CN119125527A

CN119125527A - Magnetic fluid layered micro-controlled immunoassay reagents and detection kits and their applications

Info

Publication number: CN119125527A
Application number: CN202411073197.1A
Authority: CN
Inventors: 刘海波; 刘嵽
Original assignee: Chongqing Hejian Business Information Consulting Partnership Enterprise LP
Current assignee: Chongqing Hejian Business Information Consulting Partnership Enterprise LP
Priority date: 2024-08-06
Filing date: 2024-08-06
Publication date: 2024-12-13

Abstract

本发明涉及免疫检测试剂技术领域，尤其涉及磁流体分层微控免疫分析试剂和检测试剂盒及其应用，分析试剂包括R1试剂、R2试剂和R3试剂，R1试剂包括稀释液一和分布在稀释液一中的由免疫磁珠形成的磁流体，免疫磁珠为用已知的抗原或抗体包被磁微粒构成；R2试剂包括稀释液二和分布在稀释液二中的标记结合物，标记结合物为不同荧光物质标记的被测物所对应的抗原或抗体；R3试剂为与水不相容的强疏水液体试剂。本发明选择了一种强疏水轻相物质来实现未参与免疫学反应标记物的分离，选择了固定磁场的稳定磁力牵引磁流体中的磁微粒运动集聚，选择了荧光标记的多重性，从而实现其检测结果灵敏度高，一致性和重复性好，结果稳定，操作简便，检测设备可便携等需求。The present invention relates to the technical field of immunoassay reagents, and in particular to magnetic fluid layered micro-controlled immunoassay reagents and detection kits and their applications. The analysis reagents include R1 reagent, R2 reagent and R3 reagent. R1 reagent includes a diluent one and a magnetic fluid formed by immunomagnetic beads distributed in the diluent one, and the immunomagnetic beads are composed of magnetic particles coated with known antigens or antibodies; R2 reagent includes a diluent two and a labeled conjugate distributed in the diluent two, and the labeled conjugate is an antigen or antibody corresponding to a test object labeled with different fluorescent substances; R3 reagent is a strongly hydrophobic liquid reagent incompatible with water. The present invention selects a strongly hydrophobic light phase substance to achieve the separation of markers that do not participate in immunological reactions, selects the stable magnetic force of a fixed magnetic field to pull the movement and aggregation of magnetic particles in the magnetic fluid, and selects the multiplicity of fluorescent labels, so as to achieve the requirements of high sensitivity, good consistency and repeatability of the detection results, stable results, simple operation, and portable detection equipment.

Description

Magnetic fluid layered micro-control immunoassay reagent, detection kit and application thereof

Technical Field

The invention relates to the technical field of immunodetection reagents, in particular to a magnetic fluid layering micro-control immunoassay reagent, a detection kit and application thereof.

Background

The immunological analysis technology and method used clinically at present comprise an enzyme immunoassay technology, a chemiluminescence immunoassay technology, an immunochromatography analysis technology and a fluorescence immunoassay technology, and the technologies have advantages and disadvantages, wherein the chemiluminescence immunoassay technology and the immunochromatography analysis technology are most widely applied to clinical immunological diagnosis.

The chemiluminescent immunoassay technology is a liquid phase reaction and detection technology which is carried out by reacting antigen and antibody in aqueous solution, and has the advantages of high sensitivity, good repeatability, stable detection result, high degree of automation and the like, and the basic principle method is as follows: the method comprises the steps of fixing known antigen-antibody by using magnetic microspheres, adding the detected antigen-antibody into a buffer system of an aqueous solution, then adding an antigen-antibody marker which is corresponding to a detected object and is marked with a luminescent substance, repeatedly washing and cleaning the unreacted or redundant reactant by an instrument after reacting for a certain time, adding excitation liquid to make the reactant emit light and detect the luminescence value, thus calculating the content of the detected substance.

The immunochromatography analysis technology is a detection technology formed by the free movement of antigen-antibody reaction results on a cellulose membrane through chromatography, and has the basic principle that known antigen-antibody is marked on a marker such as colloidal gold, fluorescent substances and the like, then the marker is fixed on the nitrocellulose membrane, a detected object is added to enable the antigen-antibody to react on the membrane, then the accumulation of reactants is formed on the fixed line of the other known antigen-antibody through free chromatography, and the content of the detected object is confirmed through detection of fluorescent or colloidal gold markers.

