Bacterial endotoxin detection method of natural killer cell suspension

Abstract

The invention discloses a bacterial endotoxin detection method for a natural killer cell suspension, and relates to the technical field of bacterial endotoxin detection, the bacterial endotoxin detection method for the natural killer cell suspension comprises the following steps: S1, determining a bacterial endotoxin limit value L of the natural killer cell suspension; s2, rechecking the sensitivity of the tachypleus amebocyte lysate; s3, carrying out a cell suspension pre-interference experiment, and determining the minimum non-interference dilution multiple of the natural killer cell suspension of the test sample; s4, carrying out a natural killer cell suspension interference test on the test sample; s5, detecting bacterial endotoxin in the natural killer cell suspension of the test sample; the bacterial endotoxin detection method of the natural killer cell suspension is rapid and sensitive, after the limit value and the dilution multiple of the endotoxin are determined, the to-be-detected test sample can be directly detected, the dilution multiple of the test sample does not need to be determined through a test again, the detection accuracy is guaranteed, the detection cost is reduced, and the detection efficiency is improved.

Description

Technical Field

[0001] The invention relates to the field of bacterial endotoxin detection technology, in particular to a bacterial endotoxin detection method for a natural killer cell suspension. Background Art

[0002] NK cells are natural killer cells, which are important immune cells in the body responsible for killing abnormal cells such as aging, virus-infected, and tumor cells. As the first line of defense for the human body against cancer cells and viral infections, NK cells can directly kill tumor cells non-specifically without the need for antigen sensitization and antibody participation, and without MHC restrictions. They have powerful killing functions and strong immune regulation functions, interacting with other immune cells in the body to regulate the body's immune status and immune function. Clinical studies have found that NK cell adoptive immunotherapy has good application prospects for malignant tumors, various solid tumors, and hematological malignancies.

[0003] Bacterial endotoxin is a cell wall component of Gram-negative bacteria. Endotoxin is released when the bacteria die or autolyze. It can activate neutrophils, etc., causing them to release an endogenous pyrogen, which acts on the body's temperature regulation center to cause fever. According to the "Technical Guidelines for Pharmaceutical Research and Evaluation of Immune Cell Therapy Products (Trial) 2022.05.31", the application of immune cell therapy products requires safety testing, among which the detection of bacterial endotoxin is crucial.

[0004] The inspection method in the prior art is not convenient for directly testing the test sample after determining the endotoxin limit and dilution multiple, and is not convenient for reducing the inspection cost while ensuring the accuracy of the inspection, and the inspection efficiency needs to be improved; therefore, in view of this situation, there is an urgent need to develop a bacterial endotoxin inspection method for natural killer cell suspension to meet the needs of actual use. Summary of the invention

[0005] In view of this, the present invention aims at the deficiencies in the prior art, and its main purpose is to provide a method for detecting bacterial endotoxins in a natural killer cell suspension. The method for detecting bacterial endotoxins in a natural killer cell suspension provided by the present invention is rapid and sensitive. After determining the endotoxin limit and the dilution multiple, the test sample can be directly detected without the need to conduct another test to determine the dilution multiple of the test sample. While ensuring the accuracy of the detection, the inspection cost is reduced and the inspection efficiency is improved.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for testing bacterial endotoxins in a natural killer cell suspension comprises the following steps:

[0008] S1. Determine the bacterial endotoxin limit L of natural killer cell suspension;

[0009] S2. Review of sensitivity of horseshoe crab test kit;

[0010] S3. Conduct a cell suspension pre-interference experiment to determine the minimum non-interference dilution multiple of the test article natural killer cell suspension;

[0011] S4. Conduct the natural killer cell suspension interference test of the test article;

[0012] S5. Conduct bacterial endotoxin test on the natural killer cell suspension of the test article.

[0013] As a preferred embodiment: the bacterial endotoxin limit L in step S1 is determined according to the formula: L=K / M, wherein L is the bacterial endotoxin limit of the test sample, K is the maximum acceptable endotoxin dose per kilogram of body weight per hour for a person; and M is the maximum test sample dose per kilogram of body weight per hour for a person.

