EASYStep Pro Canine Estradiol (E2) ELISA Kit
For reference only. Please follow the manual included in your kit for instructions.
Catalog Number
RDEp-E2-c
The EASYStep Pro Canine Estradiol (E2) ELISA Kit (RDEp-E2-c) is an enzyme-linked immunosorbent assay for quantitative detection of E2 in Canine/Dog samples.
This kit uses the EASYStep Pro ELISA format for colorimetric detection of natural E2 in samples including serum and plasma.
The kit is easy to use and comes with a pre-coated microplate and all necessary reagents and buffers.
The standard curve generated in the experiment is used to calculate the concentration of E2 in samples.
For research use only.
Product Specifications
Product Name
EASYStep Pro Canine Estradiol (E2) ELISA Kit
Catalog Number
RDEp-E2-c
Detection Range
15.62-1000pg/mL
Sensitivity
6.37pg/mL
Species Reactivity
Species
Canis familiaris; Canine Dog
Assay Length
1.5 hours
Experimental Method
Detection Method
Colorimetric
Sample Type
serum and plasma
Storage
2-8°C
Shelf Life
6 months
Synonyms
Oestradiol17β-EstradiolEstra-1,3,5(10)-triene-3,17β-diol
Product Details
Intended Use
This kit is a competitive inhibition enzyme immunoassay for in vitro quantitative determination of canine E2 concentrations in serum and plasma.
Test Principle
This kit employs the competitive-ELISA principle. The micro-ELISA plate provided in this kit has been pre-coated with canine E2. A competitive inhibition reaction is launched between E2 in sample (or standards) and the pre-coated E2 with Horseradish Peroxidase (HRP) linked antibody specific to E2. After incubation, the unbound conjugate is washed off. The amount of bound HRP conjugate is reverse proportional to the concentration of E2 in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of E2 in the sample.
Application Data
Reagents and Materials Provided
| Reagents | Size | Quantity |
|---|---|---|
| Micro ELISA Plate (Detachable) | 96T | 8 wells ×12 strips |
| 48T | 8 wells × 6 strips | |
| 24T | 8 wells × 3 strips | |
| 96T × 5 | 8 wells ×12 strips × 5 | |
| Reference Standard | 96T | 2 vials |
| 48T | 1 vial | |
| 24T | 1 vial | |
| 96T × 5 | 10 vials | |
| Detection Reagent Pro | 96T | 1 × 60 μL |
| 48T/24T | 1 × 30 μL | |
| 96T × 5 | 5 × 60 μL | |
| Diluent Buffer | 96T/48T/24T | 1 × 10 mL |
| 96T × 5 | 5 × 10 mL | |
| Detection Reagent Pro Diluent | 96T/48T/24T | 1 × 7 mL |
| 96T × 5 | 5 × 7 mL | |
| Wash Buffer (25×concentrate) | 96T/48T/24T | 1 × 30 mL |
| 96T × 5 | 5 × 30 mL | |
| Substrate Reagent | 96T/48T/24T | 1 × 10 mL |
| 96T × 5 | 5 × 10 mL | |
| Stop Solution | 96T/48T/24T | 1 × 10 mL |
| 96T × 5 | 5 × 10 mL | |
| Plate Sealer | 96T/48T/24T | 5 pieces |
| 96T × 5 | 25 pieces | |
| Instruction manual | 1 |
Materials Required but not Supplied
- Microplate reader with 450 ± 10 nm filter.
- Precision single or multi-channel pipettes and disposable tips.
- Eppendorf Tubes.
- Incubator capable of maintaining 37°C.
- Deionized or distilled water.
- Absorbent paper.
- Container for Wash Buffer.
Storage of the kits
- For unopened kits: All the reagents can be stored at 2-8°C for 6 months.
