Carboxylated nano-microsphere for coupling antibody as well as preparation method and application of carboxylated nano-microsphere

Abstract

The invention provides carboxylated nano-microspheres for coupling an antibody as well as a preparation method and application of the carboxylated nano-microspheres. The preparation method comprises the following steps: S1, activating a thiazoline acylating agent by using an activating agent; s2, activating the carboxylated nano microspheres by using the activated thiazoline acylating agent; and S3, purifying the activated carboxylated nano-microspheres to obtain the carboxylated nano-microspheres. Compared with the traditional one-step or two-step carboxylated nano-microsphere activation operation, the method has the advantages that the harmless thiazoline acylating agent which has higher activation efficiency and is easy to separate and remove is used as the acylating agent, the coupling efficiency of the antibody and the microspheres can be improved after activation of the activating agent, and meanwhile, the adverse effect on the antibody is avoided; the antibody sensitized nanoparticles formed by coupling have excellent detection sensitivity and a linear detection range; and the activated acylating agent can stabilize the Zeta potential on the surface of the nano-microsphere and enhance the aggregation stability of the nano-microsphere.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay, and relates to a carboxylated nano-microsphere for coupling an antibody, and a preparation method and application thereof. Background Art

[0002] Latex-enhanced turbidimetric immunoassay (PETIA) is a relatively stable and accurate homogeneous immunoturbidimetric method for detecting body fluid proteins. Compared with the enzyme-linked immunosorbent assay (ELISA), which is the mainstream in current scientific research, it eliminates the tedious steps of incubation and plate washing, and can obtain test results in a short time. Moreover, latex-enhanced turbidimetric immunoassay has higher sensitivity than colloidal gold chromatography and can be used for quantitative detection.

[0003] Compared with the traditional immunoturbidimetry, the latex-enhanced immunoturbidimetry is to crosslink monoclonal antibodies on the surface of polymer nano-microspheres. When the nano-microspheres crosslinked with antibodies bind to the antigen, they will quickly gather together in a short time, changing the reflected light performance or transmitted light performance of the reaction solution. At present, the nano-microspheres used in PETIA detection reagents are mostly carboxylated microspheres or amino-containing microspheres. In modern scientific research, the binding of nano-microspheres to antibodies mainly includes two methods: physical adsorption and chemical coupling. Since the latex microspheres have sulfonic acid groups, carboxyl groups, aldehyde groups, etc. on the surface, they are all hydrophobic microspheres and can be designed to passively physically adsorb antibodies. The hydrophobic adsorption method can only be used for hydrophobic microspheres (microspheres modified with sulfonic acid groups, carboxyl groups, and aldehyde groups on the surface). Although physical adsorption is independent of pH, the pH of the reaction buffer has a very large effect on the structure of the antibody, thereby affecting the reaction efficiency of the antibody adsorbed on the microspheres. Generally, at a pH near the isoelectric point of the adsorbed antibody, it has a higher physical adsorption efficiency. For antibodies, the hydrophobicity of the Fc end is stronger than that of the Fab end. This property can ensure that the antibody is bound to the microspheres through the Fc end, thus not affecting the specific binding of the antibody to the antigen after adsorption.

[0004] Another method for binding antibodies to nanoparticles is chemical coupling. The binding of carboxylated nanoparticles to antibodies is through covalent bonding of the carboxyl functional groups on the surface of the nanoparticles with the free amino groups on the Fc end of the antibody surface, forming a bridging chemical arm between the nanoparticles and the antibody, which reduces the steric effect, not only improving the binding rate of the antibody, but also providing the antibody with a suitable three-dimensional spatial structure, effectively protecting the active area for the antibody to bind to the antigen. In order to achieve the coupling of carboxylated nanoparticles to antibodies, a biocrosslinking agent is usually used to activate the carboxyl functional groups on the surface of the nanoparticles. Currently, commonly used biocrosslinking agents include chemical substances such as N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and N-hydroxysulfosuccinimide (Sulfo-NHS). The use of biocrosslinking agents to activate nanoparticles mainly includes two methods: a one-step method and a two-step method. First, the nanoparticles are activated with EDC / NHS, and then the excess unreacted cross-linking agent is removed through steps such as washing and centrifugation. Finally, the antibody is coupled to the activated nanoparticles to obtain antibody-modified nanoparticles.

[0005] Although the use of EDC / NHS can activate the nanosphere-coupled antibody, EDC / NHS may have an adverse effect on the normal physiological function structure of the antibody. In the current one-step / two-step method for nanosphere-coupled antibody experiments, the EDC used is non-quantitative, and the excess amount of EDC / NHS is difficult to remove with the subsequent washing and centrifugation steps. In the subsequent antibody coupling step, it will react with the free carboxyl groups on the antibody surface, causing damage to the antibody structure, affecting the normal physiological function of the antibody, and then inhibiting the detection effect of the subsequent latex-enhanced immunoturbidimetric in vitro detection reagent. In addition, when coupling the antibody subsequently, an excessive amount of antibody must be used to ensure the theoretically corresponding coupling efficiency, but this may result in excessive use of the antibody, resulting in unnecessary waste. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a carboxylated nano-microsphere for coupling antibodies and a preparation method and application thereof.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing carboxylated nanoparticles for coupling antibodies, comprising the following steps:

[0009] S1, activating the thiazoline acylating agent with an activating agent;

[0010] S2, using the activated thiazoline acylating agent to activate the carboxylated nanoparticles;

[0011] S3, purifying the activated carboxylated nano-microspheres to obtain.

