CN115932240A - D-dimer determination kit and preparation method thereof - Google Patents
D-dimer determination kit and preparation method thereof Download PDFInfo
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- 238000003756 stirring Methods 0.000 claims abstract description 54
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- 238000001514 detection method Methods 0.000 claims abstract description 21
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- NUGAFWCHXXQOQJ-UHFFFAOYSA-N 1-hydroxypyrrolidine-2,5-dione;sodium Chemical compound [Na].ON1C(=O)CCC1=O NUGAFWCHXXQOQJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims abstract description 6
- -1 1-ethyl- (3-dimethylaminopropyl) Chemical group 0.000 claims abstract description 5
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- 230000003213 activating effect Effects 0.000 claims abstract description 3
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- 238000006243 chemical reaction Methods 0.000 claims description 55
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 34
- 230000004913 activation Effects 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 18
- 230000000903 blocking effect Effects 0.000 claims description 17
- 239000002981 blocking agent Substances 0.000 claims description 15
- 239000007983 Tris buffer Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 12
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 12
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- 238000003149 assay kit Methods 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 9
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
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- 238000002835 absorbance Methods 0.000 description 22
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- 230000035931 haemagglutination Effects 0.000 description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 4
- 239000004327 boric acid Substances 0.000 description 4
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- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of biological detection, and provides a D-dimer determination kit and a preparation method thereof. The D-dimer detection kit comprises a D-dimer R1 reagent and a D-dimer R2 reagent, and the preparation method of the D-dimer R2 reagent comprises the following steps: firstly, adding an N-hydroxysuccinimide sodium sulfonate solution into a carboxyl polystyrene microsphere solution, stirring, then adding a 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine solution, and activating to obtain activated microspheres; adding a D-dimer monoclonal antibody into the activated microspheres, and coupling to obtain coupled microspheres; adding a sealing agent into the coupling microsphere, and sealing to obtain a sealed microsphere; adding the suspension solution into the sealed microspheres, and performing ultrafiltration and concentration to obtain a D-dimer R2 concentrated solution; and mixing the R2 concentrated solution with the suspension to obtain the D-dimer R2 reagent. By the technical scheme, the problem of large batch difference of the D-dimer detection kit in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a D-dimer determination kit and a preparation method thereof.
Background
The dimer (D dimer, DD) is a specific degradation product of crosslinked fibrin, the generation or increase of which reflects the activation of a coagulation or fibrinolysis system, and can be used as one of molecular markers of a high coagulation state and hyperfibrinolysis in vivo and an effective observation index of thrombolytic therapy. D-dimer is a key indicator of Deep Vein Thrombosis (DVT), pulmonary Embolism (PE), disseminated Intravascular Coagulation (DIC).
The D-dimer detection kit takes a latex immunoturbidimetry method as a principle, and the D-dimer in a sample and anti-human D-dimer monoclonal antibody latex particles generate antigen-antibody reaction. When antigen and antibody are in special dilution system, the formed soluble immune complex is separated out from liquid phase under the action of polymerization promoter (polyethylene glycol, etc.) in dilution system to form microparticles, so that the reaction solution has turbidity. When the concentration of the antibody is fixed, the amount of the formed immune complex is increased along with the increase of the amount of the antigen in the sample, the turbidity of the reaction solution is increased, and the content of the antigen in the sample can be calculated by measuring the turbidity of the reaction solution and comparing with a series of standard products.
At present, the problems of large batch-to-batch difference and unstable performance of D-dimer detection kits on the market generally exist, so that the research of a preparation method capable of reducing the batch-to-batch difference of the D-dimer detection kit is of great significance.
Disclosure of Invention
The invention provides a D-dimer detection kit and a preparation method thereof, which solve the problem of large batch difference of the D-dimer detection kit in the prior art.
The technical scheme of the invention is as follows:
a method for preparing a D-dimer R2 reagent, comprising the steps of:
s1, activation: firstly, adding a sodium N-hydroxysuccinimide sulfonate solution into a carboxyl polystyrene microsphere solution, stirring, then adding a 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution, and activating to obtain activated microspheres;
s2, coupling: adding a D-dimer monoclonal antibody into the activated microspheres, and coupling to obtain coupled microspheres;
s3, sealing: adding a sealing agent into the coupled microspheres, and sealing to obtain sealed microspheres;
s4, adding the suspension solution into the sealed microspheres, and performing ultrafiltration and concentration to obtain a D-dimer R2 concentrated solution; and mixing the R2 concentrated solution with the suspension to obtain the D-dimer R2 reagent.
