Reaction buffer solution for detecting endotoxin by recombinant C factor method and application of reaction buffer solution

Abstract

The invention discloses a reaction buffer solution for detecting endotoxin by a recombinant C factor method and application of the reaction buffer solution. The reaction buffer solution comprises the following components: a basic buffer solution, magnesium chloride, trehalose and tween-20. The invention also discloses a preparation method of the reaction buffer solution and application of the reaction buffer solution in endotoxin detection. According to the reaction buffer solution for detecting the endotoxin by the recombinant C factor method, on the basis of ensuring the reaction sensitivity of the endotoxin, the anti-interference capability is remarkably improved, and the accuracy of detecting the endotoxin in an effective dilution range is ensured, so that the situation that the concentration of the endotoxin is lower than the detection limit due to excessive dilution, and the detection result is unreliable is avoided.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a reaction buffer for detecting endotoxin using a recombinant factor C method and application thereof. Background Art

[0002] Bacterial endotoxins, the lipopolysaccharide component of the cell wall of Gram-negative bacteria, are released into the circulation after bacterial lysis and can trigger a pyrogenic reaction. When blood concentrations exceed the body's clearance threshold, they trigger a cascade of inflammatory factors, leading to clinical risks such as fever, endotoxemia, and even shock. Due to the high thermal stability and strong pathogenicity of endotoxins, endotoxin testing is listed as a key quality control indicator for drugs, medical devices, and biologics in pharmacopoeias worldwide.

[0003] Traditional endotoxin detection technology has gone through two major stages of development. The early rabbit pyrogen method assessed whether the pyrogen limit met regulations by observing changes in the animal's body temperature. However, it had shortcomings such as low sensitivity (0.5EU / mL), cumbersome operation, and non-compliance with animal ethics. The Limulus Amebocyte Lysate (LAL) method, discovered in 1964, quickly became the industry gold standard due to its high sensitivity (0.005-0.03EU / mL) and standardized operating procedures. Its core mechanism relies on the unique coagulation cascade reaction in horseshoe crab blood: after endotoxin activates factor C, it activates factor B and prothrombin in sequence, and finally cuts the prothrombin to form a gel. However, this technique also has two inherent drawbacks. First, factor G in natural horseshoe crab blood can cross-react with β-1,3-glucan, leading to false-positive results. This can be particularly problematic with samples containing plant polysaccharides, such as cellulose-purified monoclonal antibodies. Second, due to environmental degradation and overfishing, horseshoe crab populations have plummeted, with the Chinese horseshoe crab and the round-tailed horseshoe crab listed as endangered species on the 2021 National List of Key Protected Wildlife. As a Class II protected species in my country, the supply of horseshoe crab raw materials is strictly limited, reagent costs have skyrocketed, and batch-to-batch variability is difficult to control.

[0004] In order to break through the above bottlenecks, recombinant factor C (rFC) detection technology came into being. This technology obtains factor C protein that specifically binds to endotoxin through genetic engineering expression and purification. Its mechanism of action abandons the traditional cascade reaction: after endotoxin activates the serine protease activity of recombinant factor C, it directly cuts the fluorescent substrate (such as Boc-Val-Pro-Arg-AMC) to release a detectable signal. Compared with the Limulus amebocyte lysate reagent method, recombinant factor C technology has three advantages: (1) improved specificity, completely avoiding G factor interference; (2) standardized production process, significantly reducing batch differences; (3) getting rid of dependence on horseshoe crab blood resources, in line with animal protection principles. The four major pharmacopoeias in the world have fully recognized this method: the Japanese Pharmacopoeia JP18 (2021) includes a guide to recombinant protein detection, the European Pharmacopoeia EP11.5 (2024) establishes an independent chapter, and the United States Pharmacopoeia USP <86> The 2025 edition of the Chinese Pharmacopoeia (effective in 2025) specifies technical specifications, including an appendix titled "Recombinant Factor C Method." Currently, recombinant Factor C technology covers the entire pharmaceutical industry chain (raw material screening, process monitoring, and finished product release), and is particularly suitable for testing complex samples containing glucans.

