Magnetic microsphere solution for helicobacter pylori IgM / IgG antibody combined typing detection and kit and application thereof

Abstract

The invention relates to the technical field of chemiluminescence immunodetection, in particular to a magnetic microsphere solution for helicobacter pylori IgM / IgG antibody combined typing detection and a kit and application thereof. According to the detection reagent and the kit provided by the invention, the helicobacter pylori antibody can be accurately detected and subjected to typing through one-time sample introduction under the condition that a sample does not need to be manually diluted, and an infection stage is reported, so that the detection sensitivity and the accuracy of a detection result are greatly improved, the detection process is simplified, and the detection speed is increased.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescent immunoassay, and in particular to a magnetic microsphere solution for combined typing detection of Helicobacter pylori IgM / IgG antibodies, a kit and an application thereof. Background Art

[0002] In 1982, Australian scholar Marshall observed a bacterium in the gastric mucosa that was associated with the onset of chronic gastric disease. In 1989, Goodwin et al. named it Helicobacter pylori (HP), which was recognized by the academic community. In 1994, the World Health Organization / International Agency for Research on Cancer (WHO / IARC) designated Helicobacter pylori as a human carcinogen.

[0003] Helicobacter pylori is the only bacterium known to survive in the human stomach for a long time. The main routes of infection of Helicobacter pylori are oral-oral and fecal-oral transmission, such as chewing, kissing, sharing meals, food contamination, etc., so a large number of people are infected. After infection, many diseases are induced, not only stomach-related diseases (gastric acid, bloating, stomach pain, gastritis, gastric ulcer, gastric cancer, etc.), but also related to diseases outside the stomach (iron deficiency anemia, migraine, cerebral infarction, urticaria, etc.). After infection with Helicobacter pylori, the risk of gastric cancer may increase by 2.7 to 12 times. The infection rate of Helicobacter pylori in China is high, and eradication of Helicobacter pylori is a feasible measure for the treatment of peptic ulcers and the prevention of gastric cancer. Some patients infected with Helicobacter pylori will have symptoms of related gastric diseases, but some patients will not have obvious symptoms, and they can only be discovered through testing.

[0004] Currently, Helicobacter pylori can be divided into type I and type II according to the different toxin secretion conditions. Type I: Helicobacter pylori strains can produce VacA and CagA, causing widespread tissue inflammation, easily leading to gastritis and ulcers; Type II: CagA and VacA negative strains do not produce virulence factors VacA and CagA. This type of Helicobacter pylori strain is less toxic and does not show symptoms after infection or only causes mild chronic superficial gastritis. Based on the different typing results of Helicobacter pylori, it can be decided whether to perform eradication treatment on the infected person, which can save some type II infected people from eradication treatment and the side effects of antibiotics.

[0005] Currently, most of the kits on the market are for measuring serum IgG antibodies. Serum antibodies can reflect the body's immune response to Helicobacter pylori, so it is necessary to detect antibodies. However, IgG-specific antibodies will only appear in the blood several weeks after Helicobacter pylori infection, and antibodies in the blood can last for more than 6 months after bacterial eradication. Therefore, Helicobacter pylori IgG antibodies cannot confirm that the patient has active infection, and a negative result cannot rule out early infection. Therefore, this test can only be used for epidemiological screening. Combined IgM antibody detection has greater clinical value.

[0006] There are many immunological detection methods that detect Helicobacter pylori infection by measuring Helicobacter pylori antibodies in serum, including complement fixation test, agglutination test, passive hemagglutination test, immunoblotting technology and enzyme-linked immunosorbent assay (ELISA), etc. These methods have low reagent cost and simple operation, but low sensitivity and accuracy, and are only suitable for qualitative and semi-quantitative diagnosis. They also have poor freeze-thaw stability, which not only affects the test results but is also not conducive to transportation and storage. Summary of the invention

[0007] In view of this, the present invention provides a magnetic microsphere solution for combined typing detection of Helicobacter pylori IgM / IgG antibodies, a kit and an application thereof. Typing and detecting multiple Helicobacter pylori IgM / IgG antibodies using the magnetic microsphere solution of the present invention has the advantages of good freeze-thaw stability, accurate detection results, high sensitivity, and simple operation.

[0008] The present invention provides a magnetic microsphere solution, which comprises a sealing solution, magnetic microspheres and 10-200 mmol / L amino acids;

[0009] The magnetic microspheres include magnetic microspheres coated with type I Helicobacter pylori antigens and / or magnetic microspheres coated with type II Helicobacter pylori antigens;

[0010] The amino acid includes at least one of arginine, lysine, histidine, cysteine ​​and methionine.

[0011] In some embodiments, the concentration of amino acids in the magnetic microsphere solution is 10 mmol / L, 50 mmol / L, 100 mmol / L, 150 mmol / L, or 200 mmol / L.

[0012] The present invention optimizes the magnetic microsphere solution, and adds a specific concentration of amino acid (specifically arginine) in the preparation of the magnetic microsphere solution. Experiments show that after adding a specific concentration of arginine to the magnetic microsphere solution, the accuracy of the test result can be effectively improved, and the freeze-thaw stability of the test kit can be improved. The test kit containing the magnetic microsphere solution of the present invention can distinguish four antibodies of Helicobacter pylori type I HP-IgM, type I HP-IgG, type II HP-IgM, and type II HP-IgG, and report the antibody concentration at the same time, which is better suitable for the complex situation of Helicobacter pylori infected persons, reporting whether it is infected by toxic strains or non-toxic strains, and reporting whether it is a current infection or a previous infection. The method has good freeze-thaw stability, simple operation, accurate test results, and high sensitivity.

[0013] In the magnetic microsphere solution of the present invention, the concentration of Helicobacter pylori antigen is 50 μg / L to 2000 μg / L, preferably 500 to 800 μg / L, specifically 500 μg / L, 600 μg / L, 700 μg / L, and 800 μg / L.

[0014] The concentration of the magnetic microspheres is 0.1-5 mg / mL, preferably 0.6-2 mg / mL, specifically 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2.0 mg / mL. The particle size of the magnetic microspheres is 1-3 μm, specifically 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm.

[0015] In the present invention, the magnetic microspheres are micron-sized magnetic microspheres formed by the composite of nano-sized Fe2O3 and organic polymer materials, or micron-sized magnetic microspheres formed by the composite of Fe3O4 magnetic particles and organic polymer materials.

[0016] In some specific embodiments of the present invention, the sealing solution is a buffer containing 1wt% to 5wt% of a sealing agent, 0.1wt% to 0.20wt% of a surfactant, and 0.05wt% to 0.10wt% of a preservative. The buffer is Tris, HEPES, bis-Tris, MES buffer, or PBS buffer. The present invention has no special requirements for the specific types of the sealing agent and the surfactant, and any common types in the art can be used. For example, the sealing agent can be bovine serum albumin, animal serum (calf serum, fetal bovine serum, goat serum, etc.), Casein, etc. The surfactant can be TritonX-100, sodium dodecyl sulfate (SDS), CHAPS, tween, etc.

[0017] In some embodiments, the type I Helicobacter pylori antigens include CagA antigen and / or VacA antigen, and the type II Helicobacter pylori antigens include at least one of UreA antigen, UreB antigen, and HspA antigen.

[0018] The present invention also provides a kit for Helicobacter pylori IgG / IgM typing or detection, which comprises the magnetic microsphere solution of the present invention.

[0019] In some embodiments, the kit includes, in addition to the magnetic microsphere solution described in the present invention, common components of the kit, such as calibrators and / or labeled tracer solutions. Wherein, the labeled tracer solution includes a labeled tracer solution of labeled anti-human IgG antibodies and / or a labeled tracer solution of labeled anti-human IgM antibodies. Wherein, in addition to the labeled tracers of the labeled antibodies (anti-human IgG antibodies and / or anti-human IgM antibodies), the labeled tracer solution of anti-human IgG antibodies also includes a diluent. The present invention has no special requirements for the formula of the diluent, and any commonly used diluent in the art can be used, such as a buffer containing 10 mg / ml to 200 mg / ml protein (such as BSA) and 0.4 mg / ml to 1.5 mg / ml preservatives; the buffer is a Tris buffer or a PBS buffer.

[0020] In some embodiments, in the labeled tracer solution of the labeled anti-human IgG antibody, the concentration of the anti-human IgG antibody is 10-200 μg / L, preferably 60-100 μg / L, specifically 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L, 100 μg / L; the concentration of the labeled tracer is 0.1-1 mg / l, preferably 0.4-0.7 mg / l, specifically 0.4 mg / l, 0.5 mg / l, 0.6 mg / l, 0.7 mg / l.