As a further technical scheme, in the step S1, the activation reaction temperature is 20-30 ℃;
and/or in the step S2, the coupling reaction temperature is 20-30 ℃;
and/or, in the step S3, the blocking reaction temperature is 20-30 ℃.
As a further technical scheme, in the step S1, the stirring speed of the activation reaction is 240-260r/min;
and/or in the step S2, the stirring speed of the coupling reaction is 240-260r/min;
and/or in the step S3, the stirring speed of the closed reaction is 240-260r/min.
As a further technical scheme, in the step S1, adding a sodium N-hydroxysuccinimide sulfonate solution and stirring for 0.5-2min; the activation reaction time is 15-30min;
and/or in the step S2, the coupling reaction time is 1-4h;
and/or in the step S3, the blocking reaction time is 0.5-2h.
As a further technical scheme, the suspension comprises the following components: 20-40mmol/L of boric acid buffer solution, 5-15g/L of trehalose, 1-5g/L of bovine serum albumin, 1-5g/L of sodium azide and 0.1-0.3g/L of tween.
As a further technical scheme, the solvent of the carboxyl polystyrene microsphere solution is 2- (N-morpholine) ethanesulfonic acid buffer solution;
and/or the solvent of the N-hydroxysuccinimide sodium sulfonate salt solution is 2- (N-morpholine) ethanesulfonic acid buffer solution;
and/or the solvent of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution is 2- (N-morpholine) ethanesulfonic acid buffer solution.
As a further technical scheme, the concentration of the N-hydroxysuccinimide sodium sulfonate solution is 50-100mg/mL;
and/or the concentration of the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine solution is 50-100mg/mL;
and/or the MES buffer has a molar concentration of 20-50mmol/L and a pH of 6.0.
As a further technical scheme, the blocking agent is a chemical synthesis blocking agent, and the chemical synthesis blocking agent is one of CE210 and CE 510.
The invention also provides a D-dimer R2 reagent prepared by the preparation method of the D-dimer R2 reagent.
The invention also provides a D-dimer detection kit, which comprises the D-dimer R2 reagent and a D-dimer R1 reagent, wherein the D-dimer R1 reagent comprises the following components:
tris buffer 20-40mmol/L, sodium chloride 5-15g/L, sodium azide 0.4-0.6g/L, PEG20000 10-20g/L.
The invention also provides a preparation method of the D-dimer determination kit, which is characterized by comprising the following steps:
A. preparation of D-dimer R1 reagent: mixing Tris buffer solution, sodium chloride, sodium azide and PEG20000 according to the concentration of 20-40mmol/L Tris buffer solution, 5-15g/L sodium chloride, 0.4-0.6g/L sodium azide and 10-20g/L PEG20000 to obtain a D-dimer R1 reagent;
B. preparation of D-dimer R2 reagent: the D-dimer R2 reagent is prepared by the preparation method of the D-dimer R2 reagent.
The working principle and the beneficial effects of the invention are as follows:
1. according to the invention, the preparation method of the D-dimer R2 reagent is improved, when the D-dimer R2 reagent is activated, the NHS solution is added into the carboxylic polystyrene microsphere solution, the EDC solution is added after stirring, compared with the existing method that the NHS solution and the EDC solution are directly mixed and then added into the carboxylic polystyrene microsphere solution, after the prepared D-dimer R2 reagent is used for a D-dimer determination kit, the batch difference of a low-value part is within 20 percent, and the batch difference of a high-value part is within 10 percent, so that the batch difference of the D-dimer determination kit is remarkably reduced, and the problem of large batch difference of the D-dimer detection kit in the prior art is solved.
2. In the invention, trehalose is added into the D-dimer R2 reagent, and the prepared D-dimer R2 reagent is used in the D-dimer determination kit, so that the stability of the D-dimer kit is obviously improved.
3. In the invention, when the D-dimer R2 reagent is prepared, a chemically synthesized sealing agent is used as the sealing agent, and the prepared D-dimer R2 reagent is used in a D-dimer measuring kit, so that the repeatability of the D-dimer measuring kit is obviously improved.
4. In the invention, when the D-dimer R2 reagent is prepared, the activation reaction, the coupling reaction and the sealing reaction are all carried out at 20-30 ℃, so that the reaction efficiency of the activation reaction, the coupling reaction and the sealing reaction is obviously improved, and the detection sensitivity of the D-dimer determination kit is improved after the prepared D-dimer R2 reagent is used for the D-dimer determination kit, so that the determined absorbance value is higher.
