A method for detecting the in vitro bactericidal function of serum

Abstract

The present invention provides a method for detecting the in vitro bactericidal function of serum, wherein the serum contains two or more antibodies, which are respectively directed against different groups of certain bacteria, and the method comprises the following steps: 1) inducing the bacteria of the different groups to have different antibiotic resistances; 2) in the same system, mixing and incubating the serum, complement and bacteria of the different groups having different antibiotic resistances to obtain a sterilized mixed bacterial solution; 3) mixing a culture medium containing an antibiotic that a group of bacteria resists with the sterilized mixed bacterial solution, so that the bacteria of the group having the antibiotic resistance grow; this step is performed for each group of bacteria; 4) determining the bactericidal function of the serum against the bacteria of the different groups according to the growth of each group of bacteria. The present invention can detect the level of functional antibodies in serum quickly and with high throughput, improve work efficiency, and save the amount of complement and serum to be tested.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and more particularly, relates to a method for detecting the in vitro bactericidal function of serum. Background Art

[0002] Neisseria meningitidis can cause purulent meningitis through respiratory transmission, seriously threatening the health of infants and young children. Neisseria meningitidis can be divided into at least 13 serogroups, and 95% of the cases are caused by serogroups A, B, C, W135 and Y. In the 1970s and 1980s, serogroup A was the main cause of epidemic cerebrospinal meningitis in China. Since 2002, local outbreaks of cerebrospinal meningitis caused by serogroup C have occurred in Guangxi, Anhui, Guangdong and other places in China. In 1980, the polysaccharide vaccine of serogroup A was officially used in China. Clinical observations have shown that its protective rate for school-age children and adults can reach 90%. An important indicator for the laboratory evaluation of the immune response of Neisseria meningitidis vaccine is bactericidal antibody, which has now become an alternative indicator for clinical endpoint determination. As an important indicator for the evaluation of Neisseria meningitidis vaccine, bactericidal antibody reflects the protective part of the antibody, so its role is becoming increasingly important. In 1974, after the A / C polysaccharide vaccine for epidemic cerebrospinal meningitis was marketed based on the results of the Serum bactericidal assay (SBA), the SBA results have played an increasingly important role in the clinical trials of vaccines as a substitute for clinical endpoints.

[0003] The detection method of bactericidal antibody is the serum bactericidal assay in vitro. As early as 1918, Kolmer et al. had demonstrated the protective effect of antibodies and complement on meningitis. In 1969, Goldschneider et al. found in recruited soldiers that human complement SBA≥4 had a protective effect on Neisseria meningitidis serogroup C. In 1976, the WHO recommended the SBA test methods for serogroups A and C. In 1997, the US CDC improved the WHO SBA method, established the standard SBA method for serogroups A and C, and compared the SBA results among multiple laboratories. Currently, international laboratories have basically established their own SBA test methods for serogroups A and C with reference to the CDC method. There is no specific literature report on the standardized SBA test methods for serogroups W135 and Y. Due to the limitation of the source of qualified human complement, the WHO and the US CDC recommend using 3-4 week-old rabbit complement to replace human complement. The research by Borrow et al. in 2001 showed that rabbit complement SBA≥128 for serogroup C is equivalent to human complement SBA≥4. In 2014, a standardized SBA detection method for four serogroups of epidemic cerebrospinal meningitis with rabbit complement source was published in China, but because each serogroup must be detected separately, the detection time is relatively long. Summary of the Invention

[0004] To solve the problems existing in the above-mentioned prior art, the present invention provides a method for detecting the in vitro bactericidal function of serum.

[0005] Specifically, the present invention provides:

[0006] (1) A method for detecting the in vitro bactericidal function of serum, wherein the serum contains two or more antibodies, and these antibodies respectively target different groups of a certain bacterium. The method is characterized in that it includes the following steps:

[0007] 1) Inducing the bacteria of different groups to respectively have different antibiotic resistances;

[0008] 2) In the same system, mixing and incubating the serum, complement, and bacteria of different groups with different antibiotic resistances respectively to perform bactericidal action, obtaining a bactericidal mixed bacterial solution;

[0009] 3) Mixing a culture medium containing an antibiotic against which a group of bacteria is resistant with the bactericidal mixed bacterial solution, so as to make the bacteria of this group with this antibiotic resistance grow; performing this step for each group of bacteria;

[0010] 4) Determining the bactericidal function of the serum against the bacteria of different groups according to the growth conditions of each group of bacteria.

