Introduction
The enzyme linked immunosorbent assay (ELISA) is used for the detection and quantification of cytokines secreted by activated cells in culture into tissue culture
supernatant. Immobilizing a cytokine-specific capture antibody onto a high protein binding capacity ELISA plate enables capture of target cytokine. The captured cytokine is detected by a biotinylated cytokine-specific antibody which recognizes a distinct epitope. The sandwiched target cytokine is quantified using a colorimetric reaction based on activity of avidin-horseradish peroxidase (bound to the biotinylated detection antibody) on a specific soluble substrate (e.g., ABTS). The colored end-product is read by a spectrophotometer.
eBioscience Ready-SET-Go! ELISA reagent sets (with or without high-affinity binding microwell plates) contain the necessary reagents, buffers and diluents
for performing quantitative enzyme linked immunosorbent assays (ELISA). These ELISA reagent sets are specifically engineered for accurate and precise measurement of cytokine protein levels from samples including serum, plasma, and supernatants from cell cultures.
Materials Provided in the Reagent Set
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Capture Antibody: Pre-titrated, purified antibody
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Detection Antibody: Pre-titrated, biotin-conjugated antibody
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Standard: Recombinant cytokine for generating standard curve and calibrating samples
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Coating Buffer: 10X PBS ELISA Coating Buffer
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Assay Diluent: 5X concentrated
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Detection enzyme: pre-titrated Avidin-HRP
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Substrate Solution: Tetramethylbenzidine (TMB) Substrate Solution
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Certificate of Analysis: Lot-specific instructions for dilution of antibodies and standards
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96 Well Plate: NUNC Maxisorp flat-bottom or Corning Costar 9018 (included with product catalog numbers ending in suffixes -22, -44, -76, -86)
Other Materials Needed
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96-well plate (not included in sets with catalog numbers ending in suffixes -77 or -88): Corning Costar 9018 (Cat. No. 44-2504) or NUNC Maxisorp flat-bottom (Cat. No. 44-2404)
IMPORTANT NOTE: The use of ELISA plates which are not high affinity protein binding plates will result in suboptimal performance, e.g., low signal or inconsistent data. Do not use tissue culture plates or low protein absorption plates. Use only the Corning Costar 9018 or NUNC Maxisorp 96 well plates provided or suggested.
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Buffers
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Wash Buffer
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Stop Solution
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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
eBioscience ELISA sets are guaranteed to perform as specified at least 12 months from date of receipt if stored and handled as instructed according to the accompanying datasheet and Certificate of Analysis.
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 12 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 Set 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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1 overnight incubation
