A sample diluent for eliminating differences between different sample tubes, and its preparation method and application

Abstract

The present invention relates to the field of medical testing technology, and more particularly to a sample diluent for eliminating differences between different sample tubes, and a preparation method and application thereof. The sample diluent is a buffer solution containing 0.5 wt% to 2 wt% dextran sulfate sodium salt; the buffer solution is composed of Bis-Tris buffer and MES buffer. The sample diluent of the present invention is applied to immunoassays to effectively eliminate differences in sample results between different blood collection tubes, thereby enriching the sample types applicable to the kit and facilitating clinical use.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a sample diluent for eliminating differences between different sample tubes, and a preparation method and application thereof. Background Art

[0002] The main types of blood samples used in immunoassays are serum and plasma. Six types of vacuum blood collection tubes are commonly used clinically. Three types of serum tubes are standard tubes, coagulant tubes, and separation gel coagulant tubes; and three types of plasma tubes are EDTA tubes (EDTA.Na2 or EDTA.K2), sodium citrate tubes (or sodium citrate tubes), and sodium heparin tubes (or lithium heparin tubes).

[0003] Standard tubes do not contain any additives, while other tube types contain coagulants or anticoagulants. The sodium citrate tubes contain an anticoagulant to blood ratio of 1:9 to promote rapid coagulation and separation of the blood, yielding the corresponding serum or plasma. However, these coagulants or anticoagulants may interfere with the test results of some analytes, resulting in discrepancies in test results when different tubes are used for the same individual's samples, affecting the accuracy of the test results. For example, the test results of the three serum tubes were relatively consistent, as were the test results of the three plasma tubes. However, there was a certain deviation between the serum tubes and the plasma tubes.

[0004] In order to solve the problem of differences in sample test results between different blood collection tubes mentioned above, the sample types on the instructions of many test kits are often written conservatively, such as only serum, or only plasma, or even limited to a certain type of tube, such as only EDTA tubes can be used. However, the conservative sample types written in the test kits have caused great trouble for clinical use. The same patient often needs to be tested for multiple items, but if the sample types specified for different test items are different, the patient needs to draw more than one tube of blood at the same time. For example, the patient collected a sample from a coagulant tube for the first time, but when another item needs to be re-tested, it is found that another sample from an EDTA tube needs to be drawn. This brings additional pain to the patient and is very inconvenient to operate. Summary of the Invention

[0005] In view of this, the present invention provides a sample diluent and its preparation method and application. The sample diluent can effectively eliminate the differences in sample results between different blood collection tubes, enrich the sample types applicable to the kit, and facilitate clinical use.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A sample diluent, which is a buffer solution containing 0.5 wt% to 2 wt% of dextran sulfate sodium salt;

[0008] The buffer solution consists of Bis-Tris buffer and MES buffer.

[0009] The present invention adopts a unique double buffer system formed by 0.1MBis-Tris buffer and 0.1MMES buffer, which not only broadens the buffer pH range but also improves the buffering capacity.

[0010] Compared with ordinary sample diluents, the sample diluent provided by the present invention introduces dextran sulfate sodium salt (DSS) and combines DSS with a Bis-Tris-MES dual buffer system, which effectively suppresses the interference of the coagulant or anticoagulant components in different blood collection tubes on the analyte, thereby eliminating the differences in the test results of samples in different blood collection tubes.

[0011] The sample diluent of the present invention further comprises one or more of NaCl, a preservative, bovine serum albumin, a surfactant and a blocking agent.

[0012] In some embodiments, the sample diluent provided by the present invention has the following concentrations of components:

[0013]

[0014] In some specific embodiments, the concentration of each component in the sample diluent is as follows:

[0015]

[0016] In some embodiments, the preservative is at least one of P300, Bronidox preservative, sodium azide, and MIT, the surfactant is Triton X-100, and the blocking agent is HBR.

[0017] In the present invention, the ratio of Bis-Tris to MES is adjusted so that the pH of the sample diluent is controlled between 6 and 8. In some specific embodiments, the pH is 6, 6.5, 7, 7.5 or 8.

[0018] In the present invention, the molecular weight of dextran sulfate sodium salt (DSS) is preferably M.W5000-40000, specifically M.W5000, M.W6500-10000, M.W20000 or M.W40000;

[0019] The present invention also provides a method for preparing a sample diluent, comprising:

[0020] Add NaCl, preservative, bovine serum albumin, dextran sulfate sodium salt, surfactant and blocking agent to the buffer and mix well.

