Recombinant factor c method for detecting endotoxin reaction buffer and application thereof

Abstract

The application discloses a reaction buffer for detecting endotoxin by a recombinant C factor method and application thereof. The reaction buffer comprises the following components: a basic buffer, magnesium chloride, trehalose and Tween-20. The application further discloses a preparation method of the reaction buffer and application thereof in endotoxin detection. The reaction buffer for detecting endotoxin by the recombinant C factor method can significantly improve anti-interference capability on the basis of ensuring endotoxin reaction sensitivity, guarantee the accuracy of endotoxin detection in an effective dilution range, and thus avoid the situation that endotoxin concentration is lower than a detection limit due to excessive dilution, resulting in unreliable detection results.

Description

TECHNICAL FIELD

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

[0002] Bacterial endotoxin, as a component of lipopolysaccharide in the cell wall of gram-negative bacteria, can cause pyrogen reaction when released into the circulatory system after cell lysis. When the concentration in the blood exceeds the body's clearance threshold, it will trigger a cascade reaction of inflammatory factors, leading to fever, endotoxemia and even shock and other clinical risks. Due to the high thermal stability and strong pathogenicity of endotoxin, the detection of endotoxin is listed as a key quality control indicator for drugs, medical devices and biological products in pharmacopoeias of various countries.

[0003] Traditional endotoxin detection technology has gone through two major development stages. The early rabbit pyrogen method assesses whether the pyrogen limit meets the requirements by observing the body temperature changes of animals, but it has low sensitivity (0.5 EU / mL), complicated operation and does not comply with animal ethics. The Limulus Amebocyte Lysate (LAL) method discovered in 1964 has become the industry gold standard due to its high sensitivity (0.005-0.03 EU / mL) and standardized operation process. Its core mechanism relies on the unique coagulation cascade reaction in the blood of the horseshoe crab: after the activation of C factor by endotoxin, B factor and coagulogen are activated in turn, and finally coagulase is cut to form a gel. However, this technology also has two major inherent defects: first, the G factor in the blood of the horseshoe crab can have cross-reaction with β-1,3-glucan, leading to false positive results, especially for samples containing plant polysaccharides (such as cellulose-purified monoclonal antibodies); second, due to environmental degradation and overfishing, the number of horseshoe crabs has decreased sharply, and the Chinese horseshoe crab and the Tachypleus tridentatus have been listed as endangered species in the National Key Protected Wildlife List (2021). As a second-class protected animal in China, the supply of horseshoe crab raw materials is strictly limited, and the cost of reagents has increased sharply and the batch-to-batch difference is difficult to control.

[0004] To overcome the aforementioned bottlenecks, recombinant factor C (rFC) detection technology has emerged. 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 cleaves fluorescent substrates (such as Boc-Val-Pro-Arg-AMC) to release a detectable signal. Compared with the horseshoe crab reagent method, recombinant factor C technology has three advantages: (1) improved specificity, completely avoiding interference from factor G; (2) standardized production process, significantly reducing batch-to-batch differences; (3) freeing it from dependence on horseshoe crab blood resources, in line with animal protection principles. The method has been fully recognized by the four major pharmacopoeias worldwide: the Japanese Pharmacopoeia JP18 (2021) includes guidelines for recombinant protein detection, the European Pharmacopoeia EP11.5 (2024) has established an independent chapter, and the United States Pharmacopoeia USP <86> (Effective in 2025) Clear technical specifications are provided, and the 2025 edition of the Chinese Pharmacopoeia includes an appendix—"Appendix: Recombinant C Factor Method." Currently, recombinant C factor technology covers the entire pharmaceutical industry chain (raw material screening, process monitoring, and finished product release), and is particularly suitable for the detection of complex samples containing dextran.

[0005] Recombinant factor C for endotoxin detection offers high specificity, good stability, and complies with animal protection principles because it is independent of horseshoe crab blood and eliminates factor G. These advantages will drive recombinant factor C to gradually replace traditional horseshoe crab reagent detection methods. When changing endotoxin detection methods, to ensure the accuracy, reliability, and compliance of the new method, the following key indicators need to be systematically evaluated and validated: specificity, sensitivity, linearity and range, accuracy, precision, equivalence, interference, and stability. Among these, interference experiments need to verify the anti-interference ability of the factor C method in complex matrices (such as pharmaceuticals and biological products) and the recovery rate of samples at the maximum effective dilution, which should meet the requirement of 50%-200%.

