CN115078711B

CN115078711B - A cleaning solution for flow fluorescence detection

Info

Publication number: CN115078711B
Application number: CN202210682331.2A
Authority: CN
Inventors: 方剑秋; 李猛; 方云琪; 白艳军
Original assignee: Shanghai Wanzijian Biotechnology Co ltd
Current assignee: Shanghai Wanzijian Biotechnology Co ltd
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2025-05-06
Anticipated expiration: 2042-06-15
Also published as: CN115078711A

Abstract

本发明涉及医药领域，特别是涉及一种流式荧光检测用清洗液，所述清洗液包括基础洗液、蛋白质类和醇类，所述基础洗液包括缓冲盐和表面活性剂，所述基础洗液的溶剂为水，所述基础洗液中缓冲盐选自磷酸氢二钠、磷酸二氢钠、磷酸氢二钾、磷酸二氢钾、氯化钠或氯化钾中的一种或多种；所述表面活性剂为非离子表面活性剂。使用本发明的清洗液，满足流式荧光发光平台清洗效果的同时，不仅能稳定检测信号，减小微球沉降，而且兼顾了本底信号，避免非特异性吸附。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

Flow type cleaning fluid for fluorescence detection

Technical Field

The invention relates to the field of medicines, in particular to a cleaning solution for flow type fluorescence detection.

Background

The flow type fluorescence luminescence immunoassay method uses the coded microsphere as a solid phase carrier, and connects the antibody (or antigen) to the coded microsphere through a chemical bond, captures the target antigen (or antibody) in serum or plasma, and reacts with the antibody (or antigen) marked with luminescent substances. When the immune reaction is completed, the reactants are typically washed with a washing liquid to remove unreacted materials. After washing, the microsphere enters an instrument through a sampling tube, PE fluorescence on the microsphere is excited by laser, and the microsphere is input into a computer after photoelectric conversion and is analyzed and processed by software, so that the detection of the substance to be detected is realized.

Many technicians currently research the cleaning liquid, for example, the prior art discloses an electrochemiluminescence cleaning liquid, and the main components of the electrochemiluminescence cleaning liquid comprise Tris buffer solution and the like, and the electrochemiluminescence cleaning liquid is mainly applied to a Beckmann full-automatic immunoassay analyzer. The prior art also discloses a chemiluminescent cleaning solution, which mainly comprises phosphate buffer solution and the like, and is developed for the yaban chemiluminescent immunoassay analyzer. The prior art also discloses a cleaning solution, the main components of which comprise phosphate buffer solution, inorganic salt and the like, and the cleaning solution can adapt to an enzymatic chemiluminescence and direct chemiluminescence system.

However, the above cleaning solution has the defects of unstable detection signals, abnormal background signals of partial experiments, sedimentation of microspheres and the like in the flow type fluorescence luminescence method, so improvement is needed in the aspects.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a cleaning solution for flow fluorescence detection, which is used for solving the problems in the prior art.

To achieve the above and other related objects, the present invention provides a washing liquid for flow fluorescence detection, the washing liquid including a base washing liquid, proteins and alcohols, the base washing liquid including a buffer salt and a surfactant, the solvent of the base washing liquid being water.

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

And the final concentration of the buffer salt is 5-10 g/L based on the total volume of the basic washing liquid.

The surfactant is a nonionic surfactant. The nonionic surfactant is one or more of Tween-20, tween-80, span-60, triton X-45, triton X-100 or Triton X-305.

The basic lotion also comprises a preservative. The preservative is KroVin series preservative or ProClin series preservative.

The pH of the cleaning liquid is 5.5-8.5.

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, sheep serum and chicken serum.

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

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

When the alcohols are mannitol and PVP, PVA, PEG, the final concentration of the alcohols is 0.1-50g/L.

The invention also provides application of the cleaning solution in flow type fluorescence detection.

As described above, the cleaning solution for flow type fluorescence detection has the advantages that the cleaning solution can not only stabilize detection signals and reduce microsphere sedimentation, but also give consideration to background signals and avoid nonspecific adsorption while meeting the cleaning effect of a flow type fluorescence light-emitting platform.

