A rapid detection method and kit for polysaccharide conjugate vaccine

Abstract

The application discloses a rapid detection method and kit of polysaccharide conjugate vaccine, and relates to the technical field of vaccine detection. The rapid detection method of polysaccharide conjugate vaccine comprises the following steps: selecting different types of polysaccharides and respectively depolymerizing the polysaccharides into different molecular weights to obtain polysaccharide solutions to be reacted; adding the polysaccharide solutions to be reacted into holes of a multi-well plate respectively, adding reaction reagents with different multiples into the holes respectively, respectively adjusting pH values of solutions in the holes of the multi-well plate, freeze-drying and storing to obtain a kit; adding a connecting agent into the kit to make the polysaccharides in the kit have exposed amino groups, then adding ethanol and sodium chloride, storing at 2 DEG C-8 DEG C, centrifuging, removing supernatant and obtaining a kit containing polysaccharide derivatives; adding a chromogenic agent to perform a chromogenic reaction, determining absorbance values of solutions in the holes of the kit containing the polysaccharide derivatives by means of ultraviolet spectrophotometry, calculating reaction yields and completing detection.

Description

Technical Field

[0001] This application relates to the field of vaccine detection technology, and in particular to a rapid detection method and kit for polysaccharide conjugate vaccines. Background Technology

[0002] Polysaccharide conjugate vaccines are vaccines prepared by chemically binding polysaccharides covalently to a protein carrier. Currently, sugar conjugate vaccines composed of polysaccharide antigens chemically coupled to carrier proteins from Haemophilus influenzae type b (Hib), Streptococcus pneumoniae (Sp) (10 to 20 serotypes), Neisseria meningitidis (Nm) (A, C, W, Y), and Salmonella typhi VI (Vi) have been approved. During the development of polysaccharide conjugate vaccines, due to the large molecular weight, numerous reaction sites, and difficulty in detection of bacterial polysaccharides, multiple experiments are required to screen different polysaccharides and different reaction conditions for the same polysaccharide to obtain optimal experimental conditions. This contrasts with the parallel processing of multiple reactions in small molecule drug development, resulting in long development cycles, high material input, and time-consuming testing for polysaccharide conjugate vaccines. Therefore, it is necessary to develop a rapid detection method and kit for polysaccharide conjugate vaccines. Summary of the Invention

[0003] The main objective of this application is to provide a rapid detection method and kit for polysaccharide conjugate vaccines, aiming to solve the technical problem of long detection cycles in existing polysaccharide conjugate vaccines.

[0004] To achieve the above objectives, this application proposes a rapid detection method for polysaccharide conjugate vaccines, comprising the following steps:

[0005] Different types of polysaccharides were selected and depolymerized into different molecular weights to obtain polysaccharide solutions to be reacted;

[0006] The polysaccharide solution to be reacted was added to the wells of a multi-well plate, and different multiples of the reaction reagent were added to the wells of the multi-well plate. The pH value of the solution in each well of the multi-well plate was adjusted, and then the solution was lyophilized and stored to obtain the kit.

[0007] A linker is added to the kit to react and expose the amino groups on the polysaccharides in the kit. Then, ethanol and sodium chloride are added, and the kit is stored at 2℃-8℃. After centrifugation, the supernatant is removed to obtain a kit containing polysaccharide derivatives.

[0008] A colorimetric reagent is added to the kit containing the polysaccharide derivative to initiate a colorimetric reaction. The absorbance of the solution in each well of the kit is measured by ultraviolet spectrophotometry, and the reaction yield is calculated to complete the detection.

[0009] Optionally, the step of adding a linker to the kit to react and expose naked amino groups on the polysaccharides in the kit includes:

[0010] A linker is added to the kit and reacted overnight at 4°C to link the linker to the polysaccharide in the kit. The linker has amino groups.

[0011] Optionally, the linker is adipic acid dihydrazide, and the mass ratio of the linker to the polysaccharide in the kit is (4-5):1.

