Application of kit for detecting D-dimer by using space proximity chemiluminescence method
阅读说明：本技术 空间邻近化学发光法检测d-二聚体的试剂盒的应用 (Application of kit for detecting D-dimer by using space proximity chemiluminescence method ) 是由 奚伟红 朱丹丹 廖鸳鸯 于 2018-09-25 设计创作，主要内容包括：本发明涉及一种空间邻近化学发光法检测D-二聚体的试剂盒的应用。试剂盒包括：酶标记物、发光标记物、辅助剂、触发剂和校准品；其中,校准品包括不同浓度D-Dimer抗原的校准品和0.1M磷酸盐稀释液；酶标记物的组分包括过氧化物酶标记的D-Dimer检测抗体和0.05M磷酸盐缓冲液；发光标记物的组分包括9,10-二氢吖啶标记的D-Dimer捕获抗体和0.05M Tris缓冲液；辅助剂的组分包括发光辅助剂和枸橼酸盐缓冲液；触发剂选用0.05M Tris缓冲液。本发明提供的检测方法作为一种真正意义上的均相化学发光技术,无需载体,没有包被、洗涤过程,试剂盒组分少,且灵敏度高、重复性好、操作简单。(The invention relates to an application of a kit for detecting D-dimer by a spatial proximity chemiluminescence method. The kit comprises: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein the calibrator comprises calibrators of D-Dimer antigens with different concentrations and 0.1M phosphate diluent; the components of the enzyme label comprise peroxidase labeled D-Dimer detection antibody and 0.05M phosphate buffer solution; the luminescent marker comprises a 9, 10-dihydroacridine labeled D-Dimer capture antibody and 0.05M Tris buffer; the auxiliary agent comprises a luminescence auxiliary agent and a citrate buffer solution; the trigger is 0.05M Tris buffer solution. As a true homogeneous phase chemiluminescence technology, the detection method provided by the invention does not need a carrier, does not have coating and washing processes, and has the advantages of few kit components, high sensitivity, good repeatability and simple operation.)
Use of a detection kit for a D-dimer in the detection of a D-dimer by spatial proximity chemiluminescence, wherein the detection kit for a D-dimer comprises:
enzyme label, luminescent label, adjuvant, trigger and calibrator;
the raw material components of the enzyme marker comprise peroxidase-labeled D-Dimer detection antibodies, and the raw material components of the luminescent marker comprise 9, 10-dihydroacridine-labeled D-Dimer capture antibodies;
the raw material components of the enzyme marker also comprise 0.05M phosphate buffer solution;
the preparation method of the enzyme label comprises the following steps: weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing against 1mM sodium acetate buffer solution with pH4.4, and standing at 4 ℃ overnight; adding 20 μ L of 0.2M carbonate buffer solution with pH of 9.5, immediately adding 1mg of anti-D-Dimer monoclonal antibody, stirring gently at room temperature in the dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the solution into a dialysis bag, dialyzing against 0.15M PBS with pH of 7.4, and standing at 4 deg.C overnight; taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C;
the raw material component of the luminescent marker also comprises 0.05MTris buffer solution;
the preparation method of the luminescent marker comprises the following steps: the luminescent substrate Acridan was dissolved in 500. mu.L of DMF; sucking 41.3 mu L of dissolved Acridan, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-DDimer monoclonal antibody, turning and uniformly mixing for 4-5 times, and standing for 30min at room temperature; and (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and placing in a refrigerator at-20 ℃ for storage.
2. The use of claim 1, wherein the calibrator comprises calibrators of different concentrations of D-Dimer antigen and a 0.1M calibrator dilution;
the preparation method of the calibrator diluent comprises the following steps: 14.1g of monopotassium phosphate and NaH are weighed2PO4·2H2Dissolving O3.0 g in ultrapure water, adding Proclin-3000.5-1 mL, uniformly mixing, adding ultrapure water to a constant volume of 1000mL to obtain a calibrator diluent, and storing at 2-8 ℃ for later use;
the preparation method of the calibrator comprises the following steps: and the concentration of the calibrator is 0ng/mL, 10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL and 10000ng/mL, the purified D-dimer is diluted to a corresponding concentration by using the calibrator diluent, and the purified D-dimer is stored at the temperature of 2-8 ℃ for later use.
