Method for testing in-vitro bactericidal function of typhoid vaccine immune serum

Abstract

Provided is a method for testing the in-vitro bactericidal function of a typhoid vaccine immune serum, comprising: testing the in-vitro bactericidal function of a typhoid vaccine immune serum against Salmonella typhi bacteria by means of an opsonophagocytic killing assay, wherein when the phagocytic killing step of the opsonophagocytic killing assay is completed, the Salmonella typhi bacteria having undergone the phagocytic killing step are cultured at the pH of 7.5-9.5. The opsonophagocytic killing assay is applied to Gram-negative Salmonella Typhi bacteria for the first time, and the in-vitro bactericidal levels of typhoid vaccine immune serums are effectively and accurately evaluated.

Description

Method for detecting the in vitro bactericidal function of typhoid vaccine immune serum Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for detecting the in vitro bactericidal function of typhoid vaccine immune serum. Background Art

[0002] Typhoid fever, a systemic febrile illness caused by infection with the enteric bacterium Salmonella Typhi (S. Typhi), primarily affects populations in underdeveloped or developing countries and remains a significant public health problem. S. Typhi is transmitted through the fecal-oral route via contaminated water or food, often in conjunction with poor sanitation and hygiene practices.

[0003] Globally, the latest estimates place the burden of typhoid fever at 11 to 18 million cases annually, primarily affecting children of school age or younger. Typhoid fever is one of the most common causes of bacteremia in many underdeveloped or developing countries, with most cases originating in the South Asian subcontinent. However, within these countries, the burden of disease is often unevenly distributed, with areas with poor sanitation being most affected. While improvements in health infrastructure can significantly reduce the burden of disease in most developed countries, developing adequate infrastructure for improved water and sanitation requires large-scale investments, a distant goal for populations in underdeveloped or developing countries. Therefore, vaccination remains the most cost-effective solution in these areas.

[0004] The principle of non-inferiority and the use of immunological assays as alternative endpoints for determining clinical efficacy have been applied to new vaccine approvals. Therefore, the establishment of standardized, internationally recognized immunological assays is crucial for vaccine development. Commonly used immunological assays include: serum antibody concentration testing (enzyme-linked immunosorbent assay) and in vitro bactericidal activity testing of serum antibodies.

[0005] In vitro testing for the bactericidal activity of serum antibodies typically involves two methods: serum bactericidal testing and opsonophagocytosis testing. The serum bactericidal test is typically used for Gram-negative bacteria, while the opsonophagocytosis test is typically used for Gram-positive bacteria. There are certain differences in the complement activation pathways between serum bactericidal testing and opsonophagocytosis testing. Specifically, the serum bactericidal test primarily activates a series of enzyme and protein reactions through complement to form the membrane attack complex (MAC), ultimately leading to the destruction of the microbial cell membrane. This process occurs within the humoral immune system, rather than directly involving immune cells. In contrast, opsonophagocytosis testing involves immune cells recognizing and localizing external microorganisms through surface receptors. These receptors bind to molecules on the microbial surface, triggering the initiation of phagocytosis. Once the microorganism is recognized, the immune cell encapsulates it within a membrane sac called a phagocytic vesicle through cell membrane deformation. The phagocytic vesicle contains digestive substances such as proteases, nucleases, and lipases, which work synergistically to kill or decompose the microorganism by regulating an acidic environment and generating oxygen free radicals.

[0006] Salmonella enterica and Salmonella typhi are Gram-negative bacteria. Gram-negative bacteria contain lipopolysaccharide (LPS), commonly known as endotoxin, in their structure. LPS is highly antigenic and can induce a strong immune response. When opsonophagocytosis tests are applied to Gram-negative bacteria, they are rapidly engulfed and killed during the test, making it difficult to develop a stable test method. Therefore, opsonophagocytosis tests are more commonly used with Gram-positive bacteria to provide more stable control over the test process.

[0007] In this area of ​​research, no protective factors or surrogates for typhoid conjugate vaccines have been identified, nor are there methods for measuring the in vitro bactericidal activity of serum antibodies to typhoid vaccines. While determining the bactericidal index induced by typhoid vaccines is crucial, no method for measuring this index has yet been applied in actual clinical evaluation.

[0008] Summary of the Invention

[0009] In order to solve the problems existing in the above-mentioned prior art, the present invention creatively applies the opsonophagocytic bactericidal test to the Gram-negative bacteria Salmonella typhi, and by adjusting the test conditions, obtains a stable test method so as to quickly and accurately calculate the opsonophagocytic bactericidal index induced by typhoid vaccine, thereby effectively evaluating the in vitro bactericidal level of typhoid vaccine immune serum.

[0010] The present invention provides a method for detecting the in vitro bactericidal function of typhoid vaccine immune serum. Specifically, the present invention provides:

[0011] (1) A method for detecting the in vitro bactericidal function of typhoid vaccine immune serum, comprising: using an opsonization bactericidal test to detect the in vitro bactericidal function of the typhoid vaccine immune serum against Salmonella typhi, wherein after completing the phagocytosis and bactericidal step of the opsonization bactericidal test, the Salmonella typhi that has undergone the phagocytosis and bactericidal step is cultured at a pH of 7.5 to 9.5.

