Detection method of polysaccharide content and application

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a polysaccharide content detection method and application. The polysaccharide content detection method provided by the invention comprises the following steps: preparing a solution, drawing a standard curve and detecting a sample, the step of preparing the solution comprises preparation of a standard solution, preparation of a standard serum diluent and preparation of a sample diluent; wherein the specific steps of preparing the standard serum diluent are as follows: diluting polysaccharide standard serum by 3 times with a buffer solution to obtain the standard serum diluent; the preparation method of the sample diluent comprises the following specific steps: centrifuging a sample to be detected at 8000-10000 g for 15-30 minutes, taking supernate, and diluting to obtain the sample diluent. The detection method provided by the invention has the advantages of simplicity in operation, high sensitivity, good repeatability and the like, and can provide powerful support for culture condition optimization in a pneumococcus production and fermentation process and quality control of pneumococcus polysaccharide vaccines.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to a method for detecting polysaccharide content and its application. Background Art

[0002] The determination of polysaccharide content is of great significance in the field of biomedicine, especially in the research and development and production of pneumococcal vaccines. Studying the relationship between bacterial growth and capsular polysaccharide production and finding the optimal culture conditions to obtain the maximum polysaccharide concentration in the fermentation product can effectively increase polysaccharide production. In addition, as a key component of pneumococcal vaccines, the accurate determination of polysaccharide content is crucial to ensuring the quality and safety of the vaccine.

[0003] At present, the commonly used methods for determining polysaccharide content mainly include the anthrone sulfate method and the enzyme-linked immunosorbent assay (ELISA). The anthrone sulfate method uses concentrated sulfuric acid to act on sugars to produce furfural derivatives, which then react with anthrone to form a dark green compound, thereby achieving quantitative detection of polysaccharides. However, this method can only measure total carbohydrates and cannot distinguish specific types of polysaccharides. It is also easily interfered by impurities such as glucose in the fermentation broth, resulting in inaccurate measurement results. The ELISA method uses enzyme-labeled antibodies or antigens to detect polysaccharide content through the specific binding of antigens and antibodies. Although the ELISA method has high specificity and sensitivity, the operation steps are complicated, time-consuming, and limited by the detection principle, making it difficult to achieve rapid and accurate polysaccharide content determination.

[0004] Therefore, there is an urgent need to develop a simple, rapid and accurate method for determining polysaccharide content to guide the optimization of pneumococcal culture conditions and achieve quality control of pneumococcal vaccines. Summary of the invention

[0005] In order to overcome the problems of complicated operation steps, long time consumption and low sensitivity in the existing polysaccharide content determination methods, the present application provides a polysaccharide content detection method and application.

[0006] In the first aspect, the present application provides a method for detecting polysaccharide content, which adopts the following technical solution: A method for detecting polysaccharide content comprises the following steps: (1) Preparation of solution: Prepare standard solutions: dilute the polysaccharide standard solution with sample diluent to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, and 6.0 μg / mL in sequence to obtain standard solutions; Prepare standard serum dilution: dilute the polysaccharide standard serum 3 times with buffer to obtain standard serum dilution; Prepare sample diluent: centrifuge the sample to be tested at 8000-10000g for 15-30min, take the supernatant and dilute it to obtain the sample diluent; (2) Drawing a standard curve: injecting the standard solution and the standard serum dilution into the immunochemical system for detection, using the standard polysaccharide concentration as the horizontal axis and the response value as the vertical axis, performing linear regression to obtain the standard curve regression equation; (3) Sample testing: inject the sample diluent and the standard serum diluent into the immunochemical system for testing to obtain the response value of the sample to be tested; substitute the response value of the sample to be tested into the standard curve regression equation to obtain the polysaccharide content of the sample to be tested.