3. The use according to claim 12, wherein the D-dimer detection kit is used in a method for detecting D-dimer by a spatial proximity chemiluminescence method, and comprises the following steps:
s1: respectively adding 25 mu L of standard substance, 25 mu L of peroxidase-labeled D-Dimer detection antibody and 25 mu L of luminescent marker into a chemiluminescence reaction tube;
s2: reacting for 15min in a constant temperature box at 37 ℃;
s3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then respectively adding 75 mu L of trigger, shaking and uniformly mixing, immediately detecting, and reading a signal value;
s4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.
Technical Field
The invention relates to the technical field of biology, in particular to application of a kit for detecting D-dimer by a spatial proximity chemiluminescence method.
Background
D-Dimer (D-Dimer) is a degradation product of crosslinked fibrin, and the formation mechanism is as follows: under the action of thrombin, polypeptide A and polypeptide B are sequentially removed from an alpha chain and a beta chain of fibrinogen to form fibrin I and fibrin II, and the fibrin is crosslinked on a blood vessel wall under the action of factor XII and is cleaved by activated plasmin to generate various FDP fragments. Due to the cross-linking of the gamma chain, a D-dimer was produced comprising 2D fragments linked by the gamma chain, having a molecular weight of about 18 kilodaltons and a half-life of 8h in vivo.
D-dimer is one of the markers that specifically reflects secondary hyperfibrinolysis in vivo, and D-dimer elevation can be caused by various diseases. Research data show that: the level of the D-dimer in the plasma is closely related to cardiovascular diseases, the positive rate of the content of the D-dimer in the plasma of the cardiovascular diseases is aortic dissection, acute myocardial infarction, unstable angina, stable angina, congenital heart disease and arrhythmia in sequence, and the higher the illness state is, the larger the rising amplitude of the D-dimer is, and the higher the positive rate is. The aortic dissection positive rate reaches 100%, and the detection of D-dimer has a very good negative expected value, so that the method has a high application value in aortic dissection exclusion diagnosis.
Deep Vein Thrombosis (DVT) formation is a major predisposition to pulmonary embolism, with approximately 51% to 71% of the thromboembolic thromboemboli being derived from DVT. The discovery of DVT does not allow direct diagnosis of pulmonary embolism, but gives great cues. The diagnosis of DVT is facilitated by intravenous angiography, ultrasonic Doppler blood vessel examination, limb impedance plethysmography and the like, and many studies show that the detection of the content of plasma D-dimer is also an effective means for screening DVT. The study found that patients with venography confirmed DVT all had elevated plasma D-dimer levels, and if positive with D-dimer >500ng/mL, the sensitivity and specificity of the examination were 95% and 77%, respectively, with a negative predictive value of 92%. Thus, it is clinically suspected that DVT patients will help to rule out a diagnosis of VT if the D-dimer test is normal.
In the aspect of anticoagulation and thrombolysis treatment, the D-dimer has certain guiding significance, can be used as a mark of secondary hyperfibrinolysis, and can observe the anticoagulation effect and the thrombolysis condition of heparin. After thrombolytic treatment, the D-dimer level is obviously increased after 4-8 h, which is directly related to the dissolution of a large amount of fresh thrombus, and then gradually decreased and maintained to a normal level after 24 h-7D. This indicates that the treatment has no obvious effect, the change of the thrombus size is not large, if the thrombus is reduced and then increased again, the thrombus is formed again, the D-dimer level of old thrombus patients can not be increased, and if the D-dimer level is abnormally increased, the new thrombus is formed. In the thrombolytic treatment of the acute phase of cerebral infarction, the content of D-dimer in blood plasma is increased rapidly along with thrombolysis, and when the thrombus is dissolved completely and the blood vessel is communicated, the content is reduced rapidly. If the sustained higher level is not reduced, the thrombus is not completely dissolved or secondary thrombus is formed, so that the dynamic detection of the D-dimer content in the treatment of cerebral infarction, especially in the thrombolytic treatment, has important clinical significance for the diagnosis, curative effect observation and prognosis of cerebral infarction.
