Magnetofluid layered micro-control immunoassay reagent and detection kit and application thereof

Abstract

The invention relates to the technical field of immunodetection reagents, in particular to a magnetic fluid layered micro-control immunoassay reagent, a detection kit and application of the magnetic fluid layered micro-control immunoassay reagent and the detection kit. The analysis reagent comprises a reagent R1, a reagent R2 and a reagent R3, the reagent R1 comprises a diluent I and magnetic fluid distributed in the diluent I and formed by immunomagnetic beads, and the reagent R3 comprises a reagent R2 and a reagent R3; the immunomagnetic beads are formed by coating magnetic particles with known antigens or antibodies; the R2 reagent comprises a diluent II and labeled conjugates distributed in the diluent II, and the labeled conjugates are antigens or antibodies corresponding to detected substances labeled by different fluorescent substances; and the R3 reagent is a strong hydrophobic liquid reagent incompatible with water. According to the invention, a strong hydrophobic light-phase substance is selected to realize separation of markers which do not participate in immunological reaction, stable magnetic force of a fixed magnetic field is selected to pull magnetic particles in magnetic fluid to move and gather, and multiplicity of fluorescence labeling is selected, so that high sensitivity, good consistency and repeatability of detection results, stable results, simplicity and convenience in operation and low cost of detection results are realized. The detection equipment is portable and the like.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay reagents, and in particular to a magnetic fluid layered micro-controlled immunoassay reagent and a detection kit and applications thereof. Background Art

[0002] The immunological analysis technologies and methods currently used in clinical practice include enzyme immunoassay technology, chemiluminescent immunoassay technology, immunochromatographic analysis technology and fluorescent immunoassay technology. These technologies each have their own advantages and disadvantages. Among them, chemiluminescent immunoassay technology and immunochromatographic analysis technology are the most widely used in clinical immunological diagnosis.

[0003] Chemiluminescent immunoassay technology is a technology for liquid phase reaction and detection of antigen-antibody reactions in aqueous solution. It has the advantages of high sensitivity, good repeatability, stable test results, and high degree of automation. The basic principle and method are: use magnetic microspheres to fix known antigens and antibodies, add the antigens and antibodies to be tested into the buffer system of the aqueous solution, and then add antigen-antibody markers marked with luminescent substances corresponding to the test object. After a certain period of reaction, the non-reaction or excess reactants are repeatedly washed and removed by the instrument, and then the excitation liquid is added to make it glow and the luminescence value is detected, so as to calculate the content of the test substance. This technology has the disadvantage that it requires highly automated detection instruments to complete all the processes such as sample addition and washing. The instrument has high cost and large size, is not convenient to carry and use, and cannot be used for home use and field emergency detection, and has high professional and technical requirements for users.

[0004] Immunochromatographic analysis technology is a detection technology that forms the result of antigen-antibody reaction on cellulose membrane through free movement of chromatography. The basic principle is: mark the known antigen and antibody with colloidal gold, fluorescent substance and other markers, and then fix them to nitrocellulose membrane, add the analyte to make its antigen and antibody react on the membrane, and then form an accumulation of reactants through free chromatography to the fixed line of another known antigen and antibody, and confirm the content of the analyte by detecting fluorescent or colloidal gold markers. It has the advantages of being simple and fast, with small size and low cost of supporting detection instruments, which can be carried around, no technical requirements for operators, and can also be used at home. However, this method also has disadvantages such as low sensitivity, poor repeatability, and unstable clinical test results.

[0005] Therefore, it is of great significance to develop an immunoassay technology with high sensitivity, good repeatability, stable test results and simple supporting instruments. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a magnetic fluid layered micro-controlled immunoassay reagent and a detection kit and their application, so as to at least solve the problems of the existing chemiluminescence immunoassay technology, such as high instrument cost, large size, and inconvenient portability.

[0007] The present invention solves the above technical problems by the following technical means:

[0008] In a first aspect, the present invention provides a magnetic fluid layered micro-controlled immunoassay reagent, comprising:

[0009] R1 reagent, including diluent 1 and magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, wherein the immunomagnetic beads are composed of magnetic particles coated with known antigens or antibodies;

[0010] R2 reagent, including diluent 2 and a labeled conjugate distributed in the diluent 2, wherein the labeled conjugate is an antigen or antibody corresponding to the analyte labeled with different fluorescent substances;

[0011] R3 reagent is a strongly hydrophobic liquid reagent that is incompatible with water.