[0021] The present invention also provides the use of the sample diluent in eliminating the differences in immunoassay test results of different blood collection tubes.

[0022] The blood collection tubes include at least one of a coagulant tube, a separating gel tube, an EDTA tube, a sodium citrate tube, a sodium heparin tube, and a common tube. The common tubes are blood collection tubes without any additives.

[0023] The sample diluent provided by the present invention is a buffer solution containing 0.5wt%-2wt% dextran sulfate sodium salt; the buffer solution is composed of Bis-Tris buffer and MES buffer. When used in immunoassays, the sample diluent can effectively eliminate differences in sample results between different blood collection tubes, thereby enriching the sample types applicable to the kit and facilitating clinical use. DETAILED DESCRIPTION

[0024] The present invention provides a sample diluent, a preparation method thereof, and an application thereof. Those skilled in the art may refer to the contents herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0025] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0026] The present invention will be further described below in conjunction with the embodiments:

[0027] Example 1 Preparation method of sample diluent provided by the present invention

[0028] Sample diluent composition:

[0029]

[0030] Wherein, the preservative is, the surfactant is TritonX-100, and the blocking agent is HBR.

[0031] Preparation method:

[0032] (1) Use a graduated cylinder to measure 1000 mL of purified water, add 28.233 g of 1,3-bis[(trishydroxymethyl)methylamino]propane (Bis-Tris Propane) to prepare a 0.1 M Bis-Tris solution; use a graduated cylinder to measure 1000 mL of purified water, add 21.325 g of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) to prepare a 0.1 M MES solution;

[0033] (2) Mix 400 mL of the Bis-Tris solution and 600 mL of the MES solution in (1) to obtain 1000 mL of a buffer solution with a pH of 7.0 ± 0.1;

[0034] (3) Add 8.76 g NaCl, 1 mL P300, 20 g bovine serum albumin, 10 g dextran sulfate sodium salt (DSS, M.W20000), 2 mL Triton X-100, and 100 mg blocking agent to the buffer solution obtained in (2), stir thoroughly, and store at 2-8°C for later use.

[0035] Comparative Example 1 Preparation of Sample Dilution

[0036] Use a graduated cylinder to measure 1000 mL of purified water, add 28.233 g of 1,3-bis[(trishydroxymethyl)methylamino]propane, adjust the pH to 7.0±0.1 with hydrochloric acid, add 8.76 g of NaCl, 1 mL of P300, 20 g of bovine serum albumin, 2 mL of Triton X-100, and 100 mg of blocking agent, stir thoroughly, and store at 2-8°C until ready to use.

[0037] Comparative Example 2 Preparation of Sample Dilution

[0038] Use a graduated cylinder to measure 1000 mL of purified water, add 21.325 g of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES), adjust the pH to 6.5 ± 0.1 with NaOH, add 8.76 g of NaCl, 1 mL of P300, 20 g of bovine serum albumin, 2 mL of Triton X-100, and 100 mg of blocking agent, stir thoroughly, and store at 2-8°C until ready to use.

[0039] Comparative Example 3 Preparation of sample dilution

[0040] (1) Use a graduated cylinder to measure 1000 mL of purified water, add 28.233 g of 1,3-bis[(trishydroxymethyl)methylamino]propane (Bis-Tris Propane) to prepare a 0.1 M Bis-Tris solution; use a graduated cylinder to measure 1000 mL of purified water, add 21.325 g of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES) to prepare a 0.1 M MES solution;

[0041] (2) Mix 400 mL of the Bis-Tris solution and 600 mL of the MES solution in (1) to obtain 1000 mL of a buffer solution with a pH of 7.0 ± 0.1;

[0042] (3) Add 8.76 g NaCl, 1 mL P300, 20 g bovine serum albumin, 2 mL Triton X-100, and 100 mg blocking agent to the buffer obtained in (2), stir thoroughly, and store at 2-8°C for later use.

[0043] Comparative Example 4 Preparation of Sample Dilution

[0044] Use a graduated cylinder to measure 1000 mL of purified water, add 28.233 g of 1,3-bis[(trishydroxymethyl)methylamino]propane, adjust the pH to 7.0±0.1 with hydrochloric acid, add 8.76 g of NaCl, 1 mL of P300, 20 g of bovine serum albumin, 10 g of dextran sulfate sodium salt (DSS, M.W20000), 2 mL of Triton X-100, and 100 mg of blocking agent, stir thoroughly, and store at 2-8°C until ready for use.

