Sample diluent for plasma sample fluorescent microsphere test strip detection

Abstract

The invention discloses a sample diluent for plasma sample fluorescent microsphere test strip detection and a preparation method and application thereof. The sample diluent contains a buffer solution, a stabilizer, a surfactant, a preservative, fibrinolytic enzyme and metal ion salt; the buffer solution is a boric acid-borax buffer solution; the stabilizer is any one of casein and bovine serum albumin; the surfactant is Tween 20; the preservative is any one or a combination of two of sodium azide and disodium ethylene diamine tetraacetate; the fibroprotease is earthworm fibrinolytic enzyme; the metal ion salt is sodium chloride. According to the invention, it is creatively found that the detection performance of a plasma sample for fluorescent microsphere test strip detection can be improved by adding fibrinolytic enzyme into a conventional sample diluent, the detection time is shortened, the detection accuracy is improved, and the sample applicability is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and particularly relates to a sample diluent for fluorescence microsphere test strip detection of a plasma sample and a preparation method and application thereof. BACKGROUND

[0002] Earthworm fibrinolysin (EFE) is also known as lumbrukinase, which is a protein extracted from earthworms and has fibrinolysin activity and plasminogen activation activity. It is a thrombosis treatment drug with anticoagulation, fibrinolysis and improved hemorheology. In 1983, Professor Miyauchi from Japan first reported earthworm fibrinolysin at the International Society on Thrombosis and Haemostasis. Since then, many researchers have conducted research on earthworm fibrinolysin from the aspects of pharmacology, component separation and purification, enzymatic properties, biochemical characteristics, immunoreactivity, molecular structure, gene cloning and expression, and immobilization. In clinical practice, it can be used for the treatment of thrombosis diseases such as coronary heart disease, angina pectoris, cerebral thrombosis and hemiplegia stroke. It has the advantages of convenient oral administration, fast effect and small side effects.

[0003] The fluorescence microsphere test strip is a rapid detection technology based on lateral chromatography technology and using fluorescence microspheres as markers. After the sample (such as blood, urine, saliva) is added to the sample addition area, it will carry the analyte (such as virus antigen, antibody, hormone, etc.) to flow forward. First, the fluorescence microsphere markers (which have been pre-bound with biomolecules such as antibodies that can recognize the analyte) will be dissolved. If the sample contains the target, it will combine with the fluorescence microspheres to form a "target-fluorescence microsphere" complex. The liquid continues to flow forward and passes through the detection line (T line). Another biomolecule that can specifically capture the target is fixed on this line. When the "target-fluorescence microsphere" complex passes through, it will be firmly captured and gathered here, forming an invisible "fluorescence band". When plasma samples are detected, the supernatant after centrifugation contains fibrin, which can slow down the chromatography rate of the sample during detection and affect the chromatography effect. Therefore, the development of a new type of sample diluent and the addition of earthworm fibrinolysin in the sample diluent can remove the fibrin in the plasma sample before detection, which has certain significance for the fluorescence microsphere test strip detection of plasma samples. SUMMARY

[0004] Therefore, the application aims to provide a sample diluent for fluorescence microsphere test strip detection of a plasma sample and a preparation method and application thereof. The addition of fibrinolytic enzyme in the sample diluent can remove the fibrin component in the plasma sample, improve the detection performance of the fluorescence microsphere test strip detection of the plasma sample, shorten the detection time, improve the detection accuracy, and improve the sample applicability.

[0005] To achieve the above-mentioned purpose, the embodiments of the application provide the following technical solutions: An object of the present application is to provide a sample diluent for plasma sample fluorescent microsphere test strip detection, characterized in that the sample diluent contains a buffer, a stabilizer, a surfactant, a preservative, a fibrinolytic enzyme and a metal ion salt.

[0006] Preferably, the mass percentage of the buffer in the sample diluent is 0.719% to 0.8%.

[0007] Preferably, the mass percentage of the stabilizer in the sample diluent is 0.25% to 0.3%.

[0008] Preferably, the mass percentage of the surfactant in the sample diluent is 0.005 to 0.01%.

[0009] Preferably, the mass percentage of the preservative in the sample diluent is 0.022% to 0.052%.

[0010] Preferably, the mass percentage of the fibrinolytic enzyme in the sample diluent is 0.05% to 0.1%, and the specific activity of the earthworm fibrinolytic enzyme is 120000 U per mg of protein.

[0011] Preferably, the mass percentage of the metal ion salt in the sample diluent is 2% to 2.1%.

[0012] Another object of the present application is to provide a preparation method of the sample diluent for plasma sample fluorescent microsphere test strip detection, which comprises: Mixing the buffer, the stabilizer, the surfactant, the preservative, the fibrinolytic enzyme and the metal ion salt in deionized water to obtain the sample diluent.

[0013] The embodiments of the present application have the following advantages: The present application creatively discovers that adding a fibrinolytic enzyme to a conventional sample diluent can improve the detection performance of plasma samples for fluorescent microsphere test strip detection, shorten the detection time, improve the detection accuracy and enhance the sample applicability. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can be obtained from the provided drawings without creative labor.