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4½-hour incubations
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1 hour washing and analyzing samples
Antibody Pairs Used in eBioscience Ready-SET-Go! Sets
Table 1: Human Ready-SET-Go! Antibody Pairs and Sensitivity
|
Human Ready-SET-Go! Antibody Pairs and Sensitivity
|
| Human Cytokine |
ELISA Set Catalog No. |
Capture mAb |
Detection mAb |
Recombinant Standard Range (pg/ml) |
| IFN-γ
|
88-7316
|
NIB42
|
4S.B3
|
4 - 500
|
| GM-CSF
|
88-6339
|
BVD2-23B6
|
BVD2-21C11
|
2.5 - 300
|
| IL-1β
|
88-7010
|
CRM56
|
CRM57
|
4 - 500
|
| IL-2
|
88-7026
|
MQ1-17H12
|
Polyclonal
|
4 - 500
|
| IL-4
|
88-7046
|
8D4-8
|
MP4-25D2
|
2 - 200
|
| IL-5
|
88-7056
|
TRFK5
|
JES1-5A10
|
4 - 500
|
| IL-6
|
88-7066
|
MQ2-13A5
|
MQ2-39C3
|
2 - 200
|
| IL-8
|
88-7087
|
SNAP8
|
Rabbit Polyclonal
|
4 - 500
|
| IL-10
|
88-7106
|
JES3-9D7
|
JES3-12G8
|
2 - 200
|
| IL-12 p70
|
88-7126
|
B-T21
|
C8.6
|
4 - 500
|
| IL-17A
|
88-7176
|
eBio64CAP17
|
eBio64DEC17
|
4 - 500
|
| IL-23
|
88-7237
|
eBio473P19
|
C8.6
|
15 - 2000
|
| IL-29
|
88-7296
|
eB525/13D4
|
eB525/19G11
|
8 - 1000
|
| IL-21
|
88-7216
|
eBio3A3-N2
|
eBio2B2-G20
|
31 - 4000
|
| MCP-1
|
88-7399
|
5D3
|
2H5
|
8 - 1000
|
| TGF-β1
|
88-7344
|
eb412/22F6
|
eB461/25D8
|
60 - 8000
|
| TNF-α
|
88-7346
|
MAb1
|
MAb11
|
4 - 500
|
| TNF-β
|
88-7345
|
359-238-8
|
359-81-11
|
8 - 1000
|
Table 2: Mouse Ready-SET-Go! Antibody Pairs and Sensitivity
|
Mouse Ready-SET-Go! Antibody Pairs and Sensitivity
|
| Mouse Cytokine |
ELISA Set Catalog No. |
Capture mAb |
Detection mAb |
Recombinant Standard Range (pg/ml) |
| GM-CSF
|
88-7334
|
MP1-22E9
|
MP1-31G6
|
4 - 500
|
| Granzyme B
|
88-8022
|
eB288/16G6
|
eBioLUEE
|
40 - 5000
|
| IFN-γ (Femto-HS)
|
88-8314
|
AN-18
|
R4-6A2
|
0.7 - 100
|
| IFN-γ
|
88-7314
|
AN-18
|
R4-6A2
|
15 - 2000
|
| IL-1β
|
88-7013
|
B122
|
Polyclonal
|
8 - 1000
|
| IL-2
|
88-7024
|
JES6-1A12
|
JES6-5H4
|
2 - 200
|
| IL-4
|
88-7044
|
11B11
|
BVD6-24G2
|
4 - 500
|
| IL-5
|
88-7054
|
TRFK5
|
TRFK4
|
4 - 500
|
| IL-6
|
88-7064
|
MP5-20F3
|
MP5-32C11
|
4 - 500
|
| IL-10
|
88-7104
|
JES5-16E3
|
JES5-2A5
|
15 - 2000
|
| IL-12 p70
|
88-7121
|
C18.2
|
C17.8
|
15 - 2000
|
| IL-12/IL-23 (total p40)
|
88-7120
|
C15.6
|
C17.8
|
8 - 1000
|
| IL-13
|
88-7134
|
eBioMTZ4513
|
Polyclonal
|
30 - 4000
|
| IL-15
|
88-7150
|
eBioMTZ4515
|
Polyclonal
|
62 - 8000
|
| IL-17A
|
88-7374
|
eBio17CK15A5
|
eBio17B7
|
8 - 1000
|
| IL-17F
|
88-7472
|
RN17
|
Polyclonal
|
15 - 2000
|
| IL-17A/F
|
88-7272
|
eBio17B7
|
Polyclonal
|
30 - 4000
|
| IL-23
|
88-7234
|
G23-8
|
C17.8
|
30 - 4000
|
| MCP-1 (CCL2)
|
88-7391
|
4E2/MCP
|
2H5
|
15 - 2000
|
| TGF-β1
|
88-7344
|
eb412/22F6
|
eB461/25D8
|
60 - 8000
|
| TNF-α
|
88-7324
|
TN3.19
|
Polyclonal
|
8 - 1000
|
Experimental Procedure
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Coat Corning Costar 9018 or NUNC Maxisorp 96 well ELISA plate with 100 µl/well of capture antibody in Coating Buffer* (dilute as noted on Certificate of Analysis, which is included with the reagent set). Seal the plate and incubate overnight at 4°C.
*Important: Warm the 10X Coating Buffer to room temperature prior to dilution.
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Aspirate wells and wash 5 times with >250 µl/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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Dilute 1 part 5X concentrated Assay Diluent with 4 parts DI water.* Block wells with 200 µl/well of 1X Assay Diluent. Incubate at room temperature for 1 hour. Aspirate/wash as in step 2.
*Important: Do not include sodium azide in any buffers, as this will inactivate the HRP.