[0012] Optionally, the step of adding ethanol and sodium chloride, storing at 2℃-8℃, centrifuging, removing the supernatant, and obtaining the kit containing the polysaccharide derivative includes:

[0013] Add anhydrous ethanol and 0.1%-0.2% sodium chloride to the kit, store at 2℃-8℃ for 3h-5h to obtain a precipitate, then centrifuge for 25min-35min to remove the supernatant to obtain the kit containing polysaccharide derivatives.

[0014] Optionally, the color developer is trinitrobenzenesulfonic acid.

[0015] Optionally, the step of adding a colorimetric reagent to the kit containing the polysaccharide derivative to carry out a colorimetric reaction includes:

[0016] After dissolving the polysaccharide derivative in water in the kit containing the polysaccharide derivative, a colorimetric reagent and sodium tetraborate are added, and the amino group reacts with the colorimetric reagent to produce a colorimetric reaction.

[0017] Optionally, the step of determining the absorbance value of the solution in each well of the kit containing the polysaccharide derivative by ultraviolet spectrophotometry and calculating the reaction yield includes:

[0018] The absorbance values ​​of the solutions in each well of the kit containing the polysaccharide derivative were determined by ultraviolet spectrophotometry.

[0019] The absorbance value was substituted into a standard curve to obtain the concentration of the amino group;

[0020] Based on the concentration of the amino group, the connection efficiency between the polysaccharide and the linker is calculated, and the reaction yield is obtained.

[0021] Optionally, the perforated plate is a 96-well plate.

[0022] Optionally, the reaction reagent includes N-cyano-4-dimethylamino-pyridine tetrafluoroborate.

[0023] This application also proposes a rapid detection kit for polysaccharide conjugate vaccines, comprising a 96-well plate, wherein each well of the 96-well plate is filled with polysaccharides of different types and molecular weights, and the pH value of each well of the 96-well plate is different, and each well of the 96-well plate is also filled with a reaction solvent of different reaction folds.

[0024] This application addresses key influencing factors in the preparation of polysaccharide conjugate vaccines by rapidly obtaining experimental results during a single experiment. Different polysaccharide types significantly impact the immunogenicity of conjugate vaccines, and the same polysaccharide can yield different molecular weights under different depolymerization conditions. Both excessively large and small polysaccharide molecular weights affect the molecular weight of the polysaccharide-protein conjugate, thus influencing its immunogenicity. Furthermore, pH is a critical reaction condition during the polysaccharide-protein conjugation reaction. Different reactions require different pH values; some reactions, if too low (acidic), produce byproducts, while those too high (alkaline) may not react. Therefore, pH selection is necessary during polysaccharide conjugate vaccine preparation. The amount of reagents used in the conjugation reaction is also a crucial factor affecting the reaction yield; thus, the reagent ratio is an important criterion for assessing the immunogenicity of polysaccharide conjugate vaccines. Therefore, this application simultaneously incorporates different types of polysaccharides into a multi-well plate, sets different molecular weights for the same polysaccharide, and establishes different reaction pH and reagent ratios, among other key reaction conditions for polysaccharide conjugate vaccines. A linker is then used to expose the amino groups in the polysaccharides, which can undergo a specific colorimetric reaction with the chromogenic agent. The more exposed amino groups, the more pronounced the color development. By testing the absorbance values ​​of the solutions in each well of the kit containing polysaccharide derivatives, different reaction yields can be obtained. Based on the reaction yields, the optimal reaction conditions for the polysaccharide conjugate vaccine can be screened. This application allows for the simultaneous setting of multiple variables to screen key parameters of the polysaccharide conjugate vaccine, eliminating the need for multiple experiments to test the vaccine and obtain optimal experimental results. It essentially transforms a single-variable experiment into an orthogonal experiment, rapidly obtaining experimental results by measuring absorbance, saving testing time and material input, and significantly improving vaccine development efficiency. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structure of the rapid detection kit for the polysaccharide conjugate vaccine described in the embodiments of this application;

[0027] Figure 2 This is a schematic diagram illustrating the detection of the pneumococcal polysaccharide conjugate vaccine described in the embodiments of this application;

[0028] Figure 3 This is a colorimetric reaction diagram of the rapid detection kit for the polysaccharide conjugate vaccine described in the embodiments of this application.

