阅读说明：本技术 一种磁微粒化学发光清洗液及其制备方法和应用 (Magnetic particle chemiluminescence cleaning solution and preparation method and application thereof ) 是由 张春杰 葛婵婵 王静 胡晓明 杨小蕾 赵凤 赵芮 于 2021-07-02 设计创作，主要内容包括：本发明提供一种磁微粒化学发光清洗液及其制备方法和应用,属于化学发光分析技术领域。所述磁微粒化学发光清洗液包括缓冲液、盐离子、稳定剂、表面活性剂、防腐剂和消泡剂。本发明提供的磁微粒化学发光清洗液,通过对清洗液成分等的优化筛选,从而有效解决现有技术中的清洗液所存在的清洗液清洗背景值高,灵敏度、精密度不佳以及成本高等问题,从而可应用于磁微粒化学发光体系中,因此具有良好的实际应用之价值。(The invention provides a magnetic particle chemiluminescence cleaning solution, and a preparation method and application thereof, and belongs to the technical field of chemiluminescence analysis. The magnetic particle chemiluminescence cleaning solution comprises a buffer solution, salt ions, a stabilizing agent, a surfactant, a preservative and an antifoaming agent. The magnetic particle chemiluminescence cleaning solution provided by the invention effectively solves the problems of high cleaning background value, poor sensitivity and precision, high cost and the like of the cleaning solution in the prior art by optimizing and screening the components of the cleaning solution, and can be applied to a magnetic particle chemiluminescence system, thereby having good practical application value.)

1. The magnetic particle chemiluminescence cleaning solution is characterized by comprising a buffer solution, salt ions, a stabilizer, a surfactant, a preservative and an antifoaming agent.

2. A magnetic particle chemiluminescent cleaning solution as defined in claim 1 wherein the buffer is PBS buffer, preferably the buffer has a concentration of 100mM to 1000 mM.

3. The magnetic particle chemiluminescent cleaning solution of claim 1 wherein the salt ions are sodium ions, the concentration of the sodium ions being 10% to 20%.

4. The magnetic particle chemiluminescent cleaning solution as defined in claim 1 wherein the stabilizer is disodium edetate, the concentration of disodium edetate being 0.5% -1%.

5. The magnetic particle chemiluminescent cleaning solution of claim 1 wherein the surfactant is a non-ionic surfactant and an anionic surfactant, and further preferably sodium dodecyl sulfate and/or tween 20, wherein the concentration of the tween 20 is 1.0% to 3.0%, and the concentration of the SDS is 0.1% to 1.0%.

6. The magnetic particle chemiluminescent cleaning solution of claim 1 wherein the preservative is one of PC300, sodium azide or Bronidox; preferably, the preservative is sodium azide, and the concentration of the preservative is 0.05-0.15%.

7. The magnetic particle chemiluminescent cleaning solution as claimed in claim 1 wherein the defoaming agent is a silicone oil type defoaming agent or a polyether type defoaming agent, preferably, the defoaming agent is a silicone oil type defoaming agent, and the concentration of the defoaming agent is 0.05% to 0.5%;

preferably, the pH of the magnetic particle chemiluminescence cleaning solution is alkalescent, and is further 7.0-7.5;

further preferably, the magnetic particle chemiluminescence cleaning solution comprises: 500mM PBS buffer solution, 12% sodium ion, 0.8% disodium ethylene diamine tetraacetate, 0.5% SDS, 1.5% Tween 20, 0.1% sodium azide, 0.1% silicone oil type antifoaming agent, pH 7.2.

8. A method for preparing a magnetic particle chemiluminescent cleaning solution according to any one of claims 1 to 7, wherein the method comprises the following steps: and sequentially adding salt ions, a surfactant, a stabilizer and a preservative into the buffer solution, uniformly mixing, adding a defoaming agent, and adjusting the pH value to be alkalescent.

9. Use of a magnetic particle chemiluminescent cleaning solution of any one of claims 1 to 7 in a chemiluminescent immunoassay.

10. The application of claim 9, wherein the application comprises: a cleaning step of diluting the magnetic particle chemiluminescence cleaning solution and then using the diluted solution for magnetic particle chemiluminescence immunoassay, wherein the cleaning step is used for removing unbound antigen, antibody and free conjugate and stopping the continuous combination of the antigen and the antibody; the dilution ratio is 5 to 15 times, and more preferably 10 times.

