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11-dehydro thromboxane B2 homogeneous enzyme immunoassay kit and preparation method thereof

A technology of dehydrothromboxane and detection kit, which is applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of long detection cycle, inability of large-scale promotion and application of detection items, manual operation, etc.

Inactive Publication Date: 2020-07-24
深圳市安帝宝科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 11-Dehydrothromboxane B 2 It is a small molecular compound with a low content in urine, and the composition of urine is complex and there are many interfering substances. 2 Quantitative detection of the content is difficult, conventional detection methods include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), etc. However, these methods have certain limitations, such as the need for manual operation, special equipment, expensive materials, and a long detection cycle, which prevents the large-scale promotion and application of this detection item in clinical practice.

Method used

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  • 11-dehydro thromboxane B2 homogeneous enzyme immunoassay kit and preparation method thereof
  • 11-dehydro thromboxane B2 homogeneous enzyme immunoassay kit and preparation method thereof
  • 11-dehydro thromboxane B2 homogeneous enzyme immunoassay kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Glucose-6-phosphate dehydrogenase and 11-dehydrothromboxane B 2 Preparation of conjugates

[0029] Glucose-6-phosphate dehydrogenase and 11-dehydrothromboxane B were prepared as follows 2 Conjugates:

[0030] (1) 20mg 11-dehydrothromboxane B 2 Dissolve in 4ml dimethylsulfoxide (DMSO), add 8mg of N-hydroxysuccinimide (NHS), stir to dissolve completely, then add 12mg of 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride (EDC), airtight and shake at 20°C for 4 hours, then add 16ml DMSO to the mixture, accurately dilute to 20ml, divide into 20 bottles according to 1ml / bottle, and store at 4°C;

[0031] (2) Weigh 100mg of glucose-6-phosphate dehydrogenase and use 95ml containing 2mM Mg 2+ The TAPS buffer solution is configured into a 1mg / ml solution, the pH is adjusted to 8.3, and the volume is adjusted to 100ml;

[0032] (3) Mix the solution prepared in step (1) and step (2) at a ratio of 1:100, and shake and react at 20°C for 12 hours in the dark;...

Embodiment 2

[0034] Example 2: 11-dehydrothromboxane B 2 Preparation of detection reagents for homogeneous enzyme immunoassay

[0035] Prepare 11-dehydrothromboxane B as follows 2 Homogeneous enzyme immunoassay detection reagents:

[0036] (1) Preparation of detection reagent R1: Take 5 mg of the enzyme-labeled conjugate prepared according to Example 1, dissolve it in 1 L containing 5 mM Mg 2+ 50mM Tris-HCl buffer, and adjust the pH value to 8.4;

[0037](2) Preparation of detection reagent R2: take 11-dehydrothromboxane B 2 Monoclonal antibody 4mg, disodium glucose-6-phosphate 6.1g, nicotinamide adenine dinucleotide disodium salt 13.7g, dissolved in 1L containing 5mM Mg 2+ 50mM Tris-HCl buffer, and adjust the pH value to 8.4;

[0038] (3) 11-Dehydrothromboxane B 2 Standard preparation: 11-dehydrothromboxane B 2 Prepare a solution of 5000pg / ml with formic acid.

Embodiment 3

[0039] Example three: 11-dehydrothromboxane B 2 Homogeneous enzyme immunoassay detection reagent test and test results

[0040] (1) Set the reaction parameters of the automatic biochemical analyzer: set the parameters of the biochemical analyzer according to Table 1

[0041] Table 1. Reaction parameters of automatic biochemical analyzer

[0042]

[0043] (2) Obtain the standard curve

[0044] Dilute the standard with the sample diluent to 5000, 2500, 1250, 625, 312.5, and 0 pg / ml, and measure according to the above-mentioned determination procedure to obtain the standard curve, such as figure 1 shown.

[0045] (3) Sample testing

[0046] Through the standard curve obtained by the homogeneous enzyme immunoassay reagent of the present invention, the high and low quality control samples were repeatedly measured 10 times, and the detection data and data analysis are shown in Table 2.

[0047] Table 2. Test data and analysis of quality control products

[0048]

[0049]...

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Abstract

The invention belongs to the technical field of in vitro diagnosis, and specifically provides an 11-dehydro thromboxane B2 homogeneous enzyme immunoassay kit. The invention is characterized by measuring 11-dehydro thromboxane B2 with the homogeneous enzyme immunoassay, and high-throughput rapid quantitative determination of 11-dehydro thromboxane B2 in human urine in a fully automatic biochemicalanalyzer is realized. The invention also includes an enzyme-labeled conjugate used in the kit and a preparation method thereof. Compared with the existing detection kits on the market, the kit of theinvention has the advantages such as high automation degree of the detection process, high sensitivity, high specificity and accurate results. The detection cost of 11-dehydro thromboxane B2 is also greatly reduced, and large-scale clinical popularization is facilitated.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis, specifically a 11-dehydrothromboxane B 2 A homogeneous enzyme immunoassay kit, and a preparation method for the kit. Background technique [0002] 11-Dehydrothromboxane B 2 (11-dhTXB 2 ) is the final metabolite of thromboxane A2 (thromboxane B2) in human urine, 11-dehydrothromboxane B in urine 2 The content can accurately reflect the level of thromboxane A2 in the blood within a certain period of time, and the level of thromboxane A2 in human blood is closely related to the ability of platelet aggregation. Therefore, 11-dehydrothromboxane B in human urine 2 The content can be used as one of the indicators of platelet aggregation ability. The confirmation of the effect of antiplatelet aggregation therapy, especially the detection of aspirin resistance, is of great significance. In addition, for patients with cardiovascular and cerebrovascular diseases, this indicator can reflect the inciden...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/535
CPCG01N33/94G01N33/581G01N33/535
Inventor 刘佳龙张远张伯平蔡泽浪
Owner 深圳市安帝宝科技有限公司
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