[0006] In biological sample testing, various components present in the sample, such as salt ions, surfactants, proteins, organic solvents, and chelating agents, as well as pH fluctuations, can significantly interfere with the test results. These interfering factors may lead to false positives or false negatives by altering the ionic strength of the reaction system, affecting enzyme activity, binding to endotoxins, or competitively inhibiting them. For example, high concentrations of salt ions may interfere with the specific binding of Limulus amebocyte lysate (LAL) reagent to endotoxins; surfactants may disrupt the aggregation state of endotoxins; proteins may non-specifically adsorb endotoxins; organic solvents may alter the solubility of the reaction system; and chelating agents may affect enzyme reactions by chelating divalent cations; abnormal fluctuations in sample pH may directly affect the enzymatic reaction efficiency of LAL reagent.

[0007] Therefore, to ensure the accuracy and reliability of the detection 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 capabilities, effectively neutralizing or shielding the effects of various interfering substances; second, it should maintain detection sensitivity at reasonable sample dilutions to avoid endotoxin concentrations falling below the detection limit due to over-dilution; finally, the system should have broad matrix applicability, enabling accurate detection of the true endotoxin content in samples from various sources (such as cell culture media, injection solutions, and biological agents). By optimizing parameters such as the ionic composition, pH stability, and chelating agent concentration of the buffer solution, a robust detection system can be constructed, thereby achieving precise quantification of endotoxins in complex biological samples. Summary of the Invention

[0008] To address the above technical requirements, the first objective of this invention is to develop a reaction buffer solution for the recombinant C factor method for detecting endotoxins. This solution ensures the sensitivity of the endotoxin reaction while significantly improving the anti-interference capability and guaranteeing the accuracy of endotoxin detection within the effective dilution range. This avoids unreliable detection results caused by excessive dilution leading to endotoxin concentrations below the detection limit.

[0009] A second objective of this invention is to provide a method for preparing the above-mentioned reaction buffer solution.

[0010] A third objective of this invention is to provide the application of the above-mentioned reaction buffer solution in endotoxin detection, which is applicable to the detection of endotoxins in various types of samples.

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

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

[0013] In a first aspect, a reaction buffer for detecting endotoxins using the recombinant factor C method, the reaction buffer comprising the following components: a basal buffer, magnesium chloride, trehalose, and Tween-20; wherein the basal 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: 100-300mM, pH 6.8-8.0 basal buffer, 50-250mM 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 basal buffer, 200 mM magnesium chloride, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20.

[0016] Secondly, this invention provides a method for preparing the reaction buffer for the above-mentioned recombinant factor C method for detecting endotoxins. The preparation method includes: dissolving all components in water according to the prescribed concentration, mixing thoroughly, and then filtering to obtain the final buffer. Aseptic operation must be maintained during the preparation process, which is carried out entirely in a clean bench. According to a specific embodiment of this invention, the preparation process of 100 mL of reaction buffer includes the following steps: taking an appropriate volume of sterile water for injection, sequentially adding each component (each component having a final concentration of 100-300 mM, pH 6.8-8.0), 50-250 mM magnesium chloride, 1-5% (w / v) trehalose, and 0.02-0.1% (w / v) Tween-20, dissolving and mixing thoroughly, and then adjusting the volume to 100 mL.

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

[0018] Thirdly, the present invention provides the application of the reaction buffer solution for the detection of endotoxins using the above-mentioned recombinant C factor method in the detection of endotoxins.

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

[0020] Prepare a series of endotoxin standard solutions of different concentrations;

[0021] A reagent for detecting endotoxins is prepared, comprising a fluorescent substrate, the reaction buffer, and a recombinant factor C protein solution;

[0022] The reagents for detecting endotoxins were mixed with endotoxin standard solutions of each concentration and incubated at 37°C. The microplate reader readings were taken at 0h and 1h. The logarithm of the fluorescence intensity change after calibration of the negative control well was plotted as the ordinate, and the logarithm of the endotoxin standard solution concentration was plotted as the abscissa to establish a standard curve.