5. In the invention, when the stirring speeds of the activation reaction, the coupling reaction and the sealing reaction are 240-260R/min during the preparation of the D-dimer R2 reagent, the reaction efficiency of the activation reaction, the coupling reaction and the sealing reaction is obviously improved, and after the prepared D-dimer R2 reagent is used in a D-dimer measuring kit, the detection sensitivity of the D-dimer measuring kit is improved, so that the measured absorbance value is higher.
Detailed Description
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 of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.
In the following examples and comparative examples NHS was N-hydroxysuccinimide sulfonic acid sodium salt, EDC was 1-ethyl- (3-dimethylaminopropyl) carbodiimide and MES was 2- (N-morpholino) ethanesulfonic acid.
Example 1
A D-dimer R2 reagent prepared by the following method:
s1, activation
Accurately weighing 200mgNHS, dissolving with 2mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain NHS solution;
accurately weighing 50mgEDC, and dissolving with 0.5mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain EDC solution;
accurately measuring 25mL of carboxyl polystyrene microspheres with the mass concentration of 10%, putting the microspheres into a wide-mouth glass bottle, adding 25mL of MES buffer solution with the concentration of 100mmol/L and the pH value of 6.0, putting the wide-mouth glass bottle into a constant-temperature water bath, and stirring at 25 ℃ and the stirring speed of 250r/min to obtain a carboxyl polystyrene microsphere solution;
sucking the NHS solution by a liquid transfer device, dropwise adding the NHS solution into the carboxyl polystyrene microsphere solution at a constant speed, and stirring for 1min at a stirring speed of 250r/min;
sucking the EDC solution by using a liquid transfer device, dripping the solution into the solution at a constant speed, and stirring the solution at 25 ℃ for 20min at a stirring speed of 250r/min to obtain activated microspheres;
adding the activated microspheres into 250mL of MES buffer solution with the concentration of 50mmol/L and the pH value of 6.0, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 25mL; repeating the ultrafiltration step for 3 times to obtain an activated microsphere concentrated solution; and (3) drying 1mL of activated microsphere concentrated solution to test the concentration of microspheres in the activated microsphere concentrated solution, and adjusting the mass concentration of the microspheres to 5% by using MES buffer solution with the concentration of 50mmol/L and the pH of 6.0 to obtain an activated microsphere solution.
S2, coupling
Accurately measuring 40mL of activated microsphere solution with the mass concentration of 5%, putting the activated microsphere solution into a glass bottle, stirring in a constant-temperature water bath at the temperature of 25 ℃, dropwise adding 600 mu g of D-dimer monoclonal antibody at the stirring speed of 250r/min, continuously stirring for 2h after the addition of the D-dimer monoclonal antibody, performing coupling reaction, and performing ultrasonic dispersion for 5min by using an ultrasonic dispersion crusher after the reaction is completed to obtain the coupled microsphere solution.
S3, sealing
Accurately measuring 2mL of a chemical synthesis blocking agent CE210 with the mass concentration of 2%, dropwise adding the chemical synthesis blocking agent CE210 into the coupling microsphere solution, continuously stirring at 25 ℃ for 1h for blocking reaction, wherein the stirring speed is 250r/min, and obtaining a blocked microsphere solution after the reaction is finished; adding the sealed microsphere solution into suspension with the volume 10 times that of the microsphere solution, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 40mL; repeating the ultrafiltration step for 3 times, and placing in an ice bath for ultrasonic dispersion for 30min by using an ultrasonic dispersion crusher to obtain a D-dimer reagent R2 concentrated solution; wherein, the suspension comprises the following components: 30mmol of boric acid buffer solution with pH of 7.4, 10g/L of trehalose, 2g/L of bovine serum albumin, 2g/L of sodium azide and 0.2g/L of tween.
S4, microsphere dilution and incubation
Accurately measuring 1.5L of the suspension, adding the suspension into 40mL of D-dimer reagent R2 concentrated solution, and uniformly stirring by using a stirrer to obtain a D-dimer reagent R2; wherein the suspension has the same composition as step S4.
