NA/NE(Noradrenaline/Norepinephrine) ELISA Kit 说明书

NA/NE(Noradrenaline/Norepinephrine) ELISA Kit

This manual must be read attentively and completely before using this product.

If you have any problems, please contact us.

Intended Use

The ELISA kit is a competitive enzyme immunoassay technique for the quantitative determination of NE concentrations in serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.

Specification

? Sensitivity: 6 pg/mL.

? Detection Range: 12.5-200 pg/mL.

? No significant cross-reactivity or interference between NE and analogues was observed.

? Repeatability: Coefficient of variation is < 9%.

Principle of the Procedure

This ELISA kit uses the Competitive-ELISA principle. The microtiter plate strips has been pre-coated with Coating antibody for NE. A competitive inhibition reaction is launched between HRP labeled NE and free NE (Standards or samples) with anti NE antibody. After washing off unbound material, TMB substrate solution is added to all wells and incubated. An enzyme-catalyzed reaction generates a blue color in the solution, thereafter, stop solution is added to stop the substrate reaction and the color turns yellow. The yellow solution is read at a wavelength of 450nm. The concentration of NE in the samples is then calculated from the OD value by establishing a standard curve.

Limitations of the Procedure

1. This kit is for laboratory scientific research only, we will not be responsible for any consequences if this kit is used for clinical diagnosis or any other procedures.

2. Due to the uncertainty of its validity, this kit may not be suitable for testing some special experimental samples, such as gene knockout experiments.

3. Please wear lab coats, eye protection and latex gloves for protection. Please perform the experiment following the national security protocols of biological laboratories, especially when detecting blood samples or other bodily fluids.

4. This kit should be used before its expiration date, and please strictly follow the instructions for storage.

5. Different manufacturers' kits or testing the same analyte by other methods may produce inconsistent results because we do not compare our products with those of other manufacturers.

6. Since the antibodies used in the kit are usually prepared from recombinant proteins as immunogen, and recombinant proteins can be limited by different fragmentation, expression and purification systems, it is not recommended to use this kit to detect recombinant proteins

7. In order to get the best experimental results, please use only the reagents provided by the manufacturer, and do not mix reagents from different batches.

8. Due to the existing conditions and the limitations of science and technology, we cannot fully identify and analyze the raw materials provided by the supplier comprehensively. Therefore, the kit may have some quality and technical risks.

9. The possibility of interference cannot be excluded before all factors are tested in the ELISA immunoassay.

10.In order to obtain reproducible results, each step in the experiment should be controlled and variations in sample collection, handling and storage may also lead to differences in sample measurements.

11.Although each kit passes rigorous quality testing, differences in measured values between batches of kits can still be caused by factors such as shipping conditions and different laboratory equipment.

Reagents&Materials Provided

Components	Specifications	Storage
Pre-coated ELISA Plate	12wells ×8 strips	2-8℃
Standard	5×0.3mL:200,100,50,25,12.5 pg/mL	2-8℃
HRP-Conjugate reagent	1×6mL	2-8℃
Sample Diluent	1×6mL	2-8℃
Substrate Reagent A	1×6mL	2-8℃
Substrate Reagent B	1×6mL	2-8℃
Wash Buffer (30×)	1×20mL	2-8℃
Stop Solution	1×6mL	2-8℃
Plate Sealer	2 pieces
Manual	1 copy
Self-sealing bags	1 copy

Materials & Equipment Required But Not Provided

Microplate reader with 450nm wavelength filter

Incubator capable of maintaining 37℃

Single or multi-channel pipettes with high precision

disposable pipette tips

EP tubes

Container for Wash Solution

Squirt bottle, manifold dispenser, or automated microplate washer

Deionized or distilled water

Absorbent paper for blotting the microplate

Sample Collection

Water sample: After collection, it was repeatedly frozen and thawed at -20℃ for three times, and then filtered by glass fiber to be tested.

Tissue: Tissues should be extracted with butanol: methanol: water (5:25:70 V: V: V), or extract according to relevant literature, and experiments should be carried out as soon as possible after extraction or stored at -20℃ for later use.