Detailed Description

The invention provides a flow type fluorescent detection cleaning solution, which comprises a basic cleaning solution, proteins and alcohols, wherein the basic cleaning solution comprises buffer salt and a surfactant.

In certain embodiments of the invention, the buffer salt in the base wash is selected from one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, or potassium chloride.

In some embodiments of the present invention, the final concentration of the buffer salt is 5 to 10g/L, for example, 5 to 6g/L, 6 to 7g/L, 7 to 8g/L, 8 to 9g/L, 9 to 10g/L, based on the total volume of the base wash.

In certain embodiments of the invention, the surfactant is a nonionic surfactant. The nonionic surfactant is one or more of Tween-20, tween-80, span-60, triton X-45, triton X-100 or Triton X-305. The final concentration of the surfactant is from 0.05% to 0.5% v/v, for example from 0.05% to 0.1% v/v, from 0.1% to 0.2% v/v, from 0.2% to 0.3% v/v, from 0.3% to 0.4% v/v, from 0.4% to 0.5% v/v, based on the total volume of the base wash.

In certain embodiments of the invention, a preservative is also included in the base wash. The preservative is KroVin series preservative or ProClin series preservative.

The KroVin series of preservatives is KroVin100, kroVin 400, kroVin 500 or KroVin 750, for example, and the ProClin series of preservatives is ProClin50, proClin150, proClin200, proClin300, proClin950 or ProClin5000, for example.

The final concentration of the preservative is 0.05% to 0.2% v/v, for example 0.05% to 0.1% v/v, 0.1% to 0.15% v/v, 0.15% to 0.2% v/v, based on the total volume of the base wash.

The solvent of the basic washing liquid is water.

The buffer salt, the surfactant and the preservative in the basic washing liquid are used for ensuring the basic washing effect and the stability of the system. Only basic washing liquid is used as washing liquid of a flow type fluorescence luminescence method, and the stability, the accuracy, the microsphere sedimentation and the like of detection results of different projects are insufficient.

The pH of the cleaning liquid is 5.5-8.5. Preferably, the pH of the cleaning solution is 6.0-8.0.

When the proteins are BSA, casein or ovalbumin, the final concentration of the proteins in the cleaning solution is 1-50g/L based on the total volume of the cleaning solution. In a preferred embodiment, the final concentration of the protein in the wash solution is 5-40g/L, e.g. 5-10g/L, 10-20g/L, 20-30g/L or 30-40g/L.

When the protein is serum, the final concentration of the protein in the wash solution is 0.1% -5% v/v, for example 0.1-1.5% 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.

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

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%, 0.1-0.2%, 0.2-0.3%, 0.3-0.4% or 0.4-0.5% v/v.

When the alcohols are mannitol and PVP, PVA, PEG, the final concentration of the alcohols is 0.1-50g/L. The final concentration of the alcohols is, for example, 0.1-1g/L, 1-10g/L, 10-20g/L, 20-30g/L, 30-40g/L or 40-50g/L.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before further describing embodiments of the present invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention, as the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

The washing liquid disclosed by the invention consists of basic washing liquid, proteins and alcohols, wherein in 1L of the basic washing liquid, the adding amount of the proteins is added according to the final concentration of 1-50g/L, the adding amount of the alcohols is added according to the final concentration of 0.1-50g/L, and the pH value of the washing liquid is=5.5-8.5.

The basic wash formulation used in the examples below was as follows:

preparing basic lotion by taking purified water as a solvent according to the following weight and volume, wherein the total volume is 1L after the preparation:

Na₂HPO₄	1.1g
		NaCl	5.0g
KH₂PO₄	0.5g
		KCl	1.4g
TritonX-100	1mL
		ProClin300	1mL

the pH value of the prepared basic lotion is 5.5-8.5, and the prepared basic lotion is filtered by a 0.22 mu m filter membrane and then stored at room temperature.