Example 2
A D-dimer R2 reagent prepared by the following method:
s1, activation
Accurately weighing 200mgNHS, dissolving with 2mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain NHS solution;
accurately weighing 50mgEDC, and dissolving with 0.5mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain EDC solution;
accurately measuring 25mL of carboxyl polystyrene microspheres with the mass concentration of 10%, putting the microspheres into a wide-mouth glass bottle, adding 25mL of MES buffer solution with the concentration of 100mmol/L and the pH value of 6.0, putting the wide-mouth glass bottle into a constant-temperature water bath, and stirring at the temperature of 20 ℃ and the stirring speed of 240r/min to obtain a carboxyl polystyrene microsphere solution;
sucking the NHS solution by a liquid transfer device, dripping the NHS solution into the carboxylic polystyrene microsphere solution at a constant speed, and stirring for 2min at the stirring speed of 240r/min;
sucking the EDC solution by using a liquid transfer device, adding the EDC solution into the solution at a constant speed, and stirring the solution at 20 ℃ for 30min at a stirring speed of 240r/min to obtain activated microspheres;
adding the activated microspheres into 250mL of MES buffer solution with the concentration of 50mmol/L and the pH value of 6.0, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 25mL; repeating the ultrafiltration step for 3 times to obtain an activated microsphere concentrated solution; and (3) drying 1mL of activated microsphere concentrated solution to test the concentration of microspheres in the activated microsphere concentrated solution, and adjusting the mass concentration of the microspheres to 5% by using MES buffer solution with the concentration of 50mmol/L and the pH of 6.0 to obtain an activated microsphere solution.
S2, coupling
Accurately measuring 40mL of activated microsphere solution with the mass concentration of 5%, putting the activated microsphere solution into a glass bottle, stirring in a constant-temperature water bath at the temperature of 20 ℃, dropwise adding 600 mu g of D-dimer monoclonal antibody, continuously stirring for 4 hours after the addition of the D-dimer monoclonal antibody, performing coupling reaction, and performing ultrasonic dispersion for 5 minutes by using an ultrasonic dispersion crusher after the reaction is finished to obtain the coupled microsphere solution.
S3, sealing
Accurately measuring 2mL of 2% chemical synthesis blocking agent CE510, dropwise adding the blocking agent CE510 into the coupling microsphere solution, continuously stirring at 20 ℃ for 2h for blocking reaction, wherein the stirring speed is 240r/min, and obtaining the blocked microsphere solution after the reaction is finished; adding the sealed microsphere solution into suspension with the volume 10 times that of the microsphere solution, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 40mL; repeating the ultrafiltration step for 3 times, and placing in an ice bath for ultrasonic dispersion for 30min by using an ultrasonic dispersion crusher to obtain a D-dimer reagent R2 concentrated solution; wherein, the suspension comprises the following components: 40mmol of boric acid buffer solution with pH of 7.4, 15g/L of trehalose, 5g/L of bovine serum albumin, 5g/L of sodium azide and 0.3g/L of tween.
S4, microsphere dilution and incubation
Accurately measuring 1.5L of the suspension, adding the suspension into 40mL of D-dimer reagent R2 concentrated solution, and uniformly stirring by using a stirrer to obtain a D-dimer reagent R2; wherein the suspension has the same composition as step S4.
Example 3
A D-dimer R2 reagent prepared by the following method:
s1, activation
Accurately weighing 200mgNHS, dissolving with 2mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain NHS solution;
accurately weighing 50mgEDC, and dissolving with 0.5mL MES buffer solution with concentration of 50mmol/L and pH of 6.0 to obtain EDC solution;
accurately measuring 25mL of carboxyl polystyrene microspheres with the mass concentration of 10%, putting the microspheres into a wide-mouth glass bottle, adding 25mL of MES buffer solution with the concentration of 100mmol/L and the pH value of 6.0, putting the wide-mouth glass bottle into a constant-temperature water bath, and stirring at the temperature of 30 ℃ and the stirring speed of 260r/min to obtain a carboxyl polystyrene microsphere solution;
sucking the NHS solution by a liquid transfer device, dripping the NHS solution into the carboxyl polystyrene microsphere solution at a constant speed, and stirring for 0.5min at the stirring speed of 260r/min;
sucking the EDC solution by a liquid transfer device, adding the solution into the solution at a constant speed, and stirring the solution at 30 ℃ for 15min at a stirring speed of 260r/min to obtain activated microspheres;
adding the activated microspheres into 250mL of MES buffer solution with the concentration of 50mmol/L, the pH of 6.0 and the volume of 250mL, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 25mL; repeating the ultrafiltration step for 3 times to obtain an activated microsphere concentrated solution; and (3) drying 1mL of activated microsphere concentrated solution to test the concentration of microspheres in the activated microsphere concentrated solution, and adjusting the mass concentration of the microspheres to 5% by using MES buffer solution with the concentration of 50mmol/L and the pH of 6.0 to obtain an activated microsphere solution.