[0011] (2) The method according to (1), wherein the bacteria include Neisseria meningitidis, and the different groups are selected from two or more of group A, group B, group C, group W135, and group Y.

[0012] (3) The method according to (1), wherein the serum is the serum obtained after immunizing an organism with a Neisseria meningitidis vaccine.

[0013] (4) The method according to (1), wherein the induction includes: I) Culturing bacteria of different groups respectively with a culture medium containing an antibiotic, increasing the concentration of the antibiotic to at least such a concentration: at this concentration, a group of bacteria can grow, and other groups of bacteria cannot grow, thereby obtaining a group of bacteria resistant to the antibiotic; II) Performing the method described in step I) with different antibiotics respectively until the bacteria of different groups respectively have different antibiotic resistances.

[0014] (5) The method according to (1) or (4), wherein the antibiotics are selected from two or more of spectinomycin, trimethoprim, streptomycin sulfate, and erythromycin.

[0015] (6) The method according to (1), wherein during the mixing and incubation, the serum is serum inactivated by complement

[0016] (7) The method according to (1), wherein during the mixed incubation, the number of bacteria of each strain is 8 - 14 per μl.

[0017] (8) The method according to (1), wherein during the mixed incubation, the dilution factor of the serum is 2 - fold or more, the concentration of the complement is 12.5 - 25 vol% based on the total volume of the incubation system, and the mixing volume ratio of the serum, the complement, and the bacteria is 2:(0.5 - 1):1.

[0018] (9) The method according to (1), wherein the mixed incubation is carried out at 35 - 37 °C for 0.5 - 2 hours.

[0019] (10) The method according to (1), wherein in step 3), the growth of the bacteria is carried out at 35 - 37 °C for 12 - 20 hours.

[0020] The present invention has the following advantages and positive effects compared with the prior art:

[0021] The method of the present invention firstly proposes to induce resistance in the target bacteria used in the in vitro bactericidal test methods for group A, group C, group W135, and group Y meningococcal bacteria, so that the target bacteria of each group carry resistance to different antibiotics respectively. When detecting the in vitro bactericidal titer of the serum, bacteria of the four groups can be added simultaneously in the same bactericidal system. After the bactericidal process is completed, when carrying out viable bacteria culture, different groups of bacteria are distinguished by using culture media containing different antibiotics, thus ingeniously realizing the simultaneous completion of the bactericidal tests for different bacterial groups.

[0022] The method of the present invention is also applicable to the detection of the in vitro bactericidal function of serum against other bacteria.

[0023] The present invention can achieve rapid and high - throughput detection of the level of functional antibodies in serum, improve work efficiency, and can save the usage of complement and the serum to be tested (for example, it can be reduced by 4 times), which is beneficial to infants and young children who are difficult to draw blood. At present, there is no relevant report on this method at home and abroad. Detailed Embodiments

[0024] The present invention is further described below through the description of detailed embodiments, but this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0025] The main index for the clinical evaluation of Neisseria meningitidis vaccine is the in vitro bactericidal titer of serum. The existing in vitro bactericidal test methods are to separately detect different serogroups of a certain bacterium, with each serogroup being detected individually. The inventors of the present invention realized that with the emergence of multivalent vaccines, separately detecting each serogroup would consume a large amount of manpower and material resources and take a long time. Therefore, breaking the inertial thinking, they ingeniously proposed to first induce resistance in different serogroups of bacteria, and then add each serogroup into the same system for bactericidal tests, and subsequently culture them with different antibiotic media, thereby achieving the purpose of simultaneously completing the bactericidal tests for different bacterial groups.

[0026] Based on the above inventive concept, one aspect of the present invention provides a method for detecting the in vitro bactericidal function of serum, wherein the serum contains two or more antibodies, and these antibodies respectively target different serogroups of a certain bacterium, and is characterized in that the method comprises the following steps:

[0027] 1) Inducing the different serogroups of bacteria to respectively have different antibiotic resistances;

[0028] 2) In the same system, mixing and incubating the serum, complement and different serogroups of bacteria respectively having different antibiotic resistances for bactericidal action to obtain a bactericidal mixed bacterial solution;

[0029] 3) Mixing the medium containing the antibiotic against which one serogroup of bacteria is resistant with the bactericidal mixed bacterial solution, for example, inoculating the bactericidal mixed bacterial solution on a culture medium plate containing the antibiotic against which one serogroup of bacteria is resistant, so as to allow the bacteria of this serogroup having this antibiotic resistance to grow; performing this step for each serogroup of bacteria;

[0030] 4) Determining the bactericidal function of the serum against the different serogroups of bacteria according to the growth conditions of each serogroup of bacteria.