[0012] (2) The method according to (1), wherein the method comprises the following steps:

[0013] 1) inactivating complement and diluting the typhoid vaccine immune serum to obtain diluted inactivated serum;

[0014] 2) mixing the diluted inactivated serum with Salmonella typhi and incubating the mixture to obtain an opsonized mixed bacterial solution;

[0015] 3) mixing the phagocytes and complement with the conditioned mixed bacterial solution and incubating the mixture to obtain a sterilized mixed bacterial solution;

[0016] 4) culturing the sterilized mixed bacterial solution in a culture medium at 35° C. to 37° C. and a pH of 7.5 to 9.5 for 12 to 18 hours; and

[0017] 5) Counting the colonies and determining the in vitro bactericidal function of the typhoid vaccine immune serum based on the growth of the typhoid Salmonella after steps 2) to 4) above.

[0018] (3) The method according to (2), wherein in step 1), the complement inactivation temperature is 56°C and the time is 30 minutes to 60 minutes.

[0019] (4) The method according to (2), wherein in step 1), the serum is diluted 2 to 150,000 times.

[0020] (5) The method according to (2), wherein in step 2), the optical density OD of the mixed incubation of Salmonella typhi is 600 is 0.7 to 1.0, and the volume ratio of the diluted inactivated serum to the Salmonella typhi is in the range of 1.5:1 to 2.5:1.

[0021] (6) The method according to (2), wherein in step 2), the mixing incubation time is 30 minutes to 60 minutes and the temperature is 20°C to 25°C.

[0022] (7) The method according to (2), wherein in step 3), the phagocytes are HL60 cells that have been induced to differentiate.

[0023] (8) The method according to (2), wherein in step 3), the concentration of the phagocytes is 1×107 cells / mL, and the volume ratio of the phagocytes, the complement and the opsonized mixed bacterial solution is (4.1-4.3):(0.9-1.1):(2.9-3.1).

[0024] (9) The method according to (2), wherein in step 3), the mixed incubation is performed at 35°C to 37°C for 0.5 to 3 hours, preferably for 1.5 to 3 hours.

[0025] (10) The method according to (2), wherein in steps 2) and 3), the diluted inactivated serum is further diluted with a solution of Salmonella typhi, phagocytes, and complement so that the diluted inactivated serum has a final dilution multiple, and in step 5), the highest final dilution multiple of the serum that obtains a 50% bactericidal rate is used as the opsonic bactericidal index of the typhoid vaccine immune serum.

[0026] Compared with the prior art, the present invention has the following advantages and positive effects:

[0027] The method of the present invention breaks the conventional thinking in the field of typhoid vaccine serological evaluation that serum bactericidal test is routinely selected to evaluate the in vitro bactericidal function of Gram-negative bacteria. For the first time, the opsonophagocytic bactericidal test is creatively applied to the Gram-negative bacterium Salmonella typhi, and effectively and accurately evaluates the in vitro bactericidal level of typhoid vaccine immune serum, filling the gap in the global field of using only enzyme-linked immunosorbent assay (ELISA) to evaluate the effectiveness of typhoid vaccine, and providing a new in vitro bactericidal function detection method in the field of typhoid vaccine serological evaluation.

[0028] In addition, the present invention cleverly improves the conditions of the conditioning phagocytosis and sterilization test, adjusting the pH of the culture growth environment of the typhoid Salmonella that has undergone the phagocytosis and sterilization step to 7.5 to 9.5, thereby making the colony morphology clearer and enabling the colonies to be counted by the plate colony counting method. This counting method is fast, simple and easy, and can intuitively and accurately reflect the number of colonies, thereby improving the reliability, accuracy and detection speed of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows photographs of samples from opsonized phagocytosis experiments with varying phagocytosis and oscillation conditions. From right to left, each column shows CTA, CTB, Sample 5 (post-immunization), and Sample 5 (post-immunization); from top to bottom, the final serum dilutions in each column are 437,400-fold, 145,800-fold, 48,600-fold, 16,200-fold, 5,400-fold, 1,800-fold, 600-fold, and 200-fold, respectively.

[0030] Figure 2 shows photographs of samples in the opsonization and phagocytosis assay with and without the addition of tris (hydroxymethylaminomethane) (Tris base). Panel A shows the opsonization and phagocytosis assay without Tris base, and Panel B shows the opsonization and phagocytosis assay with Tris base. From right to left, each column shows CTA, CTB, Sample 3 (post-immunization), and Sample 3 (post-immunization). From top to bottom, the final serum dilution factors in each column are 437,400-fold, 145,800-fold, 48,600-fold, 16,200-fold, 5,400-fold, 1,800-fold, 600-fold, and 200-fold, respectively. DETAILED DESCRIPTION

[0031] The present invention is further illustrated below by describing specific implementation methods, but this is not intended to limit the present invention. Those skilled in the art can make various modifications or improvements based on the basic ideas of the present invention, but as long as they do not deviate from the basic ideas of the present invention, they are all within the scope of the present invention.

[0032] In opsonophagocytic bactericidal assays, opsonizing agents primarily refer to IgG antibodies and C3b complement molecules. The C3b complement molecules in the opsonizing agents bind to bacteria to form C3b-bacteria complexes, which then bind to the C3b receptors of macrophages, thereby promoting phagocytosis of bacteria by macrophages. Simultaneously, the IgG antibodies in the opsonizing agents also bind to the Fc receptors of macrophages, thereby enhancing the efficiency of phagocytosis.

