CN102788878A

CN102788878A - D-dimer detection reagent kit and preparation method thereof

Info

Publication number: CN102788878A
Application number: CN2012102908746A
Authority: CN
Inventors: 谢兵
Original assignee: BEIJING ENJIHE BIOTECHNOLOGY Co Ltd
Current assignee: BEIJING ENJIHE BIOTECHNOLOGY Co Ltd
Priority date: 2012-08-16
Filing date: 2012-08-16
Publication date: 2012-11-21

Abstract

A D-dimer detection reagent kit, the kit body of the reagent kit comprises two reagents inside: reagent R1: buffer solution, polyethylene glycol, sodium azide, sodium ethylene diamine tetracetate, and bovine serum albumin; reagent R2: buffer solution, latex particles bound with D-dimer monoclonal antibody, Tween-20, sodium azide, sodium ethylene diamine tetracetate, and bovine serum albumin. Mixing a sample with the reagents in a specific volumetric ratio to conduct a series of reactions, placing the reactant under a semi/full automatic biochemical analyzer, and detecting the change speed of light absorbance at the position of 570nm dominant wavelength, thus the concentration of D-dimer can be figured out. The reagent kit has the advantages of accuracy, stability and convenience.

Description

A kind of D dimer detection kit and preparation method thereof

Technical field

The invention belongs to the biological medicine technology field, relate in particular to the dimeric detection kit of a kind of detection people D.

Background technology

Fibrinolytic system is the most important anticoagulation system of human body.When the fibrin coagula formed, the fibrinolysis process began, and fibrin coagula dissolving is formed various solvable fragments, and the D dimer promptly is one of most important soluble fragments wherein.

The human body fibrinolytic system, it is to keeping the normal permeability of vascular wall, and flow state and the organization restoration of keeping blood play an important role.Level increases explanation and has a Secondary cases fibrinolytic process in the D dimer blood plasma.Thromboembolism treatment is meant that medicament comes activated fiber protein dissolution system.Be generally and drop into a kind of plasminogen activator such as urine enzyme, streptokinase or histiotype plasminogen activator, a large amount of fibrinolysins are generated, thereby quicken thrombosed dissolving.The D dimer generates, and then shows to reach the thrombolysis effect.

In the fibrinolytic protein catabolite, only the crosslinked fragment of D dimer can reflect the thrombolysis activity behind the thrombosis.Therefore, in theory, but the thrombolysis effect of the dimeric detection by quantitative quantitative response of D medicine and can be used for diagnosing, screening the thrombus of new formation.

The D dimer is to measure the main factor of fibrinolytic system, monitors for Clinics and Practices fibrinolytic system disease and with fibrinolytic system diseases related and thromboembolism treatment, has great significance.The D dimer is DVT (DVT), pulmonary embolism (PE), the key index of disseminated intravascular coagulation (DIC).

Since Wilson in 1971 etc. at first are used for clinical diagnosis with fibrin degradation product (FDP); The dimeric detection of D is being brought into play enormous function aspect the diagnosis pulmonary embolism; Along with research carry out and deeply, the clinical position person is deep day by day to the dimeric meaning understanding of D.

D dimer assay method is a lot, comprising: (one) radioimmunoassay (RIA), (two) enzymoimmunoassay (ELISA), (three) latex agglutination test and immunoturbidimetry, (four) fluorescence immunoassay, (five) are dripped golden immunization.These methods have the shortcoming of complex operation, length consuming time, excess waste resource, are inappropriate for routine inspection.

Summary of the invention

The purpose of this invention is to provide the dimeric detection kit of a kind of D.This kit can directly apply on the automatic clinical chemistry analyzer, accuracy is strong as a result, reagent stability good, easy to use, be convenient to large-scale promotion.

Another object of the present invention provides the production preparation and the method for application of above-mentioned detection kit.

For realizing above-mentioned purpose, kit provided by the invention consists of:

Reagent R1: damping fluid, polyglycol, Sodium azide, sodium ethylene diamine tetracetate, bovine serum albumin(BSA);

Reagent R2: damping fluid, the latex particle that is combined with D dimer monoclonal antibody, Tween-20, Sodium azide, sodium ethylene diamine tetracetate, bovine serum albumin(BSA).

It is following that the preparation method of mentioned reagent box provided by the invention and behaviour do step:

(a) prepare reagent in following ratio:

Reagent R1:

Tris damping fluid 200-220mmol/L

Polyglycol 5-10 mmol/L

Sodium azide 1-5 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L;

Reagent R2:

Tris damping fluid 200-220mmol/L

Be combined with the latex particle 3.5-5% (v/v) of D dimer monoclonal antibody

Tween-20 1-5mmol/L

Sodium azide 0.5-3 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L

(b) reagent is mixed with sample to be tested by a certain percentage, make its complete reaction;

(c) measure the absorbance changing value with half/automatic clinical chemistry analyzer;

(d) calculate the dimeric concentration of D in the sample according to the absorbance changing value.