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Aspirate/wash as in step 2.
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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/wash as in step 2. Repeat for a total of 5 washes.
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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.
References
ELISA Wash Buffer
Stop Solution
1X PBS
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80.0 g NaCl
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11.6 g Na2HPO4
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2.0 g KH2PO4
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2.0 g KCl
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DI H20 up to 10.0 L pH to 7.0
Standard Calibration
The recombinant cytokine standard of the Ready-SET-Go! is calibrated against NIBSC standards.
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Table of Standard Calibration
|
| Cytokine |
ng of eB standard |
ng of NIBSC standard |
U of NIBSC standard |
NIBSC Lot # |
| hIL-2
|
1
|
1.1
|
14.6
|
86/564
|
| hIL-4
|
1
|
2.2
|
22
|
88/656
|
| hIL-5
|
1
|
2.2
|
22
|
90/586
|
| hIL-6
|
1
|
1.7
|
170
|
89/548
|
| hIL-10
|
1
|
0.8
|
4
|
93/722
|
| hIL-12
|
1
|
0.8
|
8
|
95/544
|
| hIFN-g
|
1
|
1.1
|
22
|
87/586
|
| hTNF-a
|
1
|
0.9
|
36
|
87/650
|
| mIL-2
|
1
|
3.1
|
310
|
93/566
|
| mIL-4
|
1
|
3
|
30
|
91/656
|
| mIL-6
|
1
|
8.5
|
850
|
93/730
|
| mIFN-g*
|
1
|
|
4.5
|
Gg02-901-533
|
| mTNF-a
|
1
|
1.7
|
340
|
88/532
|
| * Mouse IFN-g is calibrated using NIH standard (Lot Gg02-901-533) and is measured in Units (U) |
ELISA Troubleshooting Guide
|
ELISA Troubleshooting Guide
|
| Problem |
Possibility |
Solution |
| A. High Background
|
1. Improper and inefficient washing
|
1. Improve efficiency of washing. Fill plates completely, soak for 1 minute per wash, as directed
|
|
|
2. Cross contamination from other specimens or positive control
|
2. Repeat ELISA, be careful when washing and pipetting
|
|
|
3. Contaminated substrate
|
3. Substrate should be colorless
|
|
|
4. Incorrect dilutions, e.g., conjugate concentration was too high
|
4. Repeat test using correct dilutions; check with the recommendations of the antibody manufacturer
|
| B. No signal
|
1. Improper, low protein binding capacity plates were used
|
1. Repeat ELISA, using recommended high binding capacity plates
|
|
|
2. Wrong substrate was used
|
2. Repeat ELISA, use the correct substrate
|
|
|
3. Enzyme inhibitor present in buffers; e.g., sodium azide in the washing buffer and Assay Diluent inhibits peroxidase activity
|
3. Repeat ELISA, make sure your system contains no enzyme inhibitor
|
| C. Very weak signal
|
1. Improper and inefficient washing
|
1. Make sure washing procedure is done correctly
|
|
|
2. Incorrect dilutions of standard
|
2. Follow recommendations of standard handling exactly as written on the certificate of analysis
|
|
|
3. Insufficient incubation time
|
3. Repeat ELISA, follow the protocol carefully for each step’s incubation time
|
|
|
4. Incorrect storage of reagents
|
4. Store reagents in the correct temperature, avoid freeze and thaw, avoid using the “frost free” freezer
|
|
|
5. Wrong filter in ELISA reader was used
|
5. Use the correct wavelength setting
|
|
|
6. Wrong plate used
|
6. Use the recommended Corning Costar 9018 or NUNC Maxisorp flat bottom 96 well plates
|
| D. Variation amongst replicates
|
1. Improper and inefficient washing
|
1. Make sure washing procedure is done correctly; see certificate of analysis
|
|
|
2. Poor mixing of samples
|
2. Mix samples and reagents gently and equilibrate to proper temperature
|
|
|
3. Plates not clean
|
3. Plates should be wiped on bottom before measuring absorbance
|
|
|
4. Improper, low binding capacity plates were used
|
4. Use recommended high binding capacity plates
|
|
|
5. Reagents have expired
|
5. Do not use if past expiration date
|