[0045] Comparative Example 5 Preparation of Sample Dilution

[0046] Use a graduated cylinder to measure 1000 mL of purified water, add 21.325 g of 2-(N-morpholino)ethanesulfonic acid monohydrate (MES), adjust the pH to 6.5±0.1 with NaOH, add 8.76 g of NaCl, 1 mL of P300, 20 g of bovine serum albumin, 10 g of dextran sulfate sodium salt (DSS, M.W20000), 2 mL of Triton X-100, and 100 mg of blocking agent, stir thoroughly, and store at 2-8°C until ready to use.

[0047] Example 2 Study on the Effect of Different Sample Dilutions on Eliminating Differences between Different Sample Tubes

[0048] Six blood collection tube samples from the same individual were simultaneously tested on an AutoLumoA2000 using the Human Epidermal Growth Factor Receptor 2 Detection Kit (Magnetic Particle Chemiluminescence Method) from Antu Biotechnology in combination with the sample diluents of Comparative Examples 1, 2, 3, 4, and 5 and the sample diluent of the present invention (Example 1). The effectiveness of the sample diluent provided by the present invention in eliminating differences between samples in different tubes was evaluated. The results are shown in Table 1.

[0049] Table 1 Evaluation of samples in different tubes of the human epidermal growth receptor 2 detection kit

[0050]

[0051]

[0052]

[0053] It can be seen from the experimental data in Table 1 that after using the diluent of the present invention, the deviations of the detection values of human epidermal growth factor receptor 2 in six blood collection tube samples from the same person are all within 10%.

[0054] Example 3 Study on the Effect of the Sample Dilution Provided by the Present Invention on Eliminating Differences Between Different Sample Tubes

[0055] Six blood collection tube samples from the same individual were simultaneously tested on an AutoLumoA2000 using Antu Bio's Pepsinogen II Detection Kit (Magnetic Particle Chemiluminescence Method) in combination with the sample diluents of Comparative Examples 1, 2, 3, 4, and 5 and the sample diluent of the present invention (Example 1). The effectiveness of the sample diluent provided by the present invention in eliminating differences between samples in different tubes was evaluated. The results are shown in Table 2.

[0056] Table 2 Assessment of samples in different tubes of the Pepsinogen II assay kit

[0057]

[0058]

[0059]

[0060] It can be seen from the experimental data in Table 2 that after using the diluent of the present invention, the deviations of the detection values of pepsinogen II in six blood collection tube samples from the same person are all within 10%.

[0061] Example 4 Study on the ratio of sample diluent and sample

[0062] Antu Biotech's human epidermal growth factor receptor 2 detection kit (magnetic microparticle chemiluminescence method) was used with the sample diluent of the present invention on the AutoLumoA2000. Six blood collection tube samples from the same individual were analyzed at sample to sample diluent ratios of 1:5, 1:2, 1:1, and 2:1, respectively. The effects of different sample to sample diluent ratios on the sample results were evaluated. The results are shown in Table 3.

[0063] Table 3 Assessment of different ratios of project samples and sample dilutions

[0064]

[0065]

[0066] It can be seen from the experimental data in Table 3 that when the sample diluent provided by the present invention is used as the diluent, the deviations of the test values of the six blood collection tube samples of the same person are all within 10%.

[0067] Example 5 Study on the Effect of Dextran Sulfate Sodium Salt (DSS) Dosage on Eliminating Differences in Samples from Different Blood Collection Tubes

[0068] Six blood collection tube samples from the same individual were simultaneously tested on the AutoLumoA2000 using the Antu Biotechnology human epidermal growth factor receptor 2 detection kit (magnetic microparticle chemiluminescence method) with a standard sample diluent and a sample diluent containing different concentrations of dextran sulfate sodium salt (DSS) of the present invention. The effect of the amount of dextran sulfate sodium salt (DSS) used in eliminating differences in samples from different blood collection tubes was evaluated. The results are shown in Table 4.