S2, coupling
Accurately measuring 40mL of activated microsphere solution with the mass concentration of 5%, putting the activated microsphere solution into a glass bottle, stirring in a constant-temperature water bath at the temperature of 30 ℃ at the stirring speed of 260r/min, dropwise adding 600 mu g of D-dimer monoclonal antibody, continuously stirring for 1h after the addition of the D-dimer monoclonal antibody, performing coupling reaction, and performing ultrasonic dispersion for 5min by using an ultrasonic dispersion crusher after the reaction is completed to obtain the coupled microsphere solution.
S3, sealing
Accurately measuring 2mL of a chemical synthesis blocking agent CE210 with the mass concentration of 2%, dropwise adding the chemical synthesis blocking agent CE210 into the coupling microsphere solution, continuously stirring at 30 ℃ for 0.5h for blocking reaction, wherein the stirring speed is 260r/min, and obtaining a blocked microsphere solution after the reaction is finished; adding the sealed microsphere solution into suspension with the volume 10 times that of the microsphere solution, performing ultrafiltration by adopting tangential flow, and concentrating the volume to 40mL; repeating the ultrafiltration step for 3 times, and placing in an ice bath for ultrasonic dispersion for 30min by using an ultrasonic dispersion crusher to obtain a D-dimer reagent R2 concentrated solution; wherein, the suspension comprises the following components: 20mmol of boric acid buffer solution with pH of 7.4, 5g/L of trehalose, 1g/L of bovine serum albumin, 1g/L of sodium azide and 0.1g/L of tween.
S4, microsphere dilution and incubation
Accurately weighing 1.5L of the suspension, adding the suspension into 40mL of D-dimer reagent R2 concentrated solution, and uniformly stirring by using a stirrer to obtain a D-dimer reagent R2; wherein the suspension has the same composition as step S4.
Example 4
A D-dimer assay kit is prepared by the following method:
A. preparation of D-dimer R1 reagent: mixing Tris buffer solution, sodium chloride, sodium azide and PEG20000 according to 30mmol/L Tris buffer solution, 10g/L sodium chloride, 0.5g/L sodium azide and 20000 g/L PEG20000 to obtain D-dimer R1 reagent;
B. preparation of D-dimer R2 reagent: prepared by the method of preparation of the D-dimer R2 reagent of example 1 to give the D-dimer R2 reagent.
Example 5
A D-dimer assay kit prepared by the following method:
A. preparation of D-dimer R1 reagent: mixing Tris buffer solution, sodium chloride, sodium azide and PEG20000 according to the concentration of 20mmol/L Tris buffer solution, 5g/L sodium chloride, 0.4g/L sodium azide and 20000 10g/L PEG to obtain D-dimer R1 reagent;
B. preparation of D-dimer R2 reagent: prepared using the procedure for preparation of the D-dimer R2 reagent of example 2 to give the D-dimer R2 reagent.
Example 6
A D-dimer assay kit prepared by the following method:
A. preparation of D-dimer R1 reagent: mixing Tris buffer solution, sodium chloride, sodium azide and PEG20000 according to the concentration of 40mmol/L Tris buffer solution, 15g/L sodium chloride, 0.6g/L sodium azide and 20g/L PEG20000 to obtain a D-dimer R1 reagent;
B. preparation of D-dimer R2 reagent: prepared by the method of preparation of the D-dimer R2 reagent of example 3 to give the D-dimer R2 reagent.
Experiment 1
2 sets of kits were prepared as follows:
kit 1: the D-dimer assay kit of example 4;
and (3) kit 2: the difference from the kit 1 is only that when the D-dimer R2 reagent is prepared, when the activation is performed in the step S1, the NHS solution and the EDC solution are mixed and then slowly added into the carboxyl polystyrene microsphere solution, and after the addition, the mixture is stirred for 15min at 30 ℃ with the stirring speed of 260R/min, so that the activated microsphere is obtained;
each group of kits is prepared into 3 batches;
an experimental instrument: BE Compact X hemagglutination instrument.
The experimental method comprises the following steps:
and (3) performing quality control product detection on the prepared 3 batches of the kit 1 and the kit 2, wherein the method for measuring the absorbance difference comprises the following steps: uniformly mixing 30 mu L of a sample to be detected with 107 mu L of a D-dimer R1 reagent, keeping the temperature for 40s, adding 63 mu L of the D-dimer R2 reagent for 20s, then measuring absorbance A1, then, incubating at 37 ℃ for 5min, then, measuring absorbance A2, and calculating the absorbance difference delta A according to the formula delta A = A2-A1; the samples to be tested are a quality control product 1 and a quality control product 2, the concentration of the quality control product 1 is 0.5 mu g/mL, the concentration of the quality control product 2 is 2 mu g/mL, and the test data is the change result of absorbance.