Embodiment

Following embodiment is for the more detailed the present invention of explanation, is confined to this but should not be construed as the present invention.

Embodiment 1:

Kit of the present invention is double reagent for example, wherein:

Reagent R1:

Tris damping fluid 200mmol/L

Polyglycol 8 mmol/L

Sodium azide 5 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L;

Reagent R2:

Tris damping fluid 200mmol/L

Be combined with the latex particle 5% (v/v) of D dimer monoclonal antibody

Tween-20 5mmol/L

Sodium azide 3 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L.

Embodiment 2: the kit method of application.

1. reagent is prepared, and reagent is liquid double reagent, and uncork i.e. usefulness, wherein:

Reagent R1:

Tris damping fluid 200mmol/L

Polyglycol 8 mmol/L

Sodium azide 5 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L;

Reagent R2:

Tris damping fluid 200mmol/L

Be combined with the latex particle 5% (v/v) of D dimer monoclonal antibody

Tween-20 5mmol/L

Sodium azide 3 mmol/L

Sodium ethylene diamine tetracetate 5mmol/L

Bovine serum albumin(BSA) 5mmol/L.

2. the full-automatic biochemical instrument parameter is provided with

(a) detected temperatures: 37 ℃

(b) detect wavelength: predominant wavelength is 570nm;

(c) reaction time: 8 minutes, wherein, 5 minutes incubation time, 10 seconds of reaction time, 3 minutes detection times.

3. detect step (all in automatic clinical chemistry analyzer, accomplishing)

(a) get 150 μ L reagent 1 and 7 μ L serum sample mixings;

(b) with the solution behind the mixing 37 ℃ of incubations 5 minutes;

(c) add 50 μ L reagent 2 again, reacted for 10 seconds after, 3 minutes absorbance changing value of monitoring under 570nm.

4. calculate the dimeric concentration of D through the absorbance changing value.

The advantage that the present invention has:

Accuracy is strong as a result, reagent stability good for this kit, easy to use, be convenient to large-scale promotion.Required detecting instrument (Biochemical Analyzer) generally uses at various big hospital and inspection center.

Claims

1. D dimer detection kit, it consists of: reagent R1, reagent R2,

Wherein:

A, reagent R1 comprise: 200-220mmol/L Tris damping fluid, 5-10 mmol/L polyglycol, 1-5 mmol/L Sodium azide, 1-5mmol/L sodium ethylene diamine tetracetate, 1-5mmol/L bovine serum albumin(BSA);

B, reagent R2:200-220mmol/L Tris damping fluid, the latex particle that is combined with D dimer monoclonal antibody, 1-5mmol/L Tween-20,0.5-3 mmol/L Sodium azide, 1-5mmol/L sodium ethylene diamine tetracetate, 1-5mmol/L bovine serum albumin(BSA).

2. the kit of claim 1; It is characterized in that: the preparation method of the latex particle of the D dimer monoclonal antibody among the reagent R2 is: the D dimer monoclonal antibody of 10.5mg/ml and 10% latex particle (diameter 50-100nm); Join in the 200-220mmol/L Tris damping fluid concussion reaction 4 hours, the centrifugal supernatant that goes; Be diluted to 0.5% with 200-220mmol/L Tris damping fluid again, and add 0.1-0.3 mmol/L Sodium azide.

3. the preparation of mentioned reagent box and method of operating, key step is:

(a) prepare reagent in following ratio:

Reagent R1:

Tris damping fluid 200-220mmol/L

Polyglycol 5-10 mmol/L

Sodium azide 1-5 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L

Reagent R2:

Tris damping fluid 200-220mmol/L

Be combined with the latex particle 3.5-5% (v/v) of D dimer monoclonal antibody

Tween-20 1-5mmol/L

Sodium azide 0.5-3 mmol/L

Sodium ethylene diamine tetracetate 1-5mmol/L

Bovine serum albumin(BSA) 1-5mmol/L

4. the method for claim 3 is characterized in that, sample and ratio of reagents should be controlled at 1:25 between the 1:35.

5. the method for claim 3 is characterized in that, temperature of reaction is 37 (± 1) ℃.

6. the method for claim 3 is characterized in that, the reaction time is 8 minutes.

7. the method for claim 3 is characterized in that, the wavelength that uses half/automatic clinical chemistry analyzer to detect is 570nm.