[0021] In the labeled tracer solution of the labeled anti-human IgM antibody, the concentration of the anti-human IgM antibody is 10-200 μg / L, preferably 60-100 μg / L, specifically 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L, 100 μg / L; the concentration of the labeled tracer is 0.1-1 mg / l, preferably 0.4-0.7 mg / l, specifically 0.4 mg / l, 0.5 mg / l, 0.6 mg / l, 0.7 mg / l.

[0022] The present invention has no special restrictions on the specific types of labeled tracers, and any common types in the art can be used. Those skilled in the art can make a selection according to the luminescence principle of the detection. For example, when the luminescence mode is enzymatic chemiluminescence, horseradish peroxidase (HRP) or alkaline phosphatase (ALP) can be used as a luminescent marker, and luminol and hydrogen peroxide or adamantane and its derivatives can be used as luminescent substrates; when the luminescence mode is direct chemiluminescence, non-enzymatic small molecule organic compounds can be used as luminescent markers, such as acridinium esters, isoluminol ABEI and its derivatives; when the luminescence mode is electrochemical luminescence, terpyridine ruthenium can be selected as a marker, which undergoes an electrochemical reaction under the action of tripropylamine and voltage to emit visible light.

[0023] In some specific embodiments, the detection reagent includes the following components 1) to 3):

[0024] 1) R1-1: a magnetic microsphere solution coated with type I HP antigen (500-800 μg / L) (magnetic microsphere concentration 0.1-5 mg / mL, amino acid concentration 100 mmol / L arginine);

[0025] 2) R1-2: a magnetic microsphere solution coated with type II HP antigen (500-800 μg / L) (magnetic microsphere concentration 0.1-5 mg / mL, amino acid is arginine at a concentration of 100 mmol / L);

[0026] 3) R3-1: labeled tracer solution of labeled anti-human IgM antibody (60-100 μg / L).

[0027] The present invention also provides a kit for Helicobacter pylori IgG / IgM typing or detection, which comprises a calibrator C1, a calibrator C2 and the detection reagent of the present invention.

[0028] Further, the calibrator C1 is a negative human serum that does not contain type I and type II HP IgG and does not contain type I and type II HP IgM;

[0029] The calibrator C2 is human serum containing at least one of type I HP IgG, type II HP IgG, type I HP IgM, and type II HP IgM.

[0030] The luminescence mode of the kit is any one of enzymatic chemiluminescence (using horseradish peroxidase (HRP) or alkaline phosphatase (ALP) as a luminescent marker, luminol and hydrogen peroxide or adamantane and its derivatives as luminescent substrates), direct chemiluminescence (using non-enzymatic small molecule organic compounds as luminescent markers, such as acridinium ester, isoluminol ABEI and its derivatives), and electrochemiluminescence (using terpyridine ruthenium as a marker, electrochemical reaction occurs under the action of tripropylamine and voltage, and visible light is emitted).

[0031] In some embodiments, the kit of the present invention further comprises a sample diluent. The present invention has no special requirements for the formulation of the diluent, and any diluent commonly used in the art can be used, for example, Tris or PBS buffer containing 10 mg / ml to 200 mg / ml protein (such as BSA) and 0.4 mg / ml to 1.5 mg / ml preservative.

[0032] The present invention also provides a method for typing or detecting Helicobacter pylori IgG / IgM, using the detection reagent or the kit of the present invention to detect or type IgG and / or IgM in a sample.

[0033] Furthermore, the detection method of the present invention comprises the following steps:

[0034] After mixing the sample to be tested with the magnetic microsphere solution, incubate at 25-40°C for 5-20 minutes;

[0035] Then, the labeled tracer solution of the labeled anti-human IgG antibody and the labeled tracer solution of the labeled anti-human IgM antibody are added, incubated at 25-40° C. for 5-20 minutes, and then washed to obtain a reaction product;

[0036] Applying an external magnetic field to precipitate the reaction product, removing the supernatant, washing, adding a chemiluminescent exciter, and detecting the relative light intensity emitted;

[0037] The typing of Helicobacter pylori in the sample to be tested and the IgG / IgM antibody concentration of each type of bacteria are calculated.

[0038] The beneficial effects of the present invention are:

[0039] The detection reagent and kit of the present invention can directly detect antibodies in the sample, type Helicobacter pylori, and optionally provide the concentrations of various IgM / IgG antibodies of various types, thereby realizing that Helicobacter pylori antibodies can be accurately detected and typed in one injection without manually diluting the sample, and the infection stage can be reported. The present invention effectively improves the freeze-thaw stability of the reagent by adding specific types of amino acids to the magnetic microsphere solution, thereby ensuring the accuracy of the test results. The detection sensitivity is improved, the detection process is simplified, and the detection speed is accelerated. And the joint test of 1-4 antibody indicators can be flexibly selected through instrument settings. The kit provided by the present invention contains only 4 components, is small in size, and has large functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the components of the Helicobacter pylori IgM / IgG antibody combined typing detection kit on the magnetic particle platform of the present invention;

[0041] Figure 2 This is a schematic flow chart of the detection method of a Helicobacter pylori IgM / IgG antibody combined typing detection kit on a magnetic particle platform provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0042] The present invention provides a Helicobacter pylori IgM / IgG antibody combined typing detection kit, detection method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0043] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0044] The Helicobacter pylori IgM / IgG antibody combined typing detection kit provided by the present invention comprises the following components:

[0045] A magnetic microsphere solution (comprising CagA antigen and / or VacA antigen) directly coated with or indirectly linked to type I Helicobacter pylori antigen, wherein the magnetic microsphere solution contains amino acids;

[0046] A magnetic microsphere solution (comprising UreA antigen, UreB antigen and / or HspA antigen) directly coated with or indirectly linked to type II Helicobacter pylori antigen, wherein the magnetic microsphere solution contains amino acids; and / or

[0047] a labeled tracer solution directly coated or indirectly linked to an anti-human IgM antibody; and a labeled tracer solution directly coated or indirectly linked to an anti-human IgG antibody.

[0048] Preferably, the magnetic microsphere solution contains amino acids, wherein the amino acids are at least one of arginine, lysine, histidine, cysteine ​​and methionine, and the concentration is 10-200 mmol / L.

[0049] Preferably, the magnetic microsphere solution directly coated or indirectly linked to the type I Helicobacter pylori antigen comprises: the magnetic microspheres are coated with avidin and then linked to the type I Helicobacter pylori antigen via biotin; or the magnetic microspheres are linked to the type I Helicobacter pylori antigen via active groups.

[0050] Preferably, the magnetic microsphere solution directly coated or indirectly linked to the type II Helicobacter pylori antigen comprises: the magnetic microspheres are coated with avidin and then linked to the type II Helicobacter pylori antigen via biotin; or the magnetic microspheres are linked to the type II Helicobacter pylori antigen via active groups.

[0051] Preferably, the particle size of the magnetic microspheres is 1 μm to 3 μm; the magnetic microspheres are micrometer-sized magnetic microspheres formed by a composite of nanometer-sized Fe2O3 and organic polymer materials, or micrometer-sized magnetic microspheres formed by a composite of Fe3O4 magnetic particles and organic polymer materials.

[0052] Preferably, the avidin is streptavidin, and the active group is any one of -NH2, -OH, -COOH, -FITC, -NHS and -Tosyl.

[0053] Preferably, the concentration of the linked Helicobacter pylori antigen in the magnetic microsphere solution linked to the Helicobacter pylori antigen is 50 μg / L to 2000 μg / L.

[0054] Preferably, the luminescence mode of the kit is any one of enzymatic chemiluminescence (using horseradish peroxidase (HRP) or alkaline phosphatase (ALP) as a luminescent marker, luminol and hydrogen peroxide or adamantane and its derivatives as luminescent substrates), direct chemiluminescence (using non-enzymatic small molecule organic compounds as luminescent markers, such as acridinium esters, isoluminol ABEI and its derivatives), and electrochemiluminescence (using terpyridine ruthenium as a marker, an electrochemical reaction occurs under the action of tripropylamine and voltage to emit visible light).

[0055] Table 1 shows the detection components of the four antibodies of the Helicobacter pylori IgM / IgG antibody combined typing detection kit on the magnetic particle platform in the embodiment of the present invention (see Figure 1 ).

[0056] Table 1

[0057] Antibody Magnetic microsphere solution Labeled tracer solution Type I HP-IgM R1-1 R3-1 Type I HP-IgG R1-1 R3-2 Type II HP-IgM R1-2 R3-1 Type II HP-IgG R1-2 R3-2

[0058] Furthermore, the concentration of each component is:

[0059] 1) R1-1: Magnetic microsphere solution coated with type I HP antigen

[0060] Type I HP antigen concentration: 500-800 μg / L; magnetic microsphere concentration: 0.1-5 mg / mL; amino acid is arginine, and the concentration is 100 mmol / L.

[0061] 2) R1-2: Magnetic microsphere solution coated with type II HP antigen

[0062] Type II HP antigen concentration: 500-800 μg / L; magnetic microsphere concentration: 0.1-5 mg / mL; amino acid is arginine, and the concentration is 100 mmol / L.

[0063] 3) R3-1: Labeled tracer solution for labeling anti-human IgM antibody

[0064] Anti-human IgM antibody concentration: 10-200 μg / L; labeled tracer concentration: 0.1-1 mg / l.

[0065] 4) R3-2: Labeled tracer solution for labeling anti-human IgG antibody

[0066] Anti-human IgG antibody concentration: 10-200 μg / L; labeled tracer concentration: 0.1-1 mg / l.

[0067] The present invention can freely select 1-4 combination types of items, a total of 15 combination types. It is usually recommended to perform the test in a 4-joint test mode.

[0068] Table 2

[0069]

[0070] Accordingly, the present invention also discloses a magnetic particle platform Helicobacter pylori IgM / IgG antibody combined typing detection method, comprising the following reaction steps:

[0071] The sample to be tested is pre-diluted or not, and then mixed with a magnetic microsphere solution directly coated or indirectly connected to type I HP antigen or a magnetic microsphere solution directly coated or indirectly connected to type II HP antigen, incubated at 37°C for 15 minutes, and then a labeled tracer directly coated or indirectly connected to anti-human IgG antibody / anti-human IgM antibody is added, incubated at 37°C for 15 minutes, and then washed to obtain a reaction product; an external magnetic field is applied to precipitate the reaction product, the supernatant is removed, and after washing, a chemiluminescent excitation material is added to detect the relative light intensity emitted, and the instrument automatically calculates the typing of Helicobacter pylori in the sample to be tested, as well as the IgG / IgM antibody concentration of each type of bacteria.

[0072] In some specific embodiments, the detection method specifically comprises the following steps (see Figure 2 ):

[0073] (I) Test steps

[0074] After preparing the test sample (calibrator or sample to be tested) and placing it correctly, click the start button to perform the calibration procedure or sample detection procedure. The instrument will perform the following operations:

[0075] (1) Transfer the sample rack to the sample suction position and load the reaction container to the sample addition position and dilution position.

[0076] (2) Dilute the sample and calibrator in a ratio of 1:9 using sample diluent.

[0077] (3) When performing the calibration procedure, complete the dispensing of 10-50 μL of calibration material and 20 μL of magnetic particle suspension.

[0078] (4) When executing the sample detection procedure, complete the dispensing of 10-50 μL of sample and 20 μL of magnetic particle suspension.

[0079] (5) Mix the reaction solution and incubate at a temperature of 20-40° C. for 5-20 minutes.

[0080] (6) After the incubation is completed, the reaction solution is washed and separated using a washing solution.

[0081] (7) Complete the injection of 50 μL of enzyme conjugate.

[0082] (8) Mix the reaction solution and incubate at a temperature of 20-40° C. for 5-20 minutes.

[0083] (9) After the incubation is completed, the reaction solution is washed and separated using a washing solution.

[0084] (10) Complete the injection of chemiluminescent excitation substances.

[0085] (11) Mix the reaction solution and detect the luminescence intensity.

[0086] The instrument automatically calculates the typing of Helicobacter pylori in the sample to be tested, as well as the IgG / IgM antibody concentration of each type of bacteria.

[0087] (II) Calculation of results

[0088] Sample S / CO value = sample luminescence value / cutoff value. The instrument automatically calculates the S / CO value of the sample to determine the positive or negative nature of the sample.