[0012] As an embodiment of the present invention, in step S1, the thiazoline acylating agent includes at least one of 5-chloro-2-methyl-4-isothiazoline-3-one, 2-thiazoline-2-thiol, 4-aminothiazoline-2-thione, benzothiazoline, and 2-methylthio-2-thiazoline.

[0013] As an embodiment of the present invention, in step S1, the activator includes at least one of 1-hydroxyphenyltriazole, oxalyl chloride, 4-trifluoromethylbenzoic anhydride, 2-methyl-6-nitrobenzoic anhydride, N,N-diisopropylformamide, potassium peroxymonosulfonate, and trifluoroacetic acid. Preferably, the activator is potassium peroxymonosulfonate.

[0014] As an embodiment of the present invention, in step S1, the mass ratio of the thiazoline acylating agent to the activating agent is 1:1 to 1:4. Preferably, the mass ratio of the thiazoline acylating agent to the activating agent is 1:3 to 1:4.

[0015] As an embodiment of the present invention, in step S1, the activation method comprises dissolving and incubating the thiazoline acylating agent and the activating agent in a solvent; the activation time is 3-10 minutes. Preferably, the concentration of the thiazoline acylating agent is 5-20 mg / ml.

[0016] Furthermore, the solvent comprises an ethanol solution. In some preferred embodiments, the mass percentage of the ethanol solution is 75%.

[0017] As an embodiment of the present invention, in step S2, the ratio of the activated thiazoline acylating agent to the carboxylated nanoparticles is 10-100 mg: 0.5-1.5 mL. In some preferred embodiments, the solid content of the carboxylated nanoparticles is 10%.

[0018] As an embodiment of the present invention, in step S2, the activation time is 15-25 minutes.

[0019] As an embodiment of the present invention, in step S3, the purification method comprises: heating the activated carboxylated nanospheres for the first time, centrifuging, resuspending the precipitate, heating for the second time, and centrifuging. In some embodiments, the purification comprises repeating the above steps 2-3 times.

[0020] Furthermore, the first heating and the second heating are both incubated in a 50-60° C. water bath for 10-30 min.

[0021] Furthermore, the centrifugal rotation speed is 10000-20000rpm.

[0022] In a second aspect, the present invention provides a carboxylated nanoparticle for coupling with an antibody obtained by the preparation method.

[0023] In a third aspect, the present invention provides an antibody-sensitized nanoparticle, which is obtained by coupling and incubating carboxylated nanospheres with antibodies.

[0024] In some preferred embodiments, the antibody comprises a myoglobin (MYO) antibody.

[0025] In a fourth aspect, the present invention provides a use of the antibody-sensitized nanoparticles in the preparation of a PETIA detection kit.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Compared with the traditional one-step or two-step activation of carboxylated nano-microspheres, the present invention abandons the use of biological cross-linking agents such as EDC / NHS that have harmful effects on the normal physiological function structure of antibodies, and instead uses harmless thiazoline acylating agents with higher activation efficiency as acylating agents for activating nano-microspheres. After activation by the activating agent, the coupling efficiency of antibodies and microspheres can be improved, avoiding adverse effects on antibodies, so that the antibody-sensitized nanoparticles formed by coupling have excellent detection sensitivity and linear detection range.

[0028] 2. In the conventional coupling method, after adding EDC / NHS to the nanosphere suspension, the nanospheres may aggregate and settle, because EDC / NHS can react with the carboxyl active groups on the surface of the nanospheres to generate unstable active ester intermediates, which consumes a large number of carboxyl active groups on the surface of the nanospheres, resulting in a decrease in the high Zeta potential on the surface of the nanospheres due to the carboxyl active groups, thereby reducing the stability of the nanospheres and causing a tendency to aggregate and settle. Therefore, after adding EDC / NHS to the nanosphere suspension, the nanospheres will aggregate and settle. In the present invention, while the acylating agent consumes the carboxyl active groups on the surface of the nanospheres, causing the surface Zeta potential of the nanospheres to decrease, making the nanospheres easy to aggregate and settle, the activator is used to enhance the surface Zeta potential of the nanospheres and alleviate the tendency of the nanospheres to aggregate and settle, so that the surface Zeta potential of the nanospheres remains stable and the aggregation stability of the nanospheres is enhanced.

[0029] 3. By utilizing the decomposition characteristics of the acylating agent and the activating agent and weakening the physical adsorption capacity of the surface of the nanospheres, the unreacted acylating agent and the activating agent can be completely removed during the separation and purification step, thereby avoiding possible adverse effects on the normal physiological function structure of the antibody.