D-dimers are specific degradation products of cross-linked fibrin and are markers of thrombosis and lysis. Its formation and increase indicate the presence of hypercoagulable state, thrombosis and secondary increased fibrinolysis in the body. There are reports in the literature: AMI has the highest thrombosis rate, SAP has the lowest thrombosis rate, and UAP thrombosis rate is between AMI and SAP. Therefore, the determination of the D-dimer has certain reference value for clinical typing of the coronary heart disease.
At present, the detection of the content of the D-Dimer in clinic mainly adopts an immunoturbidimetry method, an enzymatic chemiluminescence method and a non-enzymatic chemiluminescence method. The immunoturbidimetry has narrow linear range and low sensitivity, and is greatly influenced by blood lipids; enzymatic chemiluminescence mainly includes horseradish peroxidase (HRP), alkaline phosphatase (ALP) systems and the like, and has the common point that enzymes serving as markers in the luminescence process are not substantially consumed; non-enzymatic chemiluminescence includes acridinium ester, oxalate systems, and the like, with the common denominator being the consumption of label during luminescence. Regardless of enzymatic and non-enzymatic chemiluminescence, a solid support (e.g., microwell plate or magnetic microparticles) is required, and the reaction complex is washed after the reaction is complete to remove unbound free components. The operation is complex, and the influence factors are many, so that the detection result is unstable, and the repeatability is poor.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the application of a kit for detecting D-dimer by a spatial proximity chemiluminescence method. As a true homogeneous phase chemiluminescence technology, the method does not need a carrier, does not have coating and washing processes, and has the advantages of few kit components, high sensitivity, good repeatability and simple operation.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a kit for detecting a D-dimer, the kit comprising: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein, the raw material component of the enzyme label comprises a peroxidase-labeled DDimer detection antibody, and the raw material component of the luminescent label comprises a 9, 10-dihydroacridine-labeled D-Dimer capture antibody.
Preferably, the raw material components of the enzyme marker also comprise 0.05M phosphate buffer solution; more preferably, the preparation method comprises: weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing against 1mM sodium acetate buffer solution with pH4.4, and standing at 4 ℃ overnight; adding 20 μ L of 0.2M pH9.5 carbonate buffer solution, immediately adding 1mg of anti-D-Dimer monoclonal antibody, stirring gently at room temperature in the dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the above solution into a dialysis bag, dialyzing against 0.15M pH 7.4PBS, and standing at 4 deg.C overnight; taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.
Preferably, the raw material component of the luminescent marker further comprises 0.05M Tris buffer; more preferably, the preparation method comprises: the luminescent substrate Acridan was dissolved in 500. mu.L of DMF; sucking dissolved Acridan41.3 mu L, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-D-Dimer monoclonal antibody, turning and uniformly mixing for 4-5 times, and standing for 30min at room temperature; and (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and storing in a refrigerator at-20 ℃.
Preferably, the components of the adjuvant include a luminescence adjuvant and a citrate buffer at pH 6.0; more preferably, the preparation method comprises: weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a proper amount of luminescence auxiliary agent into citrate buffer solution, mixing uniformly, subpackaging, and placing 10mL of each bottle in a refrigerator at 4 ℃ for later use.
