阅读说明：本技术 一种11-脱氢血栓烷b2抗原及其制备方法 (11-dehydrothromboxane B2 antigen and preparation method thereof ) 是由 邹佳信 张伯平 蔡泽浪 于 2019-11-02 设计创作，主要内容包括：本发明针对现有11-脱氢-血栓烷B<Sub>2</Sub>检测技术存在的缺陷,目的在于提供一种用于免疫竞争反应的11-脱氢-血栓烷B<Sub>2</Sub>标记抗原,以及制备方法。本发明所提供的11-脱氢-血栓烷B<Sub>2</Sub>标记抗原,其主要特征是抗原分子中同时含有六聚组氨酸和生物素结构,并在制备过程中可用Ni亲和层析柱纯化。本发明所提供的11-脱氢-血栓烷B2标记抗原具有化学结构明确、易于分离纯化、与抗体亲和能力较强等特点,适合大规模生产并可提高检测系统的性能。(The present invention is directed to the existing 11-dehydro-thromboxane B 2 The detection technology has defects and aims to provide 11-dehydro-thromboxane B for immune competitive reaction 2 Labeled antigens, and methods of preparation. The 11-dehydro-thromboxane B provided by the invention 2 The marked antigen is mainly characterized in that the antigen molecules simultaneously contain a hexahistidine and biotin structure, and can be purified by using a Ni affinity chromatography column in the preparation process. The 11-dehydrogenation-thromboxane B2 labeled antigen provided by the invention has the characteristics of definite chemical structure, easiness in separation and purification, strong affinity with an antibody and the like, is suitable for large-scale production, and can improve the performance of a detection system.)

1.[1]A method for preparing 11-dehydro-thromboxane B₂The labeled antigen for quantitative detection is characterized in that the antigen molecule simultaneously contains 11-dehydro-thromboxane B₂The structural formula of the compound is as follows:

2.[2]a process according to claim [1]]The compound is used for 11-dehydro-thromboxane B₂The preparation method of the labeled antigen for quantitative detection is characterized in that a Ni affinity chromatographic column is used for purification in the preparation process, and comprises the following steps:

(1) adding 11-dehydro-thromboxane B into solvent₂And adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), mixing, adding hexahistidine, reacting for 0.5 ~ 2 hr, and purifying with Ni affinity chromatography column to obtain 11-dehydro-thromboxane B₂With a hexahistidine conjugate, wherein 11-dehydro-thromboxane B₂The feeding ratio of the hexa-histidine to the hexa-histidine is 1:1.05 ~ 1: 1.4;

(2) adding 11-dehydro-thromboxane B into solvent₂Mixing with hexahistidine conjugate and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), adding aminated biotin, reacting for 0.5 ~ 2 hr, and purifying with Ni affinity chromatography column to obtain 11-dehydro-thromboxane B₂Labelled antigen of which 11-dehydro-thromboxane B₂The feeding ratio of the hexa-polyhistidine conjugate to the aminated biotin is 1:1.2 ~ 1: 2.0.

3. [3] the chemical reaction equation of the production method according to claim [1] is:

Technical Field

The invention belongs to the field of in-vitro diagnosis, and particularly relates to an 11-dehydro-thromboxane B2 antigen and a preparation method thereof, which are applied to quantitative detection of 11-dehydro-thromboxane B2 in a human urine sample.

Background

Thromboxane A2(Thromboxane A2, TXA2) is the strongest known platelet aggregation agent in human blood, and the content of TXA2 in the blood is closely related to the formation of atherosclerotic thrombus, and the value can reflect the risk of cardiovascular and cerebrovascular accidents (such as cerebral apoplexy, myocardial infarction and the like) of a patient to a certain extent. Aspirin (Aspirin) drugs can inhibit the activity of platelet cyclooxygenase-1 (COX-1) and platelet cyclooxygenase-2 (COX-2), thereby reducing the content of TXA2 in blood of patients and reducing abnormal platelet aggregation. Therefore, aspirin becomes an important medicament for treating cardiovascular and cerebrovascular diseases at present; meanwhile, long-term administration of low-dose aspirin is also an effective means for preventing cardiovascular and cerebrovascular diseases.