[0005] Because recombinant Factor C is used for endotoxin detection without relying on horseshoe crab blood and eliminating Factor G, it has high specificity, good stability, and complies with animal protection principles. These advantages will drive recombinant Factor C to gradually replace traditional horseshoe crab reagent detection methods. When the endotoxin detection method is changed, in order to ensure the accuracy, reliability and compliance of the new method, the following key indicators need to be systematically evaluated and verified: specificity, sensitivity, linearity and range, accuracy, precision, equivalence, interference and stability. Among them, the interference experiment needs to verify the anti-interference ability of the Factor C method in complex matrices (such as drugs and biological products), and the recovery rate of the sample at the maximum effective dilution needs to be verified, which must meet 50%-200%.

[0006] During the testing of biological samples, various components present in the sample, such as salt ions, surfactants, proteins, organic solvents, chelating agents, and fluctuations in the sample's pH value, may significantly interfere with the test results. These interfering factors may cause false positive or false negative test results by changing the ionic strength of the reaction system, affecting enzyme activity, binding to endotoxins, or competitive inhibition. For example, high concentrations of salt ions may interfere with the specific binding of the limulus amebocyte lysate (LA-lysate) to endotoxins; surfactants may disrupt the aggregation state of endotoxins; proteins may nonspecifically adsorb endotoxins; organic solvents may change the solubility of the reaction system; and chelating agents may affect enzyme reactions by chelating divalent cations. Abnormal fluctuations in the sample's pH may directly affect the efficiency of the enzymatic reaction of the limulus amebocyte lysate.

[0007] Therefore, to ensure the accuracy and reliability of the test results, a robust reaction buffer system must be established. This system needs to possess the following key characteristics: First, it should have excellent anti-interference ability and be able to effectively neutralize or shield the effects of the various interfering substances mentioned above; second, it must maintain detection sensitivity at a reasonable sample dilution to avoid endotoxin concentrations below the detection limit due to excessive dilution; finally, the system should have a broad spectrum of matrix applicability and be able to accurately detect the true endotoxin content in samples from different sources (such as cell culture fluids, injections, biological preparations, etc.). By optimizing parameters such as the buffer's ionic composition, pH stability, and chelator concentration, a robust detection system can be constructed to achieve accurate quantification of endotoxins in complex biological samples. Summary of the Invention

[0008] In response to the above technical requirements, the first purpose of the present invention is to develop a reaction buffer for detecting endotoxins using the recombinant Factor C method, which not only ensures the sensitivity of endotoxin reaction but also significantly improves the anti-interference ability and ensures the accuracy of endotoxin detection within the effective dilution range, thereby avoiding excessive dilution that causes the endotoxin concentration to be below the detection limit, resulting in unreliable test results.

[0009] The second object of the present invention is to provide a method for preparing the reaction buffer.

[0010] The third object of the present invention is to provide an application of the above reaction buffer in endotoxin detection, which can be applied to endotoxin detection of various types of samples.

[0011] The fourth object of the present invention is to provide an endotoxin detection kit comprising the above-mentioned reaction buffer and its application.

[0012] To achieve the above objectives, the present invention adopts the following technical solutions.

[0013] In a first aspect, a reaction buffer for detecting endotoxins using a recombinant Factor C method is provided, the reaction buffer comprising the following components: a basic buffer, magnesium chloride, trehalose, and Tween-20; the basic buffer is selected from one of MOPS, HEPES, TES, and Tris-HCl.

[0014] Furthermore, based on the final concentration of each component, the reaction buffer comprises the following components: 100-300 mM, pH 6.8-8.0 basic buffer, 50-250 mM magnesium chloride, 1-5% (w / v) trehalose, and 0.02-0.1% (w / v) Tween-20.

[0015] Furthermore, based on the final concentration of each component, the reaction buffer comprises the following components: 150 mM, pH 7.0 basic buffer, 200 mM magnesium chloride, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20.

[0016] In the second aspect, the present invention provides a method for preparing a reaction buffer for detecting endotoxins by the above-mentioned recombinant factor C method, the preparation method comprising: dissolving all components in water according to the formula concentration for preparation, mixing thoroughly and filtering to obtain. The preparation process needs to ensure aseptic operation, and the preparation is carried out in a clean bench throughout the process. According to a specific embodiment of the present invention, the preparation process of a reaction buffer with a volume of 100mL includes the following steps: taking an appropriate volume of sterile water for injection, adding each component in sequence, and the final concentration of each component is 100-300mM, pH 6.8-8.0 TES buffer, 50-250mM magnesium chloride, 1-5% (w / v) trehalose, 0.02-0.1% (w / v) Tween-20, and the volume is adjusted to 100mL after sufficient dissolution and mixing.