Example 1 washing solution for flow-type fluorescence luminescence method

The washing liquid preparation method comprises the following steps of preparing a washing liquid by taking a basic washing liquid as a solvent according to the following weight and volume, and adjusting pH=5.5-8.5 after the preparation is completed, wherein the total volume of the washing liquid is 1L:

Bovine serum	5mL
		BSA	20g
Basic lotion	Constant volume to 1L

The washing solution prepared as above was filtered through a 0.22 μm filter and stored at 2-8 ℃.

Example 2 washing solution for flow-type fluorescence luminescence method

Sheep serum	1mL
		Casein protein	10g
Basic lotion	Constant volume to 1L

Example 3 washing solution for flow-type fluorescence luminescence method

BSA	25g
		Egg albumin	5g
Basic lotion	Constant volume to 1L

Example 4 washing solution for flow-type fluorescence luminescence method

Glycerol	2mL
		PEG6000	10g
Basic lotion	Constant volume to 1L

Example 5A washing solution for flow-type fluorescence luminescence method

PVA	5g
		PEG200	30g
Basic lotion	Constant volume to 1L

Example 6A washing solution for use in flow-type fluorescence luminescence

PVP	2g
		PEG2000	20g
Basic lotion	Constant volume to 1L

Example 7 washing solution for flow-type fluorescence luminescence method

The washing liquid preparation method comprises the following steps of preparing a washing liquid by taking a basic washing liquid as a solvent according to the following weight and volume, and adjusting pH=6.0-6.6 after the preparation is completed, wherein the total volume of the washing liquid is 1L:

Example 8 washing solution for flow-type fluorescence luminescence method

The washing liquid preparation method comprises the following steps of preparing a washing liquid by taking a basic washing liquid as a solvent according to the following weight and volume, and adjusting pH=6.6-7.3 after the preparation is completed, wherein the total volume of the washing liquid is 1L:

Egg albumin	10g
		Bovine serum	2mL
PEG1000	25g
		Mannitol (mannitol)	5g
Basic lotion	Constant volume to 1L

Example 9 washing solution for flow-type fluorescence luminescence method

The washing liquid preparation method comprises the following steps of preparing a washing liquid by taking a basic washing liquid as a solvent according to the following weight and volume, and adjusting pH=7.3-8.0 after the preparation is completed, wherein the total volume of the washing liquid is 1L:

Sheep serum	5mL
		Egg albumin	5g
PVA	1g
		PVP	2g
Glycerol	3mL
		Basic lotion	Constant volume to 1L

Example 10

Experimental results determination the following experiments were performed using the flow-type fluorescent immunoassay assays performed with the basic washes of examples 1-9, respectively:

10.1 verification of cleaning Effect

The basic wash solution and the wash solutions of examples 1 to 9 were measured by beckmann coulter flow cytometer Dxflex, respectively, and in the table, the reagent a, the reagent B and the reagent C are all reagents in the saccharide antigen 125 quantitative detection kit, the Myoglobin (MYO) detection kit and the anti-double-stranded DNA antibody (dsDNA) detection kit, and the measurement steps were performed according to the specification, approximately as follows:

The measurement items selected from saccharide antigen 125 (CA 125), myoglobin (MYO) and anti-double-stranded DNA antibody (dsDNA), and the measurement samples were 20 cases of CA125, MYO and dsDNA calibrator, and the average value of the measurement signals of each sample is shown in Table 1:

TABLE 1 detection results of background signals of the base wash and the wash of examples 1-9

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

The results in Table 1 show that the washing liquid of the present invention has good washing effect and low background signals of each item.

10.2 System Signal stability

The same set of samples, CY211, dsDNA, CK-MB, were assayed at different times using a Beckmann coulter flow cytometer Dxflex for each of the base wash solutions and wash solutions of examples 1-9, and the signal values were again read using the Beckmann coulter flow cytometer Dxflex after the first assay at room temperature for 60 minutes, with the test signal results shown in Table 2:

TABLE 2 Signal stability test results for base washes and wash systems of examples 1-9

The results in Table 2 show that the wash of the present invention performs better than the base wash in terms of system signal stability.