Preferably, the trigger is 0.05M Tris-HCl buffer solution with the pH value of 8.0; more preferably, the preparation method comprises: weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolution, adding 2.1mL of concentrated HCl, and uniformly mixing. Adding tween-202 mL into the solution, mixing, diluting to 1000mL, mixing, packaging, and storing in a refrigerator at 4 deg.C for use, wherein 200mL per bottle.
Preferably, the calibrator comprises calibrators of different concentrations of D-Dimer antigen and 0.1M phosphate buffer; more preferably, the preparation method comprises: preparing a calibrator diluent: 14.1g of potassium dihydrogen phosphate and sodium dihydrogen phosphate (2H) were weighed2O)3.0g, adding a proper amount of ultrapure water for dissolving, namely Proclin-3000.5-1 mL, uniformly mixing, adding ultrapure water for constant volume to 1000mL, namely, obtaining a calibrator diluent, and storing at 2-8 ℃ for later use; preparing a calibrator: the concentration of the calibrator is 0, 10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL and 10000ng/mL, the purified D-dimer is diluted to the corresponding concentration by using a calibrator diluent, and the purified D-dimer is stored at the temperature of 2-8 ℃ for later use.
The kit provided by the invention adopts a double-antibody sandwich method to detect the content of D-Dimer (D-Dimer) in human plasma. Reacting a sample, an anti-D-Dimer monoclonal antibody marked by horseradish peroxidase (HRP) and an anti-D-Dimer monoclonal antibody marked by 9, 10-dihydroacridine (Acridan) together to form an antigen-antibody sandwich complex, enabling the horseradish peroxidase and the 9, 10-dihydroacridine (Acridan) to be close to each other in space, and adding a luminescent auxiliary agent and a trigger to generate flash chemiluminescence; unbound free HRP-labeled antibody and Acridan-labeled antibody did not emit light. The higher the content of D-Dimer in the sample, the greater the luminescence value (RLU) measured. Therefore, within a certain concentration range, the luminous value is in positive correlation with the concentration of the sample, and the content of the DDimer in the sample can be calculated according to the luminous value of the sample by drawing a working curve through the calibrator with known concentration and the luminous value thereof.
In a second aspect, the invention provides the use of a detection kit for detecting D-dimer by spatial proximity chemiluminescence.
In a third aspect, the present invention provides a method for detecting D-dimer by using the above-mentioned detection kit in a spatial proximity chemiluminescence method, comprising the steps of: s1: respectively adding 25 mu L of standard substance, 25 mu L of peroxidase-labeled D-Dimer detection antibody and 25 mu L of luminescent marker into a reaction tube; s2: reacting for 15min in a constant temperature box at 37 ℃; s3: respectively adding 5 mu L of auxiliary agent into the reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then respectively adding 75 mu L of trigger, shaking and uniformly mixing, immediately detecting, and reading a signal value; s4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.
The technical scheme provided by the invention has the following beneficial effects:
(1) compared with the traditional chemiluminescence technology which needs a microporous plate or magnetic particles as a carrier to coat an antibody or an antigen, the kit and the detection method provided by the invention do not need a carrier and do not have coating and washing processes, and are a true homogeneous chemiluminescence technology.
(2) In the traditional enzymatic chemiluminescence technology, an analyte to be detected is combined with a capture antibody and an enzyme-labeled detection antibody in sequence and then is washed for 2-3 times to remove unbound or loosely bound non-specific substances; in the invention, after the analyte to be detected is combined with the two specific antibodies, the luminous interference is eliminated through the auxiliary agent effect, the trigger is added to generate a luminous signal, and the whole process does not need washing.
(3) The traditional enzymatic chemiluminescence technology needs an enzyme catalysis substrate, and the luminescence value is detected in about 5-10 minutes; the invention is a flash-type chemiluminescence, which generates a luminescent signal immediately after a trigger is added.
