EASYStep Pro Chicken Testosterone (T) ELISA Kit

Name: EASYStep Pro Chicken Testosterone (T) ELISA Kit RDEp-T-Ch
Brand: Reddot Biotech
SKU: RDEp-T-Ch
Price: 665.00 USD
Availability: InStock

FAQ

For reference only. Please follow the manual included in your kit for instructions.

Catalog Number

RDEp-T-Ch

EASYStep Pro Chicken Testosterone (T) ELISA Kit - Detection Range: 0.31-20ng/mL

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Product Description

The EASYStep Pro Chicken Testosterone (T) ELISA Kit (RDEp-T-Ch) is an enzyme-linked immunosorbent assay for quantitative detection of T in Chicken samples. This kit uses the EASYStep Pro ELISA format for colorimetric detection of natural T 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 T in samples. For research use only.

Product Specifications

Product Name

EASYStep Pro Chicken Testosterone (T) ELISA Kit

Catalog Number

RDEp-T-Ch

Detection Range

0.31-20ng/mL

Sensitivity

0.13ng/mL

Species Reactivity

Chicken

Species

Chicken Gallus

Assay Length

1.5 hours

Experimental Method

Competitive Inhibition

Detection Method

Colorimetric

Sample Type

serum and plasma

Storage

2-8°C

Shelf Life

6 months

Product Details

Intended Use

This kit is a competitive inhibition enzyme immunoassay for in vitro quantitative determination of chicken T 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 chicken T. A competitive inhibition reaction is launched between T in sample (or standards) and the pre-coated T with Horseradish Peroxidase (HRP) linked antibody specific to T. After incubation, the unbound conjugate is washed off. The amount of bound HRP conjugate is inversely proportional to the concentration of T in the sample. After addition of the substrate solution, the intensity of color developed is inversely proportional to the concentration of T in the sample.

Application Data

Reagents and Materials Provided

Reagents	Quantity
Micro ELISA Plate (Detachable)	8 wells × 12 strips
Reference Standard	2 vials
Detection Reagent Pro	1 × 60 μL
Diluent Buffer	1 × 20 mL
Detection Reagent Pro Diluent	1 × 14 mL
Wash Buffer (25×concentrate)	1 × 30 mL
Substrate Reagent	1 × 10 mL
Stop Solution	1 × 10 mL
Plate Sealer	5 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

Note:

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

Plasma - Collect plasma using EDTA-Na2 as 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.

Note:

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 (avoid bubbles). The concentration of the standard in the stock solution is 20 ng/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: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.62 ng/mL, 0.31 ng/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).

Note:

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 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 T 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 T 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 T is typically less than 0.13 ng/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 chicken T.
No significant cross-reactivity or interference between chicken and analogs was observed.

Note:

Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between T 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 T were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level T 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.

Note:

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.