[0033] As used in this application, the term "opsonization bactericidal index" represents the numerical value of the strength of the in vitro bactericidal function induced by the typhoid vaccine, which can reflect the strength of the specific bactericidal ability induced by the typhoid vaccine.

[0034] In order to quickly and accurately detect the in vitro bactericidal function of typhoid vaccine immune serum, the inventors of the present invention first proposed to apply the opsonophagocytic bactericidal test to Salmonella typhi, and use the opsonophagocytic bactericidal index induced by typhoid vaccine as the main indicator for evaluating the in vitro bactericidal function of typhoid vaccine immune serum.

[0035] During the present invention's research, it was discovered that Gram-negative bacteria such as Salmonella typhi exhibit instability during opsonophagocytosis tests. To address this issue, the present inventors adjusted the test conditions and cleverly adjusted the pH of the culture environment in which Salmonella typhi undergoes the phagocytosis test to between 7.5 and 9.5, significantly improving the applicability of the opsonophagocytosis test for detecting Salmonella typhi. Furthermore, the present invention achieves highly accurate detection results and is simple and easy to use.

[0036] Therefore, the present invention provides a method for detecting the in vitro bactericidal function of typhoid vaccine immune serum, which includes using an opsonic phagocytosis bactericidal test to detect the in vitro bactericidal function of typhoid vaccine immune serum against Salmonella typhi, wherein after completing the phagocytosis bactericidal step of the opsonic phagocytosis bactericidal test, the Salmonella typhi that has undergone the phagocytosis bactericidal step is cultured at a pH of 7.5 to 9.5.

[0037] In opsonophagocytosis tests, when conventional culture medium (pH approximately 7.0) is used to culture Salmonella typhi after the phagocytosis sterilization step, the growth of the bacteria is very unstable, resulting in poor reproducibility of the test results. Unexpectedly, the inventors discovered that when the pH of the culture medium is adjusted to 7.5 to 9.5, the growth of Salmonella typhi after the phagocytosis sterilization step is stable and good, with clearer colony morphology. A simple plate colony count method can be used to count the remaining colonies in the bacterial solution, and the test results are stable and accurate. When the pH of the culture medium is above 9.5, bacterial growth is inhibited.

[0038] In some embodiments of the present invention, the method for detecting the in vitro bactericidal function of typhoid vaccine immune serum comprises the following steps: 1) complement inactivating and diluting the typhoid vaccine immune serum to obtain diluted inactivated serum; 2) mixing and incubating the diluted inactivated serum with Salmonella typhi to obtain an opsonized mixed bacterial solution; 3) mixing and incubating the opsonized mixed bacterial solution with phagocytes and complement to obtain a sterilized mixed bacterial solution; 4) culturing the sterilized mixed bacterial solution in a culture medium at 35°C to 37°C and a pH of 7.5 to 9.5 for 12 to 18 hours; and 5) counting the colonies and determining the in vitro bactericidal function of the typhoid vaccine immune serum based on the growth of Salmonella typhi after steps 2) to 4) above.

[0039] In step 1), the purpose of inactivating complement in the typhoid vaccine immune serum is to prevent the complement present in the serum from acting and thus affecting the results of the simulated in vitro bactericidal test. The temperature for inactivating complement in the typhoid vaccine immune serum is preferably 56°C, and the time for inactivating complement in the typhoid vaccine immune serum is preferably 30 to 60 minutes. In a preferred embodiment, the temperature for inactivating complement in the typhoid vaccine immune serum is 56°C, and the time for inactivating complement is 30 minutes.

[0040] The complement inactivation in the typhoid vaccine immune serum can be carried out in a water bath, oil bath, incubator or any thermostatic equipment known in the art at a predetermined temperature. In a preferred embodiment, the complement inactivation in the typhoid vaccine immune serum can be carried out in a water bath at 56°C.

[0041] The complement-inactivated serum can be diluted by an appropriate factor, A, so that the concentration of the complement-inactivated serum is 1 / A of the initial concentration. The purpose of diluting the complement-inactivated serum is to control the serum dilution factor at a 50% bactericidal rate within the experimental range. Those skilled in the art can determine the appropriate dilution factor based on the bactericidal properties of the complement-inactivated serum (e.g., based on preliminary experimental results). In a preferred embodiment of the present invention, the dilution factor, A, of the complement-inactivated serum can be 2 to 150,000-fold, preferably 50 to 110,000-fold, for example, 200 to 80,000-fold or 500 to 50,000-fold. Due to individual differences in serum samples, in some cases, the serum may not be diluted, i.e., the original complement-inactivated serum may be used. When the dilution factor, A, of the complement-inactivated serum is greater than 150,000, i.e., the concentration after dilution is 1 / 150,000 of the initial concentration, the opsonophagocytic bactericidal effect on Salmonella typhi becomes less pronounced.