[0031] The inventors of the present invention have studied and explored the ideas and requirements for inducing different resistances, including: 1) Selecting antibiotics, preferably those that are not commonly used at present, but the problem to be faced is that for pathogenic bacteria, there may be a general resistance to the antibiotics that have been phased out; 2) Determining the natural resistance of different serogroups of bacteria to the selected antibiotics; 3) Conducting directional induction according to specific circumstances; 4) Detecting the resistance of the bacteria with induced resistance to the induced resistances carried by other serogroups of bacteria; 5) Ensuring that each serogroup of bacteria only has its own induced resistance and does not have the induced resistances of other serogroups of bacteria. Thus, it can be seen that successfully inducing each serogroup of bacteria to respectively have their own induced resistances requires rigorous and scientific experimental concepts and a large amount of screening work, which is the result of creative labor.

[0032] Based on the above ideas and requirements, in some embodiments of the present invention, the induction includes: I) culturing different groups of bacteria separately in a medium containing an antibiotic, increasing the concentration of the antibiotic to at least such a concentration that one group of bacteria can grow and other groups of bacteria cannot grow at this concentration, thereby obtaining a group of bacteria resistant to the antibiotic; II) performing the method described in step I) with different antibiotics until different groups of bacteria have different antibiotic resistances.

[0033] In some embodiments, the bacteria include Neisseria meningitidis, and the different groups are selected from two or more of group A, group B, group C, group W135, and group Y.

[0034] In some embodiments, the serum is the serum obtained after immunizing an organism with a Neisseria meningitidis vaccine.

[0035] Preferably, the antibiotics are selected from two or more of spectinomycin, trimethoprim, streptomycin sulfate, and erythromycin. These antibiotics can meet the requirements for inducing resistance described above.

[0036] Preferably, during the mixed incubation, the serum is serum inactivated by complement.

[0037] Preferably, during the mixed incubation, the number of bacteria of each group is 8 - 14 per μl, for example 8 - 12 per μl.

[0038] Preferably, during the mixed incubation, the dilution factor of the serum is more than 2 times, for example 2 - 15 times, the concentration of the complement is 12.5 - 25% by volume based on the total volume of the incubation system, and the volume ratio of the serum, the complement, and the bacteria is 2:(0.5 - 1):1.

[0039] Preferably, the mixed incubation is carried out at 35 - 37 °C for 0.5 - 2 hours.

[0040] When culturing bacteria after the bactericidal test, preferably, the culture is carried out at 35 - 37 °C overnight, for example, cultured for 12 - 20 hours.

[0041] The method of the present invention can complete the detection of the bactericidal titers against different groups of bacteria in the same bactericidal system with one serum, greatly improving the work efficiency and saving the test cost. Especially for infants and young children, it is not easy to collect blood. This method can reduce the amount of serum to be tested by 4 times, reducing the burden of blood collection in clinical trials.

[0042] The following further explains or illustrates the content of the present invention by way of examples, but these examples should not be construed as limiting the protection scope of the present invention.

[0043] Example

[0044] Unless otherwise specified, the experimental methods used in the following examples are all carried out using the conventional experimental procedures, operations, materials and conditions in the art.

[0045] The sources of the materials, reagents and instruments used in the examples are as follows:

[0046] a. Target bacteria: The target bacteria of the four groups are: Neisseria meningitidis group A CMCC29201 (A4), Neisseria meningitidis group C CMCC29201 (C11), Neisseria meningitidis group Y CMCC29028, Neisseria meningitidis group W135 CMCC29037. These target bacteria are obtained from Lanzhou Institute of Biological Products Co., Ltd. and are induced to have resistance to four different antibiotics respectively.

