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 TIMP-3 antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add biotinylated detection antibody. Then, it binds with TIMP-3 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 TIMP-3 in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.
Manual- Product Name
- Chicken TIMP-3(Tissue Inhibitors of Metalloproteinase 3) ELISA Kit
- Alias
- Metalloproteinase inhibitor 3 ELISA Kit, Protein MIG-5 ELISA Kit, Tissue inhibitor of metalloproteinases 3 ELISA Kit, TIMP-3 ELISA Kit, TIMP3 ELISA Kit
- Catalogue No.
- ECH0066
- Size
- 48T/96T
- Species
- Chicken
- UniProt ID
- P26652
- Sample Type
- Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples
- Detection Method
- Sandwich ELISA, Double Antibody
- Detection Wavelength
- OD450
- Reaction Duration
- 4 hours
- Range
- 1.25-80ng/ml
- Sensitivity
- 0.75ng/ml
- Storage
- 2-8°C(Sealed), Don't cryopreserve.
- Specificity
- Specifically binds with TIMP-3 , no obvious cross reaction with other analogues.
- ELISA Kit Components
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 Curve
-
This 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.(ng/ml) OD-1 OD-2 Average 0 0.128 0.132 0.13 1.25 0.173 0.179 0.176 2.5 0.204 0.21 0.207 5 0.262 0.27 0.266 10 0.429 0.441 0.435 20 0.731 0.753 0.742 40 1.334 1.372 1.353 80 2.408 2.478 2.443 - Recovery
-
Add a certain amount of TIMP-3 into the sample. Calculate the recovery by comparing the measured value with the expected amount of TIMP-3 in the sample.
Sample Type Recovery Range(%) Average(%) serum(n=10) 88-98 92 EDTA plasma(n=10) 85-104 96 Heparin plasma(n=10) 85-105 96 - Linearity
-
Dilute the sample with a certain amount of TIMP-3 at 1:2, 1:4 and 1:8 to get the recovery range.
Sample Type 1:2 1:4 1:8 serum(n=10) 90-104% 83-101% 80-98% EDTA plasma(n=10) 86-104% 84-99% 80-93% Heparin plasma(n=10) 87-104% 82-101% 81-85% - 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 (ng/ml) 2.36 10.24 39.12 2.51 9.88 40.46 Standard deviation 0.11 0.61 2.25 0.15 0.56 1.96 CV(%) 4.65 5.95 5.74 5.86 5.64 4.85 - Stability
-
Perform 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