[0029] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0030] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0031] To address the technical problems existing in the prior art, embodiments of this application provide a rapid detection method for polysaccharide conjugate vaccines, comprising the following steps:

[0032] Different types of polysaccharides were selected and depolymerized into different molecular weights to obtain polysaccharide solutions to be reacted;

[0033] The polysaccharide solution to be reacted was added to the wells of a multi-well plate, and different multiples of the reaction reagent were added to the wells of the multi-well plate. The pH value of the solution in each well of the multi-well plate was adjusted, and then the solution was lyophilized and stored to obtain the kit.

[0034] A linker is added to the kit to react and expose the amino groups on the polysaccharides in the kit. Then, ethanol and sodium chloride are added, and the kit is stored at 2℃-8℃. After centrifugation, the supernatant is removed to obtain a kit containing polysaccharide derivatives.

[0035] A colorimetric reagent is added to the kit containing the polysaccharide derivative to carry out a colorimetric reaction. The absorbance value of the solution in each well of the kit containing the polysaccharide derivative is measured by ultraviolet spectrophotometry, and the reaction yield is calculated to complete the detection.

[0036] This application addresses the time-consuming testing issues during the development of polysaccharide conjugate vaccines. Based on key influencing factors in the preparation process, it proposes a single-stage experiment to rapidly obtain experimental results. Different polysaccharide types significantly impact the immunogenicity of conjugate vaccines. For example, pneumococcal polysaccharide vaccines can utilize pneumococcal type 1, type 2, and type 3 polysaccharides. Different types of polysaccharides result in pneumococcal polysaccharide vaccines exhibiting varying degrees of immunogenicity. Furthermore, the same polysaccharide, under different depolymerization conditions, yields different molecular weights. The molecular weight of the polysaccharide varies considerably. Both excessively large and small pH values ​​affect the molecular weight of polysaccharide-protein conjugates, thereby influencing their immunogenicity. Furthermore, pH is a critical reaction condition during the polysaccharide-protein binding reaction. Different reactions require different pH values; some reactions, if too low (acidic), will produce byproducts, while those, if too high (alkaline), will not react. Therefore, in preparing polysaccharide conjugate vaccines, it is necessary to screen the reaction pH. The amount of reagents used in the binding reaction is also a significant factor affecting the reaction yield; thus, the ratio of reagents used is an important condition for assessing the immunogenicity of polysaccharide conjugate vaccines. Therefore, this application simultaneously incorporates different types of polysaccharides into a multi-well plate, sets different molecular weights for the same polysaccharide, and establishes different reaction pH and reagent ratios, among other key reaction conditions for polysaccharide conjugate vaccines. A linker is then used to expose the amino groups in the polysaccharides, which can undergo a specific colorimetric reaction with the chromogenic agent. The more exposed amino groups, the more pronounced the color development. By testing the absorbance values ​​of the solutions in each well of the kit containing polysaccharide derivatives, different reaction yields can be obtained. Based on the reaction yields, the optimal reaction conditions for the polysaccharide conjugate vaccine can be screened. This application allows for the simultaneous setting of multiple variables to screen key parameters of the polysaccharide conjugate vaccine, eliminating the need for multiple experiments to test the vaccine and obtain optimal experimental results. It essentially transforms a single-variable experiment into an orthogonal experiment, rapidly obtaining experimental results by measuring absorbance, saving testing time and material input, and significantly improving vaccine development efficiency.

[0037] As one possible implementation of this application, the step of adding a linker to the kit to react and expose amino groups on the polysaccharides in the kit includes:

[0038] A linker is added to the kit and reacted overnight at 4°C to link the linker to the polysaccharide in the kit. The linker has amino groups.

[0039] After reacting overnight at 4°C, the linker reacts with the polysaccharide, attaching to the polysaccharide molecule. The linker has amino groups, which exposes amino groups on the polysaccharide molecule. The exposed amino groups can react with the colorimetric reagent to produce a colorimetric reaction. The more exposed amino groups there are, the more obvious the color development, thus yielding the reaction yield.

[0040] As one possible implementation of this application, the linker is adipic acid dihydrazide, and the mass ratio of the linker to the polysaccharide in the kit is (4-5):1.