[0007] The present application provides a method for detecting polysaccharide content by rate turbidimetry, which has the following effects: first, the detection method is easy to operate, does not require complicated sample pretreatment steps, and greatly saves detection time. Secondly, the rate turbidimetry has high specificity, can effectively avoid the interference of other impurities in the fermentation broth, and ensure the accuracy of the test results. In addition, the method has a wide linear range and high sensitivity, and can accurately measure the polysaccharide content at a lower concentration to meet the needs of different application scenarios. Furthermore, the method has good repeatability, stable and reliable data, and is suitable for large-scale production and quality control. Finally, the detection method makes the detection process more standardized and automated through the precise preparation of standard solutions and sample diluents and the optimization of immunochemical systems, reduces human errors, and improves detection efficiency and reliability. In short, the detection method provided by the present application not only simplifies the detection process of polysaccharide content, but also significantly improves the detection accuracy and stability, and provides strong support for the optimization of culture conditions in the production and fermentation process of pneumococcus and the quality control of pneumococcal polysaccharide vaccines. Compared with traditional determination methods, the detection method provided in the present application overcomes the problems of cumbersome operation steps, long time consumption and low sensitivity of existing polysaccharide content detection methods, and can quickly and accurately quantify the polysaccharide content. This method is used for internal monitoring of the pneumococcal fermentation culture process, which is of great significance for optimizing culture conditions and increasing polysaccharide production.

[0008] The principle of the detection method provided in the present application is: using polysaccharides as antigens, in the presence of an excess of antibodies, the antigen and antibody are combined to form antigen-antibody complex aggregates, and the specific proportional relationship between the formation rate of the antigen-antibody complex aggregates and the antigen stock in the system is used to achieve accurate detection of the content of a certain polysaccharide in the sample.

[0009] Optionally, the parameter setting of the immunochemistry system includes: selecting a non-competitive turbidity mode.

[0010] Optionally, the parameter settings of the immunochemical system include: the injection volumes of the standard solution, sample diluent and standard serum diluent are all set to 21 μL, and the reaction buffer volume is 200 μL.

[0011] Optionally, the parameter settings of the immunochemical system include: reaction time of 2-3 min, and number of repetitions of 2 times.

[0012] Optionally, the polysaccharide is selected from one or more of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F and type 23F pneumococcal polysaccharides.

[0013] Optionally, the model number of the sample diluent is 447640, and the model number of the buffer solution is 447650.

[0014] Optionally, the model of the immunochemistry system is IMMAGE 800.

[0015] In a second aspect, the present application provides a method for detecting polysaccharide content for use in quality control of pneumococcal polysaccharide vaccines or in bacterial fermentation and culture of pneumococcal conjugate vaccines.

[0016] In summary, this application has the following beneficial effects: 1. The present application provides a method for detecting polysaccharide content. The method can detect polysaccharide content simply, quickly and accurately through the steps of preparing solution, drawing standard curve and sample testing. The method has the advantages of simple operation, high sensitivity and good repeatability. It can provide strong support for the optimization of culture conditions in the production and fermentation process of pneumococcus and the quality control of pneumococcal polysaccharide vaccine.

[0017] 2. The polysaccharide content detection method provided in the present application can be used to guide the fermentation culture of pneumococcal polysaccharides of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, and type 23F, which is of great significance for optimizing the culture conditions of pneumococci and increasing the polysaccharide yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the standard curve of type 6A polysaccharide obtained in Example 1; Figure 2 is the standard curve of 18C-type polysaccharide obtained in Example 2; Figure 3 is the standard curve of type 19A polysaccharide obtained in Example 3; Figure 4 is the standard curve of type 5 polysaccharide obtained in Example 4; Figure 5is the standard curve of type 6B polysaccharide obtained in Example 5; Figure 6 This is the standard curve of 23F polysaccharide obtained in Example 6. DETAILED DESCRIPTION

[0019] The present application provides a method for detecting polysaccharide content, comprising the following steps: (1) Preparation of solution: Prepare standard solutions: dilute the polysaccharide standard solution with sample diluent to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, and 6.0 μg / mL in sequence to obtain standard solutions; Prepare standard serum dilution: dilute the polysaccharide standard serum 3 times with buffer to obtain standard serum dilution; Prepare sample diluent: centrifuge the sample to be tested at 8000-10000g for 15-30min, take the supernatant and dilute it to obtain the sample diluent; (2) Drawing a standard curve: injecting the standard solution and the standard serum dilution into the immunochemical system for detection, using the standard polysaccharide concentration as the horizontal axis and the response value as the vertical axis, performing linear regression to obtain the standard curve regression equation; (3) Sample detection: inject the sample diluent and the standard serum diluent into the immunochemical system for detection to obtain the response value of the sample diluent; substitute the response value of the sample diluent into the standard curve regression equation to obtain the polysaccharide content of the sample to be tested.