[0047] b. TTC stock solution: 1.25 g of TTC (purchased from Sigma, T-8877), add water to 40 mL. After it is completely dissolved, make up the water to 50 mL, filter with a 0.22 μm filter membrane and reserve for use;

[0048] c. Gelatin solution (1%): 0.5 g of gelatin (purchased from Sigma, G-9391), add water to 50 mL, autoclave (115 °C, 30 min) and reserve for use, store at room temperature;

[0049] d. Bactericidal buffer: Sterilized H 2 O 40 mL, 10x HBSS (purchased from Invitrogen, 14065-056) (containing Ca 2+ , Mg 2+ ) 5 mL, 1% gelatin solution 5 mL, prepare and use immediately.

[0050] Example 1: Induced resistant strains

[0051] 1. Materials and methods

[0052] 1.1 Manufacturers and usage concentrations of antibiotics

[0053] Table 1-1. Antibiotic information

[0054]

[0055]

[0056] 1.2 Culture medium

[0057] THYA plate: When the total volume is 800 mL, the ratio is to add 24 g of THB (Todd-Hewitt, purchased from Becton-Dickinson, 249240), 12 g of yeast powder (purchased from Becton-Dickinson, 212750), and 12 g of agar (purchased from Becton-Dickinson, 214010). After autoclaving (115 °C, 30 min), it is cooled to 56 °C, appropriate concentration of antibiotics is added, and it is poured into square petri dishes, 20 mL for each square petri dish; culture medium plates with different resistances are obtained and stored at 4 °C for later use.

[0058] THY liquid medium: When the total volume is 800 mL, the ratio is to add 24 g of THB, 12 g of yeast powder, and it is used after being sterilized by filtration through a 0.22-μm membrane.

[0059] 2. Process of resistance induction

[0060] What needs to be done in this example is to induce resistance to different antibiotics in four different groups of Neisseria meningitidis bacteria respectively, so that they can be separated from the mixed bacteria with different antibiotics. Therefore, the screening needs to go through four steps: 1) Select antibiotics. Antibiotics that are not commonly used at present should be selected. However, there is a problem that for this pathogenic bacterium, it may be resistant to these eliminated antibiotics; 2) Determine the natural resistance of the four strains of bacteria to the selected antibiotics; 3) Conduct directional induction according to specific circumstances; 4) Detect the resistance of bacteria with different resistances to the resistances carried by the other three strains of bacteria.

[0061] 2.1 First, determine the resistance range of the four strains of bacteria to eight different antibiotics.

[0062] The four strains of bacteria are Neisseria meningitidis group A, group C, group W135, and group Y; the eight different antibiotics are shown in Table 1-1.

[0063] Dilute the antibiotics in a 96-well cell culture plate according to the following table. The diluent is THY liquid medium (30 g of THB, 5 g of yeast powder, H 2 O 1000 ml, after being sterilized by filtration through a 0.22-μm filter membrane, stored at 4 °C). The same operation is carried out in four plates respectively (i.e., in quadruplicate). Each plate corresponds to one strain of bacteria, and bacteria are added to each well. Observe in which wells each strain of bacteria can grow. The negative control is: THY liquid medium, without antibiotics and the corresponding bacteria; the positive control is: THY liquid medium and the corresponding bacteria, without antibiotics.

[0064]

[0065] The screening results are shown in Table 3

[0066]

[0067] As can be seen from the table, four or three strains among antibiotics A, E, and F all carry resistance and are not considered. Based on the situation in the table, the following judgments are made: Group Y can induce resistance to antibiotic B, named strain YB; Group W135 can induce resistance to antibiotic C, named strain WC; Group C can induce resistance to D, named strain CD; Group A can induce resistance to H, named strain AH.

[0068] 2.2 Resistance induction

[0069] Induce strains AH / CD / WC / YB under higher antibiotic resistance pressure so that they have stronger resistance to these antibiotics.

[0070] Induce with antibiotics (for each strain, from 1 μg / mL to 16 μg / mL. If it can still grow at the highest concentration, continue to increase the concentration for induction; if it cannot grow even at the lowest concentration, reduce the concentration. The range of antibiotic concentrations used is shown in Table 4, and the induction is carried out on THYA plates: During the induction, the following operations are carried out: On each antibiotic medium plate, bacteria from 4 groups are inoculated simultaneously, and each strain is inoculated on the medium with four antibiotics. Only in this way can it be ensured that the selected strains are only resistant to one of the four antibiotics and sensitive to the other three. During the screening process, if a strain is resistant to two of the antibiotics, the antibiotic concentration needs to be readjusted. If it still doesn't work, it is necessary to start from the previous step to rescreen the colonies with single resistance until success.