The results of the assay are shown in the following table:
TABLE 13 measurement results of lots of kit 1 and kit 2
The calculation formula of the relative deviation is as follows:
in the formula, B ab Represents the relative deviation of lot a and lot b, y a ，y b The measured values of the a-th and b-th batches, respectively.
As can be seen from table 1, compared with the reagent 2, the difference between the batches of the 3-batch reagent kit 1 is smaller, and the difference between the reagent kit 2 and the reagent kit 1 is only that when the D-dimer R2 reagent of the reagent kit 2 is prepared, the NHS solution and the EDC solution are mixed and then slowly added to the carboxy polystyrene microsphere solution during the activation in the step S1, which indicates that in the preparation method of the D-dimer R2 reagent of the present invention, the NHS solution is added first during the activation and then the EDC solution is added after the stirring, thereby significantly reducing the difference between the batches of the D-dimer determination reagent kit.
Experiment 2
2 sets of kits were prepared as follows:
kit 1: the D-dimer assay kit of example 4;
and (3) kit 2: the only difference from kit 1 is that trehalose is not added to the suspending agent in steps S4 and S5 when the D-dimer R2 reagent is prepared.
An experimental instrument: BE Compact X coagulometer.
The experimental method comprises the following steps:
and (3) carrying out an accelerated stability experiment on the kit 3 and the kit 4, and respectively determining the absorbance difference values on the 1 st day, the 5 th day, the 10 th day, the 15 th day, the 17 th day and the 20 th day, wherein the determination method of the absorbance difference value comprises the following steps: uniformly mixing 30 mu L of a sample to be detected with 107 mu LD-dimer R1 reagent, keeping the temperature for 40s, adding 63 mu L of D-dimer R2 reagent for 20s, then measuring absorbance A1, then incubating at 37 ℃ for 5min, and then measuring absorbance A2, and calculating the absorbance difference delta A according to the formula delta A = A2-A1; wherein, the sample to be detected is a high-concentration D-dimer sample, and the concentration of the D-dimer sample is 2.5 mug/mL;
the results of the assay are shown in the following table:
TABLE 2 determination results of accelerated stability of kit 3 and kit 4
The calculation formula of the relative deviation is the same as above.
As can be seen from table 2, compared with the kit 4, the kit 3 has a smaller change in the measurement result after being placed under the accelerated condition for 20 days, and the kit 4 is different from the kit 3 only in that when the D-dimer R2 reagent of the kit 4 is prepared, trehalose is not added to the suspending agents of steps S4 and S5, which indicates that the stability of the D-dimer kit is significantly improved by adding trehalose to the D-dimer R2 reagent.
Experiment 3
2 sets of kits were prepared as follows:
and (5) a kit: the D-dimer assay kit of example 4;
kit 6: the difference from the kit 5 is only that when the D-dimer R2 reagent is prepared, the blocking agent is BSA when in blocking in the step S3;
an experimental instrument: BE Compact X hemagglutination instrument.
The experimental method comprises the following steps:
the kit 5 and the kit 6 are used for carrying out repeatability investigation on the D-dimer quality control product with the concentration of 1 mu g/mL, and the results are shown in the following table:
TABLE 3 repeatability test results for kit 5 and kit 6
As can be seen from table 3, the CV value of the kit 5 is significantly reduced compared to the kit 6, and the kit 6 is different from the kit 5 only in that when the D-dimer R2 reagent of the kit 6 is prepared, the blocking agent is BSA during blocking in step S3, which indicates that in the present invention, when the D-dimer R2 reagent is prepared, a chemically synthesized blocking agent is used as the blocking agent, thereby significantly improving the repeatability of the D-dimer assay kit.
Experiment 4
3 sets of kits were prepared, as follows:
and (3) a kit 7: the D-dimer assay kit of example 4;
and (3) a kit 8: the difference from the kit 7 is only that when the D-dimer R2 reagent is prepared, the temperature of the activation reaction in the step S1, the coupling reaction in the step S2 and the blocking reaction in the step S3 is 2-8 ℃;
kit 9: the difference from the kit 7 is only that when the D-dimer R2 reagent is prepared, the temperature of the activation reaction in the step S1, the coupling reaction in the step S2 and the blocking reaction in the step S3 is 37 ℃;
an experimental instrument: BE Compact X hemagglutination instrument.