[0012] In combination with 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, and cyclohexane.

[0013] In combination with the first aspect, in some embodiments, each milliliter of the R1 reagent contains 0.1 to 5 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 0.1 to 10 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1 to 10 μm.

[0014] In combination with the first aspect, in some embodiments, the diluent one is a buffer solution one containing 0.5-5.0w‰ preservative, 0.1-2.0w% serum protein, 0.05-2w% amino acid, and 0.01-0.1% surfactant per unit volume; the buffer solution one is at least one of tris(hydroxymethylaminomethane) buffer, phosphate buffer, borate buffer, glycine buffer and hydroxyethylpiperazineethanesulfonic acid buffer, and the pH of the buffer solution one is 5.0-9.0.

[0015] In combination with the first aspect, in some embodiments, the amino acid is at least one of arginine, lysine, serine, and glycine.

[0016] In combination with the first aspect, in some embodiments, the diluent two is a buffer solution two containing 0.5-5.0w% protein stabilizer, 0.5-5.0w‰ preservative, 0.1-2.0w% bovine serum albumin, and 0.01-0.1% surfactant per unit volume; the buffer solution two is at least one of tris(hydroxymethylaminomethane) hydrochloric acid buffer, phosphate buffer, and borate buffer, and the pH of the buffer solution two is 6.5-9.5.

[0017] In combination with 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.

[0018] In combination with the first aspect, in some embodiments, the preservative is at least one of sodium azide, thimerosal, phenol and procline300; the surfactant is at least one of Tween-20, Tween-80, Triton 100 and SDS.

[0019] In a second aspect, the present invention further provides a detection kit, which comprises the magnetic fluid layered micro-controlled immunoassay reagent described in the first aspect.

[0020] In a third aspect, the present invention further provides an application of the detection kit of the second aspect, wherein the application is an application in immune response detection, and the application steps are: adding 10 to 150 microliters of the sample to be tested to a mixed reagent containing 20 to 500 microliters of R1 reagent and 10 to 500 microliters of R2 reagent; then adding 100 to 1000 microliters of R3 reagent, the reaction time is 5 to 60 minutes, using a magnet to migrate the magnetic particles participating in the reaction and the test object and labeled conjugate adsorbed on the magnetic particles to the layered R3 reagent for fixation, placing it in a fluorescence detection instrument, detecting the fluorescence intensity, the fluorescence intensity is proportional to the content of the test substance, and the content of the test object is calculated.

[0021] The principle of using the magnetic fluid layered micro-controlled immunoassay reagent of the present invention for detection and analysis is as follows: using magnetic microspheres to fix different known antigens or antibodies, adding the antigen or antibody to be detected into the buffer system of the aqueous solution, and then adding antigen or antibody markers corresponding to the object to be detected and marked with different fluorescent substances, after a certain reaction time, using the magnetic force formed by a special magnet to move the magnetic particles involved in the reaction and the object to be detected and the fluorescent marker adsorbed on the magnetic particles from the aqueous solution liquid to a fixed position in a strongly hydrophobic liquid reagent incompatible with water, and then calculating and confirming the content of the object to be detected by detecting the fluorescence value in the strongly hydrophobic liquid reagent.

[0022] The present invention selects a strongly hydrophobic liquid reagent and layers it with a mixed reagent of R1 reagent and R2 reagent containing magnetic fluid for immune reaction, so that the antigen or antibody reaction can smoothly carry out immunological liquid phase reaction in the mixed reagent of R1 reagent and R2 reagent like chemiluminescence, so as to ensure its high sensitivity. At the same time, a highly stable, non-toxic, harmless, non-interfering immune response, transparent and light-permeable strongly hydrophobic liquid reagent layer is layered on the upper or lower layer of the mixed reagent to ensure stable test results. A strong magnetic field is then formed through a special magnet to move the fluorescent markers and the objects to be tested that have undergone immunological reactions in the magnetic fluid and are connected to the magnetic particles along with the magnetic particles, and are moved from the mixed reagent to a specified position of the strongly hydrophobic liquid reagent layer under the traction of a constant magnetic force, and form an easily detectable shape. By exchanging or changing the position of the layered mixed reagent and the strongly hydrophobic liquid reagent layer, only substances that have participated in the immune reaction and are connected to the magnetic particles can be in a fixed detection position to avoid interference from other substances that are not connected to the magnetic particles. Since the movement is carried out under the action of a fixed magnetic field force, the consistency of the content of the magnetic particles separated each time is guaranteed, and the problem of the heterogeneity of the free movement of immunochromatography is solved, thereby ensuring the repeatability of the test results. The amount of adsorbed fluorescence at the position of the light phase magnetic particles is then detected by a fluorescence detector. The amount of fluorescence is proportional to the components in the sample being tested, thereby detecting the content of the test object. The sensitivity and repeatability of the test results are basically close to those of the chemiluminescence method, and the fluorescence detector is small, easy to carry, and low in cost, realizing the convenience of immunochromatography. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The raw materials, equipment or instruments used, if no manufacturer is specified, are all conventional products that can be purchased on the market.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The magnetic fluid layered micro-controlled immunoassay reagent of the present invention comprises R1 reagent, R2 reagent and R3 reagent, wherein R1 reagent comprises a diluent 1 and a magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, wherein the immunomagnetic beads are magnetic particles coated with known antigens or antibodies; R2 reagent comprises a diluent 2 and a labeled conjugate distributed in the diluent 2, wherein the labeled conjugate is an antigen or antibody corresponding to a test object labeled with different fluorescent substances; and R3 reagent is a strongly hydrophobic liquid reagent incompatible with water.

[0027] In the present 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.

[0028] In the present invention, each milliliter of R1 reagent contains 0.1 to 5 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 0.1 to 10 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1 to 10 μm.

[0029] The R1 reagent contains known antigens or antibodies corresponding to the possible antibodies or antigens of the object being tested, including but not limited to various infection indicator antibodies such as procalcitonin antibodies and interleukin series antibodies, various cardiovascular indicator antibodies such as troponin antibodies and brain natriuretic peptide antibodies, and various infectious disease antigens or antibodies such as hepatitis B surface antigen and brucellosis antigen. These antigens or antibodies are coated on magnetic particles with a particle size of 0.1 to 10 μm to form a magnetic fluid.

[0030] In the present invention, the diluent is a buffer solution containing 0.5-5.0w‰ preservative, 0.1-2.0w% serum protein, 0.05-2w% amino acid, and 0.01-0.1% surfactant per unit volume; the buffer solution is at least one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, borate buffer, glycine buffer, and hydroxyethylpiperazine ethanesulfonic acid buffer, 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.

[0031] In the present invention, the diluent 2 is a buffer 2 per unit volume containing 0.5-5.0w% protein stabilizer, 0.5-5.0w‰ preservative, 0.1-2.0w% bovine serum albumin, and 0.01-0.1% surfactant; the buffer 2 is at least one of tris(hydroxymethylaminomethane) hydrochloric acid buffer, phosphate buffer, and borate buffer, and the pH of the buffer is 6.5-9.5. The protein stabilizer is at least one of sorbitol, disodium ethylenediaminetetraacetic acid, trehalose, carboxymethyl cellulose, cyclodextrin, polyethylene glycol 4000, polyethylene glycol 6000, and bovine serum albumin.

[0032] In the present invention, the preservative is at least one of sodium azide, phenol and procline300; the surfactant is at least one of Tween-20, Tween-80, Triton 100 and SDS.

[0033] For details, please refer to Examples 1-4:

[0034] Example 1

[0035] In the magnetic fluid layered micro-controlled immunoassay reagent of this embodiment, the R1 reagent includes a diluent 1 and a magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, each milliliter of the R1 reagent contains 1 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 5 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1-10 μm. The diluent 1 of this embodiment is a buffer 1 per unit volume containing 3.0w‰ preservatives, 1.5w% serum protein, 1.0w% amino acids, and 0.05% surfactants; the buffer 1 is a tris(hydroxymethyl)aminomethane buffer, and the pH of the buffer 1 is 7.0. The amino acid is lysine.

[0036] The R2 reagent of this embodiment includes a diluent 2 and a labeled conjugate distributed in the diluent 2, and each milliliter of the R2 reagent contains 3 micrograms of the labeled conjugate. The diluent 2 is a buffer 2 per unit volume containing 3.0w% protein stabilizer, 2.0w‰ preservative, 1.5w% bovine serum albumin, and 0.05w% surfactant; the buffer 2 is a tris(hydroxymethyl)aminomethane hydrochloric acid buffer, and the pH of the buffer is 7.5. The protein stabilizer is sorbitol.

[0037] The preservative in this embodiment is sodium azide; the surfactant is Tween-20. The R3 reagent in this embodiment is carbon tetrachloride.

[0038] According to the conventional method of coating magnetic beads with antigens or antibodies, the known antigens or antibodies are coated on magnetic particles, and the magnetic particles coated with the known antigens or antibodies are diluted with diluent 1 to form a magnetic fluid in the diluent 1 to obtain the R1 reagent. Using conventional fluorescent labeling technology, different fluorescent substances are selected to fluorescently label the antigens or antibodies corresponding to the test object, and added to the diluent and stirred to obtain the R2 reagent. In one container, the R1 reagent and the R2 reagent are mixed and packaged, and the R3 reagent is packaged separately.

[0039] Example 2

[0040] In the magnetic fluid layered micro-controlled immunoassay reagent of this embodiment, the R1 reagent includes a diluent 1 and a magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, each milliliter of the R1 reagent contains 1.0 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 8 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1-10 μm. The diluent 1 of this embodiment is a buffer 1 per unit volume containing 2.5w‰ preservatives, 1.5w% serum protein, 1.8w% amino acids, and 0.04% surfactant; the buffer 1 is a mixture of phosphate buffer and borate buffer in a mass ratio of 1:1, and the pH of the buffer 1 is 7.5. The amino acids are a mixture of lysine and serine in a mass ratio of 1:1.

[0041] The R2 reagent of this embodiment includes diluent 2 and a labeled conjugate distributed in diluent 2, and each milliliter of R2 reagent contains 8.0 micrograms of labeled conjugate. Among them, diluent 2 is a unit volume of buffer 2 containing 1.0w% protein stabilizer, 1.0w‰ preservative, 1.8w% bovine serum albumin, and 0.08w% surfactant; buffer 2 is a mixture of phosphate buffer and borate buffer in a mass ratio of 1:1, and the pH of the buffer is 7.0. The protein stabilizer is a mixture of trehalose and carboxymethyl cellulose in a mass ratio of 1:1.

[0042] The preservative in this embodiment is a mixture of thimerosal and procline 300 in a mass ratio of 1:1; the surfactant is a mixture of Tween-20 and Triton 100 in a mass ratio of 1:1. The R3 reagent in this embodiment is a mixture of chloroform and methyl silicone oil in a mass ratio of 1:1.

[0043] According to the conventional method of coating magnetic beads with antigens or antibodies, the known antigens or antibodies are coated on magnetic particles, and the magnetic particles coated with the known antigens or antibodies are diluted with diluent 1 to form a magnetic fluid in the diluent 1 to obtain the R1 reagent. Using conventional fluorescent labeling technology, different fluorescent substances are selected to fluorescently label the antigens or antibodies corresponding to the test object, and added to the diluent and stirred to obtain the R2 reagent. In one container, the R1 reagent and the R2 reagent are mixed and packaged, and the R3 reagent is packaged separately.

[0044] Example 3

[0045] In the magnetic fluid layered micro-controlled immunoassay reagent of this embodiment, the R1 reagent includes a diluent 1 and a magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, each milliliter of the R1 reagent contains 0.1 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 10 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1 to 10 μm. The diluent 1 of this embodiment is a buffer 1 per unit volume containing 0.5w‰ preservative, 0.1w% serum protein, 0.05w% amino acid, and 0.01% surfactant; the buffer 1 is a glycine buffer, and the pH of the buffer 1 is 5.0. The amino acid is glycine.

[0046] The R2 reagent of this embodiment includes a diluent 2 and a labeled conjugate distributed in the diluent 2, and each milliliter of the R2 reagent contains 0.10 micrograms of the labeled conjugate. The diluent 2 is a buffer 2 per unit volume containing 0.5w% protein stabilizer, 0.5w‰ preservative, 0.1w% bovine serum albumin, and 0.01w% surfactant; the buffer 2 is a borate buffer, and the pH of the buffer is 7.0. The protein stabilizer is carboxymethyl cellulose.

[0047] The preservative in this embodiment is sodium azide; the surfactant is Triton 100. The R3 reagent in this embodiment is cyclohexane.

[0048] According to the conventional method of coating magnetic beads with antigens or antibodies, the known antigens or antibodies are coated on magnetic particles, and the magnetic particles coated with the known antigens or antibodies are diluted with diluent 1 to form a magnetic fluid in the diluent 1 to obtain the R1 reagent. Using conventional fluorescent labeling technology, different fluorescent substances are selected to fluorescently label the antigens or antibodies corresponding to the test object, and added to the diluent and stirred to obtain the R2 reagent. In one container, the R1 reagent and the R2 reagent are mixed and packaged, and the R3 reagent is packaged separately.

[0049] Example 4

[0050] In the magnetic fluid layered micro-controlled immunoassay reagent of this embodiment, the R1 reagent includes a diluent 1 and a magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, each milliliter of the R1 reagent contains 5 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 0.1 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1 to 10 μm. The diluent 1 of this embodiment is a buffer 1 per unit volume containing 5.0w‰ preservatives, 2.0w% serum protein, 2w% amino acids, and 0.1w% surfactant; the buffer 1 is a phosphate buffer, and the pH of the buffer 1 is 9.0. The amino acid is arginine.

[0051] The R2 reagent of this embodiment includes a diluent 2 and a labeled conjugate distributed in the diluent 2, and each milliliter of the R2 reagent contains 10 micrograms of the labeled conjugate. The diluent 2 is a buffer 2 per unit volume containing 5.0w% protein stabilizer, 5.0w‰ preservative, 2.0w% bovine serum albumin, and 0.1% surfactant; the buffer 2 is a phosphate buffer, and the pH of the buffer is 9.5. The protein stabilizer is disodium ethylenediaminetetraacetic acid.

[0052] The preservative in this embodiment is sodium azide; the surfactant is Tween-20. The R3 reagent in this embodiment is liquid paraffin.

[0053] According to the conventional method of coating magnetic beads with antigens or antibodies, the known antigens or antibodies are coated on magnetic particles, and the magnetic particles coated with the known antigens or antibodies are diluted with diluent 1 to form a magnetic fluid in the diluent 1 to obtain the R1 reagent. Using conventional fluorescent labeling technology, different fluorescent substances are selected to fluorescently label the antigens or antibodies corresponding to the test object, and added to the diluent and stirred to obtain the R2 reagent. In one container, the R1 reagent and the R2 reagent are mixed and packaged, and the R3 reagent is packaged separately.

[0054] The present invention also provides a detection kit, comprising the magnetic fluid layered micro-controlled immunoassay reagent of the present invention. The detection kit can be used in immune response detection analysis, and the application steps are: add 10 to 150 microliters of the sample to be tested to a mixed reagent containing 20 to 500 microliters of R1 reagent and 10 to 500 microliters of R2 reagent; then add 100 to 1000 microliters of R3 reagent, the reaction time is 5 to 60 minutes, use a magnet to migrate the magnetic particles involved in the reaction and the test object and the labeled binding substance adsorbed on the magnetic particles to the layered R3 reagent for fixation, place it in a fluorescence detection instrument, detect the fluorescence intensity, the fluorescence intensity is proportional to the content of the substance to be tested, and the content of the substance to be tested is calculated.

[0055] Example 5

[0056] This example uses a detection kit containing the magnetic fluid layered micro-controlled immunoassay reagent of Example 1 to perform a PCT detection test, as follows:

[0057] 100 μl of R1 reagent coated with 5 μg PCT monoclonal antibody was added to multiple reaction cups, 200 μl of R2 reagent containing 3 μg of another PCT monoclonal antibody was added and mixed, 400 μl of R3 reagent was added, and 50 μl of the tested sample S0 (PCT concentration 0 ng / ml), the tested sample S1 (PCT concentration 0.1 ng / ml), the tested sample S2 (PCT concentration 0.35 ng / ml), the tested sample S3 (PCT concentration 0.1 ng / ml) were added to each reaction cup. T concentration 2.69ng / ml), the test sample S4 (PCT concentration 11.76ng / ml), the test sample S5 (PCT concentration 33.85ng / ml) calibrator, the test sample S6 (PCT concentration 79.84ng / ml) were added to the mixed reagents of R1 and R2, reacted for 10 minutes, and the magnet outside the reaction cup was moved to move the magnetic particles in the reacted R1 reagent to the R3 reagent, and the fluorescence intensity was detected by a fluorescence detection instrument. The fluorescence values ​​obtained by the test are shown in the detection fluorescence values ​​in Table 1. The correlation coefficient R value (R = CORREL (LOG10 (fluorescence value), LOG10 (concentration))) is greater than 0.99, and the linear range of the curve is similar to the linear range of chemiluminescence.

