated cytokines was run in the IL-23 ELISA at 100 ng/ml with no cross reactivity observed; all values were at the limit of detection of the assay.
For measurement of total p40 protein levels, the Mouse IL-12/23 Total p40 ELISA Ready-SET-Go! is available (88-7120).
Components
- Pre-coated ELISA Plate, clone 5B2
- Detection Antibody: Pre-titrated, biotin-conjugated antibody, clone C17.8
- Standard: Recombinant cytokine for generating standard curve and calibrating samples
- Assay Diluent: 5X concentrated
- ELISA Wash Buffer Powder
- Detection enzyme: pre-titrated Avidin-HRP
- Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
- Stop Solution: 5 mls of 1X solution per plate
- Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
Applications Reported
For research use only, not for diagnostic or therapeutic use.
Other Materials Needed
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Pipettes and pipettors
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Refrigerator
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96-well ELISA plate reader (microplate spectrophotometer)
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ELISA plate washer
Stability
This ELISA kit is guaranteed to perform as specified at least 6 months from date of receipt if stored and handled as instructed according to this datasheet and the Certificate of Analysis, which is included with the reagents.
Storage Instructions for Cytokine Standards
The frozen cytokine standard is already aliquoted at 20 µl per vial. Upon receipt, frozen cytokine standard should be immediately stored at -80°C; stable for at least 6 months. After thawing, quick-spin vial prior to opening. Do not re-aliquot into smaller fractions. These are single use vials. Use one time and discard. For dilution of the standard, please see instructions on the Certificate of Analysis and follow these as written.
Storage Instructions for other kit reagents
Store at 4°C.
IMPORTANT NOTE: Be certain that no sodium azide is present in the solutions used in this assay, as this inhibits HRP enzyme activity.
Time Requirements
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4˝-hour incubations
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1 hour washing and analyzing samples
Experimental Procedure
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Using Assay Diluent, dilute standards as noted on the Certificate of Analysis (C of A). Add 100 µl/well of standard to the appropriate wells. Perform 2-fold serial dilutions of the top standards to make the standard curve. Add 100 µl/well of your samples to the appropriate wells. Cover or seal the plate and incubate at room temperature for 2 hours (or overnight at 4°C for maximal sensitivity).
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Aspirate wells and wash 5 times with >250 ul / well Wash Buffer (diluted to 1X). Allowing time for soaking (~ 1 minute) during each wash step increases the effectiveness of the washes. Blot plate on absorbent paper to remove any residual buffer.
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Add 100 µl/well of detection antibody diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 1 hour.
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Aspirate/wash as in step 2. Repeat for a total of 5 washes.
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Add 100 µl/well of Avidin-HRP* diluted in 1X Assay Diluent (dilute as noted on C of A). Seal the plate and incubate at room temperature for 30 minutes.
*Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
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Aspirate and wash as in step 2. In this wash step, soak wells in Wash Buffer for 1 to 2 minutes prior to aspiration. Repeat for a total of 7 washes.
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Add 100 µl/well of Substrate Solution to each well. Incubate plate at room temperature for 15 minutes.
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Add 50 µl of Stop Solution to each well.
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Read plate at 450 nm. If wavelength subtraction is available, subtract the values of 570 nm from those of 450 nm and analyze data.
Ready-SET-Go Cytokine ELISA Kit Buffers:
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Assay Diluent (5X concentrate): Dilute 1/5 in dI water.
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ELISA Wash Buffer Powder: Reconstitute in 1 L dI water
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Substrate Solution: Ready to use (1X); 100 ul per well.
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Stop Solution: Ready to use (1X); 50 ul per well
Standard Calibration
The standard of the Ready-SET-Go! is calibrated against NIBSC standards:
Table 1: Table of Standard Calibration
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Table of Standard Calibration
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| Cytokine |
ng of eB standard |
ng of NIBSC standard |
U of NIBSC standard |
NIBSC Lot # |
| hIL-2
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1
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1.1
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14.6
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86/564
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| hIL-4
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1
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2.2
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22
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88/656
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| hIL-5
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1
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2.2
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22
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90/586
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| hIL-6
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1
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1.7
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170
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89/548
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| hIL-10
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1
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0.8
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4
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93/722
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| hIL-12
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1
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0.8
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8
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95/544
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| hIFN-g
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1
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1.1
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22
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87/586
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| hTNF-a
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1
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0.9
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36
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87/650
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| mIL-2
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1
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3.1
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310
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93/566
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| mIL-4
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1
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3
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30
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91/656
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| mIL-6
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1
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8.5
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850
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93/730
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| mIFN-g*
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1
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4.5
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Gg02-901-533
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| mTNF-a
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1
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1.7
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340
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88/532
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| * Mouse IFN-g is calibrated using NIH standard (Lot Gg02-901-533) and is measured in Units (U) |
Table 2: ELISA Troubleshooting Guide
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ELISA Troubleshooting Guide
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| Problem |
Possibility |
Solution |
| A. High Background
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1. Improper and inefficient washing
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1. Improve efficiency of washing. Fill plates completely, soak for 1 minute per wash, as directed
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2. Cross contamination from other specimens or positive control
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2. Repeat ELISA, be careful when washing and pipetting
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3. Contaminated substrate
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3. Substrate should be colorless
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4. Incorrect dilutions, e.g., conjugate concentration was too high
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4. Repeat test using correct dilutions; check with the recommendations of the antibody manufacturer
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| B. No signal
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1. Wrong substrate was used
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1. Repeat ELISA, use the correct substrate
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2. Enzyme inhibitor present in buffers; e.g., sodium azide in the washing buffer and Assay Diluent inhibits peroxidase activity
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2. Repeat ELISA, make sure your system contains no enzyme inhibitor
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| C. Very weak signal
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1. Improper and inefficient washing
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1. Make sure washing procedure is done correctly
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2. Incorrect dilutions of standard
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2. Follow recommendations of standard handling exactly as written on the certificate of analysis
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3. Insufficient incubation time
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3. Repeat ELISA, follow the protocol carefully for each step’s incubation time
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4. Incorrect storage of reagents
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4. Store reagents in the correct temperature, avoid freeze and thaw, avoid using the “frost free” freezer
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5. Wrong filter in ELISA reader was used
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5. Use the correct wavelength setting
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| D. Variation amongst replicates
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1. Improper and inefficient washing
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1. Make sure washing procedure is done correctly; see certificate of analysis
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2. Poor mixing of samples
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2. Mix samples and reagents gently and equilibrate to proper temperature
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3. Plates not clean
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3. Plates should be wiped on bottom before measuring absorbance
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4. Reagents have expired
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4. Do not use if past expiration date
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