Therefore, the development of an immunoassay technology with high sensitivity, good repeatability, stable detection result and simple matched instrument has important significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a magnetic fluid layered micro-control immunoassay reagent, a detection kit and an application thereof, so as to at least solve the problems of high instrument cost, large volume, inconvenient carrying and use, etc. existing in the existing chemiluminescent immunoassay technology.

The invention solves the technical problems by the following technical means:

in a first aspect, the invention provides a magnetic fluid layered micro-controlled immunoassay reagent comprising:

The reagent R1 comprises a diluent I and magnetic fluid which is distributed in the diluent I and is formed by immunomagnetic beads, wherein the immunomagnetic beads are formed by coating magnetic particles with known antigens or antibodies;

The R2 reagent comprises a second diluent and labeled conjugates distributed in the second diluent, wherein the labeled conjugates are antigens or antibodies corresponding to detected objects marked by different fluorescent substances;

The R3 reagent is a strong hydrophobic liquid reagent which is incompatible with water.

With reference to the first aspect, in some embodiments, the strongly hydrophobic liquid reagent is at least one of carbon tetrachloride, chloroform, liquid paraffin, gasoline, edible oil, methyl silicone oil, perfluorocarbon, chlorobenzene, petroleum benzene, petroleum ether, cyclohexane.

With reference to the first aspect, in some embodiments, each milliliter of the R1 reagent contains 0.1 to 5mg of immunomagnetic beads, each milligram of magnetic particles is coated with 0.1 to 10 micrograms of antigen or antibody, and the particle size of the magnetic particles is 0.1 to 10 micrometers.

In combination with the first aspect, in some embodiments, the diluent one is at least one of a tris buffer, a phosphate buffer, a borate buffer, a glycine buffer and a hydroxyethylpiperazine ethylsulfuric acid buffer, and the diluent one contains 0.5-5.0 w% preservative, 0.1-2.0 w% serum protein, 0.05-2 w% amino acid, and 0.01-0.1% surfactant per unit volume of the buffer one, and the pH of the buffer one is 5.0-9.0.

With reference to the first aspect, in some embodiments, the amino acid is at least one of arginine, lysine, serine, glycine.

In combination with the first aspect, in some embodiments, the second diluent is at least one of a tris (hydroxymethyl) aminomethane hydrochloride buffer, a phosphate buffer and a borate buffer, wherein the second diluent contains 0.5-5.0w% of a protein stabilizer, 0.5-5.0w% of a preservative, 0.1-2.0w% of bovine serum albumin and 0.01-0.1% of a surfactant, and the pH of the second buffer is 6.5-9.5.

With reference to the first aspect, in some embodiments, the protein stabilizer is at least one of sorbitol, disodium edetate, trehalose, carboxymethyl cellulose, cyclodextrin, polyethylene glycol 4000, polyethylene glycol 6000, and bovine serum albumin.

With reference to the first aspect, in some embodiments, the preservative is at least one of sodium azide, thimerosal, phenol, and procline a, and the surfactant is at least one of tween-20, tween-80, triton 100, SDS.

In a second aspect, the invention also provides a detection kit, which comprises the magnetic fluid layering micro-control immunoassay reagent in the first aspect.

The invention further provides an application of the detection kit in the second aspect in immunoreaction detection, which comprises the steps of adding 10-150 microliters of a detected sample into a mixed reagent containing 20-500 microliters of an R1 reagent and 10-500 microliters of an R2 reagent, adding 100-1000 microliters of an R3 reagent, reacting for 5-60 minutes, transferring magnetic particles participating in the reaction, detected objects and marked combinations adsorbed on the magnetic particles into a layered R3 reagent by using a magnet, fixing the magnetic particles and the detected objects and marked combinations, placing the magnetic particles in a fluorescence detection instrument, detecting fluorescence intensity, wherein the fluorescence intensity is proportional to the content of the detected objects, and calculating the content of the detected objects.

The principle of the magnetic fluid layered micro-control immunoassay reagent for detection and analysis is that different known antigens or antibodies are immobilized by using magnetic microspheres, detected antigens or antibodies are added into a buffer system of an aqueous solution, then antigen or antibody markers which are corresponding to detected objects and are marked with different fluorescent substances are added, after a certain period of reaction, magnetic particles which participate in the reaction, the detected objects and fluorescent markers which are adsorbed on the magnetic particles are moved into fixed positions in a strong hydrophobic liquid reagent which is incompatible with water from the aqueous solution liquid by using magnetic force formed by a special magnet, and then the content of the detected objects is calculated and confirmed by detecting fluorescent values in the strong hydrophobic liquid reagent.

