阅读说明：本技术 一种d-二聚体乳胶免疫比浊法检测试剂 (D-dimer latex immunoturbidimetry detection reagent ) 是由 丁重辉 颜川 胡晓娟 杨娟 于 2019-11-19 设计创作，主要内容包括：本申请属于临床体外检测试剂领域,公开了一种D-二聚体乳胶免疫比浊法检测试剂,包括组分R1和R2,所述R1为缓冲液,所述R2由S1和S2两种试剂组成,所述试剂S1和S2分别包含包被鼠抗人D-二聚体单克隆抗体的聚苯乙烯乳胶颗粒A和B；所述乳胶颗粒A平均粒径为180nm,乳胶颗粒B平均粒径为220nm～310nm。本发明所述试剂操作安全简便、稳定性佳、重复性好、准确度高,广泛适用于医院临床及实验室常规检验用全自动凝血测试仪。本发明所述试剂的敏感性,特异性和线性检测范围良好,可作为体外诊断试剂用于医院临床检测人血浆D-二聚体,临床检测性能良好、稳定期长、批间差小、直接检测线性范围宽。(The application belongs to the field of clinical in-vitro detection reagents and discloses a D-dimer latex immunoturbidimetry detection reagent which comprises components R1 and R2, wherein R1 is a buffer solution, R2 consists of S1 and S2, and the reagents S1 and S2 respectively comprise a polystyrene latex particle A and a polystyrene latex particle B coated with a mouse anti-human D-dimer monoclonal antibody; the average particle size of the latex particles A is 180nm, and the average particle size of the latex particles B is 220 nm-310 nm. The reagent provided by the invention is safe and simple to operate, good in stability, good in repeatability and high in accuracy, and is widely suitable for fully-automatic blood coagulation testers for routine examination in hospitals and laboratories. The reagent has good sensitivity, specificity and linear detection range, can be used as an in vitro diagnostic reagent for clinically detecting human plasma D-dimer in hospitals, and has good clinical detection performance, long stability period, small batch difference and wide direct detection linear range.)

1. A D-dimer latex immunoturbidimetry detection reagent comprises components R1 and R2, wherein the R1 is a buffer solution, the R2 consists of two reagents S1 and S2, and the reagents S1 and S2 respectively comprise polystyrene latex particles A and B coated with mouse anti-human D-dimer monoclonal antibodies; the average particle size of the latex particles A is 180nm, and the average particle size of the latex particles B is 220 nm-310 nm.

2. The reagent according to claim 1, wherein the average particle size of the latex particles B is 270 nm.

3. The reagent according to claim 1 or 2, wherein the latex particles A and B are formed by activating polystyrene latex with EDC and NHS and then chemically crosslinking, and the addition amount of the activating agent of the latex particles A is 0.6-0.8 g/L of EDC and 0.9-1.2 g/L of NHS; the addition amount of an activating agent of the latex particle B is 0.2-0.3 g/L of EDC; NHS 0.3-0.45 g/L.

4. The reagent of claim 3, wherein the activator for latex particles A and B has a ratio of EDC to NHS of 1: 1.5.

5. The reagent according to any one of claims 1 to 4, wherein the ratio of the latex particles A and B in the component R2 is 1:4 to 4: 1.

6. The reagent of claim 5, wherein the ratio of latex particles A and B in component R2 is 1B:4A, 2B:3A or 4B: 1A.

7. The reagent according to any one of claims 1 to 6, wherein the component R1 contains 10 mM-35 mM Tris, 500 mM-1M sodium chloride, 0.5-1% Chaps, 0.5-5% Procline300 and has a pH of 7.85-8.25.

8. The reagent of any one of claims 1 to 7, wherein the reagent S1 in the component R2 comprises < 4.5mg/ml of polystyrene latex particles A coated with mouse anti-human D-dimer monoclonal antibody, 15 to 25mM Mopos, 0.5 to 5% Procline300, 0.5 to 1% bovine serum albumin, and pH is 7.0 to 8.0.

9. The reagent of any one of claims 1 to 8, wherein the reagent S2 in the component R2 comprises < 2.5mg/ml polystyrene latex particles B coated with mouse anti-human D-dimer monoclonal antibody, 15 to 25mM Mopos, 0.5 to 5% Procline300, 0.5 to 1% bovine serum albumin, and pH is 7.0 to 8.0.