[0042] Preferably, during the conditioning process of step 2), the optical density OD of the mixed incubated Salmonella typhi is 600 The value of OD can be 0.7 to 1.0. For example, the number of Salmonella typhi in mixed incubation can be about 40,000-96,000 / mL. 600 Refers to the optical density of the bacterial solution measured at a wavelength of 600nm using an optical densitometer. Working Salmonella typhi can be obtained, for example, by the following method: culture Salmonella typhi on a sheep blood plate, select 20 single colonies and inoculate them into THY liquid medium, harvest them during the logarithmic growth phase, and calculate the OD 600 The optical density of the mixed incubation solution is OD 0.7 to 1.0, and the number of viable bacteria is about 40,000-96,000 per mL. 600 The value of is 0.8, and the number of typhoid bacilli is about 46750 / mL.

[0043] During the conditioning process in step 2), the volume ratio of the diluted inactivated serum to Salmonella typhi is preferably in the range of 1.5:1 to 2.5:1, and more preferably 2:1. The diluted inactivated serum and Salmonella typhi can be mixed using any mixing device known in the art.

[0044] In a preferred embodiment of the present invention, during the conditioning process in step 2), the mixing and incubation time (i.e., conditioning time) is 30 minutes to 60 minutes; the mixing and incubation temperature (i.e., conditioning temperature) is room temperature, particularly 20°C to 25°C.

[0045] In some embodiments of the present invention, a shaker can be used to mix the mixture in step 2) of the present invention at a mixing speed of 400 rpm to 800 rpm, preferably 450 rpm. In a preferred embodiment, a digital shaker is used to mix the mixture in step 2) of the present invention at a mixing speed of 450 rpm for 30 minutes.

[0046] In step 3), the phagocytes are preferably HL60 cells that have been induced to differentiate. In some embodiments of the present invention, HL60 cells can be differentiated by conventional methods known in the art to obtain differentiated HL60 cells, which can be used in the opsonophagocytic bactericidal assay of the present invention.

[0047] In a preferred embodiment of the present invention, the concentration of phagocytes is 1×10 7 cells / mL, and the volume ratio of phagocytes, complement and opsonized mixed bacterial solution ranges from (4.1-4.3):(0.9-1.1):(2.9-3.1), and the preferred volume ratio is 4:1:3.

[0048] In the method for the present invention, the purpose of adding complement is that complement is necessary for the sterilization of the opsonophagocytic bactericidal test, and can control the complement conditions of the in vitro sterilization to compare and analyze the test results. In one embodiment of the present invention, the complement added in the opsonophagocytic bactericidal test can be suckling rabbit complement. Although suckling rabbit complement is used in embodiments of the present invention, those skilled in the art know that other suitable complements can also be used to carry out the method for the present invention, as long as it can promote macrophage phagocytosis and kill Salmonella typhi.

[0049] In some embodiments of the present invention, the sterilization in step 3) is performed at 35° C. to 37° C. and 5% CO₂ for 0.5 to 3 hours, preferably 1.5 to 3 hours, and more preferably 2 hours. In a preferred embodiment, the sterilization in step 3) is performed in an incubator at 37° C. and 5% CO₂ on a digital shaker at a mixing speed of 450 rpm for 2 hours.

[0050] In step 4), most preferably, the pH value of the culture medium is 8.0.

[0051] In step 5), the colonies in the bacterial solution are preferably counted using a plate colony counting method.

[0052] In some embodiments of the present invention, in steps 2) and 3), the diluted inactivated serum is further diluted with the solution of Salmonella typhi, phagocytes, and complement such that the diluted inactivated serum has a final dilution factor B, and the highest final dilution factor B of the serum that achieves a 50% bactericidal rate is used as the opsonicidal index. In a preferred embodiment of the present invention, the highest final dilution factor B of the serum that achieves a 50% bactericidal rate is used as the opsonicidal index of the typhoid vaccine immune serum.

[0053] In one embodiment of the present invention, the calculation formula of the sterilization rate is as follows:

[0054] Sterilization rate = (1-number of surviving Salmonella typhi colonies in the sample well / number of surviving Salmonella typhi colonies in the CTB) × 100%

[0055] CTB represents control sample B, which was prepared using the method for detecting the in vitro bactericidal function of typhoid vaccine immune serum according to the present invention, except that typhoid vaccine immune serum was not added during the preparation of CTB. In other words, the preparation of CTB uses Salmonella typhi, phagocytes, and complement.

[0056] In addition, the method of the present invention also evaluates the accuracy and reliability of the test results by the nonspecific bactericidal rate. The calculation formula of the nonspecific bactericidal rate is as follows:

[0057] Nonspecific bactericidal rate = (1-number of surviving Salmonella typhi colonies in CTB / number of surviving Salmonella typhi colonies in CTA) × 100%

[0058] CTB has the same definition as above, and CTA stands for Control Sample A. CTA is also prepared without typhoid vaccine serum, using Salmonella typhi, phagocytes, and inactivated complement. Test results are only valid when the nonspecific bactericidal rate is below 30%.

[0059] In some embodiments of the present invention, the typhoid vaccine immune serum is serum obtained after immunization with the typhoid vaccine.