[0030] 4. After the thiazoline acylating agent (such as 2-thiazoline-2-thiol) of the present invention activates the nano-microspheres, a light yellow stable amide intermediate 3-acyl-2-thio-thiazoline is generated. When this stable active amide intermediate combines with the free amino functional group at the Fc end of the antibody, colorless 2-thiazoline-2-thiol is regenerated. The coupling reaction of the antibody binding to the nano-microspheres is carried out by dripping the antibody in batches, thereby achieving the purpose of quantitative use of the antibody and avoiding unnecessary waste of the antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0032] Figure 1 This is the calibration curve for Experimental Example 1;

[0033] Figure 2 This is the calibration curve for Experimental Example 2;

[0034] Figure 3 This is the calibration curve for Experimental Example 3;

[0035] Figure 4 This is the calibration curve for Experimental Example 4;

[0036] Figure 5 This is the calibration curve for comparative experiment example 1;

[0037] Figure 6 This is the calibration curve for comparative experiment example 2;

[0038] Figure 7 This is the calibration curve for comparative experiment example 3;

[0039] Figure 8 This is a scatter plot of the linear range detection results of Experimental Example 1;

[0040] Fig. 9 This is a scatter plot of the linear range detection results of Experimental Example 2;

[0041] Fig.10 This is a scatter plot of the linear range detection results of Experimental Example 3;

[0042] Fig.11 This is a scatter plot of the linear range detection results of Experimental Example 4;

[0043] Fig.12 Scatter plot of the linear range detection results of comparative experiment example 1;

[0044] Fig.13 Scatter plot of the linear range detection results of comparative experiment example 2;

[0045] Fig.14 The figure is a scatter plot of the linear range detection results of comparative experiment example 3. DETAILED DESCRIPTION

[0046] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0047] Example 1

[0048] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0049] (1) Weigh 5 mg of 2-thiazoline-2-thiol and 5 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0050] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 μL of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0051] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 3 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0052] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the activated polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 0.4 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0053] (5) The MYO antibody-sensitized particles after the incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min. The supernatant was discarded. The precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min. The supernatant was discarded. The precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and water bath ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0054] Example 2

[0055] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0056] (1) Weigh 10 mg of 2-thiazoline-2-thiol and 10 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0057] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0058] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0059] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 0.7 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0060] (5) The MYO antibody-sensitized particles after the incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion. The supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion. The supernatant was discarded, and the precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and water bath ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0061] Example 3

[0062] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0063] (1) Weigh 15 mg of 2-thiazoline-2-thiol and 15 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0064] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0065] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0066] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 1.0 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0067] (5) The MYO antibody-sensitized particles after the above incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended by ultrasonication in an ice water bath (500 W power) using 10 ml of 25 mM MES buffer at pH = 6.15, ultrasonically for 2 min and rested for 1 min. After the ultrasonication, the particles were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was discarded. The precipitate was resuspended by ultrasonication in an ice water bath (500 W power) using 10 ml of 25 mM MES buffer at pH = 7.40, ultrasonically for 2 min and rested for 1 min. After the ultrasonication, the particles were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was discarded. The precipitate was resuspended by ultrasonication in an ice water bath (500 W power) using 30 ml of R2 buffer at pH = 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300), ultrasonicated in a water bath at 500 W power for 5 min and rested for 2 min. The ultrasonication operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0068] Example 4

[0069] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0070] (1) Weigh 20 mg of 2-thiazoline-2-thiol and 20 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0071] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0072] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0073] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 1 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0074] (5) The MYO antibody-sensitized particles after the incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH value of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH value of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 30 ml of R2 buffer with a pH value of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and water bath ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0075] Example 5

[0076] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0077] (1) Weigh 15 mg of 2-thiazoline-2-thiol and 30 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0078] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0079] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0080] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 1.1 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0081] (5) The MYO antibody-sensitized particles after the incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH value of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH value of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 30 ml of R2 buffer with a pH value of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and water bath ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0082] Example 6

[0083] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0084] (1) Weigh 15 mg of 2-thiazoline-2-thiol and 45 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0085] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0086] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0087] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 1.3 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0088] (5) The MYO antibody-sensitized particles after the incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH value of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH value of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after ultrasound completion, and the supernatant was discarded. The precipitate was resuspended in 30 ml of R2 buffer with a pH value of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and water bath ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0089] Example 7

[0090] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0091] (1) Weigh 15 mg of 2-thiazoline-2-thiol and 60 mg of potassium peroxymonosulfonate, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0092] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0093] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0094] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 1.3 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0095] (5) The MYO antibody-sensitized particles after the above incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0096] Example 8

[0097] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0098] (1) Weigh 5 mg of 2-thiazoline-2-thiol and 5 mg of 2-methyl-6-nitrobenzoic anhydride, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0099] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0100] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0101] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 0.4 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0102] (5) The MYO antibody-sensitized particles after the above incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0103] Example 9

[0104] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0105] (1) Weigh 5 mg of 2-thiazoline-2-thiol and 5 mg of 1-hydroxyphenyltriazole, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0106] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0107] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0108] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 0.4 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0109] (5) The MYO antibody-sensitized particles after the above incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0110] Example 10

[0111] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0112] (1) Weigh 5 mg of 2-thiazoline-2-thiol and 5 mg of N,N-diisopropylformamide, add them to 1 ml of 75% ethanol solution, dissolve and mix, and incubate on a shaker at room temperature for 5 min;

[0113] (2) Add 10 ml of 50 mM MES buffer (pH = 6.15) to a beaker, then add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.), stir evenly, and then add 200 ul of the activated 2-thiazoline-2-thiol solution dropwise while stirring. After the addition is complete, stir and incubate at room temperature for 20 min;

[0114] (3) The activated nanospheres were incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), and then incubated in a 55°C water bath for 15 min, centrifuged at 15,000 rpm for 20 min at room temperature. This operation was repeated 3 times, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for use;

[0115] (4) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), and add 100 ul / time to the polystyrene nanosphere emulsion obtained in step (2) until the emulsion changes from light yellow to milky white until the titration is completed (about 0.4 ml of antibody is consumed). After the titration is completed, continue to stir and incubate for 30 min;

[0116] (5) The MYO antibody-sensitized particles after the above incubation were centrifuged at 4°C and 18,000 rpm for 30 min, and the supernatant was collected separately. The precipitate was first resuspended in 10 ml of 25 mM MES buffer with a pH of 6.15 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer with a pH of 7.40 by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min, and then centrifuged at 4°C and 18,000 rpm for 30 min after the ultrasound was completed. The supernatant was discarded, and the precipitate was resuspended in 30 ml of R2 buffer with a pH of 7.40 (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin300) and ultrasound at 500 W power for 5 min and rest for 2 min. The ultrasound operation was repeated 3 times, and the obtained MYO antibody-sensitized nanosphere emulsion was stored at 4°C for later use.

[0117] Comparative Example 1

[0118] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0119] (1) Weigh 15 mg of NHS and dissolve it in 1 ml of ultrapure water; weigh 5 mg of EDC and dissolve it in 1 ml of pure water;

[0120] (2) Add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.) to 10 ml of 50 mM MES buffer (pH = 6.15), mix well, add 200 ul of 10 mg / ml EDC solution and 200 ul of 30 mg / ml NHS solution, and incubate on a shaker at room temperature for 30 min;

[0121] (3) Centrifugation at 15,000 rpm for 20 min at 4°C;

[0122] (4) The supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), centrifuged at 4°C, 15000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for later use;

[0123] (5) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), add all of it to the activated nanosphere suspension obtained in step (4), mix well, and incubate on a shaker at room temperature for 60 min;

[0124] (6) Centrifuge at 4°C, 18,000 rpm for 30 min, collect the supernatant separately, and first resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 6.15) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 7.40) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 30 ml of R2 buffer (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin 300) at pH = 7.40, ultrasound at 500 W power for 5 min in a water bath and rest for 2 min. Repeat the ultrasound operation 3 times, and store the obtained MYO antibody-sensitized nanosphere emulsion at 4°C for future use.

[0125] Comparative Example 2

[0126] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0127] (1) Weigh 30 mg of NHS and dissolve it in 1 ml of ultrapure water; weigh 10 mg of EDC and dissolve it in 1 ml of pure water;

[0128] (2) Add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.) to 10 ml of 50 mM MES buffer (pH = 6.15), mix well, add 200 ul of 10 mg / ml EDC solution and 200 ul of 30 mg / ml NHS solution, and incubate on a shaker at room temperature for 30 min;

[0129] (3) Centrifugation at 15,000 rpm for 20 min at 4°C;

[0130] (4) The supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), centrifuged at 4°C, 15000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for later use;

[0131] (5) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), add all of it to the activated nanosphere suspension obtained in step (4), mix well, and incubate on a shaker at room temperature for 60 min;

[0132] (6) Centrifuge at 4°C, 18,000 rpm for 30 min, collect the supernatant separately, and first resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 6.15) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 7.40) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 30 ml of R2 buffer (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin 300) at pH = 7.40, ultrasound at 500 W power for 5 min in a water bath and rest for 2 min. Repeat the ultrasound operation 3 times, and store the obtained MYO antibody-sensitized nanosphere emulsion at 4°C for future use.

[0133] Comparative Example 3

[0134] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0135] (1) Weigh 45 mg of NHS and dissolve it in 1 ml of ultrapure water; weigh 15 mg of EDC and dissolve it in 1 ml of ultrapure water;

[0136] (2) Add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.) to 10 ml of 50 mM MES buffer (pH = 6.15), mix well, add 200 ul of 10 mg / ml EDC solution and 200 ul of 30 mg / ml NHS solution, and incubate on a shaker at room temperature for 30 min;

[0137] (3) Centrifugation at 15,000 rpm for 20 min at 4°C;

[0138] (4) The supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), centrifuged at 4°C, 15000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for later use;

[0139] (5) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), add all of it to the activated nanosphere suspension obtained in step (4), mix well, and incubate on a shaker at room temperature for 60 min;

[0140] (6) Centrifuge at 4°C, 18,000 rpm for 30 min, collect the supernatant separately, and first resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 6.15) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 7.40) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 30 ml of R2 buffer (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin 300) at pH = 7.40, ultrasound at 500 W power for 5 min in a water bath and rest for 2 min. Repeat the ultrasound operation 3 times, and store the obtained MYO antibody-sensitized nanosphere emulsion at 4°C for future use.