The invention selects a strong hydrophobic liquid reagent and the mixed reagent of the magnetic fluid-containing R1 reagent and the R2 reagent for immune reaction to be layered, so that antigen or antibody reaction can smoothly perform immunological liquid phase reaction like chemiluminescence in the mixed reagent of the R1 reagent and the R2 reagent, thereby ensuring high sensitivity. Meanwhile, the upper layer or the lower layer of the mixed reagent is layered with a strong hydrophobic liquid reagent layer which is highly stable, nontoxic and harmless, does not interfere with immune reaction, is transparent and easy to transmit light, so as to ensure the stable detection result, then a strong magnetic field is formed by a special magnet, fluorescent markers which are subjected to immune reaction in magnetic fluid and are connected to magnetic particles and detected substances move together with the magnetic particles under the traction of constant magnetic force from the mixed reagent to the specified position of the strong hydrophobic liquid reagent layer, and form an easy-detection shape, and the substances which participate in immune reaction and are connected to the magnetic particles can be positioned at the fixed detection position by the position exchange or transformation of the layered mixed reagent and the strong hydrophobic liquid reagent layer, so that the interference aim of other substances which are not connected to the magnetic particles is avoided. Then the fluorescence quantity adsorbed by the light phase magnetic particle position is detected by the fluorescence detector, the fluorescence quantity is proportional to the components in the detected sample, so that the content of the detected object is detected, the sensitivity, the repeatability and the chemiluminescence method of the detection result are basically similar, the fluorescence detector is small and exquisite, the carrying is easy, the cost is low, and the convenience of immunochromatography is realized.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The following examples were conducted under conventional conditions or conditions recommended by the manufacturer, without specifying the specific conditions. The raw materials, equipment or instruments used are conventional products which are commercially available without identifying manufacturers.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The magnetic fluid layering micro-control immunoassay reagent comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the R1 reagent comprises a first diluent and magnetic fluid which is distributed in the first diluent and is formed by immunomagnetic beads, the immunomagnetic beads are formed by coating magnetic particles with known antigens or antibodies, the R2 reagent comprises a second diluent and a marked conjugate distributed in the second diluent, the marked conjugate is an antigen or an antibody corresponding to a tested object marked by different fluorescent substances, and the R3 reagent is a strong hydrophobic liquid reagent which is incompatible with water.

In the invention, the strong hydrophobic liquid reagent is at least one of carbon tetrachloride, chloroform, liquid paraffin, gasoline, edible oil, methyl silicone oil, perfluorocarbon, chlorobenzene, petroleum benzene, petroleum ether and cyclohexane.

In the invention, each milliliter of R1 reagent contains 0.1-5 mg of immunomagnetic beads, each milligram of magnetic particles is coated with 0.1-10 micrograms of antigen or antibody, and the particle size of the magnetic particles is 0.1-10 micrometers.

The R1 reagent contains known antigens or antibodies corresponding to antibodies or antigens possibly present in the test object, wherein the antigens or antibodies include, but are not limited to, various infection index antibodies such as procalcitonin antibodies, interleukin series antibodies, various cardiovascular index antibodies such as troponin antibodies and brain natriuretic peptide antibodies, and various infectious disease antigens or antibodies such as hepatitis B surface antigens and cloth disease antigens, and the antigens or antibodies are coated on magnetic particles with the particle size of 0.1-10 mu m to form magnetic fluid.

In the invention, the diluent I is at least one of a tris buffer solution, a phosphate buffer solution, a borate buffer solution, a glycine buffer solution and a hydroxyethylpiperazine-ethylsulfuric acid buffer solution, wherein the buffer solution I contains 0.5-5.0 w per mill preservative, 0.1-2.0 w% serum protein, 0.05-2 w% amino acid and 0.01-0.1% surfactant, and the pH of the buffer solution is 5.0-9.0. The amino acid is at least one of arginine, lysine, serine and glycine.

In the invention, the second diluent is at least one of a tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution, a phosphate buffer solution and a borate buffer solution, wherein the second diluent comprises 0.5-5.0w% of protein stabilizer, 0.5-5.0w% of preservative, 0.1-2.0w% of bovine serum albumin and 0.01-0.1% of surfactant, and the pH of the buffer solution is 6.5-9.5. Wherein the protein stabilizer is at least one of sorbitol, disodium ethylenediamine tetraacetate, trehalose, carboxymethyl cellulose, cyclodextrin, polyethylene glycol 4000, polyethylene glycol 6000 and bovine serum albumin.