Note for sample

1. Samples should be used within 6 days when stored at 2-8℃, otherwise samples must be stored at -20℃ (≤1month). Avoid repeated freeze-thaw cycles.

2. Please predict the concentration before assaying. If the sample concentration is not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.

3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.

Reagent Preparation

1. Bring all components to room temperature (18-25℃) before use. Follow the Microplate reader manual for set-up and preheat it for 15 min before OD measurement.

2. Wash Buffer: Dilute 20mL of Concentrated Wash Buffer with 580 mL of deionized or distilled water to prepare 600 mL of Wash Buffer.

Assay Protocol

1. Determine wells for diluted standard, sample and blank separately (blank wells don’t add sample and HRP-Conjugate reagent, other each step operation is same). Prepare 5 wells for standard points, 1 well for blank, add 50μL of standard solution to standard well. Add 10μL of sample to the sample well, then add 40μL of sample diluent, respectively.

2. Add 50μL of HRP-Conjugate antigen to each well, except blank well, mix thoroughly, cover with the plate sealer provided in the kit, incubate for 60 min at 37℃.

3. Discard the liquid from each well, add 350μL of wash buffer to each well. Soak for 30sec and decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 5 times in total.

4. Add 100μL of the equal volume mixture of Substrate Reagent A and Substrate Reagent B to each well, cover with a new plate sealer. Incubate for 15min at 37℃. Protect the plate from light.

5. Add 50 μL of Stop Solution to each well, adding the stop solution should be done in the same order as the

substrate solution.

6. Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.

Calculation of results

Average the duplicate readings for each standard and samples, then subtract the average zero standard optical density. Create a standard curve with standard concentration on the x-axis and OD values on the y-axis. Draw a best fit curve through the points and it can be determined by regression analysis. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

Example data

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), so plotting log of the data to establish a standard curve for each test is strongly recommended. Typical standard curve is provided below for reference only.

pg/mL	OD1	OD2	Average	Corrected	Standard curve
0	2.417	2.403	2.41	2.365
12.5	1.509	1.499	1.504	1.442
25	0.892	0.886	0.889	0.834
50	0.43	0.424	0.427	0.376
100	0.179	0.225	0.202	0.143
200	0.15	0.15	0.15	0.111

Sensitivity

The minimum detectable dose of NE is typically less than 6 pg/mL.

The sensitivity of this assay, or Limit of Detection (LOD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding three standard deviations to the mean OD value of twenty zero standard replicates and calculating the corresponding concentration.

Specificity

This assay has high sensitivity and excellent specificity for detection of NE. No significant cross-reactivity or interference between NE and analogues was observed. Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between NE and all the analogues, therefore, cross reaction may still exist.

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level NE were tested on one plate, 20 replicates in the plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level NE were tested on 3 different plates, 20 replicates in each plate.

CV(%) = SD/mean × 100%

Recovery

Three matrices listed below were spiked with certain level of NE, The recovery rates of NE were calculated by comparing the measured value to the expected amount of NE in samples.

Linearity

Three types of Sample were spiked with appropriate concentrations of NE and diluted into a series of concentration gradients, then the linearity of the assay was demonstrated by the percentage of comparing calculated concentrations and expected values

Summary

Troubleshooting

Problem	Possible Causes	Subsequent Actions
Poor standard curve	Improper standard dilution	Ensure that standards are dissolved and diluted in the recommended manner
	Inaccurate  pipetting	Periodically calibrate pipettes and check the pipette tips
	Evaporate the reaction solution	Seal the enzyme plate with plate sealer
	Incomplete plate washing	Adequate washing times and the amount of washing solution added
	Foreign matter in the bottom of wells	Clean the bottom of the plate before reading
Weak or no color development	Insufficient reaction of reagents	Ensure incubation time and incubate at the recommended temperature
	Inadequate reagent volumes	Check the pipette and Follow the steps strictly to operate
	Improper dilution	Check the reagent dilution process
	Inactivation of enzyme conjugate	Mix conjugate and substrate, check by color development
Low OD value	Incorrect setting of microplate reader	Check the wavelength of reader
	No stop solution added	Add appropriate amount of stop solution
	Waiting too long time to read	Read the plate in time