[0014] As a preferred solution: the sensitivity review of the horseshoe crab reagent in step S2 specifically includes the following steps:

[0015] S21, take the bacterial endotoxin standard, add bacterial endotoxin test water to reconstitute, seal and mix well;

[0016] S22, according to the indicated value (λ) of the sensitivity of the horseshoe crab test, dilute the above bacterial endotoxin standard with bacterial endotoxin test water to form endotoxin standard solutions of different concentrations;

[0017] S23, take the horseshoe crab reagent, add bacterial endotoxin test water to dissolve it, add endotoxin standard solutions of different concentrations to a part of it, and add bacterial endotoxin test water to the other part as a negative control;

[0018] S24. After sealing and mixing the horseshoe crab reagent obtained in step S23, put it into an electric constant temperature incubator at 37±1°C, keep it warm for 60±2 minutes, then take it out, observe and record the results, and calculate the measured value (λc) of the horseshoe crab reagent sensitivity according to the formula λc=lg-1(ΣX / 4), where X is the logarithm of the concentration at the reaction endpoint (lg).

[0019] As a preferred solution, the cell suspension pre-interference experiment in step S3 specifically includes the following steps:

[0020] S31, calculate the maximum effective dilution (MVD) according to the formula MVD = L / λ, where L is the bacterial endotoxin limit of the test sample, and λ is the labeled sensitivity of the horseshoe crab reagent in the gel method;

[0021] S32. Take different batches of natural killer cell suspension, dilute them with water for bacterial endotoxin test into different series of concentration solutions and mark them as test article negative control (NPC); prepare the same series of test article solutions, and mark this series of solutions as test article positive control (PPC); take horseshoe crab reagent to react with the above-mentioned NPC and PPC respectively, seal the mixture, observe the results in a 37°C water bath for 60 minutes, and determine the minimum non-interfering dilution factor.

[0022] As a preferred solution: the natural killer cell suspension interference test in step S4 specifically comprises the following steps:

[0023] S41, take a bacterial endotoxin standard, add bacterial endotoxin test water to dissolve, seal and mix, and dilute with bacterial endotoxin test water to a 4λ bacterial endotoxin working standard working solution;

[0024] S42. Take different batches of natural killer cell suspension and dilute them 4 times with water for bacterial endotoxin test as diluent, and prepare the 4λ bacterial endotoxin working standard working solution in step S41 as the positive control of the test sample. This sequence is the endotoxin test solution Et; prepare the 4λ bacterial endotoxin working standard working solution in S41 with water for bacterial endotoxin test to prepare an endotoxin aqueous solution Es; take horseshoe crab reagent to react with Et and Es respectively; calculate the geometric mean of the reaction endpoint concentration according to the formula Es=antilg(ΣX / 4); Et=antilg(ΣX / 4), where X is the logarithm of the reaction endpoint concentration.

[0025] As a preferred embodiment: the endotoxin limit L of the natural killer cell suspension in step S1 is 0.5-1.2 EU / mL.

[0026] As a preferred embodiment: the endotoxin limit L of the natural killer cell suspension in step S1 is 0.5EU / mL.

[0027] As a preferred solution: the sensitivity of the horseshoe crab reagent in step S2 is 0.06EU.

[0028] As a preferred solution: the minimum non-interference dilution factor in the cell suspension pre-interference experiment in step S3 is 4 times.

[0029] As a preferred solution: the maximum effective dilution factor of the suspension interference test in step S4 is 8 times.

[0030] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that the method for inspecting bacterial endotoxins in the natural killer cell (NK cell) cell suspension of the present invention is fast and sensitive. After determining the endotoxin limit and dilution factor, the test sample can be directly tested without the need to conduct another test to determine the dilution factor of the test sample. While ensuring the accuracy of the test, it reduces the inspection cost, improves the inspection efficiency, and facilitates the monitoring of the production process and the implementation of inspection work; it complies with the requirements of the application guidelines of the bacterial endotoxin inspection method 9251 in Part IV of the 2020 edition of the "Chinese Pharmacopoeia", and ensures the quality and safety of NK cell products.