[0017] Furthermore, the reaction buffer was filtered using a 0.1 μm filter membrane, and the endotoxin level was controlled below 0.005 EU / ml.

[0018] In a third aspect, the present invention provides the use of the reaction buffer for detecting endotoxins using the recombinant Factor C method in endotoxin detection.

[0019] Furthermore, the application includes the following steps:

[0020] Prepare a series of endotoxin standard solutions;

[0021] preparing a reagent for detecting endotoxin, which comprises a fluorescent substrate, the reaction buffer, and a recombinant Factor C protein solution;

[0022] The endotoxin detection reagent was mixed with each concentration of endotoxin standard solution, incubated at 37°C, and read on a microplate reader at 0h and 1h. The logarithm of the fluorescence intensity change value after calibration of the negative control well was taken as the vertical axis, and the logarithm of the endotoxin standard solution concentration was taken as the horizontal axis to establish a standard curve;

[0023] The sample to be tested is diluted to prepare a sample to be tested. At the same time, a spiked sample to be tested with a certain concentration of endotoxin standard solution is prepared. The endotoxin detection reagent is then mixed with the sample to be tested and the spiked sample to be tested, respectively. The samples are incubated at 37°C, and the microplate reader is read at 0h and 1h. The endotoxin content of the sample to be tested is calculated using the established standard curve, and the endotoxin content of the initial undiluted sample to be tested is calculated according to the dilution multiple.

[0024] In the endotoxin detection reagent, the volume ratio of the fluorescent substrate (e.g., Boc-Val-Pro-Arg-AMC), the reaction buffer, and the recombinant Factor C protein solution is 5:4:1.

[0025] Furthermore, the concentration of the recombinant Factor C protein solution is 20 μg / ml.

[0026] Furthermore, the volume ratio of the endotoxin detection reagent to the endotoxin standard solution is 1:1.

[0027] Furthermore, the volume ratio of the reagent for detecting endotoxin to the sample to be tested or the spiked sample to be tested is 1:1.

[0028] In a fourth aspect, the present invention provides an endotoxin detection kit comprising the above-mentioned reaction buffer.

[0029] Furthermore, the present invention also claims protection for the use of the above-mentioned endotoxin detection kit in endotoxin detection.

[0030] The beneficial effects of the present invention are as follows:

[0031] 1. When the reaction buffer of the present invention is used for endotoxin detection using the recombinant Factor C method, the minimum limit of endotoxin that can be stably detected is 0.005 EU / ml.

[0032] 2. When the reaction buffer of the present invention is used for the recombinant Factor C method to detect endotoxins, it can effectively reduce the interference of other substances in the sample (including proteins, metal ions, etc.) on the reaction. Without excessive dilution, the actual content of endotoxins in the sample can be accurately detected within the effective dilution range, and the sample spike recovery rate is 50%-200%.

[0033] 3. The reaction buffer of the present invention can be used to accurately detect the endotoxin content in biological samples such as glucose injection, dextran injection, albumin injection, compound amino acid injection, emicizumab, cell culture medium (DMEM, RPMI 1640), cell freezing solution, serum, serum substitutes, etc., and the required dilution factor is much smaller than that of the existing technology, including but not limited to the method disclosed in U.S. Patent US6645724B1. DETAILED DESCRIPTION

[0034] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0035] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional biochemical reagent manufacturers, and all experimental methods used were conventional methods in the art.

[0036] Example 1

[0037] Take an appropriate volume of sterile water for injection and add each component in sequence to a final concentration of 100 mM TES buffer pH 6.8, 250 mM magnesium chloride solution, 5% (w / v) trehalose, and 0.02% (w / v) Tween-20. After fully dissolving and mixing, adjust to volume and filter through a 0.1 μm filter membrane to obtain reaction buffer 1.

[0038] Example 2

[0039] Take an appropriate volume of sterile water for injection and add each component in sequence to a final concentration of 150 mM TES buffer pH 7.0, 200 mM magnesium chloride solution, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20. After thorough dissolution and mixing, adjust to volume and filter through a 0.1 μm filter membrane to obtain reaction buffer 2.

[0040] Example 3

[0041] Take an appropriate volume of sterile water for injection and add each component in sequence to a final concentration of 200 mM TES buffer pH 7.5, 150 mM magnesium chloride solution, 3% (w / v) trehalose, and 0.06% (w / v) Tween-20. After thorough dissolution and mixing, adjust to volume and filter through a 0.1 μm filter membrane to obtain reaction buffer 3.