[0020] In the present application, the parameter settings of the immunochemistry system include: selecting the non-competitive turbidity mode; the injection volume of the standard solution, sample diluent and standard serum diluent are all set to 21 μL, and the reaction buffer volume is 200 μL; the reaction time is 2-3 min, and the number of repetitions is 2 times.

[0021] In this application, the model of the sample diluent is 447640, purchased from Beckman; the model of the buffer is 447650, purchased from Beckman; type 5 standard polysaccharides, type 6A standard polysaccharides, type 6B standard polysaccharides, type 18C standard polysaccharides, type 19A standard polysaccharides and type 23F standard polysaccharides are all purchased from ATCC; type 5 polysaccharide standard serum, type 6A polysaccharide standard serum, type 6B polysaccharide standard serum, type 18C polysaccharide standard serum, type 19A polysaccharide standard serum and type 23F polysaccharide standard serum are all purchased from SSI; the sample to be tested is type 5 pneumococcal fermentation broth, type 6A pneumococcal fermentation broth, type 6B pneumococcal fermentation broth, type 18C pneumococcal fermentation broth, type 19A pneumococcal fermentation broth or type 23F pneumococcal fermentation broth, which is made by our company. The raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0022] The present application is further described in detail below in conjunction with embodiments, test experiments and accompanying drawings.

[0023] Example 1 Example 1 provides a method for detecting the polysaccharide content in type 6A pneumococcal fermentation broth.

[0024] The method for detecting the polysaccharide content in the above-mentioned type 6A pneumococcal fermentation broth comprises the following steps: (1) Preparation of solution: Prepare standard solutions: dilute the type 6A standard polysaccharide solution to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, and 6.0 μg / mL with sample diluent to obtain standard solutions; Prepare standard serum dilution: dilute the 6A polysaccharide standard serum 3 times with buffer to obtain standard serum dilution; Prepare sample diluent: centrifuge the sample to be tested at 8000-10000g for 15-30min, take the supernatant and dilute it to obtain the sample diluent; (2) Drawing a standard curve: injecting the standard solution and the standard serum dilution into the immunochemical system for detection, using the standard polysaccharide concentration as the horizontal axis and the response value as the vertical axis, performing linear regression to obtain the standard curve regression equation; (3) Sample detection: inject the sample diluent and the standard serum diluent into the immunochemical system for detection to obtain the response value of the sample diluent; substitute the response value of the sample diluent into the standard curve regression equation to obtain the polysaccharide content of the sample to be tested.

[0025] The parameters of the immunochemistry system were set as follows: non-competitive turbidity mode was selected; the injection volume of the standard solution, sample diluent and standard serum diluent was set to 21 μL, and the reaction buffer volume was 200 μL; the reaction time was 2 min, and the number of repetitions was 2 times.

[0026] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of type 6A polysaccharide. The standard curve of type 6A polysaccharide obtained in Example 1 is as follows: Figure 1 As shown; the standard curve regression equation is y=85.099x-66.995, and the correlation coefficient R=0.9960.

[0027] 2. The fermentation broth of 6A type pneumococcal fermentation for 1h, 2h, 3h, 4h, 5h, 6h, 6.5h, 7h, 7.5h, 8h, and 8.5h was used as the test sample, and the sample dilution was prepared according to step (1). The dilution multiple of each fermentation broth is shown in Table 1; then the method according to step (3) was used to detect, and the response value was obtained. The response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution, and the polysaccharide content in each 6A type pneumococcal fermentation broth was obtained after conversion. The results are shown in Table 1 below.

[0028] Table 1 Detection results of polysaccharide content in 6A pneumococcal fermentation broth According to the test results in Table 1, as the fermentation time of type 6A pneumococcus increases, the polysaccharide content in the fermentation broth shows a trend of first increasing and then remaining basically unchanged. Considering the comprehensive time cost, the optimal fermentation culture time of type 6A pneumococcus is 6.5-7.5h. Therefore, it is shown that the detection method of polysaccharide content provided by the present application can quickly and accurately detect the polysaccharide content in the fermentation process of type 6A pneumococcus, realize internal monitoring of the fermentation culture process of pneumococcus, and is of great significance for optimizing culture conditions and increasing polysaccharide yield.

[0029] Repeatability The fermentation broth of type 6A pneumococcus (fermentation for 8.5 h) was taken, and 6 sample dilutions were prepared in parallel. Then, the polysaccharide content in the fermentation broth was obtained according to the method of Example 1, and the relative standard deviation of the 6 measurement results was calculated.