Technical Field

The invention belongs to the technical field of chemiluminescence analysis, and particularly relates to a magnetic particle chemiluminescence cleaning solution, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

The magnetic particle chemiluminescence immunoassay is an immunoassay technology for detecting trace antigens or antibodies, which is established by combining a magnetic particle technology, luminescence analysis and immunoreaction. The method has the advantages of high sensitivity of luminescence analysis, high specificity of antigen-antibody reaction, wide detection range, short detection time and the like, can be used for detection and analysis of various antigens, antibodies, hormones, fatty acids, vitamins and the like, and is a novel in-vitro detection technology.

The detection steps of the magnetic particle chemiluminescence immunoassay technology are as follows: sample adding, incubation, washing and reading. The main purpose of washing is to remove unbound antigen, antibody and free conjugate, stop the continuous combination of antigen and antibody, and reduce the influence of non-specific adsorbed substance on the detection result.

The inventor finds that the existing magnetic particle chemiluminescence cleaning solution has poor cleaning effect and high background value, causes carrying pollution, thereby influencing the sensitivity and precision of detection and even causing false positive.

CN111607465A discloses a chemiluminescent cleaning solution, a preparation method and an application thereof, wherein the chemiluminescent cleaning solution comprises the following components: tris, sodium ion, CHAPS, Triton-100, PC300, BND, lactone of paraben and silicone oil type defoaming agent, the cleaning solution has complex components, and the used preservative comprises three types, wherein BND is difficult to dissolve in water, and the preparation process is complex. CHAPS is selected as a surfactant, is an amphoteric surfactant, can crack protein, has strong washing capacity, and can wash away antigens and antibodies which are easy to be subjected to specific binding in the detection process, so that the detection result is inaccurate.

CN112159733A discloses a cleaning solution for magnetic particle chemiluminescence immunoassay and a preparation method thereof, and the cleaning solution comprises the following components: TRIS-HCl, sodium chloride and Tween 20. The cleaning solution uses a single mild nonionic surfactant Tween 20, has weak cleaning capability, and causes false positive results due to the fact that proteins or other non-specific adsorption substances which do not participate in specific reaction cannot be completely cleaned when the cleaning solution is applied to clinical projects with low detection sensitivity. In addition, the cleaning solution does not use any preservative, and cannot meet the requirement of preservative effect.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a magnetic particle chemiluminescence cleaning solution and a preparation method and application thereof. The invention optimizes the components, the dosage, the pH value and the like of the cleaning solution for the magnetic particle chemiluminescence by screening, thereby preparing the cleaning solution which has the advantages of low cleaning background value, higher sensitivity and precision and low cost.

In a first aspect of the invention, a magnetic particle chemiluminescence cleaning solution is provided, and comprises a buffer solution, salt ions, a stabilizer, a surfactant, a preservative and an antifoaming agent.

The buffer solution is PBS buffer solution, and the PBS buffer solution is used as the buffer solution of the chemiluminescent cleaning solution, so that the cost is lower, and the cleaning effect is better.

The salt ions are sodium ions (such as sodium chloride), the addition of the salt ions can provide proper electrolyte for immune reaction, the normal operation of the immune reaction is ensured, the cost of the sodium ions is low, and the cost of the whole cleaning solution is reduced.

The stabilizer is disodium ethylene diamine tetraacetate.

The surfactant may be a nonionic surfactant and an anionic surfactant, and is more preferably Sodium Dodecyl Sulfate (SDS) and/or tween 20.

The preservative is one of PC300, sodium azide or Bronidox. Preferably, the preservative is sodium azide.

The defoaming agent is a silicone oil defoaming agent or a polyether defoaming agent.

The pH of the magnetic particle chemiluminescence cleaning solution is alkalescent, and is further 7.0-7.5.

In a second aspect of the present invention, a preparation method of the above-mentioned magnetic particle chemiluminescent cleaning solution is provided, where the preparation method includes: and sequentially adding salt ions, a surfactant, a stabilizer and a preservative into the buffer solution, uniformly mixing, adding a defoaming agent, and adjusting the pH value to be alkalescent.

In a third aspect of the invention, the application of the magnetic particle chemiluminescence cleaning solution in chemiluminescence immunoassay is provided.

Specifically, the application includes: and a cleaning step of diluting the magnetic particle chemiluminescence cleaning solution and then using the diluted solution for magnetic particle chemiluminescence immunoassay, wherein the cleaning step is used for removing unbound antigen, antibody and free conjugate and stopping the continuous combination of the antigen and the antibody.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme provides the magnetic particle chemiluminescence cleaning solution, and the problems of high cleaning background value, poor sensitivity and precision, high cost and the like of the cleaning solution in the prior art are effectively solved through optimized screening of the components of the cleaning solution, so that the cleaning solution can be applied to a magnetic particle chemiluminescence system, and has good practical application value.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As mentioned above, the existing magnetic particle chemiluminescence cleaning solution has poor cleaning effect and high background value, and causes carrying pollution, thereby affecting the sensitivity and precision of detection, and even causing false positive.

