A cleaning solution for flow fluorescence detection

Abstract

The present invention relates to the field of medicine, and in particular to a cleaning solution for flow fluorescence detection, wherein the cleaning solution comprises a basic cleaning solution, proteins and alcohols, the basic cleaning solution comprises a buffer salt and a surfactant, the solvent of the basic cleaning solution is water, and the buffer salt in the basic cleaning solution is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride; the surfactant is a nonionic surfactant. The cleaning solution of the present invention can satisfy the cleaning effect of the flow fluorescence luminescence platform, and can not only stabilize the detection signal and reduce the sedimentation of microspheres, but also take into account the background signal and avoid nonspecific adsorption.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a cleaning liquid for flow fluorescence detection. Background Art

[0002] The flow fluorescence immunoassay uses the coded microspheres as solid phase carriers, connects the antibodies (or antigens) to the coded microspheres through chemical bonds, captures the target antigens (or antibodies) in the serum or plasma, and reacts with the antibodies (or antigens) labeled with luminescent substances. When the immune reaction is over, the reactants are usually washed with a cleaning solution to remove substances that did not participate in the reaction. After washing, the microspheres enter the instrument through the sample suction tube, the laser excites the PE fluorescence on the microspheres, and after photoelectric conversion, it is input into the computer and analyzed and processed by the software, thereby realizing the detection of the substance to be tested.

[0003] At present, many technicians have studied cleaning solutions. For example, the prior art discloses an electrochemiluminescent cleaning solution, whose main components include Tris buffer, etc., which is mainly used in Beckman automatic immunoassay analyzers. There is also a prior art that discloses a chemiluminescent cleaning solution, whose main components include phosphate buffer, etc., which is a cleaning solution developed for Abbott chemiluminescent immunoassay analyzers. There is also a prior art that discloses a cleaning solution, whose main components include phosphate buffer, inorganic salts, etc., which can adapt to enzymatic chemiluminescence and direct chemiluminescence systems.

[0004] However, the above cleaning solutions all have shortcomings in terms of unstable detection signals, abnormal background signals in some experiments, and microsphere sedimentation for flow cytometry. Therefore, improvements in these aspects are urgently needed. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a cleaning solution for flow fluorescence detection, so as to solve the problems in the prior art.

[0006] To achieve the above-mentioned object and other related objects, the present invention provides a cleaning solution for flow fluorescence detection, wherein the cleaning solution comprises a basic cleaning solution, proteins and alcohols, wherein the basic cleaning solution comprises a buffer salt and a surfactant, and the solvent of the basic cleaning solution is water.

[0007] The buffer salt in the basic washing solution is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride.

[0008] Based on the total volume of the basic washing solution, the final concentration of the buffer salt is 5-10 g / L.

[0009] The surfactant is a nonionic surfactant, which is one or more of Tween-20, Tween-80, Span-60, Triton X-45, Triton X-100 or Triton X-305.

[0010] The basic lotion also includes a preservative, which is a KroVin series preservative or a ProClin series preservative.

[0011] The pH of the cleaning solution is 5.5-8.5.

[0012] In some embodiments, the protein is selected from one or more of BSA, casein, ovalbumin, or serum. The serum is selected from any one or more of bovine serum, goat serum, and chicken serum.

[0013] When the protein is BSA, casein or ovalbumin, the final concentration of the protein in the cleaning solution is 1-50 g / L based on the total volume of the cleaning solution.

[0014] The alcohol is selected from one or more of glycerol, mannitol, PVP, PVA, and PEG.

[0015] When the alcohol is mannitol, PVP, PVA, or PEG, the final concentration of the alcohol is 0.1-50 g / L.

[0016] The present invention also provides use of the cleaning solution in flow fluorescence detection.

[0017] As described above, a cleaning solution for flow fluorescence detection of the present invention has the following beneficial effects: using the cleaning solution of the present invention, while meeting the cleaning effect of the flow fluorescence luminescence platform, it can not only stabilize the detection signal and reduce the sedimentation of the microspheres, but also take into account the background signal and avoid nonspecific adsorption. DETAILED DESCRIPTION

[0018] The invention provides a cleaning solution for flow fluorescence detection. The cleaning solution comprises a basic cleaning solution, proteins and alcohols. The basic cleaning solution comprises a buffer salt and a surfactant.

[0019] In certain embodiments of the present invention, the buffer salt in the basic washing solution is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride.

[0020] In certain embodiments of the present invention, based on the total volume of the basic washing solution, the final concentration of the buffer salt is 5-10 g / L, for example, 5-6 g / L, 6-7 g / L, 7-8 g / L, 8-9 g / L, 9-10 g / L.

