CN110895283A - High-sensitivity D-dimer detection kit and use method thereof - Google Patents
High-sensitivity D-dimer detection kit and use method thereof Download PDFInfo
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- CN110895283A CN110895283A CN201911260674.4A CN201911260674A CN110895283A CN 110895283 A CN110895283 A CN 110895283A CN 201911260674 A CN201911260674 A CN 201911260674A CN 110895283 A CN110895283 A CN 110895283A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/22—Haematology
- G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
Abstract
The invention discloses a high-sensitivity D-dimer detection kit and a use method thereof, the kit comprises a kit main body, a sealing cover, a protective cover and reagents, wherein the kit main body is sequentially provided with a solid phase carrier hole, a sample hole, an antibody detection hole, a fluorescein hole, a washing hole and a reading hole; the surface of the solid phase carrier is coated with the polysaccharide compound with biocompatibility, and the polysaccharide compound can form hydrogen bond weak interaction with protein molecules, so that specific immune signals are effectively enhanced.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a high-sensitivity D-dimer detection kit and a use method thereof.
Background
The D-dimer is derived from a plasmin-solubilized crosslinked fibrin clot, is a specific degradation product generated by fibrin monomer after crosslinking by activated factor XIIIa and then hydrolyzing by plasmin, and is a specific fibrinolysis process marker.
Under physiological state, the organism keeps the dynamic balance of coagulation and fibrinolysis to ensure the timely formation and elimination of fibrin, the normal D-dimer level of human body is generally below 200 mug/l, if the coagulation and fibrinolysis system is doubly activated in vivo, the D-dimer level will be increased, therefore, the D-dimer can be used as one of the molecular markers of hypercoagulability and hyperfibrinolysis in vivo, the specificity of the D-dimer reflects the common pathological characteristics of the diseases with the processes of coagulation and fibrinolysis, thus the clinical application is very wide. D-dimer is used as a main factor for determining the fibrinolytic system and has important significance for diagnosing and treating diseases of the fibrinolytic system and diseases related to the fibrinolytic system and detecting thrombolytic therapy, so that the D-dimer is a key index of Deep Venous Thrombosis (DVT), Pulmonary Embolism (PE) and Disseminated Intravascular Coagulation (DIC). Therefore, the determination of the content of the D-dimer in the blood has important clinical significance for early diagnosis and disease course monitoring of thrombotic diseases, treatment monitoring of thrombolytic drugs and the like.
At present, the detection methods for the D-dimer mainly comprise a latex agglutination method, a double antibody sandwich method (ELISA), an immune gold labeling method, a latex particle enhanced immunoturbidimetry method and the like. The latex agglutination method is simple and rapid, is suitable for point-of-care testing (POCT), but can be only used for qualitative determination, and is mostly used for screening; the ELISA method has high sensitivity, but has poor quantitative accuracy, long operation time and low automation level, and is not suitable for the requirements of POCT; the latex particle enhanced immunoturbidimetry can be conveniently applied to a full-automatic biochemical analyzer, but needs instruments, is long in time consumption, is suitable for processing a large number of samples, and cannot meet the aim of rapid detection in emergency departments and primary hospitals; the immuno-gold labeling method is similar to the latex agglutination method, and has the advantages of simple and rapid operation, but the disadvantage of poor quantitative accuracy, especially poor repeatability, limits the clinical application of the immuno-gold labeling method, and is particularly not suitable for the quantitative detection of the humoral marker protein which needs to be accurately quantified to help the diagnosis of diseases. Therefore, the development of a D dimer detection product with accurate quantification, rapidness and convenience is still an important problem to be solved urgently in the field of clinical diagnosis product research.
Disclosure of Invention
The invention aims to provide a high-sensitivity D-dimer detection kit and a using method thereof, and the detection kit has high sensitivity and good linearity.
In order to achieve the purpose, the technical scheme of the invention provides a high-sensitivity D-dimer detection kit, which comprises a kit main body, a sealing cover, a protective cover and a reagent, wherein the kit main body is sequentially provided with a solid phase carrier hole, a sample hole, a detection antibody hole, a fluorescein hole, a washing hole and a reading hole, the inside of the solid phase carrier hole is of a T-shaped structure, the protective cover is arranged above the solid phase carrier hole and is hinged with the solid phase carrier hole, a solid phase carrier is arranged in the solid phase carrier hole, a serum sample is filled in the sample hole, a biotin-labeled detection antibody is filled in the detection antibody hole, streptavidin labeled with fluorescein is filled in the fluorescein hole, the number of the washing holes is 8, a washing buffer solution is filled in the washing holes, the bottom of the reading hole is transparent, and the sealing cover completely covers the sample hole, the detection antibody hole, the, Fluorescein well, wash well, read well orifice.