[0041] Specifically, the structural formula of adipic acid dihydrazide is:

[0042] It has amino groups at both ends, which can cross-link with polysaccharide molecules to generate relatively stable polysaccharide derivatives, thus enabling the polysaccharide molecules to carry amino groups.

[0043] As one possible implementation of this application, the step of adding ethanol and sodium chloride, storing at 2℃-8℃, centrifuging, removing the supernatant, and obtaining the kit containing the polysaccharide derivative includes:

[0044] Add anhydrous ethanol and 0.1%-0.2% sodium chloride to the kit, store at 2℃-8℃ for 3h-5h to obtain a precipitate, then centrifuge for 25min-35min to remove the supernatant to obtain the kit containing polysaccharide derivatives.

[0045] This application allows the polysaccharide derivative to precipitate by adding ethanol and sodium chloride to the kit and storing it at 2℃-8℃. After centrifugation to remove the supernatant, a high content of polysaccharide derivative can be obtained for subsequent colorimetric reactions.

[0046] As one possible implementation method of this application, the color developer is trinitrobenzenesulfonic acid.

[0047] Because trinitrobenzenesulfonic acid has strong electrophilic properties, it can react with naked amino groups to form stable products. By measuring the absorbance of these products, the concentration of amino groups can be indirectly determined, thereby reflecting the reaction yield.

[0048] As one possible implementation of this application, the step of adding a colorimetric reagent to the kit containing the polysaccharide derivative to carry out a colorimetric reaction includes:

[0049] After dissolving the polysaccharide derivative in water in the kit containing the polysaccharide derivative, a colorimetric reagent and sodium tetraborate are added, and the amino group reacts with the colorimetric reagent to produce a colorimetric reaction.

[0050] In this application, during the colorimetric reaction, in the presence of sodium tetraborate, the amino groups carried on the polysaccharide can undergo a colorimetric reaction with trinitrobenzenesulfonic acid to form a stable product. Moreover, the colorimetric reaction becomes more obvious as the number of amino groups in the complex increases. The reaction yield can then be determined by measuring the absorbance value of the product.

[0051] As one possible implementation of this application, the step of determining the absorbance value of the solution in each well of the kit containing the polysaccharide derivative by ultraviolet spectrophotometry and calculating the reaction yield includes:

[0052] The absorbance values ​​of the solutions in each well of the kit containing the polysaccharide derivative were determined by ultraviolet spectrophotometry.

[0053] The absorbance value was substituted into a standard curve to obtain the concentration of the amino group;

[0054] Based on the concentration of the amino group, the connection efficiency between the polysaccharide and the linker is calculated, and the reaction yield is obtained.

[0055] Based on the principle that amino groups react specifically with trinitrobenzenesulfonic acid, this application links adipic acid dihydrazide to a polysaccharide, exposing the amino groups on the polysaccharide. The more exposed amino groups, the more obvious the colorimetric reaction. After irradiating the kit containing the polysaccharide derivative at a certain wavelength, different absorbance values ​​can be obtained. By substituting the absorbance values ​​into a standard curve, the concentration of amino groups can be calculated. From the concentration of amino groups, the conjugation efficiency between the polysaccharide and the conjugator can be calculated. The conjugation efficiency between the polysaccharide and the conjugator is a key parameter of polysaccharide conjugate vaccines, thus reflecting the yield of the conjugation reaction. Experimental results can be obtained quickly by a single rapid absorbance test.

[0056] As one possible implementation of this application, the perforated plate is a 96-hole plate.

[0057] In one possible embodiment of this application, the reaction reagent includes N-cyano-4-dimethylamino-pyridine tetrafluoroborate. N-cyano-4-dimethylamino-pyridine tetrafluoroborate can serve as an activating agent, which can activate the hydroxyl groups on the polysaccharide, so that adipic acid dihydrazide can be linked to the polysaccharide through the activated hydroxyl groups on the polysaccharide.

[0058] An embodiment of this application also provides a rapid detection kit for a polysaccharide conjugate vaccine, comprising a 96-well plate, wherein each well of the 96-well plate is filled with polysaccharides of different types and molecular weights, and each well of the 96-well plate has a different pH value, and each well of the 96-well plate is also filled with a reaction solvent of different reaction folds.

[0059] The rapid detection kit of this application is made using a 96-well plate. By filling each well of the 96-well plate with different types of polysaccharides and polysaccharides of different molecular weights, and setting different reaction pH and different reaction reagent ratios, the kit can screen different polysaccharides and different reaction conditions of the same polysaccharide at one time, thereby quickly obtaining experimental results and screening out the optimal experimental conditions.

[0060] The technical solutions described above in this application will be explained in detail below with reference to specific embodiments.

[0061] Example 1

[0062] A rapid detection method for pneumococcal polysaccharide conjugate vaccine includes the following steps:

[0063] Pneumonia type 1 polysaccharide, pneumonia type 2 polysaccharide, and pneumonia type 3 polysaccharide were selected and ultrasonically depolymerized into different molecular weights, including 40,000, 60,000, 80,000, and 100,000, to obtain polysaccharide solutions to be reacted.

[0064] like Figure 2 As shown, the polysaccharide solution to be reacted was added to the wells of a 96-well plate, and 4, 6, and 8 times the amount of N-cyano-4-dimethylamino-pyridine tetrafluoroborate were added to the wells of the 96-well plate, respectively. The pH value of the solution in each well of the 96-well plate was adjusted accordingly. Since the conjugation reaction of pneumococcal polysaccharides is generally carried out in a weakly alkaline environment, the pH was set to alkaline. The solution was then lyophilized and stored to obtain the kit. Columns 1-4 contain pneumococcal type 1 polysaccharides, with molecular weights of 40,000 in column 1, 60,000 in column 2, 80,000 in column 3, and 100,000 in column 4. Columns 5-8 contain pneumococcal type 2 polysaccharides. Type 1 polysaccharide: Column 5 has a molecular weight of 40,000, Column 6 has a molecular weight of 60,000, Column 7 has a molecular weight of 80,000, and Column 8 has a molecular weight of 100,000; Columns 9-12 are type 3 pneumonia polysaccharides: Column 9 has a molecular weight of 40,000, Column 10 has a molecular weight of 60,000, Column 11 has a molecular weight of 80,000, and Column 12 has a molecular weight of 100,000; The pH value of rows A, B, and C is 8, the pH value of rows D, E, and F is 8.5, and the pH value of rows G, H, and I is 9; The reaction reagent multiplier in rows A, D, and G is 4 times, the reaction reagent multiplier in rows B, E, and H is 6 times, and the reaction multiplier in rows C, F, and I is 8 times.

[0065] Add aqueous solution to the kit to make the polysaccharide concentration 10 mg / mL, then add the linker adipic acid dihydrazide and react overnight at 4 °C. The mass ratio of the linker to the polysaccharide in the kit is 4:1. The adipic acid dihydrazide has an amino group and is linked to the polysaccharide in the kit. The polysaccharide has exposed amino groups. Then add 3 volumes of anhydrous ethanol and 0.1% sodium chloride to the kit and store at 5 °C for 4 h to obtain a precipitate. Centrifuge for 30 min, remove the supernatant, and obtain the kit containing the polysaccharide derivative.

[0066] In a kit containing polysaccharide derivatives, after dissolving the polysaccharide derivatives in water, the colorimetric reagents trinitrobenzenesulfonic acid and sodium tetraborate are added. The amino group reacts with trinitrobenzenesulfonic acid to produce a colorimetric reaction, such as... Figure 3 As shown, the absorbance values ​​of the solutions in each well of the kit containing the polysaccharide derivative were determined by ultraviolet spectrophotometry.

[0067] The absorbance value was substituted into the standard curve to obtain the concentration of amino groups;

[0068] Based on the concentration of amino groups, the connection efficiency between the polysaccharide and adipic acid dihydrazide was calculated, the reaction yield was obtained, and the detection was completed.

[0069] The measured absorbance values ​​are shown in Table 1 below.

[0070] Table 1. Absorbance values ​​(L / (g·cm)) for each well in the kit.