[0015] Figure 1 Schematic diagram of the canine distemper virus fluorescent microsphere antibody detection test strip used in the present embodiment 4. DETAILED DESCRIPTION

[0016] The embodiments of the present application are illustrated by specific embodiments, and those skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the present description. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0017] Embodiment 1 Sample diluent for fluorescent microsphere test strip detection of plasma samples Each 1000 mL of sample diluent contains boric acid (H3BO3) 4.33 g, borax decahydrate (Na2B4O7·10H2O) 2.86 g, sodium chloride (NaCl) 20.0 g, disodium ethylenediaminetetraacetate (EDTA-2Na) 0.12 g, casein 2.5 g, Tween-20 (Tween-20) 0.05 g, sodium azide 0.1 g, and earthworm fibrinolytic enzyme with specific activity of 120000 U per mg of protein 0.5 g. After the prepared mixture is dissolved in deionized water and stirred uniformly, it is filtered through a 0.22 µm filter membrane to remove bacteria, and then is divided into portions for use.

[0018] Embodiment 2 Sample diluent for fluorescent microsphere test strip detection of plasma samples Each 1000 mL of sample diluent contains boric acid (H3BO3) 4.33 g, borax decahydrate (Na2B4O7·10H2O) 2.86 g, sodium chloride (NaCl) 20.0 g, disodium ethylenediaminetetraacetate (EDTA-2Na) 0.12 g, casein 2.5 g, Tween-20 (Tween-20) 0.05 g, sodium azide 0.1 g, and earthworm fibrinolytic enzyme with specific activity of 120000 U per mg of protein 0.5 g. After the prepared mixture is dissolved in deionized water and stirred uniformly, it is filtered through a 0.22 µm filter membrane to remove bacteria, and then is divided into portions for use.

[0019] Embodiment 3 Sample diluent for fluorescent microsphere test strip detection of plasma samples Each 1000 mL of sample diluent contains boric acid (H3BO3) 4.43 g, borax decahydrate (Na2B4O7·10H2O) 3.57 g, sodium chloride (NaCl) 21.0 g, disodium ethylenediaminetetraacetate (EDTA-2Na) 0.22 g, casein 3.0 g, Tween-20 0.1 g, sodium azide 0.3 g, and earthworm fibrinolysin with a specific activity of 120000 U per mg of protein 1 g. After the prepared mixture is dissolved in deionized water and stirred uniformly, it is filtered with a 0.22 µm filter membrane to remove bacteria, filtered with a 0.22 µm filter membrane to remove bacteria, and then is ready for use after being divided into portions.

[0020] Sample diluent for fluorescent microsphere test strip detection of Comparative Example 1 Each 1000 mL of sample diluent contains boric acid (H3BO3) 4.33 g, borax decahydrate (Na2B4O7·10H2O) 2.86 g, sodium chloride (NaCl) 20.0 g, disodium ethylenediaminetetraacetate (EDTA-2Na) 0.22 g, casein 2.5 g, Tween-20 0.05 g, sodium azide 0.3 g. After the prepared mixture is dissolved in deionized water and stirred uniformly, it is ready for use after being divided into portions.

[0021] Example 4 Influence of sample diluent components on detection performance (1) Influence of sample diluent components on detection time Prepare sample diluent A according to any one of the above Examples 1-3; prepare sample diluent B according to the above Comparative Example 1. Use the canine distemper virus fluorescent microsphere antibody detection test strip produced by Beijing Nai Bai Biological Company to perform testing, and the structural schematic diagram is as shown in Figure 1 Fresh plasma samples confirmed positive by the “Canine Distemper Diagnosis Technology” national standard (GB / T 27532-2023) were centrifuged at 5000 r / min for 5 min, and the upper liquid was diluted with the sample diluent according to 1:50. Then, the operation was performed according to the method described in the instruction manual, and the sample detection results were all positive. The time required for the T / C value to stabilize is shown in Table 1.

[0022] Table 1 Influence of sample diluent components on detection time

[0023] As can be seen from the results in Table 1, the sample diluent with added fibrinolysin can dissolve the fibrin in the supernatant of the plasma sample, thereby shortening the chromatographic time of the sample on the test strip, saving detection time, and improving detection efficiency.

[0024] (2) Influence of sample diluent components on detection accuracy Sample diluent A was prepared according to any one of the above Examples 1-3; sample diluent B was prepared according to the above Comparative Example 1. The canine distemper virus fluorescent microsphere antibody test strip produced by Beijing Nai Bai Bio-technology Co., Ltd. was used for testing, 15 negative plasma samples and 15 positive plasma samples were respectively detected, the plasma samples were centrifuged at 5000 r / min for 5 min, the upper liquid was taken and diluted with the sample diluent at 1:50, then the operation was carried out according to the instruction method, and the test was repeated for 3 times. The sample detection result was that the T / C value ≥0.40 was judged as positive, and the T / C value <0.40 was judged as negative. The detection results are shown in Tables 2 and 3.