In an exemplary embodiment of the present invention, a magnetic particle chemiluminescent cleaning solution is provided, which comprises a buffer solution, salt ions, a stabilizer, a surfactant, a preservative and an antifoaming agent.

In another embodiment of the present invention, the buffer solution is a PBS buffer solution, and the PBS buffer solution is used as a buffer solution of the chemiluminescent cleaning solution, so that the cost is lower and the cleaning effect is better. The concentration of the buffer is 100mM-1000mM, and for example, but not limited to, 100mM, 300mM, 500mM, 800mM or 1000mM, and when the concentration of the buffer is within the above range, the buffer can function as a buffer to ensure the normal reaction.

In another embodiment of the present invention, the test proves that when the concentration of the buffer solution is 500mM, the luminous value CV is the smallest, the background value is the lowest, which indicates that the cleaning effect of the cleaning solution is the best, and the excessive components in the immune reaction can be effectively removed.

In another embodiment of the present invention, the salt ions are sodium ions (such as sodium chloride), and the addition of the salt ions can provide a suitable electrolyte for the immune reaction, so as to ensure the normal operation of the immune reaction, and the sodium ions have a lower cost, thereby reducing the cost of the whole cleaning solution. The concentration of sodium ion is 10% -20%, for example, but not limited to 10%, 12%, 14%, 16%, 18% or 20%, and when the concentration of sodium ion is within the above range, sufficient electrolyte can be provided for immune reaction.

In another embodiment of the present invention, experiments prove that when the concentration of the sodium ions is 12%, the CV value is minimum, and simultaneously, waste is avoided, and cost is effectively saved.

In another embodiment of the present invention, the stabilizer is disodium edetate, and disodium edetate is used as a complexing agent, which can chelate ions such as calcium and magnesium, and effectively remove metal ions possibly existing in the sample; meanwhile, the ethylene diamine tetraacetic acid disodium can be used as a stabilizer, so that the chemical luminescent agent can be prevented from being excited in advance; the magnetic microsphere cleaning liquid is used as an effective component of the cleaning liquid, and particularly has an excellent cleaning effect on nonspecific binding of the magnetic microsphere.

The concentration of the ethylene diamine tetraacetic acid disodium is 0.5% -1%. For example, but not limited to, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%. When the concentration of the ethylenediamine diacetic acid disodium is within the above range, the function of stabilizing the whole cleaning system can be achieved. Preferably, the concentration of ethylenediaminetetraacetic acid is 0.8%.

In another embodiment of the present invention, the surfactant may be a nonionic surfactant and an anionic surfactant, and is further preferably Sodium Dodecyl Sulfate (SDS) and/or tween 20, the concentration of tween 20 is 1.0% to 3.0%, including but not limited to 1.0%, 1.5%, 2.0%, 2.5% and 3.0%, and the concentration of SDS is 0.1% to 1.0%, including but not limited to 0.1%, 0.3%, 0.5%, 0.8% and 1.0%, and is further preferably, a method combining a nonionic surfactant and an ionic surfactant is adopted, so that the washing effect of the washing solution is better, the influence of nonspecific adsorption on the detection result can be greatly reduced, and the detection accuracy is improved. Therefore, when the concentration of the tween 20 is 1.5% and the concentration of the SDS is 0.5%, experiments prove that the cleaning effect is best, the CV is minimum, non-specifically adsorbed interfering substances in the immunoreaction can be effectively removed, the repeatability of the sample is better, the accuracy is higher, samples containing the substance to be detected and samples not containing the substance to be detected can be effectively distinguished, and the content of the substance to be detected in the reaction sample is more real.

In another embodiment of the present invention, the preservative is one of PC300, sodium azide or Bronidox. Preferably, the preservative is sodium azide, and the concentration of the preservative is 0.05-0.15%. For example, but not limited to, 0.05%, 0.08%, 0.1%, 0.12%, or 0.15%. When the preservative is within the above concentration range, long-term preservation of the magnetic particle chemiluminescence cleaning solution can be ensured.