- For opened kits: Once the kit is opened, the remaining wells and reagents should be stored according to the table below. Data Table: Reagents | Storage conditions Micro ELISA Plate (Detachable) | 2-8°C, 1 month Reference Standard | 2-8°C, use the reconstituted standard within 24 hours Detection Reagent Pro | 2-8°C (Protect from light) Diluent Buffer | 2-8°C Detection Reagent Pro Diluent | Wash Buffer (25×concentrate) | Substrate Reagent | 2-8°C (Protect from light) Stop Solution | 2-8°C
- For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this date.
- Return the unused wells to the foil pouch containing the desiccant pack and reseal along entire edge of zip-seal.
- It is highly recommended to use the remaining reagents within 1 month of opening.
Sample Collection and Storage
- Serum - Allow samples to clot for 2 hours at room temperature or overnight at 2-8°C before centrifugation for 20 minutes at 1000 × g at 2 8°C. Collect the supernatant for use in the assay. Blood collection tubes should be disposable and free of endotoxins.
- Plasma - Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2-8°C within 30 minutes of collection. Collect the supernatant for use in the assay. Hemolyzed samples are not suitable for use with this ELISA assay.
- Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (≤1 month) or -80°C (≤2 months) to avoid loss of bioactivity and/or contamination.
- Noticeable hemolysis will affect antibody-antigen reactions. Samples with any sign of hemolysis are not suitable for this assay.
- When performing the assay, bring samples to room temperature.
Reagent Preparation
- Bring all kit components and samples to room temperature (18-25°C) before use.
- Wash Solution - Dilute 30 mL of Wash Buffer (25×) with 720 mL of deionized or distilled water to prepare 750 mL of Wash Solution (1×).
- Standard - Centrifuge the standard at 10,000 × g for 1 minute. Add 1.0 mL of Diluent Buffer, let it stand for 10 minutes and invert it gently several times (not to foam). The concentration of the standard in the stock solution is 1000 pg/mL. Prepare 7 EP tubes containing 0.5 mL Diluent Buffer and use the diluted standard to produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Prepare a dilution series with 7 points, for example: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.62 pg/mL, and the last EP tube with Diluent Buffer is the blank at 0 ng/mL.
- Detection Reagent Pro working solution - Calculate the required amount before the experiment (50 μL/well). Prepare slightly more than necessary for your experiment. Centrifuge the Detection Reagent Pro at 800 × g for 1 minute, then dilute it to the working concentration with Detection Reagent Pro Diluent (1:100).
- Do not perform a serial dilution directly in the wells.
- Prepare standard within 15 minutes of performing the assay. Do not dissolve the reagents at 37°C directly.
- Carefully reconstitute Standards according to the instruction. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated.
- The reconstituted Standards can be used only once.
- If crystals have formed in the Wash Buffer (25×), warm to room temperature and mix gently until the crystals are completely dissolved.
- Any contaminated water or container used during reagent preparation will influence the detection result.
Assay Procedure Summary
- Add 50 µL standard or sample to each well, immediately add add 50 μL of Detection Reagent Pro working solution to each well. Incubate for 60 minutes at 37°C.
- Aspirate and wash the plate for 5 times.
- Add 90 µL Substrate Solution. Incubate for 15 minutes at 37°C.
- Add 50 µL Stop Solution. Read at 450 nm immediately.
Calculation of Results
There is an inverse correlation between E2 concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of E2 concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points, or it can be determined by regression analysis. Using plotting software, (for instance, curve expert 1.30), is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Product Data
Sensitivity
- The minimum detectable dose of E2 is typically less than 6.37 pg/mL.
- The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Specificity
- This assay has high sensitivity and excellent specificity for detection of canine E2.
- No significant cross-reactivity or interference between canine E2 and analogs was observed.
- Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between E2 and all analogs, therefore, cross reactivity may still exist.
Precision
- Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level E2 were tested 20 times on one plate, respectively.
- Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level E2 were tested on 3 different plates, 8 replicates in each plate.
- CV (%) = SD/mean × 100
- Intra-Assay: CV<10%
- Inter-Assay: CV<12%
Stability
- The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage conditions.
- To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.