[0025] Table 2 Influence of sample diluent components on detection accuracy of negative samples

[0026] Table 3 Influence of sample diluent components on detection accuracy of positive samples

[0027] From the results of Tables 2 and 3, it can be seen that the detection accuracy of using sample diluent A to dilute the sample for detection is higher than that of using sample diluent B. The sample diluent added with fibrinolysin can dissolve the fibrin in the supernatant of the plasma sample, which can reduce the probability of non-specific binding of fibrin and detection line when detecting negative samples; the reduction of fibrin can reduce the probability of blockage during the sample chromatography process, and at the same time, the detection background of the test strip is cleaner, thereby improving the detection rate of weak positive samples.

[0028] (3) Influence of sample diluent components on detection performance of frozen-thawed plasma samples Sample diluent A was prepared according to any one of the above Examples 1-3; sample diluent B was prepared according to the above Comparative Example 1. The canine distemper virus fluorescent microsphere antibody test strip produced by Beijing Nai Bai Bio-technology Co., Ltd. was used for testing, 15 positive frozen-thawed plasma samples were respectively detected, the plasma samples were placed at-20℃ overnight, then thawed at room temperature, centrifuged at 5000 r / min for 5 min, the upper liquid was taken and diluted with the sample diluent at 1:50, then the operation was carried out according to the instruction method, and the test was repeated for 3 times, and the detection results are shown in Table 4, and the time required for stable T / C value is shown in Table 5.

[0029] Table 4 Influence of sample diluent components on detection accuracy of frozen-thawed positive samples

[0030] Table 5 Influence of sample diluent components on detection time of frozen-thawed positive samples

[0031] From the results of Tables 4 and 5, it can be seen that the detection accuracy of the sample diluted with sample diluent A is higher than that of the sample diluted with sample diluent B. After freezing and thawing, the plasma sample will cause fibrin to precipitate, which will affect the sample detection with a certain probability. The sample diluent added with fibrinolytic enzyme can dissolve the fibrin in the supernatant of the plasma sample, increase the suitability of the sample for fluorescence test strip detection, and reduce the influence of excessive fibrin precipitation caused by freezing and thawing of the sample on the detection results.

[0032] In summary, the addition of earthworm fibrinolytic enzyme as fibrinolytic enzyme in the sample diluent can dissolve the fibrin in the plasma sample before the sample is detected, shorten the chromatography time, and avoid non-specific binding of fibrin, thereby reducing the probability of false positives during the detection of negative samples. The reduction of fibrin can also reduce the probability of blockage during the sample chromatography process, making the test strip detection background cleaner, thereby improving the detection accuracy. The sample diluent is also suitable for frozen and thawed plasma samples, and improves the suitability of the fluorescence microsphere test strip for detecting plasma samples.

[0033] Although the present application has been described in detail by general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application are within the scope of the present application.

Claims

1. A sample diluent for plasma sample fluorescent microsphere test strip detection, characterized in that, The sample diluent contains a buffer, a stabilizer, a surfactant, a preservative, a fibrinolytic enzyme and a metal ion salt.

2. The sample diluent for fluorescent microsphere test strip detection of plasma sample according to claim 1, characterized in that The buffer is a boric acid-borax buffer; the mass percentage of the buffer in the sample diluent is 0.719%-0.8%.

3. The sample diluent for plasma sample fluorescent microsphere test strip detection according to claim 1, characterized in that The stabilizer is any one of casein and bovine serum albumin; the mass percentage of the stabilizer in the sample diluent is 0.25%-0.3%.

4. The sample diluent for fluorescent microsphere test strip detection of plasma sample according to claim 1, characterized in that The surfactant is Tween 20; the mass percentage of the surfactant in the sample diluent is 0.005-0.01%.

5. The sample diluent for fluorescent microsphere test strip detection of plasma sample according to claim 1, characterized in that The preservative is any one of sodium azide and disodium ethylenediaminetetraacetate or a combination of the two; the mass percentage of the preservative in the sample diluent is 0.022%-0.052%.

6. The sample diluent for fluorescent microsphere test strip assay of plasma sample according to claim 1, wherein The fibrinolytic enzyme is earthworm fibrinolysin; the mass percentage of the fibrinolytic enzyme in the sample diluent is 0.05%-0.1%.

7. The sample diluent for fluorescent microsphere test strip assay of plasma sample according to claim 1, wherein The metal ion salt is sodium chloride; the mass percentage of the metal ion salt in the sample diluent is 2%-2.1%.

8. The method for preparing a sample diluent for fluorescent microsphere test strip detection of plasma samples according to any one of claims 1-7, characterized in that, The preparation method comprises: Mixing the buffer, the stabilizer, the surfactant, the preservative, the fibrinolytic enzyme and the metal ion salt in deionized water to obtain the sample diluent.

9. Use of the sample diluent for plasma sample detection according to any one of claims 1-7 in performing fluorescence microsphere test strip detection of a plasma sample.

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