Through screening of the concentration of the preservative, the cleaning solution is placed at room temperature for 14 months, when the concentration of the preservative sodium azide is 0.1%, the blank CV can still be within 8%, the change of the luminous value is minimum, and the antibacterial effect of the cleaning solution is best. If the bacteriostatic effect of the cleaning solution is poor, the cleaning solution can be infected with bacteria, so that the light emitting value of the blank cleaning solution is increased, the jump value phenomenon is easy to occur, and the CV exceeds 10%.

In another embodiment of the present invention, the defoaming agent is a silicone oil type defoaming agent or a polyether type defoaming agent, and the defoaming agent is added to prevent bubbles from being generated during the addition of a cleaning solution or during the washing process, so as to prevent a jump value phenomenon from occurring and improve detection precision and accuracy.

In another embodiment of the present invention, the defoaming agent is a silicone oil type defoaming agent, and the concentration of the defoaming agent is 0.05% to 0.5%. For example, but not limited to, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. Further preferably, the concentration of the defoaming agent is 0.1%. The cleaning solution added with 0.1 percent of defoaming agent has smaller CV and higher signal to noise ratio on the IL-8 project. After the defoaming agent is added into the cleaning solution, the generation of bubbles can be effectively reduced, and the precision and the accuracy of reagent detection are improved.

In another embodiment of the present invention, the pH of the magnetic particle chemiluminescence cleaning solution is weakly alkaline, and further is 7.0 to 7.5, and for example, but not limited to, 7.1, 7.2, 7.3, 7.4, or 7.5. Further preferably, when the pH of the magnetic particle chemiluminescence cleaning solution is 7.2, a suitable acid-base environment can be provided for the whole reaction system.

In another embodiment of the present invention, the magnetic particle chemiluminescent cleaning solution comprises: 500mM PBS buffer solution, 12% sodium ion, 0.8% disodium ethylene diamine tetraacetate, 0.5% SDS, 1.5% Tween 20, 0.1% sodium azide, 0.1% silicone oil type antifoaming agent, pH 7.2.

In another embodiment of the present invention, a method for preparing the magnetic particle chemiluminescent cleaning solution comprises the following steps: and sequentially adding salt ions, a surfactant, a stabilizer and a preservative into the buffer solution, uniformly mixing, adding a defoaming agent, and adjusting the pH value to be alkalescent.

In another embodiment of the present invention, there is provided an application of the magnetic particle chemiluminescence cleaning solution in chemiluminescence immunoassay.

Specifically, the application includes: and a cleaning step of diluting the magnetic particle chemiluminescence cleaning solution and then using the diluted solution for magnetic particle chemiluminescence immunoassay, wherein the cleaning step is used for removing unbound antigen, antibody and free conjugate and stopping the continuous combination of the antigen and the antibody.

The dilution factor is 5 to 15 times, including, but not limited to, 5 times, 8 times, 10 times, 12 times and 15 times, and more preferably 10 times.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1

The embodiment provides a magnetic particle chemical light concentrated cleaning solution, which comprises: 500mM PBS buffer solution, 12% sodium chloride, 0.8% disodium ethylene diamine tetraacetate, 0.5% SDS, 1% Tween 20, 0.1% sodium azide, 0.1% silicone oil type antifoaming agent, pH 7.2. After diluted by 10 times, the solution is used for cleaning a magnetic particle chemiluminescence experiment.

Comparative example 1

A magnetic particle chemical light concentrated cleaning solution, comprising: 500mM PBS buffer solution, 12% sodium chloride, 0.8% disodium ethylene diamine tetraacetate, 1% Tween 20, 0.1% sodium azide, 0.1% silicone oil type antifoaming agent, pH7.2. After diluted by 10 times, the solution is used for cleaning a magnetic particle chemiluminescence experiment.

Comparative example 2

A magnetic particle chemical light concentrated cleaning solution, comprising: 500mM PBS buffer solution, 16% sodium chloride, 0.5% disodium ethylene diamine tetraacetate, 0.5% Tween 20, 0.1% sodium azide, 0.5% silicone oil type antifoaming agent, pH 7.2. After diluted by 10 times, the solution is used for cleaning a magnetic particle chemiluminescence experiment.