[0031] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a process flow chart of the method for testing bacterial endotoxins in natural killer cell suspension. DETAILED DESCRIPTION

[0033] The present invention Figure 1 As shown, a method for testing bacterial endotoxins in a natural killer cell suspension comprises the following steps:

[0034] S1. Determine the bacterial endotoxin limit L of natural killer cell suspension;

[0035] S2. Review of sensitivity of horseshoe crab test kit;

[0036] S3. Conduct a cell suspension pre-interference experiment to determine the minimum non-interference dilution multiple of the test article natural killer cell suspension;

[0037] S4. Conduct the natural killer cell suspension interference test of the test article;

[0038] S5. Conduct bacterial endotoxin test on the natural killer cell suspension of the test article.

[0039] In step S1, the bacterial endotoxin limit L is determined according to the formula: L=K / M, wherein L is the bacterial endotoxin limit of the test article, K is the maximum acceptable endotoxin dose per kilogram of body weight per hour for a person, and M is the maximum test article dose per kilogram of body weight per hour for a person.

[0040] The sensitivity review of the horseshoe crab reagent in step S2 specifically includes the following steps:

[0041] S21, take the bacterial endotoxin standard, add bacterial endotoxin test water to reconstitute, seal and mix well;

[0042] S22, according to the indicated value (λ) of the sensitivity of the horseshoe crab test, dilute the above bacterial endotoxin standard with bacterial endotoxin test water to form endotoxin standard solutions of different concentrations;

[0043] S23, take the horseshoe crab reagent, add bacterial endotoxin test water to dissolve it, add endotoxin standard solutions of different concentrations to a part of it, and add bacterial endotoxin test water to the other part as a negative control;

[0044] S24. After sealing and mixing the horseshoe crab reagent obtained in step S23, put it into an electric constant temperature incubator at 37±1°C, keep it warm for 60±2 minutes, then take it out, observe and record the results, and calculate the measured value (λc) of the horseshoe crab reagent sensitivity according to the formula λc=lg-1(ΣX / 4), where X is the logarithm of the concentration at the reaction endpoint (lg).

[0045] The cell suspension pre-interference experiment in step S3 specifically includes the following steps:

[0046] S31, calculate the maximum effective dilution (MVD) according to the formula MVD = L / λ, where L is the bacterial endotoxin limit of the test sample, and λ is the labeled sensitivity of the horseshoe crab reagent in the gel method;

[0047] S32. Take different batches of natural killer cell suspension, dilute them with water for bacterial endotoxin test into different series of concentration solutions and mark them as test article negative control (NPC); prepare the same series of test article solutions, and mark this series of solutions as test article positive control (PPC); take horseshoe crab reagent to react with the above-mentioned NPC and PPC respectively, seal the mixture, observe the results in a 37°C water bath for 60 minutes, and determine the minimum non-interfering dilution factor.

[0048] The natural killer cell suspension interference test in step S4 specifically includes the following steps:

[0049] S41, take a bacterial endotoxin standard, add bacterial endotoxin test water to dissolve, seal and mix, and dilute with bacterial endotoxin test water to a 4λ bacterial endotoxin working standard working solution;

[0050] S42. Take different batches of natural killer cell suspension and dilute them 4 times with water for bacterial endotoxin test as diluent, and prepare the 4λ bacterial endotoxin working standard working solution in step S41 as the positive control of the test sample. This sequence is the endotoxin test solution Et; prepare the 4λ bacterial endotoxin working standard working solution in S41 with water for bacterial endotoxin test to prepare an endotoxin aqueous solution Es; take horseshoe crab reagent to react with Et and Es respectively; calculate the geometric mean of the reaction endpoint concentration according to the formula Es=antilg(ΣX / 4); Et=antilg(ΣX / 4), where X is the logarithm of the reaction endpoint concentration.

[0051] The endotoxin limit L of the natural killer cell suspension in step S1 is 0.5-1.2 EU / mL.

[0052] The endotoxin limit L of the natural killer cell suspension in step S1 is 0.5 EU / mL.

[0053] The sensitivity of the horseshoe crab reagent in step S2 is 0.06EU.

[0054] The minimum non-interference dilution factor in the cell suspension pre-interference experiment in step S3 is 4 times.

[0055] The maximum effective dilution factor of the suspension interference test in step S4 is 8 times.

[0056] Example 1: A method for testing bacterial endotoxins in a natural killer cell suspension

[0057] Reagents and consumables used:

[0058]

[0059]

[0060] S1. Determine the bacterial endotoxin limit L of natural killer cell suspension;

[0061] The bacterial endotoxin limit (L) of a drug is generally determined according to the formula: L = K / M, where L is the bacterial endotoxin limit of the test article, generally expressed in EU / ml, EU / mg or EU / U (activity unit); K is the maximum acceptable endotoxin dose per kilogram of body weight per hour, expressed in EU / (kg·h), and for injections, K = 5EU / (kg·h); M is the maximum test article dose per kilogram of body weight per hour, expressed in ml / (kg·h), mg / (kg·h) or U / (kg·h), with an average body weight of 60 kg and a human surface area of ​​1.62 m 2 Calculation. If the injection time is less than 1 hour, it will be calculated as 1 hour. In clinical research literature, the specifications of natural killer cell (NK cell) suspension are generally 100-250ml. The dose per square meter of body surface area of ​​the test article can be converted into the dose per kilogram of body weight (M) by multiplying it by 0.027. When the specification is 100ml, L = K / M = [5EU / (kg·h)-250EU / 60(kg·h)] / 100mL / 60(kg·h) = 0.5EU / mL. Similarly, when the specification is 250ml, L = 1.2EU / mL.

[0062] S2. Review of sensitivity of horseshoe crab test kit;

[0063] (1) Take one tube of bacterial endotoxin national standard (90EU / ml) containing lyophilized product, add 1ml of bacterial endotoxin test water to reconstitute, seal the tube, and mix on a vortex mixer for 15 minutes;

[0064] (2) According to the indicated value (λ) of the sensitivity of the horseshoe crab test, the above-mentioned national bacterial endotoxin standard was diluted with bacterial endotoxin test water to form four concentrations of endotoxin standard solutions of 2λ, λ, 0.5λ, and 0.25λ, and each dilution step was mixed on a vortex mixer for 30 seconds;

[0065] (3) Take 18 bottles of horseshoe crab reagent (Xiamen horseshoe crab reagent), add 0.1 ml of bacterial endotoxin test water to each bottle to dissolve it, add 0.1 ml of endotoxin standard solution of different concentrations to 16 bottles, and add 4 bottles of each concentration in parallel; add 0.1 ml of bacterial endotoxin test water to the other 2 bottles as negative control;

[0066] (4) Take 2 ampoules of horseshoe crab reagent (Zhanjiang Andus) and break them open. Add 0.65 mL of test water to each ampoule and shake gently to dissolve it. Each aliquot is 50 μl, for a total of 18 aliquots. 16 of them are added with 50 μl of endotoxin standard solution of different concentrations, and 4 aliquots are prepared for each concentration. The other 2 aliquots are added with 50 μl of bacterial endotoxin test water as a negative control.

[0067] (5) Seal the ampoule with sealing film, shake gently to mix the solution in the ampoule, and then vertically place it in an electric constant temperature incubator at 37±1℃. Keep it warm for 60±2 minutes, then take it out, observe and record the results, and calculate the value according to the formula λc=lg -1 (ΣX / 4) was used to calculate the measured value (λc) of the sensitivity of the horseshoe crab reagent, where X was the logarithmic value (lg) of the reaction endpoint concentration. The results are shown in Table 1.

[0068] Table 1: Results of the sensitivity verification test of the horseshoe crab reagent

[0069]

[0070] Note: At the end of incubation, slowly invert the ampoule 180°. If gel is formed in the ampoule and the gel does not deform or slide off the tube wall, it is positive (+). If no gel is formed or the formed gel is not solid, deforms and slides off the tube wall, it is negative (-).