[0060] In a preferred embodiment of the present invention, the method for detecting the in vitro bactericidal function of typhoid vaccine immune serum according to the present invention comprises the following steps:

[0061] 1) Placing serum from a human immunized with typhoid vaccine in a 56°C water bath for 30 minutes to inactivate complement, and diluting the inactivated serum by A times to obtain diluted inactivated serum, wherein A is 50 to 110,000;

[0062] 2) mixing the diluted inactivated serum with Salmonella typhi in a multi-well culture plate at room temperature (e.g., 20° C. to 25° C.) and incubating for 30 minutes to obtain a conditioned mixed bacterial solution. The number of Salmonella typhi in each well of the culture plate (approximately 1 mL of the mixed bacterial solution) is 40,000-96,000 cells / mL (e.g., the number of Salmonella typhi may be 46,750 cells / mL), and the volume ratio of the diluted inactivated serum to Salmonella typhi in each well is 2:1, wherein the diluted inactivated serum is further diluted by the addition of Salmonella typhi;

[0063] 3) The differentiated HL60 cells, suckling rabbit complement and the conditioned mixed bacterial solution were mixed and incubated at 37°C and 5% CO2 for 2 hours to obtain a sterilized mixed bacterial solution, wherein the concentration of the differentiated HL60 cells was 1×10 7 cells / mL, and the volume ratio of cell fluid, suckling rabbit complement, and conditioned mixed bacterial solution is 4:1:3. The diluted inactivated serum in the conditioned mixed bacterial solution is further diluted by the addition of differentiated HL60 cells and suckling rabbit complement, resulting in a final dilution factor B. At this time, the original dilution factor A is diluted another 4 times, i.e., B = 4A.

[0064] 4) culturing the sterilized mixed bacterial solution in a culture medium at 37° C., 5% CO 2 , and a pH of 8.0 for 12 hours; and

[0065] 5) Count the colonies and calculate the bactericidal rate of each well of the culture plate in the test. The highest final dilution factor B of the inactivated serum that obtains a 50% bactericidal rate is used as the opsonicidal index of the serum after immunization with the typhoid vaccine.

[0066] The present invention is further explained or illustrated below by way of examples, but these examples should not be construed as limiting the scope of protection of the present invention.

[0067] Example

[0068] Unless otherwise specified, the experimental methods used in the following examples were performed using conventional experimental procedures, operations, materials and conditions in the art.

[0069] The materials, reagents and instruments used in the examples are from the following sources:

[0070] a. Cells: HL60 cells, obtained from the American Type Culture Collection (ATTC);

[0071] b. Salmonella typhi: from China Medical Collection Center (CMCC);

[0072] c. Plate culture medium: When the total volume is 800 mL, add 24 g Todd-Hewitt broth (THB), 12 g yeast extract, 12 g agar, and TTC stock solution (1 / 2000 to 1 / 4000 of the culture volume, i.e., 400 μL to 200 μL of TTC stock solution). Autoclave (115°C, 30 minutes), then cool to 56°C. Adjust the pH to 7.5 to 9.5 with Tris base (Sigma-Aldrich, Catalog No. SLBQ4845V, 1 mol / L). Pour 13 mL to 15 mL into square Petri dishes for later use.

[0073] d. TTC stock solution: Prepare 1.25 g of 2,3,5-triphenyltetrazolium chloride (TTC) by adding water to 40 mL. Once completely dissolved, add water to 50 mL. Filter through a 0.22 μm filter and set aside. This will create the TTC stock solution. When adding TTC to the culture medium, directly take the appropriate volume of TTC stock solution.

[0074] e. CM1 medium for HL60 cell proliferation: 500 mL of RPMI 1640 supplemented with 5.7 mL of GlutaMax and 57 mL of fetal bovine serum. Store at 2–8°C.

[0075] f. CM2 medium for HL60 cell differentiation: 500 mL of RPMI1640 supplemented with 5.7 mL of GlutaMax, 57 mL of fetal bovine serum, and 4 mL of DMF. Store at 2-8°C.

[0076] g. CM3 medium for HL60 cell recovery: 500 mL of RPMI1640 supplemented with 6.4 mL of GlutaMax and 128 mL of fetal bovine serum. Store at 2-8°C.

[0077] h. Gelatin solution (1%): 0.5 g of gelatin, add water to 50 mL, autoclave (115°C, 30 minutes) and store at room temperature;

[0078] i. Opsonization Buffer (OB buffer, containing Ca + Mg 2+ ): Sterile H2O 80mL, 10xHBSS (containing Ca + Mg 2+ ) 10 mL, 1% gelatin solution 10 mL, FBS (inactivated) 5 mL, prepare and use immediately.

[0079] j. Opsonized phagocytosis buffer (OB buffer, without Ca + Mg2+ ): Sterile H2O 80mL, 10x HBSS (without Ca + Mg 2+ ) 10 mL, 1% gelatin solution 10 mL, FBS (inactivated) 5 mL, prepare and use immediately.

[0080] k. Digital shaker (mini orbital shaker, model 7644-20115, purchased from Bellco Biotechnology)

[0081] l. Complement: suckling rabbit complement was purchased from Qingdao Zhongchuanghuike Biotechnology Co., Ltd., product batch numbers: 21072203, 21072117, 21072111, 21072116, 21072202, 21072209, 21072313, 21072312, 21072214, 21072206, 21072217, 21072301, 21072310, 21072116, 21072211, 21072115, 21072307, 21072309, 21072306, 21072305, 21072302, 21072303, 21072308, and 21072311.