[0030] The repeatability test results are shown in Table 2 below: Table 2 Repeatability test results According to the test results in Table 2, when 6 groups of sample dilutions were tested in parallel using the test method provided by the present application, the relative standard deviation (RSD) of the test results was 1.2%. Therefore, it was shown that the method for detecting the polysaccharide content in the fermentation broth of type 6A pneumococcus provided by the present application had good repeatability.

[0031] accuracy Prepare the spiked solution: Take the 6A pneumococcal fermentation broth (fermentation for 1 h), add the 6A standard polysaccharide solution (10 μg / mL) in equal volume, and prepare 6 spiked solutions in parallel; The above 6 parts of spiked solutions were prepared into 6 parts of spiked sample dilution solutions (dilution factor was 10 times), and then tested according to the method of Example 1 to obtain the polysaccharide content in the spiked solution, and the measured value of the standard polysaccharide solution was obtained to calculate the standard polysaccharide recovery rate. The calculation formula is as follows: Measured value of standard polysaccharide solution = polysaccharide content of spiked solution - polysaccharide content of fermentation broth (Note: the polysaccharide content of fermentation broth is 11.30 μg / mL) Standard polysaccharide recovery rate = measured value / theoretical value × 100%.

[0032] Table 3 Accuracy test results According to the test results in Table 3, the recovery rates of the standard polysaccharides in the six spiked solutions are 83.5% to 94.5%. Therefore, it is shown that the method for detecting the polysaccharide content in the fermentation broth of type 6A pneumococcus provided in the present application has high accuracy.

[0033] Example 2 Example 2 provides a method for detecting the polysaccharide content in a 18C type pneumococcal fermentation broth.

[0034] The difference between the above embodiment and embodiment 1 is that the sample to be tested is 18C pneumococcal fermentation broth, the standard serum is 18C polysaccharide standard serum, and the standard polysaccharide solution is 18C standard polysaccharide solution.

[0035] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of 18C polysaccharide. The standard curve of 18C polysaccharide obtained in Example 1 is as follows: Figure 2 As shown; the standard curve regression equation is y=82.915x-80.804, and the correlation coefficient is R=0.9927.

[0036] 2. The fermented liquid of 18C pneumococcal fermentation for 1h, 2h, 3h, 4h, 4.5h and 5h was used as the sample to be tested, and the sample dilution was prepared according to step (1). The dilution multiple of each fermented liquid is shown in Table 4; then the method according to step (3) was used to detect, and the response value was obtained. The response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution, and the polysaccharide content in each 18C pneumococcal fermented liquid was obtained after conversion. The results are shown in Table 4 below.

[0037] Table 4 Detection results of polysaccharide content in 18C pneumococcal fermentation broth According to the test results of Table 4, as the fermentation time of 18C pneumococcus increases, the polysaccharide content in the fermentation liquid shows a trend of first increasing and then decreasing. Taking all factors into consideration, the optimal fermentation culture time of 18C pneumococcus is 4.5h. Therefore, it is shown that the detection method of polysaccharide content provided by the present application can quickly and accurately detect the polysaccharide content in the fermentation process of 18C pneumococcus, realize the internal monitoring of the fermentation culture process of 18C pneumococcus, and has important significance for optimizing culture conditions and increasing polysaccharide yield.

[0038] Example 3 Example 3 provides a method for detecting the polysaccharide content in type 19A pneumococcal fermentation broth.

[0039] The difference between the above embodiment and embodiment 1 is that the sample to be tested is type 19A pneumococcal fermentation broth, the standard serum is type 19A polysaccharide standard serum, and the standard polysaccharide solution is type 19A standard polysaccharide solution.

[0040] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of 19A type polysaccharide. The standard curve of 19A type polysaccharide obtained in Example 1 is as follows: Figure 3 As shown; the standard curve regression equation is y=72.694x-66.327, and the correlation coefficient is R=0.9914.

[0041] 2. The fermented liquid of 19A pneumococcal fermentation for 1h, 2h, 3h, 4h, 4.5h, 5h, 5.5h, 6h, and 6.5h was used as the test sample, and the sample dilution was prepared according to step (1). The dilution multiple of each fermented liquid is shown in Table 5; then the method according to step (3) was used to detect, and the response value was obtained. The response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution, and the polysaccharide content in each 19A pneumococcal fermented liquid was obtained after conversion. The results are shown in Table 5.