Technical Field

The invention belongs to the field of clinical in-vitro detection reagents, particularly relates to a latex immunoturbidimetry detection reagent, and particularly relates to a D-dimer latex immunoturbidimetry detection reagent

Background

The fibrinolytic system is the most important anticoagulant system of human body, when fibrin clot forms, plasminogen is activated and converted into plasmin in the presence of plasminogen activator, the fibrinolysis process begins, and plasmin degrades fibrin clot to form various soluble fragments. Fibrin exists in blood, and is activated and hydrolyzed to generate specific degradation products, which are called fibrin degradation products. D-Dimer (DD) is a cross-linked fibrin-specific degradation product, the simplest fibrin degradation product, whose formation or increase reflects the activation of the coagulation and fibrinolytic systems, suggesting the presence of hypercoagulable states and secondary hyperfibrinolysis in vivo. The mass concentration of the D-dimer has important significance for diagnosing and treating diseases of the fibrinolytic system (such as DIC and various thrombi) and diseases related to the fibrinolytic system (such as tumors and pregnancy syndromes) and monitoring the thrombolytic therapy. Elevated levels of the fibrin degradation product D-dimer indicate frequent fibrin degradation processes in the body. Thus, D-dimer is a key indicator of Deep Vein Thrombosis (DVT), Pulmonary Embolism (PE), Disseminated Intravascular Coagulation (DIC).

There are various methods for detecting D-dimer, including qualitative or semi-quantitative tests based on the latex agglutination principle (e.g., latex agglutination test), quantitative tests based on the ELISA principle (e.g., enzyme-linked immunosorbent assay (ELISA)), and tests based on the immunoturbidimetric or immunofluorescence principles (e.g., light-electron scattering turbidimetric assay, colloidal Gold Immunosmotic Assay (GIA), and latex immunoturbidimetric assay. The ELISA method has the advantages of high quantification and sensitivity, and the like, but is complex and time-consuming to operate and is not suitable for measuring a single sample. The photo-nephelometry has high sensitivity and specificity but requires large equipment. In addition, some latex in the existing D-dimer determination reagent is incompletely activated, so that the antibody coupling efficiency is low, the linear range of the reagent is narrow, and the sensitivity of the reagent is too low or the linear range is narrow due to improper selection of some latex.

Disclosure of Invention

In view of this, the present invention aims to provide a D-Dimer detection reagent based on the immunoturbidimetry principle, in which an antibody capable of recognizing a D-Dimer antigen is coupled to polystyrene latex particles, and which has good repeatability accuracy, high sensitivity, and a wide linear range.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

a D-dimer latex immunoturbidimetry detection reagent comprises components R1 and R2, wherein the R1 is a buffer solution, the R2 consists of two reagents S1 and S2, and the reagents S1 and S2 respectively comprise polystyrene latex particles A and B coated with mouse anti-human D-dimer monoclonal antibodies; the average particle size of the latex particles A is 180nm, and the average particle size of the latex particles B is 220 nm-310 nm.

The D-dimer latex immunoturbidimetry test reagent has a wide detection linear range (0.25-16 mug/ml), and avoids the problem that a clinical high-value sample needs to be re-diluted and then measured due to the narrow direct detection range of the reagent, so that the detection efficiency is improved; the reagent does not need to be re-diluted within the concentration detection range of 16 mu g/ml, thereby improving the accuracy of clinical test results.

In the present invention, the component R2 is composed of two reagents, S1 and S2, the reagent S1 contains polystyrene latex particles a coated with a mouse anti-human D-dimer monoclonal antibody, and the average particle diameter is 180 nm; the reagent S2 contains polystyrene latex particles B coated with mouse anti-human D-dimer monoclonal antibody, and the average particle size is 220 nm-310 nm.

In some embodiments, the latex particles B in the reagent S2 have an average particle size of 270 nm.

In the invention, the latex particles A and B are formed by activating polystyrene latex by EDC and NHS and then chemically crosslinking, and the specific steps are as follows:

1. stirring and activating the polystyrene latex at room temperature for 15-35 min by using EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution;

2. adding a mouse anti-human D-dimer monoclonal antibody into the activated latex particle solution, and stirring at room temperature for chemical crosslinking for 60-120 min;

3. adding 3-5% bovine serum albumin into the latex solution after chemical crosslinking, continuously stirring at room temperature for 60-120 min, and obtaining the final polystyrene latex particles coated with the mouse monoclonal antibody by a centrifugal ultrasonic mode.

Wherein the addition amount of the activating agent of the latex particle A is 0.6-0.8 g/L of EDC and 0.9-1.2 g/L of NHS; the addition amount of an activating agent of the latex particle B is 0.2-0.3 g/L of EDC; NHS 0.3-0.45 g/L.