[0021] In certain embodiments of the present invention, the surfactant is a nonionic surfactant. The nonionic surfactant is one or more of Tween-20, Tween-80, Span-60, Triton X-45, TritonX-100 or Triton X-305. Based on the total volume of the base wash solution, the final concentration of the surfactant is 0.05%-0.5% v / v, for example, 0.05-0.1% v / v, 0.1-0.2% v / v, 0.2-0.3% v / v, 0.3-0.4% v / v, 0.4-0.5% v / v.

[0022] In certain embodiments of the present invention, the basic lotion further comprises a preservative, which is a KroVin series preservative or a ProClin series preservative.

[0023] The KroVin series preservatives include, for example, KroVin100, KroVin 400, KroVin 500 or KroVin750; the ProClin series preservatives include, for example, ProClin50, ProClin150, ProClin200, ProClin300, ProClin950 or ProClin5000.

[0024] Based on the total volume of the basic washing solution, the final concentration of the preservative is 0.05%-0.2% v / v, for example, 0.05%-0.1% v / v, 0.1%-0.15% v / v, 0.15%-0.2% v / v.

[0025] The solvent of the basic washing solution is water.

[0026] The buffer salt, surfactant and preservative in the basic washing solution are to ensure the basic cleaning effect and the stability of the system. Using only the basic washing solution as the washing solution for flow fluorescence luminescence method has deficiencies in the stability, accuracy and microsphere sedimentation of the test results for different projects.

[0027] The pH of the cleaning solution is 5.5-8.5. Preferably, the pH of the cleaning solution is 6.0-8.0.

[0028] In some embodiments, the protein is selected from one or more of BSA, casein, ovalbumin, or serum. The serum is selected from any one or more of bovine serum, goat serum, and chicken serum.

[0029] When the protein is BSA, casein or ovalbumin, the final concentration of the protein in the cleaning solution is 1-50 g / L based on the total volume of the cleaning solution. In a preferred embodiment, the final concentration of the protein in the cleaning solution is 5-40 g / L, for example, 5-10 g / L, 10-20 g / L, 20-30 g / L or 30-40 g / L.

[0030] When the protein is serum, the final concentration of the protein in the cleaning solution is 0.1%-5% v / v, for example, 0.1-1% v / v, 1-1.5% v / v, 1.5-2% v / v, 2-2.5% v / v, 2.5-3% v / v, 3-3.5% v / v, 3.5-4% v / v, 4-4.5% v / v, 4.5-5% v / v.

[0031] In one embodiment, the alcohol is selected from one or more of glycerol, mannitol, PVP, PVA, and PEG. Specifically, the PEG is selected from PEG200, PEG1000, PEG1500, PEG2000, PEG6000, and PEG8000.

[0032] When the alcohol is glycerol, the final concentration of glycerol is 0.05%-0.5% v / v. The final concentration of glycerol is, for example, 0.05-0.1% v / v, 0.1-0.2% v / v, 0.2-0.3% v / v, 0.3-0.4% v / v or 0.4-0.5% v / v.

[0033] When the alcohol is mannitol, PVP, PVA, or PEG, the final concentration of the alcohol is 0.1-50 g / L. The final concentration of the alcohol is, for example, 0.1-1 g / L, 1-10 g / L, 10-20 g / L, 20-30 g / L, 30-40 g / L, or 40-50 g / L.

[0034] The present invention also provides use of the cleaning solution in flow fluorescence detection.

[0035] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0037] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0038] The washing liquid of the present invention is composed of a basic washing liquid, proteins and alcohols; wherein, in 1L of the basic washing liquid, the amount of protein substances added is 1-50g / L at a final concentration, and the amount of alcohols added is 0.1-50g / L at a final concentration; the pH of the washing liquid is 5.5-8.5.

[0039] The basic lotion preparation scheme used in the following examples is as follows:

[0040] Use purified water as solvent to prepare the basic washing solution according to the following weight and volume. The total volume after preparation is 1L:

[0041] <![CDATA[Na2HPO4]]> 1.1g NaCl 5.0g <![CDATA[KH2PO4]]> 0.5g KCl 1.4g TritonX-100 1mL ProClin300 1mL

[0042] The pH value of the basic washing solution prepared as above is 5.5-8.5. The prepared basic washing solution is filtered through a 0.22 μm filter membrane and stored at room temperature.