In the invention, the preservative is at least one of sodium azide, phenol and procline, and the surfactant is at least one of Tween-20, tween-80, triton 100 and SDS.

For details, please refer to examples 1-4:

Example 1

In the magnetic fluid layered micro-control immunoassay reagent of the embodiment, the R1 reagent comprises a diluent I and magnetic fluid formed by immunomagnetic beads distributed in the diluent I, wherein each milliliter of the R1 reagent contains 1mg of immunomagnetic beads, 5 micrograms of antigen or antibody is coated on each milligram of magnetic particles, and the particle size of the magnetic particles is 0.1-10 mu m. The first diluent of this example was a buffer solution one containing 3.0 w% preservative, 1.5w% serum protein, 1.0w% amino acid, and 0.05% surfactant, wherein the buffer solution one was a tris buffer solution, and the pH of the buffer solution one was 7.0. Wherein the amino acid is lysine.

The R2 reagent of this example comprises diluent two and labeled conjugate distributed in diluent two, wherein each milliliter of R2 reagent contains 3 micrograms of labeled conjugate. The second diluent is buffer solution II with unit volume and contains 3.0w% of protein stabilizer, 2.0 w% of preservative, 1.5w% of bovine serum albumin and 0.05w% of surfactant, and the buffer solution II is buffer solution of tris hydrochloride with pH of 7.5. Wherein the protein stabilizer is sorbitol.

The preservative in this example is sodium azide and the surfactant is tween-20. The R3 reagent of this embodiment is carbon tetrachloride.

The known antigen or antibody is coated on the magnetic particles according to the conventional method of coating the magnetic beads with the antigen or antibody, the magnetic particles coated with the known antigen or antibody are diluted by a diluent I, and magnetic fluid is formed in the diluent I, so that the R1 reagent is obtained. And (3) adopting a conventional fluorescent labeling technology, selecting different fluorescent substances to perform fluorescent labeling on antigens or antibodies corresponding to the detected substances, and adding the substances into a diluent for stirring to obtain an R2 reagent. In one container, the R1 reagent and the R2 reagent are mixed and packaged, and the R3 reagent is packaged separately.

Example 2

In the magnetic fluid layered micro-control immunoassay reagent of the embodiment, the R1 reagent comprises a diluent I and magnetic fluid formed by immunomagnetic beads distributed in the diluent I, wherein each milliliter of the R1 reagent contains 1.0mg of immunomagnetic beads, 8 micrograms of antigen or antibody is coated on each milligram of magnetic particles, and the particle size of the magnetic particles is 0.1-10 mu m. The first diluent of this example is a mixed solution of phosphate buffer and borate buffer in a mass ratio of 1:1, and the pH of the first diluent is 7.5, wherein the buffer contains 2.5w per mill preservative, 1.5w percent serum protein, 1.8w percent amino acid and 0.04 percent surfactant. Wherein the amino acid is a mixture of lysine and serine in a mass ratio of 1:1.

The R2 reagent of this example includes diluent two and labeled conjugate distributed in diluent two, with 8.0 micrograms of labeled conjugate per milliliter of R2 reagent. Wherein the second diluent is buffer solution II with unit volume and contains 1.0w% of protein stabilizer, 1.0w% of preservative, 1.8w% of bovine serum albumin and 0.08w% of surfactant; the buffer solution II is a mixture of phosphate buffer solution and borate buffer solution with the mass ratio of 1:1, and the pH value of the buffer solution is 7.0. The protein stabilizer is a mixture of trehalose and carboxymethyl cellulose in a mass ratio of 1:1.

The preservative in this example is a mixture of thiomersal and procline, in a mass ratio of 1:1, and the surfactant is a mixture of tween-20 and triton 100, in a mass ratio of 1:1. The R3 reagent of this example was a mixture of chloroform and methyl silicone oil in a mass ratio of 1:1.

Example 3

In the magnetic fluid layered micro-control immunoassay reagent of the embodiment, the R1 reagent comprises a diluent I and magnetic fluid formed by immunomagnetic beads distributed in the diluent I, wherein each milliliter of the R1 reagent contains 0.1mg of immunomagnetic beads, 10 micrograms of antigen or antibody is coated on each milligram of magnetic particles, and the particle size of the magnetic particles is 0.1-10 micrometers. The first diluent of this example is a buffer solution one containing 0.5 w/w preservative, 0.1w% serum protein, 0.05w% amino acid, and 0.01% surfactant, wherein the buffer solution one is a glycine buffer solution, and the pH of the buffer solution one is 5.0. Wherein the amino acid is glycine.