Preferably, the solid phase carrier is a quartz needle.
Preferably, the protective cover is a protective aluminum foil.
Preferably, the lower end part of the solid phase carrier is coated with a polysaccharide complex, and the polysaccharide complex is a deionized water solution containing 0.5-2.5% of chitosan, 0.1-0.5% of glycerol and 0.5-1.5% of polyethylene glycol 1000 by mass.
Preferably, the washing buffer solution is PBS buffer solution (0.01M, pH7.4) containing 0.15-0.35M NaCl, 0.05-0.1% vol triton X-100 and 3-6 g/L sodium dodecyl sarcosinate.
Preferably, the diameter of each of the sample well, the detection antibody well and the fluorescein well is d1, the diameter of the washing well is d2, the diameter of the reading well is d3, the diameter of the solid phase carrier well is d4, the maximum diameter of the solid phase carrier is d5, and d5 > d3 > d1 is d4 > d 2.
A method for using a high-sensitivity D-dimer detection kit comprises the following steps: injecting a serum sample into a sample hole, putting a solid phase carrier coated with a capture antibody into the sample hole, taking out after 10-60s, sequentially oscillating and cleaning in a first washing hole, a second washing hole and a third washing hole, putting into a detection antibody hole after cleaning, taking out after 10-60s, sequentially oscillating and cleaning in a fourth washing hole and a fifth washing hole, putting into a streptavidin labeled with fluorescein after cleaning for 10-60s, taking out, sequentially oscillating and cleaning in a sixth washing hole, a seventh washing hole and an eighth washing hole, airing, putting into a reading hole, and detecting by adopting a fluorescence analysis detector.
The invention has the following beneficial effects: the surface of the quartz needle is covered with a biocompatible polysaccharide compound, the polysaccharide compound can form a hydrogen bond weak interaction with protein molecules, so that nonspecific adsorption is reduced, the polysaccharide compound can effectively enhance a specific immune signal, and the amplification of a detection signal is realized by forming a plurality of layers of fluorescein-streptavidin molecular layers on the surface of the polysaccharide compound; according to the invention, SDS and Triton-X100 with strong washing capacity are added into the washing buffer solution, the ionic strength of the washing solution is improved, the nonspecific interaction between molecules is effectively controlled, the low background and high signal-to-noise ratio of the detection result are further realized, and the sensitivity and accuracy of the detection are improved.
Drawings
FIG. 1 is a schematic structural diagram of a kit according to the present invention;
FIG. 2 is another schematic view of the structure of the kit of the present invention;
FIG. 3 is a standard curve diagram of the kit of the present invention for detecting different D-dimer standards.
In the figure: 1-solid support well, 2-protective cap, 3-solid support, 4-sample well, 5-detection antibody well, 6-fluorescein well, 7-first wash well, 8-second wash well, 9-third wash well, 10-fourth wash well, 11-fifth wash well, 12-sixth wash well, 13-seventh wash well, 14-eighth wash well, 15-reading well, 16-sealing cap.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below.
Fluorescein-labeled streptavidin was purchased from Solarbio under the cat # SF068-0.1 ml.
Example 1
A high-sensitivity D-dimer detection kit comprises a kit main body, a sealing cover, a protective cover and a reagent, wherein the kit main body is sequentially provided with a solid phase carrier hole, a sample hole, a detection antibody hole, a fluorescein hole, a washing hole and a reading hole, the interior of the solid phase carrier hole is of a T-shaped structure, a protective cover is arranged above the solid phase carrier hole and is hinged with the solid phase carrier hole, a solid phase carrier is arranged in the solid phase carrier hole, the sample hole is filled with a serum sample, the detection antibody hole is filled with a biotin-labeled detection antibody, the fluorescein holes are filled with streptavidin marked by fluorescein, the number of the washing holes is 8, the washing buffer solution is filled in the washing holes, the bottom of the reading hole is transparent, and the sealing cover completely covers the orifices of the sample hole, the detection antibody hole, the fluorescein hole, the washing hole and the reading hole.