[0043] Example 1 A washing solution for flow fluorescence luminescence method

[0044] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0045] Bovine serum 5mL BSA 20g Basic lotion Make up to 1L

[0046] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0047] Example 2 A washing solution for flow fluorescence luminescence method

[0048] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0049] Sheep serum 1mL Casein 10g Basic lotion Make up to 1L

[0050] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0051] Example 3 A washing solution for flow fluorescence luminescence method

[0052] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0053] BSA 25g Ovalbumin 5g Basic lotion Make up to 1L

[0054] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0055] Example 4 A washing solution for flow fluorescence luminescence method

[0056] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0057] glycerin 2mL PEG6000 10g Basic lotion Make up to 1L

[0058] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0059] Example 5 A washing solution for flow fluorescence luminescence method

[0060] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0061] PVA 5g PEG200 30g Basic lotion Make up to 1L

[0062] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0063] Example 6 A washing solution for flow cytometry

[0064] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 5.5-8.5 after the preparation, and the total volume of the washing solution is 1L:

[0065] PVP 2g PEG2000 20g Basic lotion Make up to 1L

[0066] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0067] Example 7 A washing solution for flow cytometry

[0068] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 6.0-6.6 after the preparation, and the total volume of the washing solution is 1L:

[0069]

[0070]

[0071] The washing solution prepared as above was filtered through a 0.22 μm filter membrane and stored at 2-8°C.

[0072] Example 8 A washing solution for flow fluorescence luminescence method

[0073] The preparation method of the washing solution is as follows: Use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 6.6-7.3 after the preparation, and the total volume of the washing solution is 1L:

[0074] Ovalbumin 10g Bovine serum 2mL PEG1000 25g Mannitol 5g Basic lotion Make up to 1L

[0075] Example 9 A washing solution for flow cytometry

[0076] The preparation method of the washing solution is as follows: use the basic washing solution as the solvent, prepare the washing solution according to the following weight and volume, adjust the pH to 7.3-8.0 after the preparation, and the total volume of the washing solution is 1L:

[0077] Sheep serum 5mL Ovalbumin 5g PVA 1g PVP 2g glycerin 3mL Basic lotion Make up to 1L

[0078] Example 10

[0079] Experimental results: Flow fluorescence immunoassay was performed using Examples 1-9 and the basic washing solution, and the following experiments were performed respectively:

[0080] 10.1 Verification of cleaning effect

[0081] Beckman Coulter flow cytometer Dxflex was used to measure the basic washing solution and the washing solution of Examples 1-9 respectively. Reagent A, reagent B, and reagent C in the table are reagents in the carbohydrate antigen 125 quantitative detection kit, myoglobin (MYO) detection kit, and anti-double-stranded DNA antibody (dsDNA) detection kit. The measurement steps were carried out according to the instructions, which are roughly as follows:

[0082]

[0083] The measurement items selected were carbohydrate antigen 125 (CA125), myoglobin (MYO), and anti-double-stranded DNA antibody (dsDNA). The test samples were 20 CA125, MYO, and dsDNA calibrators. The average value of the detection signal of each sample is shown in Table 1:

[0084] Table 1: Background signal detection results of basic washing solution and washing solution of Examples 1-9

[0085] Background signal (RLU) CA125 MYO dsDNA Basic lotion 2293 2192 1916 Example 1 1213 1052 664 Example 2 1256 1024 865 Example 3 1125 995 542 Example 4 1610 1332 1033 Example 5 1584 1361 1201 Example 6 1609 1488 994 Example 7 1027 894 655 Example 8 939 912 571 Example 9 1028 985 601

[0086] The results in Table 1 show that the cleaning effect of the cleaning solution of the present invention is good, and the background signal of each item is low.

[0087] 10.2 System Signal Stability

[0088] Beckman Coulter flow cytometer Dxflex was used to measure the same group of samples at different times for the basic washing solution and the washing solution of Examples 1-9. The samples were calibrators of CY211, dsDNA, and CK-MB. After the first measurement, the samples were placed at room temperature for 60 minutes and the signal values ​​were read again using the Beckman Coulter flow cytometer Dxflex. The detection signal results are shown in Table 2:

[0089] Table 2: Signal stability test results of the basic lotion and the lotion systems of Examples 1-9

[0090]

[0091]

[0092] The results in Table 2 show that the washing solution of the present invention performs better than the basic washing solution in terms of system signal stability.

[0093] 10.3 Sedimentation experiment

[0094] The basic washing solution and the working solution of the magnetic balls coated with antigen / antibody in Examples 7-9 were respectively used, mixed, and divided into 4 portions, each of 1 mL. 200 μL of the supernatant was taken at intervals and the number of balls was read using the Beckman Coulter flow cytometer Dxflex. The test was repeated 3 times, and the average value was taken for statistical results. The test results are shown in Table 3:

[0095] Table 3: Determination of natural sedimentation rate of basic lotion and lotion of Examples 7-9

[0096]

[0097] The results in Table 3 show that the sedimentation rate of the magnetic spheres coated with antigen / antibody in the basic washing solution is faster than that in the washing solution of Examples 7-9, and there is a significant difference at 20 minutes. In summary, it can be considered that the washing solution of the present invention is less likely to cause the microspheres to settle under natural gravity than the basic washing solution.

