This kit is based on Double antibody-Sandwich ELISA detection method and takes 4h assay time. The microplate provided in this kit has been precoated with anti IL-23 antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add biotinylated detection antibody. Then, it binds with IL-23 bound to precoated antibody. Wash unbound components and add HRP-Streptavidin Conjugate (SABC). Wash unbound components again and add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Calculate the concentration of IL-23 in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.
Kit Components | Item | Size(48T) | Size(96T) | Storage Condition for Opened Kit |
---|---|---|---|---|
E001 | ELISA Microplate(Dismountable) | 8×6 | 8×12 | Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C |
E002 | Lyophilized Standard | 1vial | 2vial | Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C |
E003 | Biotin-labeled Antibody(Concentrated, 100X) | 60ul | 120ul | 2-8°C (Avoid Direct Light) |
E034 | HRP-Streptavidin Conjugate(SABC, 100X) | 60ul | 120ul | |
E024 | TMB Substrate | 5ml | 10ml | |
E039 | Sample Dilution Buffer | 10ml | 20ml | 2-8°C |
E040 | Antibody Dilution Buffer | 5ml | 10ml | |
E049 | SABC Dilution Buffer | 5ml | 10ml | |
E026 | Stop Solution | 5ml | 10ml | |
E038 | Wash Buffer(Concentrated, 25X) | 15ml | 30ml | |
E006 | Plate Sealer | 3 pieces | 5 pieces | |
E007 | Product Description | 1 copy | 1 copy |
Required Instruments and Reagents
- Microplate reader (wavelength: 450nm)
- 37°C incubator (CO2 incubator for cell culture is not recommenced.)
- Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
- Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
- Sterile tubes and Eppendorf tubes with disposable tips
- Absorbent paper and loading slot
- Deionized or distilled water
Assay Procedure Summary
- Step 1: Add 100ul standard or sample into each well, seal the plate and statically incubate for 90 minutes at 37°C.
- Washing: Wash the plate twice without immersing.
- Step 2: Add 100ul biotin-antibody working solution, seal the plate and statically incubate for 60 minutes at 37°C.
- Washing: Wash the plate three times and immerse for 1min each time.
- Step 3: Add 100ul HRP-Streptavidin Conjugate (SABC) working solution, seal the plate and statically incubate for 30 minutes at 37°C.
- Washing: Wash the plate five times and immerse for 1min each time.
- Step 4: Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
- Step 5: Add 50ul stop solution. Read at 450nm immediately and calculate.
Standard CurveThis product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.)
Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.
STD.(pg/ml) | OD-1 | OD-2 | Average |
---|---|---|---|
0 | 0.16 | 0.164 | 0.162 |
39.062 | 0.216 | 0.222 | 0.219 |
78.125 | 0.353 | 0.363 | 0.358 |
156.25 | 0.505 | 0.519 | 0.512 |
312.5 | 0.716 | 0.736 | 0.726 |
625 | 0.967 | 0.995 | 0.981 |
1250 | 1.48 | 1.522 | 1.501 |
2500 | 2.275 | 2.341 | 2.308 |
RecoveryAdd a certain amount of IL-23 into the sample. Calculate the recovery by comparing the measured value with the expected amount of IL-23 in the sample.
Sample Type | Recovery Range(%) | Average(%) |
---|---|---|
serum(n=10) | 84-104 | 93 |
EDTA plasma(n=10) | 86-105 | 93 |
Heparin plasma(n=10) | 87-101 | 95 |
LinearityDilute the sample with a certain amount of IL-23 at 1:2, 1:4 and 1:8 to get the recovery range.
Sample Type | 1:2 | 1:4 | 1:8 |
---|---|---|---|
serum(n=10) | 89-105% | 86-99% | 82-90% |
EDTA plasma(n=10) | 87-96% | 84-100% | 87-104% |
Heparin plasma(n=10) | 90-101% | 82-100% | 87-99% |
Precision(%)Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.
Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
Item | Intra-assay Precision | Inter-assay Precision | ||||
---|---|---|---|---|---|---|
Sample | 1 | 2 | 3 | 1 | 2 | 3 |
n | 20 | 20 | 20 | 20 | 20 | 20 |
Mean (pg/ml) | 77.11 | 306.7 | 1220 | 74 | 311.8 | 1228 |
Standard deviation | 3.88 | 13.31 | 53.56 | 3.18 | 13.47 | 53.05 |
CV(%) | 5.03 | 4.34 | 4.39 | 4.3 | 4.32 | 4.32 |
StabilityPerform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.
ELISA kit(n=5) | 37°C for 1 month | 2-8°C for 6 months |
Average(%) | 80 | 95-100 |
Vendor | FineTest |
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