Radioimmunoassay Kit-Endothelin Radioimmunoassay Kit (Endothelin Radioimmunoassay Kit)

1. Overview and clinical significance

Endothelin is a kind of bioactive peptide synthesized by endothelial cells and consists of 21 amino acids. Mammals can synthesize four endothelins, including ET-1, ET-2, ET-3 and mouse endothelin. They all have the functions of strongly constricting blood vessels, strengthening myocardial contractility, enhancing smooth muscle contraction and promoting neuroendocrine functions. Among them, ET-1 (hereinafter referred to as ET) has the strongest activity. Endothelin is distributed in vascular smooth muscle, kidney, heart, lung, brain, spinal cord gray matter, nerve, liver, spleen, intestine, trachea, and reproductive system, and has high affinity with specific receptors on these organs. Many physical, chemical and biological factors can stimulate a large amount of ET release and participate in various pathophysiological processes of the body. It has been confirmed that the release of ET is closely related to cardiovascular diseases, neuroendocrine diseases, respiratory diseases, digestive system and nervous system diseases. At the same time, it also plays an important role in the process of trauma, infection and shock. At present, domestic and foreign countries have begun to use ET level as an important biochemical indicator for clinical observation of the occurrence and development of the above system diseases.
The kit is used for measuring the content of ET in human plasma and belongs to an in vitro radioimmunoassay kit.

Radioimmunoassay Kit-Endothelin Radioimmunoassay Kit (Endothelin Radioimmunoassay Kit)

Second, the measurement principle

Applying the principle of competition mechanism, the ET in the standard or sample and the added 125I-ET together produce a competitive immune response with a certain amount of specific antibodies. The amount of 125I-ET bound to the antibody is a function of the ET content in the standard or sample. After separating the binding part (B) from the free part (F) with an immunoseparation reagent (PR), the radioactive intensity of the binding part was measured, and the corresponding binding rate B / B0 was calculated. Draw the known standard ET content and the corresponding binding rate to obtain the standard inhibition curve. From the standard curve, check the content of ET in the test sample corresponding to the binding rate.

3. Composition of Pill Box

4. Preparation, usage and storage of reagents

1. ET standard: dissolve it with 1ml of buffer before use, shake well. The lyophilized product is stored at 2-8 ° C in the dark, and is stable within the validity period of the label; after reconstitution, it is stored at -20 ° C and is stable during the validity period of the batch of kits.

2. 125I-ET: Dissolve in 5.5ml buffer solution before use, shake well. If more than 50 tubes are added at one time, the two bottles are dissolved and used together. The lyophilized product is stored at 2-8 ° C in the dark, and is stable within the validity period of the label; after reconstitution, it is stored at -20 ° C and is stable during the validity period of the batch of kits.

3. Rabbit anti-ET antibody: dissolve in buffer (100 tubes ml, 50 tubes ml) before use, shake well. The lyophilized product is stored at 2 ~ 8 ° C and is stable within the validity period of the label; after reconstitution, it is stored at 2 ~ 8 ° C and is stable during the validity period of the batch of kits.

4. Donkey anti-rabbit immuno-isolating agent: Store at 2 ~ 8 ℃, stable within the validity period of the label. Shake well before adding samples!

5. ET buffer: Store at 2 ~ 8 ℃, stable within the validity period of the label.

6. EDTA anticoagulant: liquid, used directly. If there is any precipitation, shake it up and put it in the incubator to heat it until it dissolves. It can be stored at 2 ~ 8 ℃ or room temperature, and can be used within the validity period of the logo.

Note: Standards and markers should be stored frozen at -20 ℃ in time after reconstitution and sample addition to ensure the reliability of the next use.

Radioimmuno kit-Endothelin Radioimmunoassay Kit

V. Sample processing

Take 2ml of venous blood, and after removing the needle, slowly inject a tube containing 10% EDTA 30ul, preferably with a stopper, along the wall of the tube, upside down twice, and mix well. After leaving it at room temperature for about 1 hour or 4 ° C for about 2 hours, centrifuge at 3000 rpm for 10 minutes (either at room temperature or 4 ° C) to separate the plasma for testing. If you do n’t measure immediately, you can seal the sample and store it at 2 ~ 8 ℃ or -20 ℃ for storage. The storage at 2 ~ 8 ℃ should not exceed 12 hours, the storage at -20 ℃ should not exceed 2 months, and -70 ℃ Not more than 6 months. Before measuring a frozen sample, allow the sample to thaw in room temperature or normal temperature water, and shake the sample thoroughly before measuring.

6. Measurement steps

Take a few round-bottom polystyrene test tubes, use a marker or special pencil to number T, NSB, S0 ~ S5 and the sample tube to be tested, etc., then use the micro-sampler to add samples according to the table below. It is best to equilibrate all reagents including plasma samples to room temperature and shake well before loading.

Operating procedure table Unit: μl

Control

Reagent

Total T

NSB

S0

S1 ~ S5

Sample to be tested

Buffer

—

200

100

—

—

ET Standard

—

—

—

100

—

Sample to be tested

—

—

—

—

100

125I-ET

100

100

100

100

100

Rabbit anti-ET antibody

—

—

100

100

100

Shake well and incubate at 4-8 ° C for 18-24 hours.

Donkey anti-rabbit immune separation agent

—

500

500

500

500

Shake well, leave at room temperature for 15 minutes, centrifuge at 3500 rpm for 15 minutes, aspirate the supernatant, and measure the radioactivity count (cpm) of each precipitation tube.

Seven, data processing

1. Online processing: The result is automatically processed by the computer. Note that due to different principles of radioimmunoassay and immunoradiotherapy, the processing software is also different. Users must use suitable processing software for data processing. It is recommended that users use logit-log or four-parameter data processing mode.

2. Hand work drawing or calculator processing:

1). Percentage binding rate calculation: S0 tube count is B0, each standard tube or sample tube count is B, non-specific tube count is NSB, then the percentage binding rate calculation formula is: B / B0 = (B－NSB ) / (B0－NSB) × 100%

2). Logit calculation: The formula for calculating the logit value of each standard point or sample tube is: logit = ln (B / B0) / (1－B / B0)

3). Hand work diagram: take the log value of the standard concentration as the abscissa, the corresponding logit value as the ordinate on the ordinary coordinate paper or the standard concentration as the abscissa, and the corresponding B / B0 as the ordinate in the logit-log coordinate Draw a standard curve (a straight line when idealized) on paper. According to the B / B0 of the sample to be tested, the concentration value of the sample can be found from the coordinate paper. Note: If you use ordinary graph paper, the detected value should take the logarithm to be the final concentration value.

4). Calculator processing: using a calculator with linear fitting function, the sample concentration value can be calculated more conveniently, please refer to the instruction manual of the calculator for the specific operation method.

Examples of standard curves (for reference only, not for actual calculations).

project

B / T

B / B0 (concentration unit ng / ml)

NSB

Zero standard

20

60

180

540

1620

%

1.5

55.6

88.4

74.1

51.5

29.1

13.4

8. The main technical indicators of the pill box

1. Standard range: 20 ~ 1620pg / ml
2. Sensitivity: 5pg / ml
3. Coefficient of variation within batches (CV): <10%; Coefficient of variation between batches (CV): <15%.
4. Use period: see the product packaging.

Nine, refer to the normal value:

Reference normal value: 53.14 ± 15.23pg / ml
Due to different regions and instruments used, there will be some differences in the measured normal values, so the normal values ​​provided in this manual are for reference only. It is best for each laboratory to establish a range of normal values ​​that meets the requirements of this laboratory.