[0058] sample Sample concentration (ng / ml) Detection of fluorescence value 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

[0059] Table 1

[0060] The PCT monoclonal antibody was used to perform precision detection test, and the detection kit containing the magnetic fluid layered micro-controlled immunoassay reagent of Example 1 was used to detect the low-concentration sample 1 and the high-concentration sample 2 ten times, respectively. The detection results are shown in Table 2. The larger the test concentration variation coefficient and the fluorescence value variation coefficient, the worse the repeatability of the detection; if the test concentration variation coefficient and the fluorescence value variation coefficient are smaller, the better the repeatability of the detection, and further the higher the precision of the detection, the precision of this test is less than 5%, which is better than the precision of more than 10% of the immunochromatography method.

[0061]

[0062] Table 2

[0063] A PCT detection sensitivity test was performed using a detection kit comprising the magnetic fluid layered micro-controlled immunoassay reagent of Example 1. The test results are shown in Table 3.

[0064]

[0065]

[0066] Table 3

[0067] The data in Tables 1, 2 and 3 indicate that, from multiple detection tests of PCT monoclonal antibodies, it can be seen that the detection kit of the present invention has a high detection accuracy, reliable detection results, good repeatability, high precision, and high sensitivity, and can detect samples with PCT monoclonal antibodies below 0.05 ng / ml, and its sensitivity is higher than that of the immunochromatography method and close to that of the chemiluminescence method.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention. The techniques, shapes, and structural parts not described in detail in the present invention are all known technologies.

Claims

1. A magnetic fluid layered micro-controlled immunoassay reagent, characterized in that: include: R1 reagent, including diluent 1 and magnetic fluid formed by immunomagnetic beads distributed in the diluent 1, wherein the immunomagnetic beads are composed of magnetic particles coated with known antigens or antibodies; R2 reagent, including diluent 2 and a labeled conjugate distributed in the diluent 2, wherein the labeled conjugate is an antigen or antibody corresponding to the analyte labeled with different fluorescent substances; R3 reagent is a strongly hydrophobic liquid reagent that is incompatible with water.

2. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 1, characterized in that: 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.

3. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 1, characterized in that: Each milliliter of the R1 reagent contains 0.1 to 5 mg of immunomagnetic beads, and each milligram of magnetic particles is coated with 0.1 to 10 micrograms of antigen or antibody; the particle size of the magnetic particles is 0.1 to 10 μm.

4. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 1, characterized in that: The diluent is a buffer solution containing 0.5-5.0w‰ preservative, 0.1-2.0w% serum protein, 0.05-2w% amino acid, and 0.01-0.1% surfactant per unit volume; The buffer solution one is at least one of a tris(hydroxymethylaminomethane) buffer solution, a phosphate buffer solution, a borate buffer solution, a glycine buffer solution and a hydroxyethylpiperazineethanesulfonic acid buffer solution, and the pH of the buffer solution one is 5.0 to 9.

0.

5. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 4, characterized in that: The amino acid is at least one of arginine, lysine, serine and glycine.

6. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 1, characterized in that: The diluent 2 is a buffer 2 containing 0.5-5.0w% protein stabilizer, 0.5-5.0w‰ preservative, 0.1-2.0w% bovine serum albumin, and 0.01-0.1% surfactant per unit volume; The buffer solution 2 is at least one of a tris(hydroxymethylaminomethane) hydrochloric acid buffer solution, a phosphate buffer solution, and a borate buffer solution, and the pH of the buffer solution 2 is 6.5 to 9.

5.

7. The magnetic fluid layered micro-controlled immunoassay reagent according to claim 6, characterized in that: 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 according to claim 4 or 6, characterized in that: The preservative is at least one of sodium azide, thimerosal, phenol and procline300; The surfactant is at least one of Tween-20, Tween-80, Triton 100 and SDS.

9. A detection kit, characterized in that: The detection kit comprises the magnetic fluid layered micro-controlled immunoassay reagent as described in any one of claims 1 to 8.

10. Use of the detection kit according to claim 9, characterized in that: The application is an application in immune response detection, and the application steps are: adding 10 to 150 microliters of the sample to be tested to a mixed reagent containing 20 to 500 microliters of R1 reagent and 10 to 500 microliters of R2 reagent; then adding 100 to 1000 microliters of R3 reagent, the reaction time is 5 to 60 minutes, using a magnet to migrate the magnetic particles participating in the reaction and the test object and the labeled conjugate adsorbed on the magnetic particles to the layered R3 reagent for fixation, placing it in a fluorescence detection instrument, detecting the fluorescence intensity, and calculating the content of the test object.

Images