[0042] Example 4

[0043] Take an appropriate volume of sterile water for injection and add each component in sequence. The final concentration of each component is 250mM TES buffer pH 7.8, 100mM magnesium chloride solution, 2% (w / v) trehalose, and 0.08% (w / v) Tween-20. After fully dissolving and mixing, adjust the volume and filter with a 0.1μm filter membrane to obtain reaction buffer 4.

[0044] Example 5

[0045] Take an appropriate volume of sterile water for injection and add each component in sequence to a final concentration of 300 mM TES buffer pH 8.0, 50 mM magnesium chloride solution, 1% (w / v) trehalose, and 0.1% (w / v) Tween-20. After fully dissolving and mixing, adjust to volume and filter with a 0.1 μm filter membrane to obtain reaction buffer 5.

[0046] Comparative Example 1

[0047] Take an appropriate volume of sterile water for injection and add each component in sequence to a final concentration of 50 mM, pH 8.0 Tris-HCl, 0.1 M sodium chloride, and 50 mM calcium chloride. After thorough dissolution and mixing, adjust to volume and filter with a 0.1 μm filter membrane to obtain comparative reaction buffer 1 (prepared with reference to the recipe of the reaction buffer disclosed in US6645724B1).

[0048] Comparative Example 2

[0049] Take an appropriate volume of sterile water for injection and add each component in sequence. The final concentration of each component is 100mM TES buffer pH 7.5, 50mM magnesium chloride solution, and 1% (w / v) trehalose. After fully dissolving and mixing, adjust the volume and filter with a 0.1μm filter membrane to obtain comparative reaction buffer 2.

[0050] Comparative Example 3

[0051] Take an appropriate volume of sterile water for injection and add each component in sequence. The final concentration of each component is 100mM TES buffer pH 7.5, 50mM magnesium chloride solution, and 0.02% (w / v) Tween-20. After fully dissolving and mixing, adjust the volume and filter with a 0.1μm filter membrane to obtain comparative reaction buffer 3.

[0052] Comparative Example 4

[0053] Take an appropriate volume of sterile water for injection and add each component in sequence. The final concentration of each component is 100 mM TES buffer pH 7.5, 1% (w / v) trehalose, and 0.02% (w / v) Tween-20. After thorough dissolution and mixing, adjust the volume and filter with a 0.1 μm filter membrane to obtain comparative reaction buffer 4.

[0054] Example 6: Preparation of endotoxin standard solution, preparation of reagents for endotoxin detection and drawing of standard curve

[0055] Take a disposable pyrogen-free glass tube and dissolve the endotoxin standard to 20 EU / ml. Then, perform a gradient dilution (5, 0.5, 0.05, 0.005 EU / ml) to obtain endotoxin standard solutions of various concentrations. Take 100 μl of each concentration of endotoxin standard and endotoxin-free water and add it to an endotoxin-free ELISA plate. Each reaction is performed in 2-3 replicates and preheated in a 37°C incubator. Then, according to a volume ratio of 5:4:1, the fluorescent substrate (Boc-Val-Pro-Arg-AMC), the reaction buffer prepared in Examples 1-5 of the present invention, and the recombinant Factor C protein solution (the concentration of the recombinant Factor C protein solution is 20 μg / ml) are mixed to prepare the endotoxin detection reagent. After adding 100 μl of the endotoxin detection reagent to the endotoxin standard solution of different concentrations or endotoxin-free water, immediately read the zero fluorescence value. Then, place the ELISA plate in a 37°C incubator, incubate for one hour, and read the value on the microplate reader. After subtracting the zero-hour reading from the one-hour reading, the final ΔRFU was obtained by subtracting the difference between the readings of 5, 0.5, 0.05, and 0.005 EU / ml from the difference between the readings of 0 EU / ml. The logarithm of the result was taken to draw a standard curve and the correlation coefficient R was calculated. 2 , compare the sensitivity and linearity differences in endotoxin detection using different reaction buffers.