The R2 reagent of this example comprises diluent two and labeled conjugate distributed in diluent two, wherein each milliliter of R2 reagent contains 0.10 micrograms of labeled conjugate. Wherein the second diluent is buffer solution II with unit volume and contains 0.5w% of protein stabilizer, 0.5w% of preservative, 0.1w% of bovine serum albumin and 0.01w% of surfactant, and the buffer solution II is borate buffer solution with pH of 7.0. Wherein the protein stabilizer is carboxymethyl cellulose.

The preservative in this example is sodium azide and the surfactant is triamcinolone acetonide 100. The R3 reagent of this example is cyclohexane.

Example 4

In the magnetic fluid layered micro-control immunoassay reagent of the embodiment, the R1 reagent comprises a diluent I and magnetic fluid formed by immunomagnetic beads distributed in the diluent I, wherein each milliliter of the R1 reagent contains 5mg of immunomagnetic beads, each milligram of magnetic particles is coated with 0.1 microgram of antigen or antibody, and the particle size of the magnetic particles is 0.1-10 micrometers. The first diluent of this example is a buffer solution one containing 5.0w% preservative, 2.0w% serum protein, 2w% amino acid, and 0.1w% surfactant, wherein the buffer solution one is a phosphate buffer solution, and the pH of the buffer solution one is 9.0. Wherein the amino acid is arginine.

The R2 reagent of this example comprises diluent two and labeled conjugate distributed in diluent two, wherein 10 micrograms of labeled conjugate is contained in each milliliter of R2 reagent. The second diluent is buffer solution II with unit volume and pH of 9.5, and contains 5.0w% protein stabilizer, 5.0w% preservative, 2.0w% bovine serum albumin and 0.1% surfactant. Wherein the protein stabilizer is disodium ethylenediamine tetraacetate.

The preservative in this example is sodium azide and the surfactant is tween-20. The R3 reagent of this example is liquid paraffin.

The invention also provides a detection kit which comprises the magnetic fluid layering micro-control immunoassay reagent. The detection kit can be used in immune reaction detection analysis, and comprises the steps of adding 10-150 microliters of a detected sample into a mixed reagent containing 20-500 microliters of an R1 reagent and 10-500 microliters of an R2 reagent, adding 100-1000 microliters of an R3 reagent, reacting for 5-60 minutes, transferring magnetic particles participating in the reaction, detected objects and marked combinations adsorbed on the magnetic particles into a layered R3 reagent by using a magnet for fixation, placing the layered R3 reagent into a fluorescent detection instrument, detecting fluorescent intensity, wherein the fluorescent intensity is proportional to the content of the detected objects, and calculating the content of the detected objects.

Example 5

In this example, PCT assay was performed using a detection kit comprising the magnetic fluid layered micro-controlled immunoassay reagent of example 1, as follows:

100 microliters of R1 reagent coated with 5 micrograms of PCT monoclonal antibody is added into a plurality of reaction cups respectively, 200 microliters of fluorescent marker R2 reagent containing 3 micrograms of another bead of PCT monoclonal antibody is added into the reaction cups and mixed evenly, 400 microliters of R3 reagent is added into each reaction cup, 50 microliters of sample S0 (PCT concentration 0 ng/ml) to be tested, sample S1 (PCT concentration 0.1 ng/ml) to be tested, sample S2 (PCT concentration 0.35 ng/ml) to be tested, sample S3 (PCT concentration 2.69 ng/ml) to be tested, sample S4 (PCT concentration 11.76 ng/ml) to be tested, sample S5 (PCT concentration 33.85 ng/ml) to be tested are added into the mixed reagent of R1 and R2, the magnet is moved outside the reaction cups to enable magnetic particles in the reacted R1 reagent to move into the R3 reagent for 10 minutes, fluorescence intensity of the fluorescent particles is detected by a fluorescent detection instrument respectively, and fluorescence values obtained by the detection are shown in a table 1. The correlation coefficient R value (R= CORREL (LOG 10 (fluorescence value)), LOG10 (concentration)) is more than 0.99, and the linear range of the curve is similar to the linear range of chemiluminescence.