Comparative example 3

As described in CN111896731A patent, the present comparative example provides a cleaning solution for chemiluminescence immunoassay comprising:

PBS	30mM
		tween 20	0.1wt％
Sodium caseinate	0.75wt％
		Ethylenediaminetetraacetic acid disodium salt	0.1wt％
Deoxycholic acid sodium salt	0.05wt％
		Sodium azide	0.05wt％

Comparative example 4

As described in the CN110862881A patent, the present comparative example provides a cleaning solution specially used for a full-automatic chemiluminescence apparatus:

sodium chloride	35g
		Potassium chloride	185g
Disodium hydrogen phosphate dodecahydrate	53.72g
		Potassium dihydrogen phosphate	0.13g
Tween 20	24g
		Sodium monthly silicate	0.5g
Proclin300	10g
		Bronidox	0.4g
Dilution factor when in use	15

Comparative example 5

Original beckmann Access wash buffer.

Comparative example

Performance testing

1. Linear testing

The cleaning solution of the present invention of example 1 and the cleaning solutions 1 to 5 of comparative examples were diluted in the proportions indicated in the specification, and then 6 cleaning solutions were compared and verified by using a Shinei2900 type full-automatic chemiluminescence apparatus, and the linearity of the item was determined by selecting interleukin 8(IL-8) which is a sandwich method item.

TABLE 1 IL-8 calibration curves using the cleaning solutions of example 1 and comparative examples 1-5

Concentration of	Example 1	Comparative example 1	Comparative example 2	Comparative example 3	Comparative example 4	Comparative example 5
							pg/mL	RLU	RLU	RLU	RLU	RLU	RLU
0	632	876	942	1042	972	1003
							2.5	1327	1664	1790	1980	1847	2012
10	5176	5825	6264	6929	6464	7032
							50	25363	27379	29442	32568	30380	31380
250	121741	131421	141323	156325	145823	155833
							1000	477226	515170	553985	612794	571627	571877
3000	1369639	1478539	1440360	1593264	1486231	1586230
							7500	3355617	3430211	3197599	3712306	3433194	3533194
r	0.9999	0.9995	0.9984	0.9993	0.9995	0.9987

As can be seen from Table 1, the IL-8 calibration curve obtained by washing the reaction composite with the washing solution of example 1 and comparative examples 1-5 has better linear correlation and can be used for other performance verification.

2. Precision verification

The cleaning solution of the invention of example 1 and the cleaning solutions 1 to 5 of the comparative examples were diluted according to the ratio indicated in the specification, and then 6 cleaning solutions were compared and verified by using a Shinei2900 type full-automatic chemiluminescence apparatus, and the precision of the two items was determined by selecting interleukin 8(IL-8) as a sandwich method item and thyroglobulin antibody (Anti-Tg) as a competition method item, and 1 sample was measured for each item, and 10 replicates were performed for each sample.

TABLE 2 precision of IL-8 and Anti-Tg Using washes of example 1 and comparative examples 1-5

The precision of the precision detection items of the cleaning liquid is within 2 percent, the precision of the cleaning liquid of other comparative examples is within 2 to 4 percent, and the results show that the precision and the reactivity of the detection of the cleaning liquid are good, and in the reaction system, the cleaning liquid of the invention is obviously superior to the comparative cleaning liquid.

3. Stability verification

The cleaning solution of the present invention of example 1 and the cleaning solutions of comparative examples 1 to 5 were respectively dispensed into 4 bottles, and left at room temperature for 0 day, 6 months, 12 months, and 14 months for testing. Before the detection, the cleaning solution is diluted according to the proportion shown in the specification, then a Shinei2900 type full-automatic chemiluminescence apparatus is used for blank measurement and IL-8 item measurement of 6 cleaning solutions respectively, the detection is repeated for 10 times, and the experimental results are shown in tables 3 and 4.

Table 3 blank test results for long term stability of example 1 cleaning solution and comparative examples 1 to 5 cleaning solution

TABLE 4 precision test results for IL-8 long term stability of example 1 cleaning solution and comparative examples 1-5 cleaning solution

The results show that: at room temperature, the background of comparative examples 1 to 5 is increased at month 12, the blank luminescence value of the cleaning solution is increased, and the precision is poor, because the cleaning solution may be infected with bacteria after being stored for a long time, and the blank value detection of the cleaning solution provided by the invention is still stable after being placed for 14 months, which shows that the cleaning solution provided by the invention can still be stable within 14 months. On the item IL-8 test, the invention provides a cleaning solution CV of around 2% at 14 months, with a significant increase in comparative cleaning solution CV. The cleaning liquid of the invention still keeps stable within 14 months and has better performance than the comparative example.

Experimental data of examples and comparative examples show that the buffer solution is superior to the comparative examples in cleaning solution effect, storage stability and project performance tests, and the problems of poor cleaning effect, high background and poor stability of the existing cleaning solution are solved.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.