The experimental method comprises the following steps:
and (3) respectively testing the D-dimer quality control product 3, the quality control product 4 and the quality control product 5 with different concentrations by using the kit 7, the kit 8 and the kit 9, and determining the absorbance value. Wherein, the concentration of the quality control substance 3 is 0.5 mug/mL, the concentration of the quality control substance 4 is 1 mug/mL, the concentration of the quality control substance 5 is 2 mug/mL, and the determination result is shown in the following table:
TABLE 4 kit 7-9 for testing absorbance value data of quality control products with different concentrations
| Quality control product | Kit 7 | Kit 8 | Kit 9 |
| 3 | 0.042 | 0.036 | 0.025 |
| 4 | 0.120 | 0.087 | 0.078 |
| 5 | 0.245 | 0.168 | 0.0145 |
It can be seen from table 4 that, compared with the kit 7, the absorbance values of the quality control products 3 to 5 measured by the kits 8 to 9 are significantly reduced, and the difference between the kits 8 to 9 and 7 is only that the temperatures of the step S1 activation reaction, the step S2 coupling reaction and the step S3 sealing reaction are different when the D-dimer R2 reagent of the kits 8 to 9 is prepared, which indicates that in the present invention, when the temperatures of the step S1 activation reaction, the step S2 coupling reaction and the step S3 sealing reaction are all 20 to 30 ℃ during the preparation of the D-dimer R2 reagent, the reaction efficiencies of the activation reaction, the coupling reaction and the sealing reaction are significantly improved, thereby improving the detection sensitivity of the D-dimer measurement kit and making the measured absorbance values higher.
Experiment 5
4 sets of kits were prepared as follows:
kit 10: the D-dimer assay kit of example 4;
kit 11: the difference from the kit 10 is only that when the D-dimer R2 reagent is prepared, the stirring speeds of the activation reaction in the step S1, the coupling reaction in the step S2 and the blocking reaction in the step S3 are all 150R/min;
kit 12: the difference from the kit 10 is only that when the D-dimer R2 reagent is prepared, the stirring speed of the activation reaction in the step S1, the coupling reaction in the step S2 and the blocking reaction in the step S3 is 350R/min;
kit 13: the difference from the kit 10 is only that when the D-dimer R2 reagent is prepared, the stirring speeds of the activation reaction in the step S1, the coupling reaction in the step S2 and the blocking reaction in the step S3 are all 450R/min;
an experimental instrument: BE Compact X hemagglutination instrument.
The reagent kit 10, the reagent kit 11, the reagent kit 12 and the reagent kit 13 are respectively used for testing the quality control substances 6, 7 and 8 with different concentrations, and the absorbance value is measured. Wherein, the concentration of the quality control product 6 self-made by the company is 0.5 mug/mL, the concentration of the quality control product 7 is 1 mug/mL, the concentration of the quality control product 8 is 2 mug/mL, and the determination result is shown in the following table:
TABLE 5 kit 10-13 for testing absorbance value data of quality control products with different concentrations
| Quality control product | Kit 10 | Kit 11 | Kit 12 | Kit 13 |
| 6 | 0.048 | 0.030 | 0.032 | 0.027 |
| 7 | 0.127 | 0.089 | 0.099 | 0.076 |
| 8 | 0.242 | 0.165 | 0.179 | 0.151 |
As can be seen from table 5, compared with the kit 10, the absorbance values of the quality control materials 6 to 8 measured by the kits 11 to 13 are significantly reduced, and the difference between the kits 11 to 13 and the kit 10 is only that the stirring speeds of the step S1 activation reaction, the step S2 coupling reaction and the step S3 blocking reaction are different when the D-dimer R2 reagent of the kits 11 to 13 is prepared, which indicates that in the present invention, when the stirring speeds of the step S1 activation reaction, the step S2 coupling reaction and the step S3 blocking reaction are all 250R/min during the preparation of the D-dimer R2 reagent, the reaction efficiencies of the activation reaction, the coupling reaction and the blocking reaction are significantly improved, thereby improving the detection sensitivity of the D-dimer measurement kit and making the measured absorbance values higher.
Experiment 6
2 sets of kits were prepared, as follows:
kit 14: the D-dimer assay kit of example 4, batch 3, labeled 1#, 2#, 3#;
kit 15: the D Dimer detection kit is purchased from 3 batches of reagents of Wuhan Seillers Biotechnology GmbH, wherein the batches are respectively C1DD22101, C1DD22302 and C1DD22501 and are marked as BE1, BE2 and BE3.
An experimental instrument: BE Compact X hemagglutination instrument.
Detecting a sample: calibrator, lot 220507.