[0056] Example 7: Dilute the sample to be tested and perform a spike experiment to detect the recovery rate

[0057] Based on the standard test in Example 6, the sample to be tested was diluted in a gradient, and 100 μl was added to an endotoxin-free ELISA plate. At the same time, a spiked sample well was set, that is, another 100 μl of the sample to be tested was added to 10 μl of 5 EU / ml endotoxin and then added to the ELISA plate, which was placed in a 37°C incubator for preheating. Then, 100 μl of the reaction solution was added to the sample to be tested and the spiked sample, and the zero-point fluorescence value was immediately read. The ELISA plate was then placed in a 37°C incubator and incubated for one hour, and the microplate reader read the value again. After subtracting the zero-hour reading from the one-hour reading, the difference was subtracted from the 0 EU / ml difference in the standard curve to obtain ΔRFU. The endotoxin content of the sample to be tested and the spiked sample was then calculated based on the standard curve. The recovery rate was (spiked sample - sample to be tested) / spiked endotoxin content × 100%. When the recovery rate was between 50-200%, it was considered that the sample had no interference with the endotoxin reaction and the result was reliable. The endotoxin content of the sample solution to be tested is the endotoxin content measured in the sample dilution multiplied by the dilution factor. Compare the recovery rates and the corresponding sample dilution factors (i.e., the anti-interference ability) when detecting endotoxin content in different reaction buffers.

[0058] Experimental Example 1: Testing the Sensitivity of Different Reaction Buffers to Recombinant Factor C Endotoxin

[0059] Endotoxin reactions were performed using the reaction buffers prepared in Examples 1-5 and Comparative Examples 1-4. The method described in Example 6 was followed, ensuring that all components except the reaction buffer were consistent. Table 1 compares the effects of different reaction buffers on endotoxin reaction sensitivity.

[0060] Table 1 Effects of different reaction buffers on the sensitivity of recombinant Factor C endotoxin detection

[0061] ΔRFU 0.005EU / ml 0.05EU / ml 0.5EU / ml 5EU / ml <![CDATA[R 2 ]]> Comparative Example 1 -1.5 29 369 3031 0.9887 Comparative Example 2 1 51.5 496.5 4450 0.9769 Comparative Example 3 -0.5 57 665 5617.5 0.9759 Comparative Example 4 -27.5 127.5 303 1103 0.8528 Example 1 8.5 106.5 1158 8598 0.9973 Example 2 15 130.5 1208.5 8837 0.9995 Example 3 10 110.5 1080.5 8463 0.9988 Example 4 15.5 140.5 1376.5 8138.5 0.9972 Example 5 20 196 1871 8005 0.9902

[0062] From the above results, it can be seen that magnesium chloride, trehalose and Tween-20 all have a significant effect on improving the sensitivity of the reaction and the linearity of the standard curve. The minimum detection limit of the reaction buffers of Examples 1-5 is lower than that of Comparative Example 1, reaching 0.005 EU / ml, and the reaction buffer of Example 2 has the best reading and linearity.

[0063] Experimental Example 2: Detecting the Anti-Interference Ability of Different Reaction Buffers on the Reaction of Recombinant Factor C with Endotoxin

[0064] Endotoxin reactions were performed using the reaction buffer prepared in Examples 1-5 and Comparative Example 1. The method described in Example 7 was followed, ensuring that all components except the reaction buffer were consistent. Table 2 compares the effects of different reaction buffers on the anti-interference ability of PBS solutions in the endotoxin reaction.

[0065] Table 2 Effects of different reaction buffers on the anti-interference of endotoxin detection by recombinant factor C

[0066] PBS solution Dilution multiple Recovery rate Endotoxin content EU / ml Limulus amebocyte lysate turbidimetric method 1 134％ 0.010 Comparative Example 1 1 34％ <0.050 Comparative Example 1 2 48％ <0.100 Comparative Example 1 5 59％ <0.250 Example 1 1 64％ 0.012 Example 2 1 110％ 0.013 Example 3 1 89％ 0.016 Example 4 1 114％ 0.010 Example 5 1 144％ 0.011

[0067] As shown in Table 2, reaction buffers 1-5 of the present invention were capable of performing recombinant Factor C endotoxin reactions in PBS solutions without sample dilution, with recoveries ranging from 50% to 200%. Furthermore, the endotoxin content of the detected PBS solutions was accurate (the accuracy of endotoxin detection in the present invention is based on the assumption that a qualified recovery rate is considered reliable if the difference between the recovery rate and the result of the Limulus amebocyte lysate assay is less than 2 times). This demonstrates their excellent anti-interference ability. Comparative Example 1, on the other hand, required at least 5-fold dilution to achieve a qualified recovery rate, indicating poor anti-interference ability.