[0071] Finally, four strains with single and relatively high resistance are obtained: AH / CD / WC / YB.

[0072] 2.3 Confirmation of resistance range

[0073] When the above four strains are induced to a relatively high antibiotic resistance concentration, it is also necessary to confirm the resistance concentration range, that is, to find the resistance range in which only bacteria from one group can grow. The results are as follows:

[0074]

[0075]

[0076]

[0077]

[0078] In the table, "+" indicates bacterial growth and "-" indicates no bacterial growth. From the above results, it can be seen that: for Group A, the concentration exploration of antibiotic H with resistance ranges from 0.02 to 1.6 μg / mL, and the final determined concentration range is 0.08 - 0.4 μg / mL. The upper limit value of 0.4 μg / mL of this antibiotic concentration is lower than the upper limit value of the screening concentration shown in Table 3 because the culture medium used is changed from liquid to solid, resulting in a change in the growth conditions of Group A and a change in the concentration of antibiotic H tolerated, which is a normal phenomenon. The conditions obtained after confirming the resistance range in this step are stable conditions; for Group C, the concentration exploration of antibiotic D with resistance ranges from 1 to 400 μg / mL, and the final determined concentration range is 50 - 200 μg / mL; for Group W135, the concentration exploration of antibiotic C with resistance ranges from 1 to 240 μg / mL, and the final determined concentration range is 8 - 240 μg / mL and above; for Group Y, the concentration exploration of antibiotic B with resistance ranges from 1 to 480 μg / mL, and the final determined concentration range is 32 - 480 μg / mL and above. Therefore, the use concentration of H is set at 0.18 μg / mL; the use concentration of D is set at 100 μg / mL; the use concentration of C is set at 30 μg / mL; the use concentration of B is set at 120 μg / mL.

[0079] 2.4 Resistance cross - test:

[0080] The above - determined concentrations are tested with three concentrations, namely the determined concentration and twice the upper and lower concentrations of it. The results are as follows:

[0081]

[0082] The results show that the determined working concentration of the antibiotic is correct.

[0083] Example 2: In vitro bactericidal test of meningococcal polyserogroup sera

[0084] 1. Sera are obtained after immunizing human bodies with Neisseria meningitidis vaccines. The serum samples are shown in the following table. Inactivate the complement in the sera:

[0085] 1.1 Take out the samples from the - 80 °C freezer and place them at room temperature. Adjust the water bath temperature to 56 °C.

[0086] 1.2 After the samples are completely melted, mix them thoroughly and place them in a 56 °C water bath for 30 minutes.

[0087] 1.3 Take out the samples from the water bath and cool them to room temperature.

[0088] 1.4 Store the samples at 4 °C for short - term (<7 days) and at - 80 °C for long - term.

[0089]

[0090] 2. Serum dilution:

[0091] 2.1 In a 96-well cell culture plate, add 30 μl of bactericidal buffer to rows A to H in columns 1 and 2; add 30 μl of bactericidal buffer to rows A to G in columns 3 to 12.

[0092] 2.2 In plate A, add 45 μl of heat-inactivated and pre-diluted (at least 2-fold) serum sample 1 to rows A to H in columns 3 and 4, add 45 μl of heat-inactivated and pre-diluted (at least 2-fold) serum sample 2 to rows A to H in columns 5 and 6, and continue to add serum samples 5 in this form as shown in the following table. The remaining serum samples are added to plates B, C, and D in the same manner as shown in the following table.

[0093] Set up controls A and B in plate A as shown in the following table.

[0094]

[0095] 2.3 From column 3 to 12, perform 3-fold serial dilutions: Transfer 15 μl of serum from row H to row G, mix gently to avoid generating bubbles, then transfer 15 μl from row G to row F, and so on. After transferring 15 μl from row B to row A and mixing, aspirate 15 μl of the liquid from row A and discard it.

[0096] Plate A

[0097] 1 2 3 4 5 6 7 8 9 10 11 12 A Control A Control B Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 Dilution 8 B Control A Control B Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 Dilution 7 C Control A Control B Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 Dilution 6 D Control A Control B Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 Dilution 5 E Control A Control B Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 Dilution 4 F Control A Control B Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 Dilution 3 G Control A Control B Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 Dilution 2 H Control A Control B Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Dilution 1 Sample 1 Sample 1 Sample 2 Sample 2 Sample 3 Sample 3 Sample 4 Sample 4 Sample 5 Sample 5

[0098] Plates B / C / D

[0099]

[0100] 3. Quickly thaw 4 working bacterial strains and wash the bacteria:

[0101] 1) Gently rotate the bacterial strain tube in a 37 °C water bath to melt it.