[0071] It can be seen from the data in Table 1 that the maximum concentration 2λ tubes are all positive (+), the minimum concentration 0.25λ tubes are all negative, and the negative control tube is negative, indicating that the values ​​of the sensitivity of the horseshoe crab reagent in this test are all within the range of 0.5λ to 2.0λ, which meets the requirements. Substituting the logarithmic value (X) of the reaction endpoint concentration in Table 1 into the formula of the above horseshoe crab reagent sensitivity measurement value λc=antilg(ΣX / 4), λc=antilg(ΣX / 4)=antilg(lgλ4 / 4)=0.06EU / ml is obtained, and the sensitivity of the horseshoe crab reagents of the two horseshoe crab reagent manufacturers meets the use requirements.

[0072] S3. Pre-interference experiment of natural killer cell (NK cell) suspension to determine the minimum non-interference dilution multiple of the test article natural killer cell suspension;

[0073] (01) The maximum effective dilution factor (MVD) was calculated according to the formula MVD = L / λ, where L is the bacterial endotoxin limit of the test sample, and λ is the labeled sensitivity of the horseshoe crab reagent in the gel method. L = 0.5EU / ml, λ = 0.0625; then MVD = L / λ = 0.5 / 0.0625 = 8 times, and the maximum dilution factor does not exceed 8 times. The dilution factors of the pre-interference experiment of the test sample are 2, 4, 6, and 8 times. The pre-interference experiment was carried out using horseshoe crab reagents from two different manufacturers.

[0074] (02) Take three different batches of natural killer cell (NK cell) suspension and dilute them with water for bacterial endotoxin test into a series of solutions with concentrations of 2, 4, 6, and 8, marked as the negative control (NPC) of the test article. Prepare another series of test article solutions with the same concentration, and make sure that each concentration of the test article solution contains 0.25EU / mL of bacterial endotoxin. This series of solutions is marked as the positive control (PPC) of the test article. Take 0.06EU / mL of horseshoe crab reagent to react with the above NPC and PPC respectively. Make 2 tubes in parallel for each concentration, and set up 2 negative control tubes (NC) for bacterial endotoxin test water and 2 bacterial endotoxin positive control tubes (PC) containing 0.25EU / mL of endotoxin. After mixing, seal the tubes, bathe in 37℃ water for 60 minutes, and observe the results to determine the minimum non-interfering dilution factor. The results are shown in Table 2.

[0075] Table 2: Results of pre-interference experiment of natural killer cell (NK cell) cell suspension

[0076]

[0077]

[0078] Note: At the end of incubation, slowly invert the ampoule 180°. If gel is formed in the ampoule and the gel does not deform or slide off the tube wall, it is positive (+). If no gel is formed or the formed gel is not solid, deforms and slides off the tube wall, it is negative (-).

[0079] (03) All parallel tubes in the NPC and NC groups were negative, and all parallel tubes in the PC group were positive, indicating that the test was valid. Three batches of natural killer cell (NK cell) suspensions were tested using Zhanjiang Andus and Xiamen Limulus reagents with a marked sensitivity of 0.06EU / mL. The minimum non-interfering dilution factor was 4 times, so the interference experiment could be performed using dilution factors of 4, 6, and 8 times.

[0080] S4. Conduct the test article natural killer cell (NK cell) cell suspension interference test;

[0081] (01) The maximum effective dilution (MVD) was 8 times and the horseshoe crab reagents from two different manufacturers were used for pre-interference experiments.

[0082] (02) Take one tube of bacterial endotoxin national standard (90EU / ml), add 1ml of bacterial endotoxin test water to dissolve it, seal it, mix it on a vortex mixer for 15 minutes, and then dilute it with bacterial endotoxin test water to make 4λ bacterial endotoxin working standard working solution.