The experimental method comprises the following steps:
1#, 2#, 3# kits were used to measure the absorbances of D-dimer calibrators with concentrations of 0.1. Mu.g/mL, 0.2. Mu.g/mL, 0.4. Mu.g/mL, 0.8. Mu.g/mL, 1.6. Mu.g/mL, 3.2. Mu.g/mL, respectively, the test method was the same as experiment 1;
respectively measuring the absorbances of D-Dimer calibrators with the concentrations of 0.1 mu g/mL, 0.2 mu g/mL, 0.4 mu g/mL, 0.8 mu g/mL, 1.6 mu g/mL and 3.2 mu g/mL by BE1, BE2 and BE3 kits, and carrying out the measuring method according to the instruction of a D Dimer detection kit;
the test results are given in the following table:
TABLE 6 kit 14 and kit 15 test run for run-to-run differences for calibrators of different concentrations
As can be seen from table 6, the 3 batches of kit 14 had a batch to batch difference within 20% in the low value part and within 10% in the high value part; and the 3 batches of the kit 15 have the batch difference of more than 20 percent in the low value part and more than 10 percent in the high value part, which shows that compared with the existing D Dimer detection kit on the market, the method provided by the invention has the advantage that the batch difference of the D-Dimer detection kit is obviously reduced.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a D-dimer R2 reagent is characterized by comprising the following steps:
s1, activation: firstly, adding a sodium N-hydroxysuccinimide sulfonate solution into a carboxyl polystyrene microsphere solution, stirring, then adding a 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution, and activating to obtain activated microspheres;
s2, coupling: adding a D-dimer monoclonal antibody into the activated microspheres, and coupling to obtain coupled microspheres;
s3, sealing: adding a sealing agent into the coupling microsphere, and sealing to obtain a sealed microsphere;
s4, adding the suspension solution into the sealed microspheres, and performing ultrafiltration and concentration to obtain a D-dimer R2 concentrated solution; and mixing the R2 concentrated solution with the suspension to obtain the D-dimer R2 reagent.
2. The method for preparing a D-dimer R2 reagent as claimed in claim 1, wherein in the step S1, the activation reaction temperature is 20-30 ℃;
and/or in the step S2, the coupling reaction temperature is 20-30 ℃;
and/or, in the step S3, the blocking reaction temperature is 20-30 ℃.
3. The method for preparing a D-dimer R2 reagent according to claim 1, wherein in the step S1, the stirring speed of the activation reaction is 240-260R/min;
and/or in the step S2, the stirring speed of the coupling reaction is 240-260r/min;
and/or in the step S3, the stirring speed of the closed reaction is 240-260r/min.
4. The method for preparing a D-dimer R2 reagent according to claim 1, wherein in the step S1, the N-hydroxysuccinimide sodium sulfonate solution is added and stirred for 0.5-2min; the activation reaction time is 15-30min;
and/or in the step S2, the coupling reaction time is 1-4h;
and/or in the step S3, the blocking reaction time is 0.5-2h.
5. The method of claim 1, wherein the suspension comprises the following components: 5-15g/L trehalose, 1-5g/L bovine serum albumin, 1-5g/L sodium azide and 0.1-0.3g/L tween 20-40 mmol/L.
6. The method for preparing the D-dimer R2 reagent according to claim 1, wherein the solvent of the carboxylic polystyrene microsphere solution is 2- (N-morpholine) ethanesulfonic acid buffer solution;
and/or the solvent of the N-hydroxysuccinimide sodium sulfonate salt solution is 2- (N-morpholine) ethanesulfonic acid buffer solution;
and/or the solvent of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution is 2- (N-morpholine) ethanesulfonic acid buffer solution.
7. The method for preparing a D-dimer R2 reagent according to claim 1, wherein the blocking agent is a chemically synthesized blocking agent.
8. A D-dimer R2 reagent prepared by the method of any one of claims 1 to 7.
9. A D-dimer detection kit comprising the D-dimer R2 reagent of claim 8 and further comprising a D-dimer R1 reagent, said D-dimer R1 reagent comprising the following components:
tris buffer 20-40mmol/L, sodium chloride 5-15g/L, sodium azide 0.4-0.6g/L, PEG20000 10-20g/L.
10. A preparation method of a D-dimer assay kit is characterized by comprising the following steps:
A. preparation of D-dimer R1 reagent: mixing Tris buffer solution, sodium chloride, sodium azide and PEG20000 according to the concentration of 20-40mmol/L Tris buffer solution, 5-15g/L sodium chloride, 0.4-0.6g/L sodium azide and 10-20g/L PEG20000 to obtain a D-dimer R1 reagent;
B. preparation of D-dimer R2 reagent: the D-dimer R2 reagent prepared by the preparation method of the D-dimer R2 reagent as claimed in any one of claims 1 to 7.
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