[0071]

[0072]

[0073] As shown in Table 1, the highest absorbance value was 0.95, which means that when the type 2 pneumonia polysaccharide was selected and depolymerized into polysaccharides with a molecular weight of 60,000, the reaction pH was 8, and the reaction reagent ratio was 4, the connection efficiency between the polysaccharide and adipic acid dihydrazide was the highest, resulting in the highest reaction yield.

[0074] Example 2

[0075] A rapid detection method for polysaccharide conjugate vaccines includes the following steps:

[0076] Different types of polysaccharides were selected and depolymerized into different molecular weights to obtain polysaccharide solutions to be reacted;

[0077] The polysaccharide solution to be reacted was added to the wells of a 96-well plate, and different multiples of N-cyano-4-dimethylamino-pyridine tetrafluoroborate were added to the wells of the 96-well plate. The pH value of the solution in each well of the 96-well plate was adjusted, and then the solution was lyophilized and stored to obtain the kit.

[0078] Add the linker adipic acid dihydrazide to the kit and react overnight at 4°C. The mass ratio of the linker to the polysaccharide in the kit is 4:1. The adipic acid dihydrazide has an amino group and is linked to the polysaccharide in the kit. The polysaccharide has exposed amino groups. Then add anhydrous ethanol and 0.1% sodium chloride to the kit and store at 2°C for 35 h to obtain a precipitate. Centrifuge for 25 min and remove the supernatant to obtain the kit containing the polysaccharide derivative.

[0079] After dissolving the polysaccharide derivative in water in the kit containing the polysaccharide derivative, trinitrobenzenesulfonic acid and sodium tetraborate, a colorimetric reagent, are added. The amino group reacts with the trinitrobenzenesulfonic acid to produce a colorimetric reaction. The absorbance value of the solution in each well of the kit containing the polysaccharide derivative is determined by ultraviolet spectrophotometry.

[0080] The absorbance value was substituted into a standard curve to obtain the concentration of the amino group;

[0081] Based on the concentration of the amino group, the connection efficiency between the polysaccharide and the linker is calculated, the reaction yield is obtained, and the detection is completed.

[0082] Example 3

[0083] A rapid detection method for polysaccharide conjugate vaccines includes the following steps:

[0084] Different types of polysaccharides were selected and depolymerized into different molecular weights to obtain polysaccharide solutions to be reacted;

[0085] The polysaccharide solution to be reacted was added to the wells of a 96-well plate, and different multiples of N-cyano-4-dimethylamino-pyridine tetrafluoroborate were added to the wells of the 96-well plate. The pH value of the solution in each well of the 96-well plate was adjusted, and then the solution was lyophilized and stored to obtain the kit.

[0086] Add the linker adipic acid dihydrazide to the kit and react overnight at 4°C. The mass ratio of the linker to the polysaccharide in the kit is 5:1. The adipic acid dihydrazide has an amino group and is linked to the polysaccharide in the kit. The polysaccharide has exposed amino groups. Then add anhydrous ethanol and 0.2% sodium chloride to the kit and store at 8°C for 3 hours to obtain a precipitate. Centrifuge for 35 minutes, remove the supernatant, and obtain the kit containing the polysaccharide derivative.

[0087] After dissolving the polysaccharide derivative in water in the kit containing the polysaccharide derivative, trinitrobenzenesulfonic acid and sodium tetraborate, a colorimetric reagent, are added. The amino group reacts with the trinitrobenzenesulfonic acid to produce a colorimetric reaction. The absorbance value of the solution in each well of the kit containing the polysaccharide derivative is determined by ultraviolet spectrophotometry.

[0088] The absorbance value was substituted into a standard curve to obtain the concentration of the amino group;

[0089] Based on the concentration of the amino group, the connection efficiency between the polysaccharide and the linker is calculated, the reaction yield is obtained, and the detection is completed.

[0090] Example 4

[0091] A rapid test kit for a polysaccharide conjugate vaccine, such as Figure 1As shown, the plate includes a 96-well plate, each well of which is filled with polysaccharides of different types and molecular weights, and each well of which has a different pH value. The well of the 96-well plate is also filled with different multiples of N-cyano-4-dimethylamino-pyridine tetrafluoroborate.