[0083] (03) Take 3 different batches of natural killer cell (NK cell) suspension and dilute it 4 times with bacterial endotoxin test water, as diluent, prepare 4λ bacterial endotoxin working standard solution in (02), so that it contains 2.0λ, 1.0λ, 0.5λ, 0.25λ of bacterial endotoxin test sample positive control, this sequence is endotoxin test sample solution Et; use bacterial endotoxin test water to prepare 4λ bacterial endotoxin working standard solution in (02), and prepare endotoxin aqueous solution Es containing 2.0λ, 1.0λ, 0.5λ, 0.25λ of endotoxin. Take 0.06EU / mL horseshoe crab reagent to react with the above Et and Es respectively, and repeat 4 tubes for each concentration, and set up 2 tubes each of negative control tube (NC) of bacterial endotoxin test water and negative control tube (NPC) of test sample containing 1:8 times diluted NK cell suspension. After mixing, seal the tube and place it in a 37°C water bath for 60 minutes to observe the results. The geometric mean of the reaction endpoint concentration was calculated according to the formula Es = antilg (ΣX / 4); Et = antilg (ΣX / 4). The results are shown in Table 3.

[0084] Table 3: Results of natural killer cell (NK cell) cell suspension interference experiment

[0085]

[0086] Note: At the end of incubation, slowly invert the ampoule 180°. If gel is formed in the ampoule and the gel does not deform or slide off the tube wall, it is positive (+). If no gel is formed or the formed gel is not solid, deforms and slides off the tube wall, it is negative (-).

[0087] (04) The interference test results of (03) showed that the Es of horseshoe crab reagents from the two manufacturers were all between 0.5λ and 2λ, and the Et of the test samples of the three batches of NK cell suspensions were all within the range of 0.5Es-2Es, indicating that the NK cell suspension had no interference effect on the bacterial endotoxin test after being diluted 1:8, and bacterial endotoxin detection could be carried out.

[0088] S5. Bacterial endotoxin detection of natural killer cell (NK cell) cell suspension

[0089] (1) Take one tube of the national standard for bacterial endotoxin (90 EU / ml) of freeze-dried product, add 1 ml of bacterial endotoxin test water to dissolve it, seal it, and mix it on a vortex mixer for 15 minutes;

[0090] (2) Dilution: dilute the dissolved bacterial endotoxin working standard in (1) in a gradient (the dilution gradient should not be greater than 10 times) to 4;

[0091] (3) Six batches of NK cell suspension were taken respectively, and each batch of test samples was tested using the Xiamen Limulus Reagent and the Zhanjiang Andus Limulus Reagent according to Table 4;

[0092] Table 4: Preparation and grouping of bacterial endotoxin test solutions

[0093]

[0094] Note: At the end of incubation, slowly invert the ampoule 180°. If gel is formed in the ampoule and the gel does not deform or slide off the tube wall, it is positive (+). If no gel is formed or the formed gel is not solid, deforms and slides off the tube wall, it is negative (-).

[0095] (4) The bacterial endotoxin test results of 6 batches of NK cell suspension samples were tested using the Xiamen Limulus Reagent and the Zhanjiang Andus Limulus Reagent. See Table 5 for the bacterial endotoxin test results.

[0096] Table 5: Bacterial endotoxin test results

[0097]

[0098] (5) The above results show that the results of the two manufacturers' horseshoe crab reagents with a sensitivity of 0.06EU / ml for the NK cell suspension test sample are in compliance with the regulations. The bacterial endotoxin detection limit of the NK cell suspension test sample is 0.5EU / ml.

[0099] The design focus of the present invention is that the method for inspecting bacterial endotoxins in the natural killer cell (NK cell) cell suspension of the present invention is rapid and sensitive. After determining the endotoxin limit and dilution multiple, the test sample can be directly tested without the need to conduct another test to determine the dilution multiple of the test sample. While ensuring the accuracy of the test, it reduces the inspection cost, improves the inspection efficiency, and facilitates the monitoring of the production process and the implementation of inspection work; it complies with the requirements of the application guidelines of the bacterial endotoxin inspection method 9251 in Part IV of the 2020 edition of the "Chinese Pharmacopoeia", ensuring the quality and safety of NK cell products.