[0082] m. Serum samples before and after typhoid vaccine immunization: derived from the clinical trial of the drug with approval number 2006L01307.

[0083] Example 1: Opsonophagocytic bactericidal test of serum from people immunized with typhoid vaccine

[0084] 1. Complement inactivation and dilution of serum from humans immunized with typhoid vaccine

[0085] Serum samples from people immunized with typhoid vaccine were thawed at room temperature and then placed in a 56°C water bath for 30 minutes to inactivate complement in the serum.

[0086] The serum samples of people immunized with the complement-inactivated typhoid vaccine were added to a 96-well U-bottom tissue culture plate according to the distribution scheme shown in Table 1 and diluted with OB buffer by the corresponding multiples. The diluted serum samples added to the 96-well U-bottom tissue culture plate were 14 μL per well.

[0087] Table 1. Sample dilution and distribution scheme in a 96-well U-bottom tissue culture plate (This experiment used serum samples from five individuals, designated samples 1 to 5, with each sample divided into pre-immune serum and post-immune serum)

[0088] Note: Columns 3-12 show the dilution multiples of each serum sample; CTA is control sample A: containing Salmonella typhi + phagocytes + inactivated rabbit complement (rabbit complement was inactivated by placing it in a 56°C water bath for 30 minutes); CTB is control sample B: containing Salmonella typhi + phagocytes + non-inactivated rabbit complement.

[0089] 2. Treatment and conditioning of Salmonella typhi

[0090] Take out Salmonella typhi (500 μL / tube) from -80℃ freezer, immediately put it in 37℃ water bath to thaw, centrifuge after thawing (12500rpm, 2min), discard the supernatant, add 500 μL OB buffer (containing Ca 2+ , Mg 2+ )Resuspend.

[0091] 46750 Salmonella typhi cells / mL (OD value of about 0.8) were added to the 96-well U-bottom tissue culture plate prepared in step 1 at 7 μL / well and incubated at 450 rpm for 30 minutes using a digital shaker.

[0092] 3. Phagocytosis and sterilization

[0093] HL60 cells were treated and differentiated according to the following steps:

[0094] (1) Processing and storage of HL60 primary cells

[0095] Monitor CO2 levels and humidity in the tissue culture incubator to maintain proper growth of HL60 cells. Primary HL60 cells are stored long-term in liquid nitrogen tanks by controlled-rate freezing.

[0096] Acceptable criteria for primary HL60 cell stocks are as follows:

[0097] a. Free from contamination, including mycoplasma culture.

[0098] b. The culture conforms to the microscopic characteristics of HL60 cells (such as cell size, morphological changes, etc.).

[0099] c. Keep the source documents properly.

[0100] (2) Use of working HL60 cells

[0101] To properly maintain the culture and integrity of working HL60 cells, the cell concentration and quality were strictly controlled as follows: HL60 cells were discarded after 4 months (approximately 48 passages) and new frozen HL60 primary cells were thawed for use in experiments.

[0102] a. Monday: Add 40 mL of fresh CM1 (proliferation medium) to each 150 cm2 cell culture flask to a final volume of 120 mL / flask. 80 mL should be in each flask before injection. The cell concentration should be 3 × 10 5 cells / mL.

[0103] b. Wednesday: Mix the contents of the flasks and remove 80 mL from each flask (40 mL remaining in each flask). Pool the removed cells for differentiation. Add 80 mL of fresh CM1 to the remaining cells in each flask, bringing the final volume to 120 mL per flask. The cell concentration should be approximately 3 × 10 5 cells / mL.

[0104] c. Friday: Mix the contents of the culture flasks and remove 100 mL (leaving 20 mL in each flask). Pool the removed cells for differentiation. Add 60 mL of fresh CM1 to the remaining cells in each flask, bringing the final volume to 80 mL per flask. The cell concentration should be 2 × 10 5 cells / mL.

[0105] (3) Differentiation of HL60 cells

[0106] HL60 cells were collected as described above and gently resuspended. The concentration was adjusted to 4 × 10 cells / mL using CM2 medium. 5 cells / mL.

[0107] Perform differentiation on Wednesday or Friday. HL60 cells on day 5 or 6 of differentiation are used in the OPA assay (a method for detecting the in vitro bactericidal activity of typhoid vaccine immune serum). HL60 cells that have been differentiated for more than 6 days should be discarded.

[0108] The survival rate of differentiated HL60 cells was detected by trypan blue or propidium iodide staining. The surface markers (CD11b, CD35 and CD71) expressed by normal HL60 cells and differentiated HL60 cells were monitored by flow cytometry.

[0109] The acceptance criteria for differentiated HL60 cells as effector cells are as follows:

[0110] a. The culture conforms to the microscopic characteristics of differentiated HL60 cells (e.g., cell size, morphological changes, etc.).

[0111] b. Cell viability ≥90% (Trypan blue staining method) or ≥65% (Propidium iodide staining method).

[0112] c. ≥55% of the total cells express CD35.

[0113] d. ≤20% of the total cells expressed CD71.

[0114] e. Apoptotic cells, defined as Annexin V+ / PI-, were ≤ 25% of the total cells in flow cytometry.