[0141] Comparative Example 4

[0142] A myoglobin (MYO) antibody-sensitized polystyrene nanosphere is obtained by the following steps:

[0143] (1) Weigh 60 mg of NHS and dissolve it in 1 ml of ultrapure water; weigh 20 mg of EDC and dissolve it in 1 ml of pure water;

[0144] (2) Add 0.2 ml of carboxylated polystyrene nanospheres (purchased from Beijing Boerma Biotechnology Co., Ltd.) to 10 ml of 50 mM MES buffer (pH = 6.15), mix well, add 200 ul of 10 mg / ml EDC solution and 200 ul of 30 mg / ml NHS solution, and incubate on a shaker at room temperature for 30 min;

[0145] (3) Centrifugation at 15,000 rpm for 20 min at 4°C;

[0146] (4) The supernatant was discarded, and the precipitate was resuspended in 10 ml of 50 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min), centrifuged at 4°C, 15000 rpm for 20 min, the supernatant was discarded, and the precipitate was resuspended in 10 ml of 25 mM MES buffer (pH = 6.15) by water bath ultrasound (500 W power, ultrasound for 2 min) for later use;

[0147] (5) Take 0.5 ml of MYO antibody (purchased from Nanjing Okai Biotechnology Co., Ltd.) and mix with 1 ml of 25 mM MES buffer (pH = 6.15), add all of it to the activated nanosphere suspension obtained in step (4), mix well, and incubate on a shaker at room temperature for 60 min;

[0148] (6) Centrifuge at 4°C, 18,000 rpm for 30 min, collect the supernatant separately, and first resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 6.15) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 10 ml of 25 mM MES buffer (pH = 7.40) by ice-water bath ultrasound (500 W power), ultrasound for 2 min and rest for 1 min. After the completion of ultrasound, centrifuge at 4°C, 18,000 rpm for 30 min, discard the supernatant, and resuspend the precipitate in 30 ml of R2 buffer (10 mM phosphate buffer + 1% sucrose + 0.1% Proclin 300) at pH = 7.40, ultrasound at 500 W power for 5 min in a water bath and rest for 2 min. Repeat the ultrasound operation 3 times, and store the obtained MYO antibody-sensitized nanosphere emulsion at 4°C for future use.

[0149] Experimental Example 1

[0150] This experimental example provides a myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection kit, and the composition of each component is shown in Table 1.

[0151] Table 1 Components of Myoglobin (MYO) latex-enhanced immunoturbidimetric assay kit

[0152]

[0153]

[0154] The various ingredients were added sequentially at room temperature and stirred magnetically until completely dissolved.

[0155] The myoglobin (MYO) latex enhanced immunoturbidimetric in vitro detection kit described in this experimental example is suitable for various types of fully automatic biochemical analyzers. This experiment uses the Dirui CS-600B fully automatic biochemical analyzer for determination. The detection conditions are shown in Table 2 and the test method is shown in Table 3.

[0156] Table 2 Myoglobin (MYO) detection conditions

[0157] wavelength 546nm(main) specimen 10ul Reaction method Two-point endpoint method Colorimeter light path 1cm R1 150ul Calibration Type Nonlinear Reaction temperature 37℃ R2 50ul Reaction direction up

[0158] Table 3 Myoglobin (MYO) assay parameters

[0159]

[0160] Experimental Example 2

[0161] In this experimental example, except that the myoglobin (MYO) sensitized nanoparticles in the R2 reagent were prepared in Example 5, the rest were the same as those in Experimental Example 1.

[0162] Experimental Example 3

[0163] In this experimental example, except that the myoglobin (MYO) sensitized nanoparticles in the R2 reagent were prepared in Example 6, the rest were the same as those in Experimental Example 1.

[0164] Experimental Example 4

[0165] In this experimental example, except that the myoglobin (MYO) sensitized nanoparticles in the R2 reagent were prepared in Example 7, the rest were the same as those in Experimental Example 1.

[0166] Comparative Experiment Example 1

[0167] In this experimental example, except that the myoglobin (MYO) sensitized nanoparticles in the R2 reagent were prepared in Comparative Example 2, the rest were the same as Experimental Example 1.

[0168] Comparative Experiment Example 2

[0169] In this experimental example, except that the myoglobin (MYO) sensitized nanoparticles in the R2 reagent were prepared in Comparative Example 4, the rest were the same as those in Experimental Example 1.

[0170] Comparative Experiment 3

[0171] A commercially available myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection kit (purchased from Fujirebio Inc.).

[0172] Performance Test Example 1

[0173] 1. Stability

[0174] The stability of the nanospheres can be shown by the Zeta potential on the surface of the nanospheres. When EDC is used as a biocrosslinking reagent, the stability of the nanospheres will decrease after the addition of EDC. This may be due to the decrease in the Zeta potential on the surface of the nanospheres caused by the activation of the nanospheres by EDC. Therefore, the samples before and after the activation of the nanospheres in Examples 3 and Examples 5-7 can be collected for comparison by Zeta potential measurement.

[0175] Zeta potential analyzer (Shanghai Powerchip Scientific Instrument Co., Ltd.) was used to measure the Zeta potential of the nanospheres in Example 3, Examples 5-7 and Comparative Example 3 before and after activation according to the following experimental steps, and the average value was calculated.

[0176] (1) Turn on the power of the Zeta potential analyzer, let it stand for 30 minutes, and turn on the computer to perform instrument self-test;

[0177] (2) Establish the measurement procedure: select the material name and the dispersed medium, set the measurement temperature to 4°C, measure each sample 3 times, set the sample volume to 1 ml, and measure the sample after the settings are completed;

[0178] (3) 1 ml of each sample of the nanospheres before and after activation in Example 3, Examples 5-7 and Comparative Example 3 was taken to measure the Zeta potential (unit: mV). The measurement results are shown in Table 4.