[0102] 2) In a microcentrifuge, centrifuge at 12,000 g for 2 minutes.

[0103] 3) Carefully remove the supernatant and discard it.

[0104] 4) Add 1 ml of bactericidal buffer to each tube to suspend the bacteria and mix well.

[0105] 5) Centrifuge at 12,000 g for 2 minutes.

[0106] 6) Carefully remove the supernatant and discard it.

[0107] 7) Resuspend the bacterial pellet with the initial volume of bactericidal buffer (e.g., 0.5 ml), and perform colony counting:

[0108] 4. Take a certain volume of each of the four strains of bacteria and add it to 30 ml of sterilizing buffer to make the final concentration of each strain of bacteria approximately 32,000 - 48,000 CFU / ml, obtaining a mixed bacterial solution. Add 15 μl of the mixed bacterial solution to each well with diluted serum, including all control wells;

[0109] 5. Take out the complement (complement from 3 - 4 week old rabbits, purchased from Pel-freez Biologicals, 31061) from the refrigerator. Approximately 5 ml of active complement and 0.15 ml of heat-inactivated complement are required. The complement is thawed under a laminar flow hood and immediately placed on ice for later use;

[0110] 6. Add inactivated complement to the first column of plate A, 15 μl / well, and add active complement to the remaining wells, 15 μl / well;

[0111] 7. Vortex the microtiter plate on a small shaker and place it in an incubator at 37 °C with 5% CO 2 for 60 minutes. To maintain the concentration of CO 2 , do not open the incubator door during the process;

[0112] 8. After incubation, place the plate in an ice bath for about 5 minutes to terminate the reaction;

[0113] 9. Take 10 μl from each well of each reaction mixture and add it to four THYA plates containing antibiotics H, D, C, and B resistance respectively. The antibiotic concentrations are as described above. Use a multi-channel pipette to mix the mixtures in each well. Take 10 μl of liquid from 8 wells in each column and drip it from the left side of the THYA plate. Immediately tilt the plate so that the dropped liquid forms a streak, about 2 - 3 cm long. Drip the 32 wells in four columns side by side on one plate. When tilting the plate to make the liquid droplets flow, place the lid of the plate under the culture medium plate. The lid is marked with grids indicating the range where each liquid droplet can flow, so that the liquid droplets can be completely prevented from flowing together.

[0114] 10. Incubate at room temperature for 10 - 20 minutes to allow the liquid to be absorbed into the agar plate;

[0115] 11. Place the plate upside down and incubate at 37 °C with 5% CO 2 for 16 - 18 hours;

[0116] 12. Use an automatic colony counter to count the number of surviving colonies.

[0117] 13. Data analysis: Take the number of colonies in the normal complement control (control B) as the standard and calculate the bactericidal rate for each well in the experiment: Bactericidal rate = [1 - (number of colonies in control B - number of colonies in the well of the test plate) / number of colonies in control B] * 100%. The bactericidal index is the highest serum dilution multiple when the bactericidal rate is 50%. This multiple is calculated using the linear interpolation method to calculate the bactericidal titer.

[0118] Result:

[0119] The bactericidal titers of each serum sample against Neisseria meningitidis serogroups A, C, W135, and Y are as follows:

[0120]

[0121] Note: The appearance of "131220" indicates that the initial dilution factor of the serum in the bactericidal test is too low. At the highest dilution, most or all of the bacteria are killed, so the calculation cannot be performed. The initial dilution factor can be increased for adjustment.

[0122] This method takes 4 times less time than the conventional method for separate detection of different serogroups, greatly improving work efficiency and significantly saving time, labor, and material costs. It is very suitable for industrial application.