10.3 Sedimentation experiments

Working solutions of antigen/antibody coated magnetic beads were prepared using the basic wash solution and examples 7 to 9, respectively, and mixed uniformly in 4 parts with 1mL each. 200 μl of the supernatant was taken at intervals, the number of balls was read by beckmann coulter flow cytometer Dxflex, the detection was repeated 3 times, the average value was taken and the statistics of the results were taken, and the detection results are shown in table 3:

TABLE 3 determination of the Natural sedimentation Rate of the base washes and the washes of examples 7-9

The results in Table 3 show that the antigen/antibody coated magnetic spheres have a faster sedimentation rate in the basic wash than in the washes of examples 7-9, and have a more pronounced difference at 20 min. In summary, it is considered that the washing liquid of the present invention is less likely to cause sedimentation of microspheres under natural gravity than the base washing liquid.

10.4 Accuracy experiment

The basic wash solution and the wash solutions of examples 7 to 9 were each measured using a beckmann coulter flow cytometer Dxflex, 20 samples of the same group were each measured, the samples were calibrants of CA125, CEA, NSE, CY211, proGRP, and the average value of the detection signals was as shown in table 4:

TABLE 4 accuracy test results for base wash and wash of examples 7-9

As can be seen from Table 4, the measured values of the 5 indexes are all close, each two groups of data are subjected to t-test, the difference has no statistical significance (p > 0.05), and the R ² values are all larger than 0.990, so that the positive correlation is high. The above results show that the accuracy of the cleaning liquid test is good, and the detection accuracy is not affected by the addition of components.

10.5 Precision experiments

Three batches of cleaning solution were prepared according to examples 7-9, batch 1, batch 2 and batch 3, respectively. Taking AFP samples with the concentration of 40ng/ml, measuring three batches of detection results of examples 7-9 by adopting a Beckmann coulter flow cytometer Dxflex, repeatedly detecting each batch of cleaning liquid for 20 times, and calculating intra-batch and inter-batch variation coefficients, wherein the detection results are shown in Table 5:

TABLE 5 results of examples 7-9 washing reagent precision test

The results in Table 5 show that the washing solutions of the present invention have an intra-batch Coefficient of Variation (CV) <10%, an inter-batch Coefficient of Variation (CV) <15%, and meet the requirements of a typical diagnostic reagent having an intra-batch coefficient of variation of less than 10% and an inter-batch coefficient of variation of less than 15%. The above results demonstrate that the accuracy and reproducibility of the detection of the wash solution of the present invention are good.

10.6 Storage stability of the lotion

1.4L of cleaning solution was prepared as in examples 7-9, and each bottle was divided into 7 bottles of about 200mL, and the bottles were left for 0 day for 2 months, 4 months, 6 months, 9 months, 12 months, and 15 months at 2-8deg.C, respectively, and then tested. The measurement was repeated 20 times using CEA samples at a concentration of 5.+ -. 0.5ng/mL, and the coefficient of variation was calculated, and the measurement results are shown in Table 5:

TABLE 5 storage stability test results for base lotions and lotions of examples 7-9

The results in Table 5 show that the wash solutions prepared in examples 7-9 have CV values of less than 5% for samples stored for less than 15 months at 2-8 ℃. From the reagent status, the 3 example washes were clear and transparent, and no precipitation occurred, indicating that the wash of the invention has good storage stability.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. Use of the following flow fluorescence detection cleaning solution in flow fluorescence luminescence immunoassay detection, characterized in that the flow fluorescence detection cleaning solution comprises a basic cleaning solution, the basic cleaning solution comprises a buffer salt and TritonX-100, the solvent of the basic cleaning solution is water, and the flow fluorescence detection cleaning solution 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 method according to claim 1, wherein 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 to 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 the basic washing liquid also includes a preservative.

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 lotion, 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.