[0115] The differentiated HL60 cells were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 50 mL of Ca-free 2+ , Mg 2+ The OBs were centrifuged (1500 rpm, 5 min), the supernatant was discarded, and the OBs were resuspended in 50 mL of Ca 2+ , Mg 2+ Take 0.6 mL of resuspended cells from OB and dilute them 10-fold before counting. Centrifuge (1500 rpm, 5 min) and discard the supernatant. 7 The cells were suspended in OB buffer (containing Ca 2+ , Mg 2+ ) to obtain cell suspension. Room temperature-thawed suckling rabbit complement was mixed with the cell suspension at a volume ratio of 1:4. 35 μL / well of the mixture was added to the above-mentioned 96-well U-bottom tissue culture plate. The plate was placed in a 37°C, 5% CO2 incubator and incubated at 450 rpm on a digital shaker for 2 hours.

[0116] At the same time, CTA control and CTB control were prepared by the same operation. The difference was that serum was not added to CTA control, the rabbit complement added in the phagocytic sterilization step was inactivated complement, and CTA control contained Salmonella typhi, phagocytes and inactivated complement; serum was not added to CTB control, and CTB control contained Salmonella typhi, phagocytes and rabbit complement; an equal volume of OB buffer (containing Ca) was used as the serum. 2+ , Mg 2+ ) to ensure that all samples had the same volume.

[0117] After the phagocytic sterilization step, each well of the 96-well plate actually contained 14 μL of diluted serum, 7 μL of Salmonella typhi, and 35 μL of a mixture of suckling rabbit complement and phagocytes (containing 7 μL of suckling rabbit complement and 28 μL of phagocytes), for a total volume of 56 μL. Therefore, the initial volume of 14 μL of diluted serum was further diluted 4-fold, and the final dilution factor of the serum is shown in Table 2 below.

[0118] Table 2. Final dilution of serum in 96-well U-bottom tissue culture plates

[0119] 4. Survival Bacteria Culture

[0120] Use an 8-channel pipette to dispense 10 μL of the sterilized bacterial mixture into each well of the plate (pH 8) and spot-inoculate the mixture. Slant the mixture, keeping the length of the strips approximately 1 to 1.5 cm and avoiding contact. A total of 32 strips per plate are placed. Once the strips have dried, invert the plate and incubate in a 37°C, 5% CO2 incubator overnight (approximately 12 hours).

[0121] 5. Colony Counting and Data Analysis

[0122] The next day, the plate was taken out after incubation and the colonies on the plate were counted using a fully automatic colony analyzer (model: ProtoCOL, purchased from Synbiosis). The sterilization rate of each sample was calculated using the sterilization rate calculation formula. Table 3 shows the remaining colony count statistics for samples 1-5 (at least two parallel experiments were performed for each sample).

[0123] Table 3. Remaining colony values ​​measured by the fully automatic colony analyzer

[0124] According to the results of the remaining colony counts shown in Table 3, the sterilization rate was calculated using the following formula.

[0125] Sterilization rate = (1-number of surviving Salmonella typhi colonies in the sample well / number of surviving Salmonella typhi colonies in the CTB) × 100%

[0126] The highest final dilution factor of the serum that achieved a 50% bactericidal rate was taken as the opsonic bactericidal index of the typhoid vaccine immune serum.

[0127] Table 4 shows the calculated bactericidal rate and opsonization bactericidal index for Sample 1 (post-immunization). In this experiment, using CTB as a control sample, the remaining colony count measured by an automated colony analyzer was 69.25. For serum samples from Sample 1 (post-immunization), where the remaining colony count exceeded 70 at certain dilutions, those skilled in the art will recognize this as an error inherent in the colony growth and counting experiments. In these cases, the bactericidal rate was recorded as ~0%, indicating no significant inhibitory effect on colony growth.

[0128] If the final dilution of the serum sample includes a data point with a bactericidal rate of 50%, the final dilution factor value of the serum can be used as the opsonicidal index. If the final dilution of the serum sample does not include a data point with a bactericidal rate of 50%, the opsonicidal index is calculated by linear interpolation. Taking sample 1 (after immunization) as an example, first select two final dilution factor values ​​of 5400 and 16200 with a bactericidal rate of more than 50% (5400 times, a bactericidal rate of 78.26%) and less than 50% (16200, a bactericidal rate of ~0%), and take the logarithm of these two values ​​to obtain the corresponding Log (Log value of the final dilution factor). With Log as the abscissa and bactericidal rate as the ordinate, a point graph is drawn. A linear regression is performed on the two points to obtain a straight line. On this straight line, the abscissa Log value of the data point with a bactericidal rate of 50% is determined. The antilogarithm of this Log value is taken to obtain the final dilution factor value of the serum at which the bactericidal rate is 50%, which is the opsonicidal index.

[0129] Table 4. Calculation results of opsonization bactericidal index of sample 1 (after immunization)

[0130] The conditioning bactericidal index of samples 2 to 5 was calculated using the same calculation method as sample 1, and the results are listed in Table 5.

[0131] Table 5. Conditioning and sterilization index results obtained according to the method of the present invention

[0132] According to the results in Table 5, the opsonic bactericidal index of each post-immunization serum sample was significantly higher than that of the corresponding pre-immunization serum sample. Visual observation of in vitro bactericidal activity (colony growth) before and after immunization, as well as the calculated opsonic bactericidal index, showed a significant difference between the post-immunization and pre-immunization opsonic bactericidal indices, indicating that this method can be used to determine functional antibody levels in typhoid vaccine clinical trials.