[0100] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for testing bacterial endotoxins in a natural killer cell suspension, characterized in that: The steps include: S1. Determine the bacterial endotoxin limit L of natural killer cell suspension; S2. Review of sensitivity of horseshoe crab test kit; S3. Conduct a cell suspension pre-interference experiment to determine the minimum non-interference dilution multiple of the test article natural killer cell suspension; S4. Conduct the natural killer cell suspension interference test of the test article; S5. Conduct bacterial endotoxin test on the natural killer cell suspension of the test article.

2. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: In step S1, the bacterial endotoxin limit L is determined according to the formula: L=K / M, wherein L is the bacterial endotoxin limit of the test sample, K is the maximum acceptable endotoxin dose per kilogram of body weight per hour for a person, and M is the maximum test sample dose per kilogram of body weight per hour for a person.

3. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The sensitivity review of the horseshoe crab reagent in step S2 specifically includes the following steps: S21, take the bacterial endotoxin standard, add bacterial endotoxin test water to reconstitute, seal and mix well; S22, according to the indicated value (λ) of the sensitivity of the horseshoe crab test, dilute the above bacterial endotoxin standard with bacterial endotoxin test water to form endotoxin standard solutions of different concentrations; S23, take the horseshoe crab reagent, add bacterial endotoxin test water to dissolve it, add endotoxin standard solutions of different concentrations to a part of it, and add bacterial endotoxin test water to the other part as a negative control; S24. After sealing and mixing the horseshoe crab reagent obtained in step S23, put it into an electric constant temperature incubator at 37±1°C, keep it warm for 60±2 minutes, then take it out, observe and record the results, and calculate the measured value (λc) of the horseshoe crab reagent sensitivity according to the formula λc=lg-1(ΣX / 4), where X is the logarithm of the concentration at the reaction endpoint (lg).

4. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The cell suspension pre-interference experiment in step S3 specifically includes the following steps: S31, calculate the maximum effective dilution (MVD) according to the formula MVD = L / λ, where L is the bacterial endotoxin limit of the test sample, and λ is the labeled sensitivity of the horseshoe crab reagent in the gel method; S32. Take different batches of natural killer cell suspension, dilute them with water for bacterial endotoxin test into different series of concentration solutions and mark them as test article negative control (NPC); prepare the same series of test article solutions, and mark this series of solutions as test article positive control (PPC); take horseshoe crab reagent to react with the above-mentioned NPC and PPC respectively, seal the mixture, observe the results in a 37°C water bath for 60 minutes, and determine the minimum non-interfering dilution factor.

5. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The natural killer cell suspension interference test in step S4 specifically includes the following steps: S41, take a bacterial endotoxin standard, add bacterial endotoxin test water to dissolve, seal and mix, and dilute with bacterial endotoxin test water to a 4λ bacterial endotoxin working standard working solution; S42. Take different batches of natural killer cell suspension and dilute them 4 times with water for bacterial endotoxin test as diluent, and prepare the 4λ bacterial endotoxin working standard working solution in step S41 as the positive control of the test sample. This sequence is the endotoxin test solution Et; prepare the 4λ bacterial endotoxin working standard working solution in S41 with water for bacterial endotoxin test to prepare an endotoxin aqueous solution Es; take horseshoe crab reagent to react with Et and Es respectively; calculate the geometric mean of the reaction endpoint concentration according to the formula Es=antilg(ΣX / 4); Et=antilg(ΣX / 4), where X is the logarithm of the reaction endpoint concentration.

6. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The endotoxin limit L of the natural killer cell suspension in step S1 is 0.5-1.2 EU / mL.

7. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 6, characterized in that: The endotoxin limit L of the natural killer cell suspension in step S1 is 0.5EU / mL.

8. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The sensitivity of the horseshoe crab reagent in step S2 is 0.06EU.

9. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The minimum non-interference dilution factor in the cell suspension pre-interference experiment in step S3 is 4 times.

10. The method for testing bacterial endotoxins in a natural killer cell suspension according to claim 1, characterized in that: The maximum effective dilution factor of the suspension interference test in step S4 is 8 times.

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