[0179] Table 4 Zeta potential of nanospheres before and after activation in Example 3, Examples 5-7, and Comparative Example 3

[0180]

[0181]

[0182] By comparing the measurement results of the Zeta potential of the nanospheres before and after activation in Comparative Example 3, it was found that adding EDC / NHS crosslinking agent to the nanosphere suspension to activate the nanospheres would lead to a decrease in the Zeta potential on the surface of the nanospheres, thereby weakening the stability of the nanospheres, resulting in the aggregation and sedimentation of the nanospheres after adding EDC / NHS to activate the nanospheres. A comprehensive comparison of the Zeta potential measurement results of the nano-microspheres before and after activation of Example 3 and Example 5-7 found that when 2-thiazoline-2-thiol and potassium peroxymonosulfonate were used as coupling reagents, the Zeta potential of the surface of the nano-microspheres after activation was higher than that when EDC / NHS was used as a cross-linking agent to activate the nano-microspheres, and as the amount of potassium peroxymonosulfonate used in the coupling reagent increased, the Zeta potential also increased, but due to the limitations of factors such as the content of carboxyl functional groups on the surface of the carboxylated nano-microspheres, there was a system maximum value. In the reaction system used in this experiment, when the mass ratio of 2-thiazoline-2-thiol to potassium peroxymonosulfonate reached 1:3, the effect was best. At this time, the Zeta potential of the nano-microspheres did not change substantially before and after activation, and further increasing the mass of potassium peroxymonosulfonate in the system could not cause the Zeta potential of the nano-microspheres to continue to increase after activation, that is, under this experimental condition, the stability of the nano-microspheres did not change substantially before and after activation.

[0183] 2. Antibody coupling efficiency

[0184] A key point in determining the performance of latex-enhanced immunoturbidimetric reagents is the amount of antibody that is chemically coupled to the surface of the nanospheres. The traditional method is to detect the antibody protein remaining in the supernatant after coupling by enzyme-linked immunosorbent reaction (ELISA) or protein quantification method (Bradford, BCA method, etc.), and evaluate the efficiency of antibody coupling to the surface of the nanospheres based on the remaining amount of antibody in the supernatant. The BCA kit (Shanghai Biotech Co., Ltd.) was used to perform the experiment using the Read Max 1000F light absorption microplate reader (Shanghai Flash Biotech Co., Ltd.) according to the steps described in the kit instructions.

[0185] (1) Take 1.2 ml of standard protein preparation solution and add it to 30 mg of BSA to fully dissolve to prepare a 25 mg / ml standard protein solution; take 40 ul of 25 mg / ml standard protein solution and add 960 ul of PBS diluent to prepare a 1.0 mg / ml protein standard solution;

[0186] (2) Prepare BCA working solution according to the number of samples (mix reagent A and reagent B in a volume ratio of 50:1 and mix thoroughly);

[0187] (3) Add 1.0 mg / ml protein standard solution to the sample wells of a 96-well plate at the following levels: 0ul, 1ul, 2ul, 4ul, 8ul, 12ul, 16ul, and 20ul, and then add the standard diluent to make up to 20ul. That is, the concentrations of the standard are 0 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, and 1.0 mg / ml, respectively;

[0188] (4) 20ul of sample was taken from the supernatant collected after the antibody in Examples 1-4, Example 6, Examples 8-10, and Comparative Examples 1-4 was bound to the nanospheres and added to the sample wells of a 96-well plate;

[0189] (5) Add 200 μl of BCA working solution to each well, and measure each standard and experimental sample in parallel three times. Place the 96-well plate in a 37°C constant temperature incubator and incubate for 20-30 min.

[0190] (6) A562 was measured using a Read Max 1000F microplate reader, and the absorbance values ​​of the standards and samples were collected and collated. The results are shown in Table 5;

[0191] (7) Draw a standard curve based on the absorbance measurement results of the standard, and calculate the amount of antibody remaining in the supernatant based on the absorbance of the experimental sample and the sample volume used.

[0192] Table 5 Absorbance values ​​measured for each sample well

[0193]

[0194]

[0195] The standard curve y=Ax+B was drawn with the concentration of each standard as the X-axis and the absorbance of each standard as the Y-axis. The amount of antibody remaining in the sample was calculated based on the absorbance value of the measured sample, and the coupling efficiency of the antibody and microspheres in each sample was evaluated.

[0196] According to the standard curve y=0.0002x+0.2572 (R2=0.9963) obtained in the experiment, the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 1 was 84.00ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 2 was 107.30ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 3 was 90.70ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 4 was 87.30ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 6 was 74.00ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres were coupled to the antibodies in Example 8 was The remaining antibody concentration is 182.30ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Example 9 is 158.70ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Example 10 is 212.30ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Comparative Example 1 is 580.70ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Comparative Example 2 is 360.70ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Comparative Example 3 is 617.30ug / ml; the remaining antibody concentration in the supernatant after the nano-microspheres are coupled to the antibodies in Comparative Example 4 is 784.00ug / ml.