In the invention, the activation time in the step 1 is 30 min; the chemical crosslinking in the step 2 is 90 min; and in the step 3, stirring at room temperature is carried out for 90 min.

In the present invention, the amount ratio of EDC to NHS in the activators of the latex particles a and B is 1: 1.5.

In the invention, the ratio of the latex particles A and B in the S1 and S2 in the component R2 is 1: 4-4: 1.

In some embodiments, the ratio of latex particles a and B in component R2 is 1B: 4A. In some embodiments, the ratio of latex particles a and B in component R2 is 2B: 3A. In some embodiments, the ratio of latex particles a and B in component R2 is 4B: 1A.

In the invention, the component R1 comprises 10 mM-35 mM Tris, 500 mM-1M sodium chloride, 0.5-1 per mill Chaps and 0.5-5 per mill Procline300, and the pH value is 7.85-8.25. In some embodiments, the working concentration of each component in component R1 is 25mM Tris, 750mM sodium chloride, 0.8% Chaps, 1% Procline 300.

In the invention, a reagent S1 in the component R2 comprises polystyrene latex particles A which are less than 4.5mg/ml and coated with mouse anti-human D-dimer monoclonal antibodies, 15-25 mM Mopos, 0.5-5% Procline300, 0.5-1% bovine serum albumin, and the pH is 7.0-8.0. In some embodiments, the working concentration of each component of reagent S1 in component R2 is 3.5mg/ml of polystyrene latex particle a coated with murine anti-human D-dimer monoclonal antibody, 20mM Mopos, 750mM sodium chloride, 0.5% bovine serum albumin, 1% Procline 300.

In the invention, a reagent S2 in the component R2 contains polystyrene latex particles B which are less than 2.5mg/ml and coated with mouse anti-human D-dimer monoclonal antibodies, 15-25 mM Mopos, 0.5-5% Procline300, 0.5-1% bovine serum albumin, and the pH is 7.0-8.0. In some embodiments, the working concentration of each component of reagent S2 in component R2 is 2.2mg/ml, 20mM Mopos, 750mM sodium chloride, 0.5% bovine serum albumin, 1% Procline 300.

According to the technical scheme, the D-dimer latex immunoturbidimetry detection reagent comprises components R1 and R2, wherein R1 is a buffer solution, R2 is composed of two reagents S1 and S2, and the reagents S1 and S2 respectively comprise polystyrene latex particles A and B coated with mouse anti-human D-dimer monoclonal antibodies; the average particle size of the latex particles A is 180nm, and the average particle size of the latex particles B is 220 nm-310 nm. The D-dimer latex immunoturbidimetry detection reagent is safe and simple to operate, good in stability, good in repeatability and high in accuracy, and is widely applicable to full-automatic blood coagulation testers for routine examination in hospital clinics and laboratories. The D-dimer latex immunoturbidimetry test reagent has good sensitivity, specificity and linear detection range, and the accuracy and controllability of the test value of a clinical sample of the D-dimer in the detection concentration range of 16 mu g/ml are good. The D-dimer latex immunoturbidimetry detection reagent is in a liquid type, can be used as an in vitro diagnostic reagent for clinically detecting human plasma D-dimer in hospitals, has good clinical detection performance, long stability period, small batch difference and wide direct detection linear range, provides reliable detection data for clinically diagnosing and treating thrombus and hemostasis diseases, and is favorable for further popularization and use in the market.

Detailed Description

The invention discloses a D-dimer latex immunoturbidimetry detection reagent. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

The reagent component R2 of the invention is composed of polystyrene latex particles coated with mouse monoclonal antibody, preservative and buffer solution, and the reagent component R1 is matched for detection. The polystyrene latex particles coated with the mouse monoclonal antibody are formed by chemically crosslinking polystyrene latex and a mouse anti-human D-dimer monoclonal antibody, and mainly comprise the following steps:

1. stirring and activating the polystyrene latex at room temperature for 15-35 min by using EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solution;

2. adding a mouse anti-human D-dimer monoclonal antibody into the activated latex particle solution, and stirring at room temperature for chemical crosslinking for 60-120 min;

3. adding 3-5% bovine serum albumin into the latex solution after chemical crosslinking, continuously stirring at room temperature for 60-120 min, and obtaining the final polystyrene latex particles coated with the mouse monoclonal antibody by a centrifugal ultrasonic mode.