[0089] Validity determination

[0090] When setting the Cutoff value, the average luminescence value of calibration sample 1 / the average luminescence value of calibration sample 0 should be greater than 10, otherwise the test should be repeated. The instrument automatically completes the validity judgment.

[0091] Positive judgment value

[0092] When S / CO≥1.00, the result is considered positive; when S / CO<1.00, the result is considered negative.

[0093] Correspondingly, the present invention also discloses the application of the magnetic particle platform Helicobacter pylori IgM / IgG antibody combined typing detection kit on a chemiluminescence immunoassay analyzer.

[0094] The present invention will be further described below in conjunction with embodiments:

[0095] Example 1 Kit of the present invention

[0096] This embodiment is used to type H.pylori and mainly includes the following components:

[0097] 1) R1-1: Magnetic microsphere solution coated with type I HP antigen

[0098] HP type I antigen concentration: 600 μg / L; magnetic microsphere concentration: 0.8 mg / mL; amino acid is arginine, and the concentration is 100 mmol / L.

[0099] 2) R1-2: Magnetic microsphere solution coated with type II HP antigen

[0100] Type II HP antigen concentration: 600 μg / L; magnetic microsphere concentration: 0.8 mg / mL; amino acid is arginine, and the concentration is 100 mmol / L.

[0101] 3) R3-2: Labeled tracer solution for labeling anti-human IgG antibody

[0102] Anti-human IgG antibody concentration: 60 μg / L; labeled tracer concentration: 0.7 mg / l.

[0103] 4) Calibrator C1 without HP type I / II IgG.

[0104] 5) Calibrator C2: contains type I HP IgG concentration: 2.5S / CO; contains type II HP IgG concentration: S / CO value is 2.5.

[0105] Example 2 Kit of the present invention

[0106] The second embodiment of the Helicobacter pylori IgM / IgG antibody combined typing detection kit provided by the present invention is described in detail below. This embodiment is used to determine the infection period of H. pylori and includes the following components:

[0107] 1) R1-1: Magnetic microsphere solution coated with type I HP antigen

[0108] HP type I antigen concentration: 700 μg / L Magnetic microsphere concentration: 0.6 mg / mL Amino acid is arginine, concentration is 100 mmol / L

[0109] 2) R3-1 and R3-2: Labeled tracer solution for labeling anti-human IgM and IgG antibodies

[0110] Anti-human IgG antibody concentration: 90 μg / L Label tracer concentration: 0.7 mg / l;

[0111] Anti-human IgM antibody concentration: 90 μg / L Label tracer concentration: 0.7 mg / l;

[0112] 3) Calibrator C1 does not contain HP type I IgM / IgG antibodies.

[0113] 4) Calibrator C2: Contains HP type I IgM concentration: S / CO=2.5; contains HP type I IgG concentration: S / CO=2.5.

[0114] Example 3 Kit of the present invention

[0115] The kit is used to type H.pylori and determine the infection period, and includes the following components (see Figure 1 ):

[0116] 1) R1-1: Magnetic microsphere solution coated with HP antigen

[0117] HP type I antigen concentration: 800 μg / L Magnetic microsphere concentration: 2 mg / mL Amino acid is arginine, concentration is 100 mmol / L;

[0118] 2) R1-2: HP type II antigen concentration: 800 μg / L Magnetic microsphere concentration: 2 mg / mL Amino acid is arginine, concentration is 100 mmol / L;

[0119] 3) R3-1: a labeled tracer solution for labeling anti-human IgM antibodies;

[0120] R3-2: labeled tracer solution for labeling anti-human IgG antibody;

[0121] Anti-human IgG antibody concentration: 100 μg / L, labeled tracer concentration: 0.4 mg / L;

[0122] Anti-human IgM antibody concentration: 100 μg / L, labeled tracer concentration: 0.4 mg / L;

[0123] 4) Calibrator C1 without type I / II HP IgM / IgG antibodies;

[0124] 5) Calibrator C2: contains type I HP IgM (concentration: 2.5S / CO, i.e. S / CO value is 2.5); contains type I HP IgG (concentration: 2.5S / CO); contains type II HP IgM (concentration: 2.5S / CO); contains type II HP IgG (concentration: 2.5S / CO).

[0125] The magnetic microsphere solution directly coated with or indirectly connected to the type I Helicobacter pylori antigen comprises: the magnetic microspheres are connected to the type I Helicobacter pylori antigen via active groups.

[0126] The magnetic microsphere solution directly coated with or indirectly connected to the type II Helicobacter pylori antigen is: or the magnetic microspheres are connected to the type II Helicobacter pylori antigen via active groups.

[0127] The particle size of the magnetic microspheres is 2 μm; the magnetic microspheres are nano-scale or micron-scale magnetic microspheres that are composited with Fe3O4 magnetic particles and organic polymer materials.

[0128] The active group is -COOH.

[0129] The labeled tracer includes a chemiluminescent marker, and the chemiluminescent marker is horseradish peroxidase.

[0130] Example 4 Detection method of the present invention

[0131] This embodiment provides a magnetic microparticle platform Helicobacter pylori IgM / IgG antibody combined typing detection method (see Figure 2 ), comprising the following reaction steps:

[0132] The sample to be tested is pre-diluted, and then mixed with a magnetic microsphere solution directly coated or indirectly connected to type I HP antigen or a magnetic microsphere solution directly coated or indirectly connected to type II HP antigen, incubated at 37°C for 15 minutes, and then a labeled tracer directly coated or indirectly connected to anti-human IgG antibody / anti-human IgM antibody is added, incubated at 37°C for 17 minutes, and then washed to obtain a reaction product; an external magnetic field is applied to precipitate the reaction product, the supernatant is removed, and after washing, a chemiluminescent excitation material is added to detect the relative light intensity emitted, and the instrument automatically calculates the typing of Helicobacter pylori in the sample to be tested, as well as the IgG / IgM antibody concentration of each type of bacteria.

[0133] In order to simplify the components, the present invention does not provide a sample diluent, and only retains a universal sample diluent (not included in the kit, as a common consumable). In order to allow the sample and magnetic beads to react fully after mixing, the sample reaction volume is increased. The volumes of the first step reaction components are shown in the table below:

[0134] Table 3

[0135] Magnetic beads Sample after dilution outside the well Sample diluent Mode 1 20μL 10μL 50μL Mode for the Invention 20μL 50μL /

[0136] Test Example 1 Comparison of the results of the two detection modes in Table 3

[0137] The kit of Example 3 was used according to the method of Example 4 and the mode of the present invention in Table 3 to test 15 positive samples and 5 negative samples. The results are shown in Table 4.

[0138] Table 4

[0139]

[0140] Note: / means that the concentration deviation of negative samples is not calculated and the test is negative.

[0141] Table 5

[0142]

[0143]

[0144] The results showed that the deviations of S / CO of positive samples in both modes were within 10%, and negative samples were all detected as negative, indicating that the results of the detection mode of the present invention without diluting samples are accurate and reliable and can be widely used.

[0145] Test Example 2 Test kit accuracy test

[0146] The kit of Example 3 has 4 channels, and 15 positive samples and 5 negative samples were tested using the single test mode and the joint test mode. The results showed that the 4 test channels did not interfere with each other, and the test results were highly accurate.

[0147] Table 6

[0148]

[0149]

[0150] Table 7

[0151]

[0152] Test Example 3: Test kit sensitivity and specificity

[0153] 100 samples that have been tested by other clinical reagents were selected and tested using the kit of Example 3. S / CO ≥ 1 was considered positive. The statistical results are shown in the following table:

[0154] Table 8

[0155]

[0156]

[0157] According to the table above, among the 100 samples, 23% were toxic for current type I infection, 8% were non-toxic for current type II infection, 11% were previously infected, and 58% were negative. 23 people were recommended to undergo eradication therapy for Helicobacter pylori.

[0158] The compliance rates of the various indicators of the kit (i.e., the test reagent) of Example 3 of the present invention and the commercial reagents are shown in the following table:

[0159] Table 9

[0160]

[0161] According to the above table, the total compliance rate of the four antibodies is above 93%, and the highest compliance rate reaches 100%. The results show that the test results of the kit of the present invention are accurate and reliable, with an accuracy of more than 93%, and can be used for clinical testing.

[0162] Test Example 4 Freeze-thaw stability test of the test kit

[0163] Control kit: Compared with the kit of Example 3 of the present invention, no amino acid is added, and other components are the same.

[0164] The study found that during use, the control kit without amino acid addition was frozen and thawed several times in an extreme environment such as -20℃, and the stability of the control kit stored at 2-8℃ was compared. The results showed that the reactivity of different single positive samples was inconsistent, that is, some increased and some decreased, and the deviation was greater than 10%. Take HP I type-IgG antibody as an example:

[0165] Table 10

[0166]

[0167] The same 15 positive samples were tested using the kit 3 stored at 2-8° C. without freezing and thawing and the kit 3 frozen and thawed three times. The results are shown in Table 11.

[0168] The results showed that the deviation of the two test results was within 10%, indicating that after adding 100 mmol / L arginine to the magnetic microsphere solution, the kit of Example 3 of the present invention eliminated the matrix effect between different samples, effectively improved the freeze-thaw stability, and had a high application value.

[0169] Table 11

[0170]

[0171]

[0172] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetic microsphere solution, characterized in that: Including sealing solution, magnetic microspheres and 10-200mmol / L amino acids; The magnetic microspheres include magnetic microspheres coated with type I Helicobacter pylori antigens and / or magnetic microspheres coated with type II Helicobacter pylori antigens; The amino acid includes at least one of arginine, lysine, histidine, cysteine ​​and methionine.

2. The magnetic microsphere solution according to claim 1, characterized in that: In the magnetic microsphere solution, the concentration of amino acids is 100 mmol / L.

3. The magnetic microsphere solution according to claim 1 or 2, characterized in that: In the magnetic microsphere solution, the concentration of the Helicobacter pylori antigen is 50 μg / L to 2000 μg / L, preferably 800 μg / L.

4. The magnetic microsphere solution according to any one of claims 1 to 3, characterized in that: In the magnetic microsphere solution, the concentration of the magnetic microspheres is 0.1-5 mg / mL, preferably 2 mg / mL; The particle size of the magnetic microspheres is 1 to 3 μm; The magnetic microspheres are micron-sized magnetic microspheres formed by the composite of nano-sized Fe2O3 and organic polymer materials, or micron-sized magnetic microspheres formed by the composite of Fe3O4 magnetic particles and organic polymer materials.

5. The magnetic microsphere solution according to any one of claims 1 to 4, characterized in that: The sealing solution is a buffer containing 1wt% to 5wt% of a sealing agent, 0.1wt% to 0.20wt% of a surfactant and 0.05wt% to 0.10wt% of a preservative; the sealing agent is at least one of bovine serum albumin, animal serum and Casein; the surfactant is at least one of TritonX-100, sodium dodecyl sulfate, CHAPS and Tween; the buffer is Tris, HEPES, bis-Tris, MES buffer or PBS buffer.

6. The magnetic microsphere solution according to any one of claims 1 to 5, characterized in that: The type I Helicobacter pylori antigens include CagA antigen and / or VacA antigen, and the type II Helicobacter pylori antigens include at least one of UreA antigen, UreB antigen, and HspA antigen.

7. A kit for Helicobacter pylori IgG / IgM typing or detection, characterized in that: The method comprises the magnetic microsphere solution according to any one of claims 1 to 6 and one or more of the following components: Calibrator, labeled tracer solution for labeling anti-human IgG antibody, labeled tracer solution for labeling anti-human IgM antibody.

8. The kit according to claim 7, characterized in that In the labeled tracer solution of the labeled anti-human IgG antibody, the concentration of the anti-human IgG antibody is 10-200 μg / L, and the concentration of the labeled tracer is 0.1-1 mg / L; In the labeled tracer solution for labeling anti-human IgM antibodies, the concentration of anti-human IgM antibodies is 10-200 μg / L, and the concentration of the labeled tracer is 0.1-1 mg / L.

9. The kit according to claim 7, characterized in that The calibrator C1 is human negative serum / plasma or calibrator diluent that does not contain type I and type II HP IgG and does not contain type I and type II HP IgM; Calibrator C2 is a sample obtained by diluting the high value of human serum / plasma containing at least one of type I HP IgG, type II HP IgG, type I HP IgM, and type II HP IgM, or a calibrator diluent.

10. A method for non-diagnostic typing or non-diagnostic detection of Helicobacter pylori IgG / IgM, characterized in that: The IgG and / or IgM in the sample is detected or typed using the kit according to any one of claims 7 to 9.

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