Preferably, the solid phase carrier is a quartz needle.
Preferably, the protective cover is a protective aluminum foil.
Preferably, the lower end part of the solid phase carrier is coated with a polysaccharide complex, and the polysaccharide complex is a deionized water solution containing 0.5-2.5% of chitosan, 0.1-0.5% of glycerol and 0.5-1.5% of polyethylene glycol 1000 by mass.
Preferably, the washing buffer solution is PBS buffer solution (0.01M, pH7.4) containing 0.15-0.35M NaCl, 0.05-0.1% vol triton X-100 and 3-6 g/L sodium dodecyl sarcosinate.
Preferably, the diameter of each of the sample well, the detection antibody well and the fluorescein well is d1, the diameter of the washing well is d2, the diameter of the reading well is d3, the diameter of the solid phase carrier well is d4, the maximum diameter of the solid phase carrier is d5, and d5 > d3 > d1 is d4 > d 2.
The kit comprises all reagent components required for completing immunoassay, the end part of a solid phase carrier loaded in the kit is coated with a capture antibody, and the solid phase carrier is immersed into each hole in the kit, so that the combination of target antigens in a detected sample and subsequent detection steps can be completed, and the manufacturing and maintenance costs are greatly reduced.
Example 2
First, use method of kit
Injecting a serum sample into a sample hole, putting a solid phase carrier coated with a capture antibody into the sample hole, taking out after 10-60s, sequentially oscillating and cleaning in a first washing hole, a second washing hole and a third washing hole, putting into a detection antibody hole after cleaning, taking out after 10-60s, sequentially oscillating and cleaning in a fourth washing hole and a fifth washing hole, putting into a streptavidin labeled with fluorescein after cleaning for 10-60s, taking out, sequentially oscillating and cleaning in a sixth washing hole, a seventh washing hole and an eighth washing hole, airing, putting into a reading hole, and detecting by adopting a fluorescence analysis detector.
The target antigen in the sample is firstly combined with the capture antibody on the surface of the solid phase carrier, the solid phase carrier is then sequentially immersed into the detection antibody containing the biotin label and the streptavidin labeled with the fluorescein for combination reaction, the surface of the solid phase carrier is coated with the polysaccharide complex with biocompatibility, the nonspecific combination can be effectively resisted, and the amplification of the signal is realized by forming the Cy5-SA molecular layers which are overlapped layer by layer on the surface of the inert polysaccharide matrix. This cyclic labeling reaction can be repeated to further amplify the signal.
Secondly, analyzing the detection result
1. Linearity: the kit establishes a calibration curve by taking the concentration value of the standard substance as an X axis and the luminous value of the standard substance as a Y axis, and calculates the corresponding concentration value according to the luminous value intensity of the sample to be detected, as shown in fig. 2, the linear equation is that Y is 0.7545X-3.7028, R2 is 0.9979, and the minimum detection limit is 0.5 ng/mL.
Taking a D-dimer standard substance with the concentration of 20ng/ml for 10 times of experiments, and calculating a detection concentration value by using a linear equation y which is 0.7545 x-3.7028; as shown in Table 1, the measured average values were 20.113ng/ml, the standard deviation was 0.209, and the intra-batch coefficient of variation was 1.03%.
TABLE 1
2. Stability: the kit is placed for 18 months (the effective period is 12 months) at 4 ℃, the relative deviation between the detection value and the standard value is less than 5 percent, and the kit is relatively stable when stored for 18 months at 4 ℃.
The kit has high accuracy of detection results, good linear relation and good stability, and can be used as an important tool for assisting in judging thrombotic diseases.