[0042] Table 5 Detection results of polysaccharide content in 19A pneumococcal fermentation broth According to the test results in Table 5, as the fermentation time of 19A pneumococcus increases, the polysaccharide content in the fermentation broth shows a trend of gradually increasing. Taking all factors into consideration, the optimal fermentation culture time of 19A pneumococcus is 6.5h. Therefore, it is shown that the detection method of polysaccharide content provided by the present application can quickly and accurately detect the polysaccharide content in the fermentation process of 19A pneumococcus, realize the internal monitoring of the fermentation culture process of 19A pneumococcus, and has important significance for optimizing culture conditions and increasing polysaccharide yield.

[0043] Example 4 Example 4 provides a method for detecting the polysaccharide content in type 5 pneumococcal fermentation broth.

[0044] The difference between the above embodiment and embodiment 1 is that the sample to be tested is type 5 pneumococcal fermentation broth, the standard serum is type 5 polysaccharide standard serum, and the standard polysaccharide solution is type 5 standard polysaccharide solution.

[0045] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of type 5 polysaccharide. The standard curve of type 5 polysaccharide obtained in Example 1 is as follows: Figure 4 As shown; the standard curve regression equation is y=92.936x-97.613, and the correlation coefficient R=0.9914.

[0046] 2. The fermented liquids of 5 types of pneumococcal fermentation for 1h, 2h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, and 6.5h were used as test samples, and sample dilutions were prepared according to step (1). The dilution multiples of each fermented liquid were as shown in Table 6; then the method according to step (3) was used to detect, and a response value was obtained. The response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution, and the polysaccharide content in each type 5 pneumococcal fermented liquid was obtained after conversion. The results are shown in Table 6 below.

[0047] Table 6 Detection results of polysaccharide content in type 5 pneumococcal fermentation broth According to the test results of Table 6, as the fermentation time of type 5 pneumococcus increases, the polysaccharide content in its fermentation liquid shows a trend of gradually increasing. Taking all factors into consideration, the optimal fermentation culture time of type 5 pneumococcus is 6.5h. Therefore, it is shown that the detection method of polysaccharide content provided by the application can detect the polysaccharide content in the fermentation process of type 5 pneumococcus quickly and accurately, realize the internal monitoring of the fermentation culture process of type 5 pneumococcus, and is of great significance to optimizing the culture conditions and increasing the polysaccharide yield.

[0048] Example 5 Example 5 provides a method for detecting the polysaccharide content in type 6B pneumococcal fermentation broth.

[0049] The difference between the above embodiment and embodiment 1 is that the sample to be tested is type 6B pneumococcal fermentation broth, the standard serum is type 6B polysaccharide standard serum, and the standard polysaccharide solution is type 6B standard polysaccharide solution.

[0050] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of type 5 polysaccharide. The standard curve of type 6B polysaccharide obtained in Example 1 is as follows: Figure 5 As shown; the standard curve regression equation is y=28.317x-19.625, and the correlation coefficient R=0.9970.

[0051] 2. The fermented liquid of 6B type pneumococcal fermentation for 1h, 2h, 3h, 4h, 4.5h, 5h, and 5.5h was used as the sample to be tested, and the sample dilution was prepared according to step (1). The dilution multiple of each fermented liquid is shown in Table 7; then the method according to step (3) was used to detect, and the response value was obtained, and the response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution. After conversion, the polysaccharide content in each 6B type pneumococcal fermented liquid was obtained. The results are shown in Table 7 below.

[0052] Table 7 Detection results of polysaccharide content in 6B pneumococcal fermentation broth According to the test results of Table 7, as the fermentation time of 6B type pneumococcus is prolonged, the polysaccharide content in its fermentation liquid shows a trend of gradually increasing. Taking all factors into consideration, the optimal fermentation culture time of 6B type pneumococcus is 5h. Therefore, it is shown that the detection method of polysaccharide content provided by the application can detect the polysaccharide content in the fermentation process of 6B type pneumococcus quickly and accurately, realize the internal monitoring of the fermentation culture process of 6B type pneumococcus, and is of great significance to optimizing culture conditions and increasing polysaccharide yield.

[0053] Example 6 Example 6 provides a method for detecting the polysaccharide content in 23F type pneumococcal fermentation broth.