[0197] According to the calculation results of the residual antibody concentration in the supernatant after the nano-microspheres are coupled with antibodies in Examples 1-4 and Example 6, since the present invention adopts the color development effect to quantitatively add antibodies for coupling reaction, the residual antibody concentration in the supernatant after the antibodies are coupled is almost the same; according to the amount of antibody used in the coupling reaction, combined with the amount of 2-thiazoline-2-thiol and potassium peroxymonosulfonate used when activating the microspheres, it can be concluded that the activation effect of carboxylated nano-microspheres increases with the increase of the concentration of 2-thiazoline-2-thiol and potassium peroxymonosulfonate, but due to the limitations of factors such as the particle size of the nano-microspheres, the solid content and the content of carboxyl functional groups on the surface of the nano-microspheres, the activation efficiency of 2-thiazoline-2-thiol and potassium peroxymonosulfonate for the carboxylated nano-microspheres has a maximum use concentration under the specified nano-microsphere conditions. Comparing the experimental results of Example 1 and Examples 8-10, it was found that the nanoparticles and antibodies had a higher coupling efficiency when potassium peroxymonosulfonate was used as an activator, indicating that the activation efficiency of the carboxylated nanoparticles was better when potassium peroxymonosulfonate was used as an activator.

[0198] By comprehensively comparing the test results of the embodiments and the comparative examples, it is found that excessive use of EDC / NHS when using a biocrosslinker to activate carboxylated nano-microspheres will lead to a decrease in the coupling efficiency of the nano-microspheres and antibodies. The reason may be that the excess EDC / NHS is difficult to remove in the subsequent cleaning step, so that it reacts with the free carboxyl groups on the surface of the antibody, resulting in the antibody being unable to couple with the nano-microspheres, thus causing the problem of reduced efficiency of nano-microsphere coupling with antibodies, resulting in a higher concentration of antibodies remaining in the supernatant after the nano-microspheres are coupled with antibodies in the comparative example. The accuracy and actual effect of quantitatively titrating antibodies after activating nano-microspheres with 2-thiazoline-2-thiol and potassium peroxymonosulfonate still need to be supplemented by the test results of the reagents prepared by the subsequent use of the present invention.

[0199] Performance Test Example 2

[0200] The following experiments were performed using Experimental Examples 1-4 and Comparative Experimental Examples 1-3.

[0201] 1. Calibration

[0202] The calibration results of Experimental Examples 1-4 and Comparative Experimental Examples are shown in Tables 6 to 12. The calibration curves of Experimental Examples 1-4 and Comparative Experimental Examples 1-3 are shown in Tables 6 to 12. Figure 1-7 shown.

[0203] Table 6 Calibration results of Experimental Example 1

[0204]

[0205] Table 7 Calibration results of Experimental Example 2

[0206]

[0207] Table 8 Calibration results of Experimental Example 3

[0208]

[0209] Table 9 Calibration results of Experimental Example 4

[0210]

[0211] Table 10 Calibration results of comparative experimental example 1

[0212]

[0213] Table 11 Calibration results of comparative experimental example 2

[0214]

[0215] Table 12 Calibration results of comparative experimental example 3

[0216]

[0217] 2. Reagent detection sensitivity

[0218] Serum was diluted with 0.9% NaCl solution to a concentration of 2.5 ng / ml, and this was used as the mother solution. 0.9% NaCl solution was used to dilute the serum to 1 / 2, 1 / 4, 1 / 8, 1 / 16, and 1 / 32 at a ratio of 50%, and the mother solution with a concentration of 2.5 ng / ml was used to test the reagents prepared in the above experimental examples and comparative experimental examples. Each sample was tested 5 times, and the average value and SD value were calculated based on the test results. Starting from the lowest concentration, the value of the average value - 2SD above the zero average value + 2SD is the detection sensitivity of the reagent. The test results are shown in Tables 13 to 19.

[0219] Table 13 Experimental Example 1 Detection sensitivity test results

[0220]

[0221] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Experimental Example 1 is 0.625 ng / ml.

[0222] Table 14 Experimental Example 2 Detection Sensitivity Detection Results

[0223]

[0224]

[0225] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Experimental Example 2 is 0.3125 ng / ml.

[0226] Table 15 Detection sensitivity test results of experimental example 3

[0227]

[0228] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Experimental Example 3 is 0.078125 ng / ml.

[0229] Table 16 Experimental Example 4 Detection Sensitivity Detection Results

[0230]

[0231]

[0232] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Experimental Example 4 is 0.078125 ng / ml.

[0233] Table 17 Comparative Experimental Example 1 Detection Sensitivity Detection Results

[0234]

[0235] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Comparative Experimental Example 1 is 0.15625 ng / ml.

[0236] Table 18 Comparative Experimental Example 2 Detection Sensitivity Detection Results

[0237]

[0238]

[0239] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Comparative Experimental Example 2 was 1.25 ng / ml.

[0240] Table 19 Comparative Experimental Example 3 Detection Sensitivity Detection Results

[0241]

[0242] According to the above measurement results, the detection sensitivity of the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared in Comparative Experimental Example 3 is 0.15625 ng / ml.