(4) The kit provided by the invention has the advantages of few components, simple production process, low production cost and easy amplification production; and the detection process is convenient and fast, and the full automation is easy to realize.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
The invention provides a detection kit for a D-dimer, which comprises: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein the components of the enzyme label comprise peroxidase-labeled D-Dimer detection antibody and 0.05M phosphate buffer solution; the luminescent marker comprises a 9, 10-dihydroacridine labeled D-Dimer capture antibody and 0.05M Tris buffer; the adjuvant comprises a luminescence adjuvant and a citrate buffer solution with pH of 6.0; the trigger is 0.05M Tris-HCl buffer solution with the pH value of 8.0; the calibrator comprises calibrators of D-Dimer antigens with different concentrations and 0.1M calibrator diluent.
Preparation of calibrator
(1) Preparing a calibrator diluent: 14.1g of potassium dihydrogen phosphate and sodium dihydrogen phosphate (2H) were weighed2O)3.0g, adding ultrapure water for dissolving, Proclin-3000.5-1 mL, mixing uniformly, adding ultrapure water for constant volume to 1000mL to obtain a calibrator diluent, and storing at 2-8 ℃ for later use.
(2) Preparing a calibrator: the concentration of the calibrator is 0, 10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL and 10000ng/mL, the purified D-dimer is diluted to the corresponding concentration by using a calibrator diluent, and the purified D-dimer is stored at the temperature of 2-8 ℃ for later use.
Secondly, preparation of enzyme label
(1) 5mg of HRP was weighed and dissolved in 1mL of distilled water, 0.2mL of newly prepared 0.1M sodium periodate solution was added to the supernatant, and the mixture was stirred at room temperature in the dark for 20min, and the solution was packed in a dialysis bag and dialyzed against 1mM pH4.4 sodium acetate buffer at 4 ℃ overnight.
(2) Adding 20 μ L of 0.2M pH9.5 carbonate buffer solution, immediately adding 1mg of anti-D-Dimer monoclonal antibody, stirring gently at room temperature in the dark for 2h, adding new 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the above solution into dialysis bag, dialyzing against 0.15M pH 7.4PBS, and standing at 4 deg.C overnight.
(3) Taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.
Preparation of luminescent markers
(1) The luminogenic substrate Acridan was dissolved in 500. mu.L of DMF.
(2) Sucking 41.3 mu L of dissolved Acridan, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-D-Dimer monoclonal antibody, overturning and uniformly mixing for 4-5 times, and standing at room temperature for 30 min.
(3) And (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and storing in a refrigerator at-20 ℃.
Preparation of adjuvant
Weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a luminescence auxiliary agent into a citrate buffer solution, mixing uniformly, subpackaging, and storing in a refrigerator at 4 ℃ for later use; among them, 10mL per bottle is more preferable.
Preparation of trigger
Weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolving, adding 2.1mL of concentrated HCl, and uniformly mixing; adding tween-202 mL, mixing, diluting to 1000mL, packaging, and storing in a refrigerator at 4 deg.C; among them, more preferably 200mL per bottle.
The invention also provides a method for detecting D-dimer by adopting the D-dimer detection kit in a space proximity chemiluminescence method, which comprises the following steps:
s1: add 25. mu.L of standard, 25. mu.L of peroxidase-labeled D-Dimer detection antibody, and 25. mu.L of luminescent label, respectively, to the chemiluminescent reaction tube.
S2: the reaction was carried out at 37 ℃ for 15 min.
S3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then, 75 mu L of trigger is added respectively, the mixture is shaken and mixed evenly, and the signal value is read immediately.
S4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.
The technical solution provided by the present invention is further illustrated below with reference to specific examples.
Example one
This embodiment provides a detection kit for D-dimer, comprising: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein the calibrator comprises not less than 6 calibrators of D-Dimer antigens with different concentrations and 0.1M phosphate buffer; the enzyme label comprises peroxidase-labeled D-Dimer detection antibody and 0.05M phosphate buffer solution; the luminescent markers comprise a 9, 10-dihydroacridine labeled D-Dimer capture antibody and a 0.05M Tris buffer solution; the adjuvant comprises a luminescence adjuvant and a citrate buffer solution with pH of 6.0; the trigger is 0.05M Tris-HCl buffer solution with the pH value of 8.0.
Preparation of calibrator
(1) Preparing a calibrator diluent: 14.1g of potassium dihydrogen phosphate and sodium dihydrogen phosphate (2H) were weighed2O)3.0g, adding ultrapure water for dissolving, Proclin-3000.8mL, mixing uniformly, adding ultrapure water for constant volume to 1000mL to obtain a calibrator diluent, and storing at 4 ℃ for later use.
(2) Preparing a calibrator: the concentration of the calibrator is 0, 10ng/mL, 50ng/mL, 500ng/mL, 1000ng/mL, 10000ng/mL, the purified D-dimer is diluted to the corresponding concentration by the calibrator diluent, and the calibrator is stored at 4 ℃ for later use.
Secondly, preparation of enzyme label
(1) Weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution into the upper solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing against 1mM sodium acetate buffer solution with pH4.4, and standing at 4 ℃ overnight;
(2) adding 20 μ L of 0.2M carbonate buffer solution with pH of 9.5, immediately adding 1mg of anti-D-Dimer monoclonal antibody, stirring gently at room temperature in the dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing well, standing at 4 ℃ for 2h, filling the solution into a dialysis bag, dialyzing against 0.15MpH 7.4.4 PBS, and standing at 4 ℃ overnight;
(3) taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.
Preparation of luminescent markers
(1) The luminescent substrate Acridan was dissolved in 500. mu.L of DMF;
(2) sucking 41.3 mu L of dissolved Acridan, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-D-Dimer monoclonal antibody, turning and uniformly mixing for 5 times, and standing for 30min at room temperature;
(3) the labeled reaction tube was placed on a shaker, mixed overnight at 4 ℃ and taken out, added with an appropriate amount of glycerol and stored in a refrigerator at-20 ℃.
Preparation of adjuvant
Weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a luminescence auxiliary agent into a citrate buffer solution, uniformly mixing, subpackaging, and storing 10mL of each bottle in a refrigerator at 4 ℃ for later use.
Preparation of trigger
Weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolving, adding 2.1mL of concentrated HCl, and uniformly mixing; adding tween-202 mL, mixing, diluting to 1000mL, packaging, and storing 200mL per bottle in a refrigerator at 4 deg.C for use.
Example two
The present embodiment provides a method for detecting D-dimer by using a D-dimer detection kit in a spatial proximity chemiluminescence method, comprising the following steps:
s1: add 25. mu.L of standard, 25. mu.L of peroxidase-labeled D-Dimer detection antibody, and 25. mu.L of luminescent label, respectively, to the chemiluminescent reaction tube.
S2: the reaction was carried out at 37 ℃ for 15 min.
S3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then, 75 mu L of trigger is added respectively, the mixture is shaken and mixed evenly, and the signal value is read immediately.
S4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.
The method adopts a space proximity chemiluminescence method to detect the D-dimer, and the sensitivity can reach 10 ng/mL; the specificity of D-dimer for diagnosis of acute cardiovascular disease was 86.3%, and particularly the positivity for aortic dissection diagnosis was 100% (23/23); the sensitivity of the diagnosis of acute pulmonary embolism can reach more than 90%.
Of course, other conditions and parameters in the preparation process are possible in addition to those recited in example one and example two.
The applicant finds out after creative work that: as a true homogeneous phase chemiluminescence technology, the detection method provided by the invention does not need a carrier, does not have coating and washing processes, and has the advantages of few kit components, high sensitivity, good repeatability and simple operation.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains. Unless specifically stated otherwise, the relative steps, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the present invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.
In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention, and all of the technical solutions are covered in the protective scope of the present invention.