[0092] In use, add adipic acid dihydrazide to the kit and react overnight at 4°C to allow the adipic acid dihydrazide to attach to the polysaccharide, resulting in exposed amino groups on the polysaccharide. Then, add anhydrous ethanol and sodium chloride to the kit to precipitate the polysaccharide derivative. Centrifuge to remove the supernatant, then add trinitrobenzenesulfonic acid and sodium tetraborate to allow the exposed amino groups to react with the trinitrobenzenesulfonic acid in a colorimetric reaction. The colorimetric results are shown below. Figure 3 As shown, the absorbance values ​​of each well in the kit were determined by ultraviolet spectrophotometry, the reaction yield was calculated, and the detection was completed.

[0093] The above description is merely an optional embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the inventive concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A rapid detection method for polysaccharide conjugate vaccines, characterized in that, Includes the following steps: Different types of polysaccharides were selected and depolymerized into different molecular weights to obtain polysaccharide solutions to be reacted; The polysaccharide solution to be reacted was added to the wells of a multi-well plate, and different multiples of the reaction reagent were added to the wells of the multi-well plate. The pH value of the solution in each well of the multi-well plate was adjusted, and then the solution was lyophilized and stored to obtain the kit. A linker is added to the kit to react and expose the amino groups on the polysaccharides in the kit. Then, ethanol and sodium chloride are added, and the kit is stored at 2℃-8℃. After centrifugation, the supernatant is removed to obtain a kit containing polysaccharide derivatives. A colorimetric reagent is added to the kit containing the polysaccharide derivative to initiate a colorimetric reaction. The absorbance of the solution in each well of the kit is measured by ultraviolet spectrophotometry, and the reaction yield is calculated to complete the detection.

2. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The step of adding a linker to the kit and reacting it to expose naked amino groups on the polysaccharides in the kit includes: A linker is added to the kit and reacted overnight at 4°C to link the linker to the polysaccharide in the kit. The linker has amino groups.

3. The rapid detection method for polysaccharide conjugate vaccines according to claim 2, characterized in that, The linker is adipic acid dihydrazide, and the mass ratio of the linker to the polysaccharide in the kit is (4-5):

1.

4. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The steps of adding ethanol and sodium chloride, storing at 2℃-8℃, centrifuging, removing the supernatant, and obtaining the kit containing the polysaccharide derivative include: Add anhydrous ethanol and 0.1%-0.2% sodium chloride to the kit, store at 2℃-8℃ for 3h-5h to obtain a precipitate, then centrifuge for 25min-35min to remove the supernatant to obtain the kit containing polysaccharide derivatives.

5. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The colorimetric agent is trinitrobenzenesulfonic acid.

6. The rapid detection method for polysaccharide conjugate vaccines according to claim 5, characterized in that, The step of adding a colorimetric reagent to the kit containing the polysaccharide derivative to carry out a colorimetric reaction includes: After dissolving the polysaccharide derivative in water in the kit containing the polysaccharide derivative, a colorimetric reagent and sodium tetraborate are added, and the amino group reacts with the colorimetric reagent to produce a colorimetric reaction.

7. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The step of determining the absorbance value of the solution in each well of the kit containing the polysaccharide derivative by ultraviolet spectrophotometry and calculating the reaction yield includes: The absorbance values ​​of the solutions in each well of the kit containing the polysaccharide derivative were determined by ultraviolet spectrophotometry. The absorbance value was substituted into a standard curve to obtain the concentration of the amino group; Based on the concentration of the amino group, the connection efficiency between the polysaccharide and the linker is calculated, and the reaction yield is obtained.

8. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The perforated plate is a 96-well plate.

9. The rapid detection method for polysaccharide conjugate vaccines according to claim 1, characterized in that, The reaction reagents include N-cyano-4-dimethylamino-pyridine tetrafluoroborate.

10. A rapid detection kit for a polysaccharide conjugate vaccine, characterized in that, The invention includes a 96-well plate, wherein each well of the 96-well plate is filled with polysaccharides of different types and molecular weights, and each well of the 96-well plate has a different pH value. Each well of the 96-well plate is also filled with a reaction solvent of different reaction ratios.

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