Sample of	Sample concentration (ng/ml)	Detecting fluorescence values
			S0	0	1199
S1	0.1	21804
			S2	0.35	47464
S3	2.69	167497
			S4	11.76	622393
S5	33.85	1791133
			S6	79.84	4230183

TABLE 1

The PCT monoclonal antibody was used for the precision detection test, and the detection kit containing the magnetic fluid layered micro-control immunoassay reagent of example 1 was used to detect the sample 1 with low concentration and the sample 2 with high concentration ten times, respectively, and the detection results are shown in table 2. The higher the test concentration variation coefficient and the fluorescence value variation coefficient, the worse the detection repeatability, and if the test concentration variation coefficient and the fluorescence value variation coefficient are smaller, the better the detection repeatability is, and further the higher the detection precision is, the lower the precision of the test is, and the higher the precision is, which is better than the precision of the immunochromatography method by more than 10%.

TABLE 2

PCT detection sensitivity tests were performed using a detection kit comprising the magnetic fluid layered micro-controlled immunoassay reagent of example 1, with the test results shown in table 3.

TABLE 3 Table 3

As can be seen from the data in tables 1, 2 and 3, the detection kit of the present invention has the advantages of high detection accuracy, reliable detection result, good repeatability, high precision and high sensitivity, and can detect samples with PCT monoclonal antibodies lower than 0.05ng/ml, which are higher than immunochromatography and are close to chemiluminescence.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.

Claims

1. The magnetic fluid layering micro-control immunoassay reagent is characterized by comprising the following components:

2. The magnetic fluid layered micro-control immunoassay reagent according to claim 1, wherein the strongly hydrophobic liquid reagent is at least one of carbon tetrachloride, chloroform, liquid paraffin, gasoline, edible oil, methyl silicone oil, perfluorocarbon, chlorobenzene, petroleum benzene, petroleum ether, and cyclohexane.

3. The magnetic fluid layering micro-control immunoassay reagent according to claim 1, wherein each milliliter of the R1 reagent contains 0.1-5 mg of immunomagnetic beads, each milligram of magnetic particles is coated with 0.1-10 micrograms of antigen or antibody, and the particle size of the magnetic particles is 0.1-10 micrometers.

4. The magnetic fluid layered micro-control immunoassay reagent according to claim 1, wherein the diluent first is buffer first with unit volume, and contains 0.5-5.0 w per mill preservative, 0.1-2.0 w% serum protein, 0.05-2 w% amino acid, and 0.01-0.1% surfactant;

the buffer solution I is at least one of a tris buffer solution, a phosphate buffer solution, a borate buffer solution, a glycine buffer solution and a hydroxyethyl piperazine ethyl thioic acid buffer solution, and the pH value of the buffer solution I is 5.0-9.0.

5. The magnetic fluid layered micro-control immunoassay reagent according to claim 4, wherein the amino acid is at least one of arginine, lysine, serine, glycine.

6. The magnetic fluid layered micro-control immunoassay reagent according to claim 1, wherein the second diluent is buffer solution II with unit volume, and contains 0.5-5.0w% of protein stabilizer, 0.5-5.0w% of preservative, 0.1-2.0w% of bovine serum albumin and 0.01-0.1% of surfactant;

the second buffer solution is at least one of a tris (hydroxymethyl) aminomethane hydrochloride buffer solution, a phosphate buffer solution and a borate buffer solution, and the pH of the second buffer solution is 6.5-9.5.

7. The magnetic fluid layered micro-control immunoassay reagent of claim 6, wherein the protein stabilizer is at least one of sorbitol, disodium edetate, trehalose, carboxymethyl cellulose, cyclodextrin, polyethylene glycol 4000, polyethylene glycol 6000, and bovine serum albumin.

8. The magnetic fluid layered micro-controlled immunoassay reagent of claim 4 or 6, wherein the preservative is at least one of sodium azide, thimerosal, phenol, and procline a;

the surfactant is at least one of Tween-20, tween-80, triton 100 and SDS.

9. A test kit comprising the magnetic fluid layered micro-controlled immunoassay reagent of any one of claims 1-8.

10. The application of the detection kit according to claim 9, wherein the application is in immunoreaction detection, and the application steps comprise adding 10-150 microlitres of a detected sample into a mixed reagent containing 20-500 microlitres of R1 reagent and 10-500 microlitres of R2 reagent, adding 100-1000 microlitres of R3 reagent, reacting for 5-60 minutes, transferring the magnetic particles participating in the reaction, the detected object and the marked conjugate adsorbed on the magnetic particles into a layered R3 reagent by utilizing a magnet, fixing, placing into a fluorescence detection instrument, detecting fluorescence intensity, and calculating to obtain the content of the detected object.