[0133] Examples 2 to 14: Effects of Phagocytic Killing Time and Oscillation Conditions on Opsonophage Killing Assays

[0134] Examples 2 to 14 (using immunized serum sample 5) were performed using the same opsonization and phagocytosis test steps and conditions as in Example 1, except that the time for phagocytosis and whether oscillation was performed was changed. The specific conditions (including those in Example 1) are shown in Table 6.

[0135] Table 6. Opsonophagocytosis and sterilization test with varying phagocytosis and sterilization time and oscillation conditions

[0136] As shown in Figure 1, in the conditioning and phagocytosis sterilization tests of Examples 2 to 8, oscillation was not performed during phagocytosis and sterilization, which would result in less accurate results and poorer sterilization effects. In the conditioning and phagocytosis sterilization tests of Examples 1, 9 to 14, oscillation was performed during phagocytosis and sterilization, and the phagocytosis and sterilization times were different. Studies have found that oscillation makes the test results more accurate and the sterilization effect better; the sterilization time has an impact on the test results. If the sterilization time is too short, the reaction is incomplete, and the test results are not true. Therefore, the time point at which the sterilization effect begins to remain unchanged is selected as the sterilization time of the test. Through the above examples, it was found that the best effect can be achieved by phagocytosis and sterilization for 2 hours, and no significant changes in the results were observed by extending the sterilization time. Therefore, it is preferred to perform phagocytosis and sterilization for 2 hours.

[0137] Example 15: Effect of pH during culture of viable bacteria on opsonophagocytic bactericidal assay

[0138] Example 15 (using immunized serum sample 3) was performed using the same opsonization and phagocytosis test steps and conditions as Example 1, except that no Tris base was added to the plate culture medium used for culturing surviving bacteria, and the pH of the plate culture medium was 7.02-7.22.

[0139] As shown in FIG2 , according to the opsonophagocytic bactericidal test of Example 15, if the pH of the plate culture medium is not adjusted to 8 using Tris base, the clarity of the colony outline becomes poor, making it difficult to identify the colony using a colony counter.

[0140] In contrast, samples adjusted to pH 8 exhibited clearer outlines of individual colonies, enabling the colony counter to more accurately identify them, thereby improving the accuracy of the test results.

Claims

1. A method for detecting the in vitro bactericidal function of typhoid vaccine immune serum, including detecting the in vitro bactericidal function of the typhoid vaccine immune serum against Salmonella typhi by using an opsonophagocytic killing assay, wherein after the phagocytic killing step of the opsonophagocytic killing assay is completed, the Salmonella typhi after the phagocytic killing step is cultured at a pH of 7.5 to 9.

5.

2. The method according to claim 1, wherein the method comprises the following steps: 1) Inactivating and diluting the complement of the typhoid vaccine immune serum to obtain diluted inactivated serum; 2) Mixing and incubating the diluted inactivated serum with Salmonella typhi to obtain an opsonized mixed bacterial solution; 3) Mixing and incubating phagocytes, complement with the opsonized mixed bacterial solution to obtain a bactericidal mixed bacterial solution; 4) Culturing the bactericidal mixed bacterial solution in a culture medium at 35°C to 37°C and a pH of 7.5 to 9.5 for 12 hours to 18 hours; and 5) Counting the colonies, and determining the in vitro bactericidal function of the typhoid vaccine immune serum according to the growth of Salmonella typhi after the above steps 2) to 4).

3. The method according to claim 2, wherein in step 1), the temperature for complement inactivation is 56°C and the time is 30 minutes to 60 minutes.

4. The method according to claim 2, wherein in step 1), the serum is diluted 2 to 150,000 times.

5. The method according to claim 2, wherein in step 2), the optical density OD of the Salmonella typhi undergoing the mixed incubation 600 is 0.7 to 1.0, and the volume ratio range of the diluted inactivated serum to the Salmonella typhi is 1.5:1 to 2.5:

1.

6. The method according to claim 2, wherein in step 2), the time for the mixing and incubation is 30 minutes to 60 minutes and the temperature is 20°C to 25°C.

7. The method according to claim 2, wherein in step 3), the phagocytes are HL60 cells after induced differentiation.

8. The method according to claim 2, wherein in step 3), the concentration of the phagocytes is 1×10 7 cells / mL, and the volume ratio of the phagocytes, the complement and the opsonized mixed bacterial solution is (4.1-4.3):(0.9-1.1):(2.9-3.1).

9. The method according to claim 2, wherein in step 3), the mixing and incubation is carried out at 35°C to 37°C for 0.5 hour to 3 hours, preferably 1.5 hours to 3 hours.

10. The method according to claim 2, wherein in steps 2) and 3), the diluted inactivated serum is further diluted by the solution of Salmonella typhi, phagocytes, complement, so that the diluted inactivated serum has a final dilution factor, and in step 5), the highest final dilution factor of the serum when a 50% bactericidal rate is obtained is used as the opsonophagocytic bactericidal index of the typhoid vaccine immune serum.

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