Claims

1. A method for detecting the in vitro bactericidal function of serum, wherein the serum contains two or more antibodies that respectively target different groups of a certain bacterium. Characterized in that, The method comprises the following steps: 1) Inducing the bacteria of different groups to respectively have different antibiotic resistances; 2) In the same system, mixing and incubating the serum, complement and bacteria of different groups with different antibiotic resistances to perform bactericidal action, obtaining a bactericidal mixed bacterial solution; 3) Mixing a culture medium containing an antibiotic against which the bacteria of one group are resistant with the bactericidal mixed bacterial solution, so that the bacteria of this group with this antibiotic resistance grow; performing this step for each group of bacteria; 4) Determining the bactericidal function of the serum against the bacteria of different groups according to the growth conditions of the bacteria of each group; Wherein the bacterium is Neisseria meningitidis, and the different groups are selected from two or more of group A, group C, group W135 and group Y; the antibiotics are selected from two or more of spectinomycin, trimethoprim, streptomycin sulfate and erythromycin; Wherein the induction includes: I) Simultaneously culturing the bacteria of different groups in a solid culture medium plate containing a gradient concentration of a certain antibiotic; for erythromycin, the gradient concentration range is 0.02 μg / mL - 1.6 μg / mL; for streptomycin sulfate, the gradient concentration range is 1 μg / mL - 400 μg / mL; for trimethoprim, the gradient concentration range is 1 μg / mL - 240 μg / mL; for spectinomycin, the gradient concentration range is 1 μg / mL - 480 μg / mL; performing this step with different antibiotics respectively; II) According to the growth conditions of the bacteria in step I), confirming the antibiotic resistance and concentration range, finding a culture medium containing an antibiotic in which only the bacteria of one group grow while the bacteria of other groups do not grow, and determining the concentration range of this antibiotic, so as to determine the induction of the following two or more antibiotic resistances: the resistance of group A Neisseria meningitidis to erythromycin; the resistance of group C Neisseria meningitidis to streptomycin sulfate; the resistance of group W135 Neisseria meningitidis to trimethoprim; the resistance of group Y Neisseria meningitidis to spectinomycin; wherein, for group A Neisseria meningitidis, determining its erythromycin resistance concentration range is 0.08 - 0.4 μg / mL; for group C Neisseria meningitidis, determining its streptomycin sulfate resistance concentration range is 50 - 200 μg / mL; for group W135 Neisseria meningitidis, determining its trimethoprim resistance concentration range is 8 - 240 μg / mL; for group Y Neisseria meningitidis, determining its spectinomycin resistance concentration range is 32 - 480 μg / mL.

2. The method according to claim 1, wherein the serum is the serum obtained after immunizing an organism with a Neisseria meningitidis vaccine.

3. The method according to claim 1, wherein the induction also includes: i) Before step I), determine the natural resistance of the different groups of bacteria to each antibiotic, including: performing the following operations on each group of bacteria: adding a gradient-diluted antibiotic and bacteria into the wells of a 96-well microplate, and observing which wells the bacteria can grow in; for spectinomycin and trimethoprim, the concentration range of the gradient dilution is from 1500 μg / mL to 5.86 μg / mL; for streptomycin sulfate, the gradient concentration range is from 125 μg / mL to 0.49 μg / mL; for erythromycin, the gradient concentration range is from 200 μg / mL to 0.78 μg / mL. ii) According to the growth of bacteria in the wells in step i), determine two or more of the following conclusions: Neisseria meningitidis group A has natural resistance to erythromycin; Neisseria meningitidis group C has natural resistance to streptomycin sulfate; Neisseria meningitidis group W135 has natural resistance to trimethoprim; Neisseria meningitidis group Y has natural resistance to spectinomycin.

4. The method according to claim 1, wherein the induction further includes: iii) After step II), verify the resistance of the bacteria with induced resistance to the induced resistance of other groups of bacteria, so as to ensure that each group of bacteria only has its own induced resistance within a certain antibiotic concentration range and does not have the induced resistance of other groups of bacteria.

5. The method according to claim 1, wherein during the mixed incubation, the serum is serum inactivated by complement.

6. The method according to claim 1, wherein during the mixed incubation, the number of bacteria in each group is 8 - 14 per μl.

7. The method according to claim 1, wherein during the mixed incubation, the dilution factor of the serum is more than 2 times, the concentration of the complement is 12.5 - 25 vol% based on the total volume of the incubation system, and the volume ratio of the serum, the complement, and the bacteria is 2:(0.5 - 1):

1.

8. The method according to claim 1, wherein the mixed incubation is carried out at 35 - 37 °C for 0.5 - 2 hours.

9. The method according to claim 1, wherein in step 3), the growth of the bacteria is carried out at 35 - 37 °C for 12 - 20 hours.

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