[0068] Experimental Example 3: Testing the anti-interference ability of the optimal reaction buffer for different samples

[0069] According to the method described in Example 7, the endotoxin reaction was carried out using the reaction buffer prepared in Example 2 and Comparative Example 1, and the sample was diluted step by step. When the recovery rate met the requirements for the first time within the effective dilution factor, that is, between 50% and 200%, the corresponding dilution factor and the endotoxin content of the sample were recorded, as shown in Table 3.

[0070] Table 3 Results of endotoxin content detection of samples with recombinant Factor C endotoxin reaction buffer

[0071]

[0072]

[0073] As shown in Table 3, the anti-interference ability of Comparative Example 1 is poor, and the recovery rates of serum, serum substitutes, and albumin injection are unqualified within the effective dilution multiple, and the endotoxin content cannot be detected. However, Example 2 has a lower dilution multiple and can make the recovery rate between 50-200%, has excellent anti-interference ability, and can accurately measure the endotoxin content of various samples.

[0074] The reaction buffers prepared in other examples also have the same excellent properties as the reaction buffer in Example 2, and are not listed here one by one.

[0075] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A reaction buffer for detecting endotoxins using a recombinant Factor C method, characterized in that: The reaction buffer comprises the following components: basic buffer, magnesium chloride, trehalose and Tween-20; The basic buffer is selected from one of MOPS, HEPES, TES, and Tris-HCl.

2. The reaction buffer according to claim 1, wherein Based on the final concentration of each component, the reaction buffer comprises the following components: 100-300 mM, pH 6.8-8.0 basic buffer, 50-250 mM magnesium chloride, 1-5% (w / v) trehalose, and 0.02-0.1% (w / v) Tween-20.

3. The reaction buffer according to claim 1, wherein The reaction buffer contained the following components based on the final concentration of each component: 150 mM basic buffer solution at pH 7.0, 200 mM magnesium chloride, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20.

4. The method for preparing the reaction buffer for detecting endotoxin by the recombinant Factor C method according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: dissolving all components in water according to the formula concentration for preparation, fully mixing and then filtering to obtain the product.

5. The preparation method according to claim 4, characterized in that Filter using a 0.1 μm filter membrane; Preferably, the endotoxin level of the reaction buffer is controlled below 0.005 EU / ml.

6. Use of the reaction buffer for detecting endotoxins using the recombinant Factor C method according to any one of claims 1 to 3 in endotoxin detection.

7. The use according to claim 6, characterized in that The application comprises the following steps: Prepare a series of endotoxin standard solutions; preparing a reagent for detecting endotoxin, which comprises a fluorescent substrate, the reaction buffer, and a recombinant Factor C protein solution; The endotoxin detection reagent was mixed with each concentration of endotoxin standard solution, incubated at 37°C, and read on a microplate reader at 0h and 1h. The logarithm of the fluorescence intensity change value after calibration of the negative control well was taken as the vertical axis, and the logarithm of the endotoxin standard solution concentration was taken as the horizontal axis to establish a standard curve; The sample to be tested is diluted to prepare a sample to be tested. At the same time, a spiked sample to be tested with a certain concentration of endotoxin standard solution is prepared. The endotoxin detection reagent is then mixed with the sample to be tested and the spiked sample to be tested, respectively. The samples are incubated at 37°C, and the microplate reader is read at 0h and 1h. The endotoxin content of the sample to be tested is calculated using the established standard curve, and the endotoxin content of the initial undiluted sample to be tested is calculated according to the dilution multiple.

8. The use according to claim 7, characterized in that In the reagent for detecting endotoxin, the volume ratio of the fluorescent substrate, the reaction buffer, and the recombinant Factor C protein solution is 5:4:1; Preferably, the concentration of the recombinant Factor C protein solution is 20 μg / ml; Preferably, the volume ratio of the endotoxin detection reagent to the endotoxin standard solution is 1:1; Preferably, the volume ratio of the reagent for detecting endotoxin to the sample to be tested or the spiked sample to be tested is 1:

1.

9. An endotoxin detection kit, characterized in that: A reaction buffer for detecting endotoxins using the recombinant Factor C method according to any one of claims 1 to 3.

10. Use of the endotoxin detection kit according to claim 9 in endotoxin detection.

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