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상품 상세설명
Intended use
This ELISA kit applies to the in vitro quantitative determination of Rat HSP-70/HSPA9 concentrations in serum, plasma and other biological fluids.
Test principle
This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat HSP-70/HSPA9. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat HSP-70/HSPA9 and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat HSP-70/HSPA9, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat HSP-70/HSPA9. You can calculate the concentration of Rat HSP-70/HSPA9 in the samples by comparing the OD of the samples to the standard curve.
| Assay type | Sandwich |
| Format | 96T |
| Assay time | 3.5h |
| Reactivity | Rat |
| Detection Method | Colormetric |
| Detection Range | 0.31—20 ng/mL |
| Sensitivity | 0.19 ng/mL |
| Sample Volume Required Per Well | 100μL |
| Sample Type | Serum, plasma and other biological fluids |
Specificity
This kit recognizes Rat HSP-70/HSPA9 in samples. No significant cross-reactivity or interference between Rat HSP-70/HSPA9 and analogues was observed.
Typical data
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
|---|---|---|---|
| 20 | 2.414 2.462 | 2.438 | 2.37 |
| 10 | 1.512 1.538 | 1.525 | 1.457 |
| 5 | 0.952 0.914 | 0.933 | 0.865 |
| 2.5 | 0.434 0.436 | 0.435 | 0.367 |
| 1.25 | 0.237 0.229 | 0.233 | 0.165 |
| 0.63 | 0.172 0.16 | 0.166 | 0.098 |
| 0.31 | 0.117 0.119 | 0.118 | 0.05 |
| 0 | 0.058 0.078 | 0.068 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat HSP-70/HSPA9 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat HSP-70/HSPA9 were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 1.00 | 2.40 | 9.50 | 1.00 | 2.50 | 9.50 |
| Standard deviation | 0.10 | 0.10 | 0.40 | 0.10 | 0.10 | 0.40 |
| C V (%) | 10.00 | 4.17 | 4.21 | 10.00 | 4.00 | 4.21 |
Recovery
The recovery of Rat HSP-70/HSPA9 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
|---|---|---|
| Serum (n=5) | 90-105 | 98 |
| EDTA plasma (n=5) | 93-105 | 99 |
| Cell culture media (n=5) | 94-105 | 100 |
Linearity
Samples were spiked with high concentrations of Rat HSP-70/HSPA9 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
|---|---|---|---|---|
| 1:2 | Range (%) | 96-110 | 88-101 | 86-100 |
| Average (%) | 104 | 93 | 92 | |
| 1:4 | Range (%) | 98-115 | 83-93 | 94-107 |
| Average (%) | 106 | 88 | 99 | |
| 1:8 | Range (%) | 93-106 | 83-94 | 95-108 |
| Average (%) | 100 | 89 | 101 | |
| 1:16 | Range (%) | 98-109 | 83-96 | 92-105 |
| Average (%) | 104 | 89 | 99 |
Kit components & Storage
An unopened kit can be stored at 2-8°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
|---|---|---|
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 μL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 μL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 2-8°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 2-8°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 2-8°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the volume marked on the label, please use accurate measuring equipment instead of directly pouring into the vial(s).
Other supplies required
- Microplate reader with 450 nm wavelength filter
- High-precision transfer pipette, EP tubes and disposable pipette tips
- Incubator capable of maintaining 37°C
- Deionized or distilled water
- Absorbent paper
- Loading slot for Wash Buffer
Assay Procedure
 | 1. Add 100μL standard or sample to the wells. Incubate for 90 min at 37°C |
 | 2. Discard the liquid, immediately add 100μL Biotinylated Detection Ab working solution to each well. Incubate for 60 min at 37°C |
 | 3. Aspirate and wash the plate for 3 times |
 | 4. Add 100μL HRP conjugate working solution. Incubate for 30 min at 37°C. Aspirate and wash the plate for 5 times |
 | 5. Add 90μL Substrate Reagent. Incubate for 15 min at 37°C |
 | 6. Add 50μL Stop Solution |
 | 7. Read the plate at 450nm immediately. Calculation of the results |
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