[0069] Table 4 Study on the effect of dextran sulfate sodium salt (DSS) dosage on eliminating differences between different sample tubes

[0070]

[0071]

[0072] From the data in Table 4, it can be seen that adding 0.5%-2% dextran sulfate sodium salt (DSS) to the sample diluent is effective in eliminating the concentration differences between different tubes.

[0073] Example 6 Study on the Effect of Molecular Weight of Dextran Sulfate Sodium Salt (DSS) on Eliminating Differences in Samples from Different Blood Collection Tubes

[0074] Six blood collection tube samples from the same individual were simultaneously tested on the AutoLumoA2000 using the Antu Biotechnology human epidermal growth factor receptor 2 detection kit (magnetic microparticle chemiluminescence method) with a standard sample diluent and a sample diluent containing dextran sulfate sodium salt (DSS) of different molecular weights. The effect of DSS dosage on eliminating differences in blood collection tube samples was evaluated. The results are shown in Table 5.

[0075] Table 5 Study on the effect of different molecular weight DSS on eliminating the differences between different sample tubes

[0076]

[0077]

[0078] From the data in Table 5, it can be seen that adding dextran sulfate sodium salt (DSS) with a molecular weight of M.W 5000-40000 to the sample diluent is effective in eliminating the concentration differences between different tubes.

[0079] Example 7 Stability of the Sample Dilution Provided by the Present Invention

[0080] The sample diluent of the present invention was placed at 2-8°C, 37°C for 10 days, repeatedly frozen and thawed 7 times, and stored in a vehicle at 37°C for 7 days; it was placed on the carrier plate of the Antu AutoLumoA2000 for 28 days. After the placement, the calibrators S0-S5 and 6 clinical samples were tested simultaneously on the AutoLumoA2000 using the Antu Biological Human Epidermal Growth Factor Receptor 2 Detection Kit (Magnetic Particle Chemiluminescence Method) to evaluate the stability of the sample diluent provided by the present invention. The results are shown in Table 6.

[0081] Table 6 Stability assessment of sample dilutions

[0082]

[0083]

[0084] From the data in Table 6, it can be seen that the deviations of the thermal acceleration, freeze-thaw, vehicle-mounted and aircraft-mounted stability signal values of the sample dilution solution are all within 10%, which meets the requirements.

[0085] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A blood sample diluent, characterized in that: It is a buffer solution containing 0.5 wt%-2 wt% dextran sulfate sodium salt; The buffer consists of Bis-Tris buffer and MES buffer; The molecular weight of the dextran sulfate sodium salt is 5000-40000.

2. The blood sample diluent according to claim 1, characterized in that It also includes one or more of NaCl, a preservative, bovine serum albumin, a surfactant, and a blocking agent.

3. The blood sample diluent according to claim 2, characterized in that Includes the following components: NaCl 0.1 wt%-0.9 wt%; Preservatives 0.05 wt%-0.2 wt%; Bovine serum albumin 1 wt%-3 wt%; Dextran sulfate sodium salt 0.5 wt%-2 wt%; Surfactant 0.1 vol%-0.5 vol%; Blocking agent 50-1000 ug / mL; Bis-Tris buffer 0.1 M; MES buffer 0.1 M.

4. The blood sample diluent according to claim 3, characterized in that Includes the following components: NaCl 0.876 wt%; Preservatives 0.05 wt%-0.2 wt%; Bovine serum albumin 2wt%; Dextran sulfate sodium salt 1 wt%; Surfactant 0.2 vol%; Blocking agent 100 μg / mL; Bis-Tris buffer 0.1 M; MES buffer 0.1 M.

5. The blood sample diluent according to any one of claims 2 to 4, characterized in that: The preservative is at least one of P300, Bronidox preservative, sodium azide, and MIT; the surfactant is Triton X-100; and the blocking agent is HBR.

6. The blood sample diluent according to claim 1, characterized in that Its pH is 6~8.

7. The method for preparing a blood sample diluent according to any one of claims 2 to 6, characterized in that: include: Add NaCl, preservative, bovine serum albumin, dextran sulfate sodium salt, surfactant and blocking agent to the buffer and mix well.

8. Use of the blood sample diluent according to any one of claims 1 to 6 in eliminating differences in immunoassay test results from different blood collection tubes.

9. The use according to claim 8, characterized in that The blood collection tube includes at least one of a coagulant tube, a separating rubber tube, an EDTA tube, a sodium citrate tube, a sodium heparin tube, and an ordinary tube.

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