[0054] The difference between the above embodiment and embodiment 1 is that the sample to be tested is 23F type pneumococcal fermentation broth, the standard serum is 23F type polysaccharide standard serum, and the standard polysaccharide solution is 23F type standard polysaccharide solution.

[0055] Test results 1. Standard curve: First, perform the test according to the method of step (1-2) to draw the standard curve of 23F type polysaccharide. The standard curve of 23F type polysaccharide obtained in Example 1 is as follows: Figure 6 As shown; the standard curve regression equation is y=83.413x-81.024, and the correlation coefficient is R=0.9937.

[0056] 2. The fermented liquid of 23F type pneumococcal fermentation for 1h, 2h, 3h, 4h, 4.5h, 5h, and 5.5h was used as the sample to be tested, and the sample dilution was prepared according to step (1). The dilution multiple of each fermented liquid is shown in Table 8; then the method according to step (3) was used to detect, and the response value was obtained. The response value was brought into the standard curve regression equation to obtain the polysaccharide content in each sample dilution, and the polysaccharide content in each 23F type pneumococcal fermented liquid was obtained after conversion. The results are shown in Table 8 below.

[0057] Table 8 Detection results of polysaccharide content in 23F pneumococcal fermentation broth According to the test results of Table 8, as the fermentation time of 23F type pneumococcus is prolonged, the polysaccharide content in its fermentation liquid shows a trend of gradually increasing. Taking all factors into consideration, the optimal fermentation culture time of 23F type pneumococcus is 5.5h. Therefore, it is shown that the detection method of polysaccharide content provided by the application can detect the polysaccharide content in the fermentation process of 23F type pneumococcus quickly and accurately, realize the internal monitoring of the fermentation culture process of 23F type pneumococcus, and is of great significance to optimizing culture conditions and increasing polysaccharide yield.

[0058] In summary, the polysaccharide content detection method provided in the present application can be used to guide the fermentation culture of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, and type 23F pneumococcal polysaccharides, which is of great significance for optimizing the culture conditions of pneumococci and increasing the polysaccharide yield.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting polysaccharide content, characterized in that: The following steps are involved: (1) Preparation of solution: Prepare standard solutions: dilute the polysaccharide standard solution with sample diluent to concentrations of 1.0 μg / mL, 2.0 μg / mL, 3.0 μg / mL, 4.0 μg / mL, 5.0 μg / mL, and 6.0 μg / mL in sequence to obtain standard solutions; Prepare standard serum dilution: dilute the polysaccharide standard serum 3 times with buffer to obtain standard serum dilution; Prepare sample diluent: centrifuge the sample to be tested at 8000-10000g for 15-30min, take the supernatant and dilute it to obtain the sample diluent; (2) Drawing a standard curve: inject the standard solution and the standard serum dilution into the immunochemical system for detection, use the standard polysaccharide concentration as the horizontal axis and the response value as the vertical axis, perform linear regression, and obtain the standard curve regression equation; (3) Sample testing: Inject the sample diluent and the standard serum diluent into the immunochemical system for testing to obtain the response value of the sample diluent; substitute the response value of the sample diluent into the standard curve regression equation to obtain the polysaccharide content of the sample to be tested.

2. The method for detecting polysaccharide content according to claim 1, characterized in that: The parameter setting of the immunochemical system includes: selecting a non-competitive turbidity mode.

3. The method for detecting polysaccharide content according to claim 1, characterized in that: The parameter settings of the immunochemical system include: the injection volumes of the standard solution, sample diluent and standard serum diluent are all set to 21 μL, and the reaction buffer volume is 200 μL.

4. The method for detecting polysaccharide content according to claim 1, characterized in that: The parameter settings of the immunochemical system include: reaction time of 2-3 minutes and repetition times of 2 times.

5. The method for detecting polysaccharide content according to claim 1, characterized in that: The polysaccharide is selected from one or more of type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F and type 23F pneumococcal polysaccharides.

6. The method for detecting polysaccharide content according to claim 1, characterized in that: The model of the sample diluent is 447640, and the model of the buffer is 447650.

7. The method for detecting polysaccharide content according to claim 1, characterized in that: The model of the immunochemistry system is IMMAGE 800.

8. Use of the detection method according to any one of claims 1 to 7 in the quality control of pneumococcal polysaccharide vaccines or in the bacterial fermentation and culture process of pneumococcal conjugate vaccines.

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