Example 3
A preparation method of a kit for detecting D-dimer in blood comprises the following steps:
(1) solid phase support coated capture antibody: soaking the lower end of a quartz needle in a polysaccharide complex for 1-3 minutes, freeze-drying at 4 ℃, diluting a D-dimer capture antibody to 1mg/mL by using 0.01M PBS buffer solution, placing the dried lower end of the quartz needle in the D-dimer capture antibody solution, reacting for 2 hours at 24 ℃ for labeling, washing for 3 times by using 0.1M PBS buffer solution after labeling is finished, and then placing in a confining liquid for confining for 8-12 hours, wherein the confining liquid is Tris-HCl buffer solution containing 1% BSA, 0.1% casein and 3% sucrose, so as to prepare a solid phase carrier for coating the capture antibody;
(2) biotin-labeled detection antibody: d-dimer detection antibody was diluted to 1mg/mL with 0.01M PBS buffer, and the mass ratio of antibody: adding biotin according to the ratio of 1:3-1:5, reacting at 24 ℃ for 8-12 hours for labeling, dialyzing with 0.1M PBS buffer solution for 16-24 hours after labeling, and changing the solution for 3 times to prepare a biotin-labeled detection antibody;
(3) washing buffer solution: 3.58g of Na are weighed2HPO4·12H2O，0.4g KCl，0.54gKH2PO4Adding 800mL of double distilled water for dissolution, adjusting the pH value to 7.4, dissolving to 1000mL to prepare a PBS buffer solution, and calculating and weighing the amount of each raw material required for preparing 1L of the washing buffer solution according to the following concentration: NaCl 0.35M, triton X-100 0.1% vol, sarcosyl 4g/LAnd sodium is weighed and then is placed into the prepared PBS buffer solution to be dissolved and uniformly mixed, so that the washing buffer solution is obtained.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A high-sensitivity D-dimer detection kit is characterized by comprising a kit main body, a sealing cover, a protective cover and a reagent, wherein a solid phase carrier hole, a sample hole, a detection antibody hole, a fluorescein hole, a washing hole and a reading hole are sequentially arranged on the kit main body, a T-shaped structure is arranged inside the solid phase carrier hole, the protective cover is arranged above the solid phase carrier hole and connected with the solid phase carrier hole in a hinged mode, a solid phase carrier is arranged in the solid phase carrier hole, a serum sample is filled in the sample hole, a biotin-labeled detection antibody is filled in the detection antibody hole, a fluorescein-labeled streptavidin is filled in the fluorescein hole, the number of the washing holes is 8, a washing buffer solution is filled in the washing holes, the bottom of the reading hole is transparent, and the sealing cover completely covers the sample hole, the detection antibody hole, the fluorescein hole, the washing hole, The orifice of the reading well.
2. The highly sensitive D-dimer detection kit according to claim 1, wherein said solid support is a quartz needle.
3. The highly sensitive D-dimer detection kit according to claim 1, wherein said protective cover is a protective aluminum foil.
4. The highly sensitive D-dimer detection kit according to claim 1, wherein the lower end of the solid phase carrier is coated with a polysaccharide complex, and the polysaccharide complex is a deionized water solution containing 0.5-2.5% by mass of chitosan, 0.1-0.5% by mass of glycerol, and 0.5-1.5% by mass of polyethylene glycol 1000.
5. The kit for detecting D-dimer according to claim 1, wherein the washing buffer is PBS (0.01M, pH7.4) containing 0.15-0.35M NaCl, 0.05-0.1% vol Triton X-100, and 3-6 g/L sarcosyl.
6. The highly sensitive D-dimer detection kit of claim 1, wherein the diameter of each of the sample well, the detection antibody well and the fluorescein well is D1, the diameter of the washing well is D2, the diameter of the reading well is D3, the diameter of the solid phase carrier well is D4, and the maximum diameter of the solid phase carrier is D5, D5 > D3 > D1 ═ D4 > D2.
7. The use method of the high-sensitivity D-dimer detection kit is characterized by comprising the following steps: injecting a serum sample into a sample hole, putting a solid phase carrier coated with a capture antibody into the sample hole, taking out after 10-60s, sequentially oscillating and cleaning in a first washing hole, a second washing hole and a third washing hole, putting into a detection antibody hole after cleaning, taking out after 10-60s, sequentially oscillating and cleaning in a fourth washing hole and a fifth washing hole, putting into a streptavidin labeled with fluorescein after cleaning for 10-60s, taking out, sequentially oscillating and cleaning in a sixth washing hole, a seventh washing hole and an eighth washing hole, airing, putting into a reading hole, and detecting by adopting a fluorescence analysis detector.
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| US20020132370A1 (en) * | 2000-02-23 | 2002-09-19 | Lassen Michael Rud | Detection of a blood coagulation activity marker in a body fluid sample |
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Application publication date: 20200320 |