However, the effect of aspirin in inhibiting platelet aggregation is not effective in all people. Relevant statistics show that about 25% of patients take regular doses of Aspirin for a long period of time, but are not effective in reducing the amount of TXA2 production in vivo, a phenomenon known as "Aspirin Resistance". Therefore, there is a need for a reliable way for clinicians to analyze an individual's specific response to aspirin to assist the physician in determining treatment conditions and, thus, adjust the individual's treatment regimen. Unfortunately, TXA2 has a half-life in blood of only 30 seconds, and it is clinically very difficult to analyze the TXA2 content in blood.

11-dehydro-thromboxane B2 (11-hydro-thromboxane B2,11-dhTXB2) is a metabolite of TXA2 in urine, and the content of 11-dhTXB2 in the urine can accurately reflect the biosynthesis level of TXA2 in a patient. Therefore, the level of platelet activation of a patient can be evaluated by measuring the 11-dhTXB2 content in urine, and a doctor is helped to determine the anti-platelet aggregation treatment effect; furthermore, the risk of the patient suffering from cerebrovascular accidents such as cerebral apoplexy and the like can be predicted so as to achieve the aim of taking treatment measures in time.

11-dhTXB2 is a small molecular compound with extremely low concentration in urine, the clinical determination mainly depends on an immune competition method, and the core quantitative link of the detection is the competition reaction of a labeled antigen and an antigen to be detected which are captured by an antibody. The labeled antigen used for detection is generally a conjugate prepared by coupling 11-dhTXB2 with an enzyme. However, the use of such labelled antigens presents several problems during manufacture and use: (1) the activity of the labeled enzyme may be reduced during the coupling reaction and antigen purification; (2) the coupling reaction site has uncertainty, the chemical structures of the prepared labeled antigens are inconsistent, the batch difference is large, and the batch difference of the detection reagent (kit) is difficult to regulate and control; (3) a plurality of 11-dhTXB2 molecules are inevitably linked on the labeled antigen, more than 1 equivalent of 11-dhTXB2 antibody is inevitably consumed in the detection process, and the analysis sensitivity of a detection system cannot reach a high level easily; (4) the relative molecular mass of the antigen based on the enzyme is higher, the steric hindrance and the diffusion behavior of the antigen are obviously different relative to the small molecule 11-dhTXB2, the affinity of the antibody to the antigen and the antibody can generate a difference, and the linear range of a detection system is limited.

Disclosure of Invention

In view of the above technical drawbacks, the present invention provides a 11-dehydro-thromboxane B₂Labeled antigen for 11-dehydro-thromboxane B and its preparation method₂The quantitative detection method is characterized in that the antigen molecules simultaneously contain a hexahistidine structure and a biotin structure. The 11-dehydro-thromboxane B provided by the invention₂The labeled antigen has the characteristics of definite chemical structure, easiness in separation and purification, strong affinity with an antibody and the like, is suitable for large-scale production, and can improve the performance of a detection system.

Specifically, the content of the invention comprises:

[1]a method for preparing 11-dehydro-thromboxane B₂The labeled antigen for quantitative detection is characterized in that the antigen molecule simultaneously contains 11-dehydro-thromboxane B₂The structural formula of the compound is as follows:

；

[2]a process according to claim [1]]The compound is used for 11-dehydro-thromboxane B₂The preparation method of the labeled antigen for quantitative detection is characterized in that a Ni affinity chromatographic column is used for purification in the preparation process, and comprises the following steps:

(1) adding 11-dehydro-thromboxane B into solvent₂And adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), mixing, adding hexahistidine, reacting for 0.5 ~ 2 hr, and purifying with Ni affinity chromatography column to obtain 11-dehydro-hemocyteThromboxane B₂With a hexahistidine conjugate, wherein 11-dehydro-thromboxane B₂The feeding ratio of the hexa-histidine to the hexa-histidine is 1:1.05 ~ 1: 1.4;

(2) adding 11-dehydro-thromboxane B into solvent₂Mixing with hexahistidine conjugate and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), adding aminated biotin, reacting for 0.5 ~ 2 hr, and purifying with Ni affinity chromatography column to obtain 11-dehydro-thromboxane B₂Labelled antigen of which 11-dehydro-thromboxane B₂The feeding ratio of the hexa-polyhistidine conjugate to the aminated biotin is 1:1.2 ~ 1: 2.0;

[3] the chemical reaction equation of the production method according to claim 1 is:

drawings

FIG. 1 chemical equation for labeled antigen synthesis reaction

FIG. 2 chemical structural formula of labeled antigen

FIG. 3 correlation detection in example 3

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples.