A specific protein detection reagent and preparation method thereof

Abstract

The present invention belongs to the technical field of specific protein detection, and specifically relates to a specific protein detection reagent and a preparation method thereof. The specific protein detection reagent includes a capture protein directly labeled with an AIE small molecule compound, and the capture protein and the specific protein can specifically bind to each other. The beneficial effects are: the reagent preparation process is simple (antibodies are directly labeled with AIE materials, and there is no need to prepare nanoparticles); in the field of microbial detection, compared with nucleic acid detection reagents, the production environment requirements are low and the risk of infection is low; the detection limit is low (1ng / mL) and the detection accuracy is high; it is easy to use and has low technical requirements for operators; and the detection is rapid (only 15min).

Description

Technical Field

[0001] The present invention belongs to the technical field of specific protein detection, and in particular relates to a specific protein detection reagent and a preparation method thereof. Background Art

[0002] In Vitro Diagnosis (IVD) refers to obtaining diagnostic information through testing biological samples such as blood, body fluids, and tissues, and then judging the body's diseases or functions. Rapid, sensitive, and quantitative detection of biological samples is the current development direction of in vitro diagnosis. Biomedical testing based on immunochromatographic test strips has great social and economic benefits in the early diagnosis and prevention of diseases due to its advantages of rapidity, simplicity, intuitiveness, and low cost. Immunochromatographic Assay (ICA) is based on the principle of antigen-antibody specific immunological reaction and chromatographic reaction. The indicator labeled with the antibody will specifically recognize and bind to specific analytes in biological samples such as urine, blood, or saliva, and will be retained and colored in the test strip detection area. It can also be combined with a reader for quantitative detection to achieve the purpose of rapid and accurate detection of the analyte.

[0003] There are currently two commonly used methods for diagnosing COVID-19 in terms of molecular biology: one is viral gene sequencing, and the other is nucleic acid testing. Because the second-generation sequencing can detect trace substances, it can be applied to more types of body fluids. However, most hospitals do not have sequencing equipment. Especially for the second-generation sequencing, not only equipment is needed, but also a bioinformatics team is needed to assist in the interpretation of sequencing results. However, even so, the optimization of the process and reagent ratio in the research and development stage, the establishment of Yangshen and Yinshen, and pre-clinical trials are also needed. Moreover, because the cost of the second-generation sequencing is high and the detection cycle is relatively long, it may not be suitable for this test.

[0004] Therefore, the current diagnostic method is mainly based on nucleic acid testing (PCR or real-time PCR), which is mainly subject to: the quality of the test kit (not the production capacity), the number of P3 laboratories (biosafety level 3 laboratories) that can undertake clinical testing, the accuracy and stability of P3 laboratory equipment, and the number of staff who can perform skilled and stable operations. In actual clinical applications, in addition to high-quality test kits, the type of samples, the collection, transportation and storage methods of samples, the nucleic acid (RNA) extraction method, the accuracy and stability of the detection equipment (PCR instrument or real-time PCR instrument), and the skills of the operators will affect the actual detection effect. In addition, considering that the samples tested are highly contagious, the laboratory used for this test should theoretically be a P3 laboratory. Strict protection is required inside the testing laboratory, and purification and negative pressure are the most basic, and there are also strict regulations for the operating procedures. If the laboratory level is not reached, there may be hidden dangers of laboratory infection.

[0005] Immunochromatographic Assay (ICA) is based on the principles of antigen-antibody specific immunological reaction and chromatographic reaction. Antibodies labeled with indicators will specifically recognize and bind to specific analytes in biological samples such as urine, blood or saliva, and will be retained and colored in the detection area of ​​the test strip. It can also be combined with a reader for quantitative detection, thereby achieving the purpose of quickly and accurately detecting the analyte.

[0006] In the prior art, colloidal gold and fluorescent nanoparticles of organic dye molecules are usually used as indicators. Colloidal gold has been used in the fields of early pregnancy detection, food safety, environment, and pesticide residues. However, the detection accuracy and sensitivity of colloidal gold are both low, so it can only be used in qualitative or quantitative detection fields with low accuracy requirements.

[0007] Compared with colloidal gold indicators, fluorescent nanoparticles of organic dye molecules have improved detection accuracy and sensitivity, but they are still limited in areas with high requirements such as infectious diseases and contagious diseases. The reason is that organic dyes have good fluorescence properties in dilute solution, but these compounds have the defect of aggregation-induced quenching (ACQ) in thin film state or high concentration state. ACQ severely limits the application of organic dyes in immunochromatographic test strips. During use, quenching will occur if the concentration is too high, but too low a concentration will lead to weak fluorescence signals and serious background signal interference. Therefore, a lot of time has to be spent to find a balance value.

[0008] The advent of aggregation-induced emission (AIE) materials has fundamentally solved the drawback of ACQ in traditional fluorescent materials in practical applications, and has brought people's understanding of organic luminescent materials to a new level. Compared with traditional organic fluorescent dyes, AIE materials have shown some significant advantages in fluorescence detection and bioimaging: the high luminescence efficiency in the aggregated state makes the fluorescence intensity gradually increase with the increase of concentration, indirectly reducing the interference of background signals and achieving high-contrast fluorescence detection; it has good photostability and can effectively solve photobleaching; it is highly modifiable and can be used to design responsive fluorescent indicators.

[0009] The Chinese invention patent application with application number 201910918011.0 discloses an AIE immunochromatographic test paper, the technical solution of which is to use AIE nanoparticles to mark and capture proteins or detect antigens. Specifically, nanoparticles are made of tetraphenylethylene AIE derivatives, silole AIE derivatives, 1,4-stilbene AIE derivatives, five-membered heterocyclic AIE derivatives, and organic boron AIE derivatives, and then marked on specific proteins. However, AIE nanoparticles have the following problems:

[0010] 1. The production of AIE nanoparticles from small molecule AIE materials requires additional synthesis steps, which will increase costs and be detrimental to industrial promotion;

[0011] 2. Compared with pure small molecule AIE materials, the uniformity of nanoparticle size is poor. Under the same concentration conditions, the repeatability and color sensitivity of nanoparticles are lower than those of small molecule AIE materials.

[0012] By using AIE immunochromatography to label specific proteins, you can choose to label antibodies to detect the presence of antigens, or you can choose to label antigens to capture the presence of proteins.

[0013] Existing testing reagents usually focus more on the diagnosis of cases. However, due to the long incubation period of the new coronavirus 2019-nCoV and the fact that asymptomatic infected people are also contagious, in order to prevent asymptomatic virus carriers from infecting others in the crowd, we need a detection method that can directly and quickly detect whether the virus exists and then screen out virus carriers.

[0014] Based on this, there is an urgent need for a detection reagent with low production cost, low requirements on production conditions, no infectious risk, low detection limit, high accuracy, easy use and easy metabolism in the body. Summary of the invention

[0015] In order to solve the above problems, the purpose of the present invention is to provide a specific protein detection reagent that can meet the requirements of no infection risk in the production process, low requirements on production conditions, easy use and accurate detection.

[0016] The invention discloses a specific protein detection reagent, comprising a capture protein directly labeled with an AIE small molecule compound or a polymer, wherein the capture protein and the specific protein can be specifically bound to each other.

[0017] Furthermore, the AIE small molecule compound adopts a molecular structure based on a D-π-A structure, and the repeating unit of the AIE polymer at least includes a structure based on a D-π-A structure.

[0018] Furthermore, the AIE small molecule compound or polymer includes at least one carboxyl group (-COOH).

[0019] Preferably, the AIE small molecule compound adopts a molecular structure represented by general formula (I) or at least one of its salts, hydrates and chelates:

[0020]

[0021] Wherein, R1 is at least one of the following structures or its derivatives:

[0022]

[0023] Preferably, the AIE polymer adopts a molecular structure represented by general formula (II) or at least one of its salts, hydrates and chelates:

[0024]

[0025] Wherein, R2 adopts at least one of the following structures or its derivatives:

[0026]

[0027] Furthermore, in terms of the amount of substance, the added amount of the capture protein is 1 eq, and the added amount of the AIE small molecule compound is 200-1000 eq.

[0028] Furthermore, the specific protein detection reagent can effectively detect more than 1 ng of specific protein.

[0029] Furthermore, the specific protein includes at least one of bacteria, viruses, fungi, and tumor markers.

[0030] The present invention also discloses a method for preparing a specific protein detection reagent, comprising the following steps:

[0031] The captured protein is dispersed in PBS buffer, 1% DMSO is added, and the AIE molecule or polymer, N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are also added to the reaction system and incubated at a temperature suitable for the activity of the captured protein for 1 to 2 hours to obtain a complex in which the AIE molecule or polymer is directly connected to the antibody, which is the labeling reagent.

[0032] Preferably, when the AIE molecule or polymer contains a carboxyl group, the method for preparing a specific protein detection reagent comprises the following steps:

[0033] Step 1, activation of carboxyl groups: reacting the AIE molecule or polymer with N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in DMSO for 2 to 3 hours to prepare AIE-NHS, which is then washed and frozen for later use;

[0034] Step 2, disperse the captured protein in PBS buffer, add AIE-NHS and incubate for 1 to 2 hours at a temperature suitable for the activity of the captured protein, block with 1% bovine serum albumin (BSA), and obtain a complex of AIE molecules or polymers directly connected to antibodies, which is the detection reagent.

[0035] The invention also discloses a novel specific protein detection device, which utilizes the specific protein detection reagent.

[0036] Furthermore, the novel specific protein detection device comprises a test paper, wherein the test paper comprises a conjugate pad, and the specific protein detection reagent is arranged on the conjugate pad.

[0037] Furthermore, in the novel specific protein detection device, the test paper also includes a test line and a quality control line, the test line is coated with a capture antibody for capturing the target in the sample to be detected, and the quality control line is coated with a secondary antibody for capturing the protein.

[0038] Furthermore, in the novel specific protein detection device, the test paper also includes a base plate, a sample pad, a nitrocellulose membrane and a water absorbent pad, and the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorbent pad are overlapped in sequence on the base plate along the length direction of the base plate, and the test line and the quality control line are arranged in sequence on the nitrocellulose membrane along the sample chromatography direction.

[0039] Furthermore, the sample pad is a glass fiber membrane or a polyester fiber membrane, the conjugate pad is a glass fiber membrane, and the bottom plate is a polyvinyl chloride rubber plate.

[0040] Furthermore, the novel specific protein detection device also includes a shell, on which a first hole and a second hole are provided, wherein the first hole is used for dripping the sample to be detected, and the second hole is used for observing or reading the color development of the signal; the shell is arranged outside the test paper.

[0041] Furthermore, a quantitative detection device is also provided on the housing, and the quantitative detection device includes a light-emitting device, a receiving device, a display device, and a power supply; the power supply provides electrical energy to the light-emitting device, the receiving device, and the display device; the light-emitting device is arranged in the second hole close to the side wall, and the receiving device is arranged outside the second hole; the light-emitting device is a light source provided with a filter; the receiving device adopts a photoelectric converter with an external filter; and the receiving device is connected to the display device by electrical signals.

[0042] Furthermore, the wavelength of light emitted by the light emitting device is 250-1000nm.

[0043] Preferably, the wavelength of light emitted by the light emitting device is 365nm.

[0044] Furthermore, the novel specific protein detection device can be used to detect whether at least one sample to be detected, including throat swabs, nasal swabs, sputum, anal swabs, feces, blood, saliva, alveolar lavage fluid, urine or tissue fluid, contains specific proteins, and can also be used to detect whether samples collected in the environment contain specific proteins.

[0045] Furthermore, the specific protein detection reagent can be used to detect whether at least one sample to be detected, including throat swabs, nasal swabs, sputum, anal swabs, feces, blood, saliva, alveolar lavage fluid, urine or tissue fluid, contains specific proteins, and can also be used to detect whether samples collected in the environment contain specific proteins.

[0046] Since this technical solution uses targeted capture proteins to directly detect target specific proteins, compared with existing nucleic acid detection methods, this technology does not involve genetic material when applied to the field of microbial detection, is safer during production and use, and requires lower laboratory conditions for production. Especially when using labeled secondary antibodies to detect antibodies such as viruses, the blood of the organism is used, and there is no contact with viruses and other microorganisms at all, and there is no risk of infection from production to use.

[0047] At the same time, the technical solution of the present invention can also be used to detect whether there are target microorganisms such as specific viruses in the ambient air, water, and even on the surface of objects, which is helpful to determine whether the target microorganisms exist in the environment that the target microorganisms have been in contact with, and prevent the target microorganisms from spreading through the "aerosol" route.

[0048] In the field of tumor marker detection, it can be used to detect tumor markers in the blood, or injected into the body to mark and observe the location of tumor cells. Since this technology uses AIE molecules to directly mark proteins without the need to make nanoparticles first, it is metabolized faster in the body and has low damage to the organism.

[0049] During qualitative testing, the sample to be tested is dropped onto the test paper through the first hole, and after irradiation with excitation light, it is observed through the second hole, and it is found that the test line and the quality control line are colored at the same time, proving that the specific protein to be tested is detected.

[0050] During quantitative testing, the sample to be tested is dropped onto the test paper through the first hole. After being irradiated by the light-emitting device, if the sample contains the specific protein to be tested, the test line and the quality control line will be colored at the same time. The receiving device receives the fluorescence, and the fluorescence intensity is linearly related to the target microorganism content. The receiving device will compare the fluorescence intensity with the pre-input standard curve (such as the attached Figure 9-10 ) is converted into an electrical signal and transmitted to the display device to display a number, which is the concentration of the specific protein to be tested.

[0051] The beneficial effects of the present invention are:

[0052] 1. The reagent preparation process is simple (using AIE materials to directly label antibodies without the need to prepare nanoparticles);

[0053] 2. In the field of microbial detection, compared with nucleic acid detection reagents, it has low production environment requirements and low infection risks; in the fields of tumor marker detection, it can be used in vivo.

[0054] 3. Low detection limit (1ng / mL), high detection accuracy, can be used for quantitative detection;

[0055] 4. Easy to use, low technical requirements for operators;

[0056] 5. Rapid detection (only 15 minutes). BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 A schematic diagram of the structure of the test paper of the present invention;

[0058] Figure 2 A schematic diagram of the housing structure of the present invention;

[0059] Figure 3 A schematic diagram of the assembly structure of the test paper and the housing of the present invention;

[0060] Figure 4 A schematic diagram of the structure of the quantitative detection device of the present invention;

[0061] Figure 5 Detection limit test diagram of Example 1 of the present invention;

[0062] Figure 6 Detection limit test diagram of Example 13 of the present invention;

[0063] Figure 7 The test diagram of the effective ratio of antibodies and AIE molecules in Example 1 of the present invention;

[0064] Figure 8 The test diagram of the effective ratio of antibodies and AIE molecules in Example 1 of the present invention;

[0065] Fig. 9 The linear relationship diagram of the fluorescence intensity of the T line of the present invention and the concentration of the antigen protein;

[0066] Fig.10 The linear relationship diagram of the fluorescence intensity of the T line of the present invention and the antibody concentration. DETAILED DESCRIPTION

[0067] The specific implementation modes of the present invention are further described below in conjunction with examples. The following examples are only used to more clearly illustrate the technical embodiments of the present invention and are not intended to limit the protection scope of the present invention.

[0068] Example 1

[0069] A specific protein detection reagent comprises a capture protein directly labeled with an AIE small molecule compound or a polymer, wherein the capture protein adopts a target microorganism IgG antibody.

[0070] In this embodiment, the AIE small molecule compound has the following molecular structure:

[0071]

[0072] As described in this embodiment, the capture protein uses 2019-nCoV novel coronavirus IgG antibody.

[0073] In this embodiment, based on the amount of substance, the added amount of the capture protein is 1 eq, and the added amount of the AIE small molecule compound is 200 eq.

[0074] The present invention also discloses a method for preparing a specific protein detection reagent, comprising the following steps:

[0075] Step 1, activation of carboxyl groups: reacting the AIE molecule with N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in DMSO for 2-3 h to prepare AIE-NHS, which was then washed and frozen for later use;

[0076] Step 2: Take 2.5 μg of 2019-nCoV novel coronavirus IgG antibody and disperse it in 100 μL PBS buffer, add (200 eq of AIE-NHS and incubate at 37°C for 1 to 2 hours, and block with 1% bovine serum albumin (BSA) to obtain the AIE-IgG complex of the IgG antibody, which is the detection reagent.

[0077] The AIE-IgG complex prepared above emits a light signal under the irradiation of excitation light, wherein the wavelength of the excitation light is 300-800 nm and the irradiation power is 10-300 mW / cm 2 . And the fluorescence intensity of AIE-IgG on the chromatography test paper is much stronger than that in the dilute solution. Utilizing the above characteristics, the reagent is used for the detection of the new coronavirus 2019-nCoV.

[0078] The invention also discloses a novel specific protein detection device, which utilizes the specific protein detection reagent.

[0079] The novel specific protein detection device described in this embodiment uses a test paper 1, which includes a conjugate pad 13, and the specific protein detection reagent is arranged on the conjugate pad 13.

[0080] In the novel specific protein detection device described in this embodiment, the test strip 1 further comprises a test line 16 and a quality control line 17. The test line 16 is coated with a capture antibody for capturing the target in the sample to be detected, and the quality control line 17 is coated with a secondary antibody for capturing the protein.

[0081] In the novel specific protein detection device described in this embodiment, the test paper 1 also includes a base plate 11, a sample pad 12, a nitrocellulose membrane 14 and a water absorbent pad 15, and the sample pad 12, the conjugate pad 13, the nitrocellulose membrane 14 and the water absorbent pad 15 are overlapped on the base plate 11 in sequence along the length direction of the base plate 11; the test line 16 and the quality control line 17 are arranged in sequence on the nitrocellulose membrane 14 along the sample chromatography direction.

[0082] In this embodiment, the sample pad 12 is made of glass fiber membrane or polyester fiber membrane, the conjugate pad 13 is made of glass fiber membrane, and the bottom plate 11 is made of polyvinyl chloride rubber plate.

[0083] The novel specific protein detection device described in this embodiment also includes a shell 2, and the shell 2 is provided with a first hole 21 and a second hole 22, the first hole 21 is used for dripping the sample to be detected, and the second hole 22 is used for observing or reading the color of the signal; the shell 2 is arranged outside the test paper 1.

[0084] In the present embodiment, a quantitative detection device 23 is further provided on the housing 2, and the quantitative detection device 23 includes a light-emitting device 231, a receiving device 232, a display device 233, and a power supply 234; the power supply 234 provides electrical energy for the light-emitting device 231, the receiving device 232, and the display device 233; the light-emitting device 231 is arranged in the second hole 22 near the side wall, and the receiving device 232 is arranged outside the second hole 22; the light-emitting device 231 is a light source provided with a filter; the receiving device 232 adopts a photoelectric converter with an external filter; the receiving device 232 is electrically connected to the display device 233.

[0085] In this embodiment, the wavelength of light emitted by the light emitting device 233 is 250-1000 nm.

[0086] Preferably, the wavelength of light emitted by the light emitting device 233 is 365 nm.

[0087] In this embodiment, the sample to be tested is at least one of a throat swab, a nasal swab, sputum, anal swab, feces, blood, saliva, alveolar lavage fluid, urine or tissue fluid.

[0088] Embodiment 2-10

[0089] The detection reagents and preparation methods described in Examples 2-10 are similar to those in Example 1, and the AIE molecules are selected from compounds described in general formula (1):

[0090]

[0091] Wherein, R1 is at least one of the following structures or its derivatives:

[0092]

[0093] The difference lies in the replacement of the parameters described in the following table (Table 1):

[0094] Table 1 Parameters of AIE small molecule compound examples

[0095]

[0096] Examples 11-12

[0097] The detection reagents and preparation methods thereof described in Examples 11-12 are similar to those in Example 1, and the AIE polymers adopt at least one of the molecular structures represented by general formula (II):

[0098]

[0099] Wherein, R2 adopts at least one of the following structures or its derivatives:

[0100]

[0101] The difference lies in the replacement of the parameters described in the following table (Table 2):

[0102] Table 2 AIE polymer example parameter table

[0103]

[0104] Embodiment 13

[0105] A specific protein detection reagent includes a detection antibody directly labeled with an AIE small molecule compound or a polymer, wherein the capture antigen is a 2019-nCoV novel coronavirus specific protein.

[0106] In this embodiment, the AIE small molecule compound has the following molecular structure:

[0107]

[0108] As described in this embodiment, the capture antigen uses the 2019-nCoV novel coronavirus specific protein.

[0109] In this embodiment, based on the amount of substance, the added amount of the detection antibody is 1 eq, and the added amount of the AIE small molecule compound is 200 eq.

[0110] The present invention also discloses a method for preparing a novel antibody detection reagent, comprising the following steps:

[0111] Step 1, activation of carboxyl groups: reacting the AIE molecule with N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in DMSO for 2-3 h to prepare AIE-NHS, which was then washed and frozen for later use;

[0112] Step 2, take 2.5 μg of secondary antibody against IgG in human serum or whole blood and disperse it in 100 μL PBS buffer, add 200 eq of AIE-NHS and incubate at 37°C for 1 to 2 hours, block with 1% bovine serum albumin (BSA), and obtain the AIE-antibody complex of the AIE-linked secondary antibody, which is the detection reagent.

[0113] The AIE-antigen protein complex prepared above emits a light signal under the irradiation of excitation light, wherein the wavelength of the excitation light is 300-800nm ​​and the irradiation power is 10-300mW / cm 2. And the fluorescence intensity of the AIE-antibody on the chromatography test paper is much stronger than that in the dilute solution. Utilizing the above characteristics, the reagent is used for the detection of 2019-nCoV novel coronavirus serum antibodies.

[0114] In this embodiment, the sample to be detected is at least one of serum and whole blood.

[0115] Examples 14-22

[0116] The detection reagents and preparation methods described in Examples 2-10 are similar to those in Example 13. The AIE molecules are selected from the compounds described in the general formula (1), except that the parameters described in the following table (Table 3) are replaced:

[0117] Table 3 Parameters of AIE small molecule compound examples

[0118]

[0119] Embodiment 23-24

[0120] The detection reagents and preparation methods described in Examples 23-24 are similar to those in Example 13, and the AIE polymers adopt at least one of the molecular structures represented by the general formula (II), except that the parameters described in the following table (Table 3) are replaced:

[0121] Table 3 Parameters of AIE polymer examples

[0122]

[0123] In order to further illustrate the beneficial effects of the present invention, the following experiments were specially set up to test the products of Example 1 (directly detecting antigens using labeled antibodies) and Example 13 (detecting antibodies using labeled secondary antibodies) respectively:

[0124] Detection limit (detection accuracy) test:

[0125] The test strips prepared in Example 1 were tested with different amounts of the antigen to be tested (2019-nCoV novel coronavirus antigen);

[0126] The test strips prepared in Example 13 were tested with different amounts of the antibody to be tested (2019-nCoV novel coronavirus IgG antibody);

[0127] The following data were obtained: Figure 5-6 , attached Figure 5 From left to right in the figure are test groups 1-4; Figure 6 From right to left in the middle are test groups 5-8)

[0128] Table 3 Detection limit test table

[0129] Group Antibody concentration (μg / mL) Amount of antigen added to be tested (ng) Is there a signal on the T line? Is the T line clear? Test Group 1 0.25 0 none — Test Group 2 0.25 1 have no Test Group 3 0.25 5 have yes Test Group 4 0.25 10 have yes Test Group 5 0.25 1 yes yes Test Group 6 0.25 5 have yes Test Group 7 0.25 10 have yes Test Group 8 0.25 15 have yes

[0130] Through the above tests, it can be seen that Example 1 of the present invention can still be detected when the added amount of the antigen to be tested (2019-nCoV new coronavirus antigen) is 1 ng.

[0131] Through the above tests, it can be seen that Example 13 of the present invention can still be detected when the added amount of the antibody to be tested (2019-nCoV new coronavirus IgG antibody) is 1 ng.

[0132] Selection of the ratio of antibody to AIE molecule

[0133] Set the comparison ratio, where:

[0134] The preparation method of Comparative Example 1 is similar to that of Example 1, except that the concentration of the added antibody is different;

[0135] The preparation method of Comparative Example 2 is similar to that of Example 13, except that the concentration of the added antibody is different;

[0136] Test data is shown in the following table: Figure 7-8 Attached Figure 7 From left to right in the figure are Comparative Example 1 and Example 1; Figure 8 From right to left in the figure are respectively Example 13 and Comparative Example 2)

[0137] Table 4 AIE small molecule compound and antibody ratio change test table

[0138]

[0139]

[0140] By comparing Example 1 with Comparative Example 1, when no antigen to be tested is added, Comparative Example 1 still shows a T line, indicating that excessively coated antibodies may result in false positives, and the ratio of coated antibodies to AIE molecule labeled antibodies has a certain range requirement.

[0141] By comparing Example 13 with Comparative Example 2, when no antibody to be tested is added, Comparative Example 2 still shows a T line, indicating that a false positive will occur when the T line is coated with excessive antigen, and the ratio of antigen to AIE-labeled antibody has a certain range requirement.

[0142] This also proves that in the technical solution of the present invention, the selection range of the ratio of AIE labeled protein to coating protein requires creative work of the inventor to obtain, and is not a routine choice of those skilled in the art.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A specific protein detection reagent, characterized in that: It includes a capture protein directly labeled with an AIE small molecule compound, wherein the capture protein and the specific protein can specifically bind to each other; The AIE small molecule compound has the following molecular structure: The capture protein adopts 2019-nCoV novel coronavirus anti-human IgG secondary antibody.

2. The specific protein detection reagent according to claim 1, characterized in that: Calculated by the amount of substance, the added amount of the capture protein is 1 eq, and the added amount of the AIE small molecule compound is 200-1000 eq.

3. The specific protein detection reagent according to claim 1, characterized in that: The specific protein to be tested can be effectively detected at a concentration of more than 1 ng / mL.

4. The specific protein detection reagent according to claim 1, characterized in that: Can be used for quantitative detection.

5. The specific protein detection reagent according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Step 1, activation of carboxyl groups: reacting the AIE molecule with N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in DMSO for 2-3 h to prepare AIE-NHS, which is then washed and frozen for later use; Step 2, disperse the captured protein in PBS buffer, add AIE-NHS and incubate for 1 to 2 hours at a temperature suitable for the activity of the captured protein, block with 1% bovine serum albumin, and obtain a complex of AIE molecules directly connected to antibodies, which is the detection reagent.

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