[0243] Comprehensive comparison of the above experimental results shows that under the condition of nano-microspheres with the same particle size, the myoglobin (MYO) latex-enhanced immunoturbidimetric in vitro detection reagent prepared by the present invention has better detection sensitivity, and when the nano-microsphere-binding antibody coupling method described in the present invention is used, the actual usage of the antibody can be reduced while ensuring the coupling efficiency and the detection sensitivity of the subsequent test kit, thereby avoiding the phenomenon of large-scale waste of antibodies generated when using EDC / NHS-activated nano-microspheres to couple antibodies. While ensuring the detection sensitivity of the latex-enhanced immunoturbidimetric in vitro detection reagent, the amount of antibody used is greatly reduced when the nano-microsphere-binding antibody coupling method described in the present invention is used.

[0244] 3. Reagent linear detection range

[0245] Take a high-value sample with a concentration of 500ng / ml, use 0.9% NaCl solution as a diluent, and dilute it into 6 points according to 0.05, 0.1, 0.2, 0.4, 0.6, and 0.8, and detect it together with high-value samples of 500ng / ml, 600ng / ml and 800ng / ml according to the standard experimental procedures. Each sample is measured in parallel 3 times, and the average value is calculated respectively. The theoretical dilution concentration of the sample is used as the independent variable, and the average value of the actual measured concentration is used as the dependent variable. The linear regression equation and correlation coefficient are calculated, and the estimated concentration value of the sample is recalculated according to the linear regression equation. The deviation between the theoretical dilution concentration of the sample and the calculated estimated concentration is used to determine the linear detection range of the kit described in the experimental example. The experimental results are shown in Table 20. The scatter points of the linear range detection results of Experimental Examples 1-4 and Comparative Experimental Examples 1-3 are shown as follows: Figure 8-14 shown.

[0246] Table 20 Linear detection range measurement results of Experimental Examples 1-4 and Comparative Experimental Examples 1-3

[0247]

[0248] The results of the determination show that the test kits of Experimental Example 2, Experimental Example 3, Experimental Example 4, Comparative Experimental Example 1, and Comparative Experimental Example 3 can all reach a linear maximum value of 500ng / ml, with a good linear detection range. It is worth noting that the test kits described in Experimental Example 3 and Experimental Example 4 prepared by the present invention can reach a linear maximum value of 800ng / ml, which has a larger linear detection range than the test kits described in Comparative Experimental Example 1 and Comparative Experimental Example 3. Consistent with the above experimental results, under the same nano-microsphere experimental conditions (the same particle size of carboxylated nano-microspheres, the same content of carboxyl active groups, and the same solid content of nano-microspheres), the antibody-sensitized nanoparticles obtained by the coupling method of the present invention are used to prepare latex-enhanced immunoturbidimetric in vitro detection reagents, which have good reagent detection sensitivity and linear detection range, and the coupling method has an optimal concentration under specified conditions to ensure the experimental detection effect. Compared with traditional biocrosslinking agents such as EDC / NHS, the amount of antibody used in the latex-enhanced immunoturbidimetric in vitro detection reagent prepared by the method of the present invention is less when the same detection sensitivity and linear detection range are reached. The antibody coupling efficiency experiment, the detection sensitivity determined by the reagent prepared by the present invention, and the linear detection range experimental results show that the present invention can quantitatively use the antibody when the nano-microspheres are coupled with the antibody, thereby avoiding unnecessary waste of the antibody.

[0249] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A method for preparing carboxylated nanoparticles for coupling antibodies, characterized in that: The following steps are involved: S1, activating the thiazoline acylating agent with an activating agent; S2, using the activated thiazoline acylating agent to activate the carboxylated nanoparticles; S3, purifying the activated carboxylated nano-microspheres to obtain.

2. The preparation method according to claim 1, characterized in that: In step S1, the thiazoline acylating agent includes at least one of 5-chloro-2-methyl-4-isothiazoline-3-one, 2-thiazoline-2-thiol, 4-aminothiazoline-2-thione, benzothiazoline, and 2-methylthio-2-thiazoline; the activating agent includes at least one of 1-hydroxyphenyltriazole, oxalyl chloride, 4-trifluoromethylbenzoic anhydride, 2-methyl-6-nitrobenzoic anhydride, N,N-diisopropylformamide, potassium peroxymonosulfonate, and trifluoroacetic acid.

3. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of the thiazoline acylating agent to the activating agent is 1:1 to 1:

4.

4. The preparation method according to claim 1, characterized in that: In step S1, the activation method comprises dissolving and incubating a thiazoline acylating agent and an activating agent in a solvent; the solvent comprises an ethanol solution; and the activation time is 3-10 minutes.

5. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of the thiazoline acylating agent to the activating agent is 1:1 to 1:

4.

6. The preparation method according to claim 1, characterized in that: In step S2, the ratio of the activated thiazoline acylating agent to the carboxylated nanospheres is 10-100 mg: 0.5-1.5 mL.

7. The method according to claim 1, characterized in that In step S3, the purification method includes: heating the activated carboxylated nanospheres for the first time, centrifuging, resuspending the precipitate, heating for the second time, and centrifuging.

8. A carboxylated nanoparticle for coupling an antibody obtained by the preparation method according to any one of claims 1 to 7.

9. An antibody-sensitized nanoparticle, characterized in that: The antibody-sensitized nanoparticles are obtained by coupling and incubating the carboxylated nanospheres according to claim 8 with antibodies.

10. Use of the antibody-sensitized nanoparticles according to claim 9 in preparing a PETIA detection kit.

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