[0098] 10.4 Accuracy Experiment

[0099] Beckman Coulter flow cytometer Dxflex was used to measure the basic washing solution and the washing solution of Examples 7-9, respectively, and 20 samples of the same group were measured. The samples were calibrators of CA125, CEA, NSE, CY211, and ProGRP. The average results of the detection signals are shown in Table 4:

[0100] Table 4: Accuracy test results of basic lotion and lotion of Examples 7-9

[0101]

[0102]

[0103] As shown in Table 4, the measured values ​​of the five indicators are close, and the t-test of the two groups of data for each item shows no statistically significant difference (p>0.05), and R 2 The values ​​are all greater than 0.990, showing a high positive correlation. The above results show that the accuracy of the cleaning solution test of the present invention is good, and the addition of components does not affect the detection accuracy.

[0104] 10.5 Precision Experiment

[0105] According to Examples 7-9, three batches of cleaning solutions were prepared, namely, the first batch, the second batch and the third batch. A 40 ng / ml AFP sample was taken, and the Beckman Coulter flow cytometer Dxflex was used to measure the test results of the three batches of Examples 7-9, and each batch of cleaning solution was tested 20 times, and the intra-batch and inter-batch coefficients of variation were calculated. The test results are shown in Table 5:

[0106] Table 5: Example 7-9 washing liquid precision test results

[0107]

[0108] The results in Table 5 show that the intra-batch coefficient of variation (CV) of the washing solution of the present invention is less than 10%, and the inter-batch coefficient of variation (CV) is less than 15%, which meets the requirements of the intra-batch coefficient of variation of general diagnostic reagents being less than 10% and the inter-batch coefficient of variation being less than 15%. The above results show that the washing solution of the present invention has good precision and repeatability in detection.

[0109] 10.6 Storage stability of washing solution

[0110] According to Examples 7-9, 1.4L of cleaning solution was prepared and divided into 7 bottles, each bottle of about 200mL, and placed at 2-8°C for 0 days, 2 months, 4 months, 6 months, 9 months, 12 months, and 15 months before testing. The CEA sample with a concentration of 5±0.5ng / mL was used for repeated testing 20 times, and the coefficient of variation was calculated. The test results are shown in Table 5:

[0111] Table 5: Storage stability test results of basic lotion and lotions of Examples 7-9

[0112]

[0113]

[0114] The results in Table 5 show that the CV values ​​of the samples of the washing solutions prepared in Examples 7-9 are all within 5% when stored at 2-8°C for less than 15 months. From the reagent state, the washing solutions of the three examples are clear and transparent without precipitation, indicating that the washing solutions of the invention have good storage stability.

[0115] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. The use of the cleaning solution for flow fluorescence detection as described below in flow fluorescence immunoassay detection, characterized in that: The cleaning solution for flow fluorescence detection includes a basic cleaning solution, the basic cleaning solution includes a buffer salt and TritonX-100, the solvent of the basic cleaning solution is water, and the cleaning solution for flow fluorescence detection is selected from any of the following formulas: 1) Basic washing solution, 10 g / L casein, 25 g / L BSA, 20 g / L PEG1500, and 0.1% glycerol by volume; 2) basic washing solution, 10 g / L ovalbumin, 0.2% bovine serum, 25 g / L PEG1000, and 5 g / L mannitol; 3) Basic washing solution, 0.5% volume concentration of goat serum, 5g / L of ovalbumin, 1g / L of PVA, 2g / L of PVP, and 0.3% volume concentration of glycerol.

2. The use according to claim 1, characterized in that The buffer salt in the basic washing solution is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride or potassium chloride.

3. The use according to claim 1, characterized in that Based on the total volume of the basic washing solution, the final concentration of the buffer salt is 5-10 g / L.

4. The use according to claim 1, characterized in that Based on the total volume of the basic washing solution, the final concentration of TritonX-100 is 0.05%-0.5% v / v.

5. The use according to claim 1, characterized in that: A preservative is also included in the base lotion.

6. The use according to claim 5, characterized in that The preservative is KroVin preservative or ProClin preservative.

7. The use according to claim 5, characterized in that Based on the total volume of the basic washing solution, the final concentration of the preservative is 0.05%-0.2% v / v.

8. The use according to claim 1, characterized in that The pH of the cleaning solution is 5.5-8.5.

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