[0023] The sample to be tested was diluted to prepare a test sample. At the same time, a spiked test sample was prepared by adding a certain concentration of endotoxin standard solution. The reagent for detecting endotoxin was then mixed with the test sample and the spiked test sample, respectively, and incubated at 37°C. The ELISA reader readings were taken at 0h and 1h. The endotoxin content of the test sample was calculated using the established standard curve. The endotoxin content of the initial undiluted test sample was calculated based on the dilution factor.

[0024] In the reagent for detecting endotoxins, 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 reagent for detecting endotoxins to the endotoxin standard solution is 1:1.

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

[0028] Fourthly, the present invention provides an endotoxin detection kit, comprising the above-mentioned reaction buffer solution.

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

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

[0031] 1. When the reaction buffer of the present invention is used for the detection of endotoxins by the recombinant C factor method, the minimum amount 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 detection of endotoxins by the recombinant C factor method, it can effectively reduce the interference of other substances (including proteins, metal ions, etc.) in the sample on the reaction. There is no need for excessive dilution. Within the effective dilution range, the true content of endotoxins in the sample can be accurately detected, 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, emecizumab, cell culture medium (DMEM, RPMI 1640), cell cryopreservation solution, serum, and serum substitutes, and the required dilution factor is much smaller than that of the prior art, including but not limited to the method disclosed in US Patent US6645724B1. Detailed Implementation

[0034] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0035] Unless otherwise specified, all reagents and consumables used in the following embodiments 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, add each component in sequence, and the final concentration of each component is 100mM TES buffer pH 6.8, 250mM magnesium chloride solution, 5% (w / v) trehalose, and 0.02% (w / v) Tween-20. After fully dissolving and mixing, bring the volume to a final level and filter through a 0.1μm filter membrane to obtain reaction buffer 1.

[0038] Example 2

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

[0040] Example 3

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

[0042] Example 4

[0043] Take an appropriate volume of sterile water for injection, add each component in sequence, and 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, bring the volume to a final level and filter through a 0.1μm filter membrane to obtain reaction buffer 4.

[0044] Example 5

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

[0046] Comparative Example 1

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

[0048] Comparative Example 2

[0049] Take an appropriate volume of sterile water for injection, add each component in sequence, and 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, bring the volume to a final level and filter through a 0.1μm filter membrane to obtain control reaction buffer 2.

[0050] Comparative Example 3

[0051] Take an appropriate volume of sterile water for injection, add each component in sequence, and 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, bring the volume to a final level and filter through a 0.1μm filter membrane to obtain control reaction buffer 3.

[0052] Comparative Example 4

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

[0054] Example 6: Preparation of endotoxin standard solution, preparation of reagents for detecting endotoxin and plotting of standard curve.

[0055] Take a disposable pyrogen-free glass tube, dissolve the endotoxin standard to 20 EU / ml, and then continue to perform serial dilutions (5, 0.5, 0.05, 0.005 EU / ml) to obtain endotoxin standard solutions of various concentrations. Add 100 μl of each concentration of endotoxin standard and endotoxin-free water to an endotoxin-free ELISA plate, with each reaction performed in 2-3 replicates, and preheat in a 37°C incubator. Then, mix the fluorescent substrate (Boc-Val-Pro-Arg-AMC), the reaction buffer prepared in Examples 1-5 of this invention, and the recombinant factor C protein solution (concentration of recombinant factor C protein solution is 20 μg / ml) at a volume ratio of 5:4:1 to prepare the endotoxin detection reagent. After adding 100 μl of the endotoxin detection reagent to different concentrations of endotoxin standard solutions or endotoxin-free water, immediately read the zero-point fluorescence value, and then place the ELISA plate in a 37°C incubator for one hour, and then read the value again using the ELISA reader. Subtract the zero-hour reading from the one-hour reading, then subtract the 0 EU / ml difference from the differences in readings of 5, 0.5, 0.05, and 0.005 EU / ml to obtain the final ΔRFU. Take the logarithm of this result and plot a standard curve, then calculate the correlation coefficient R. 2 The sensitivity and linearity differences of different reaction buffers in the detection of endotoxins were compared.

[0056] Example 7: Dilute the test sample and perform a spiked experiment to detect the recovery rate.

[0057] Based on the standard detection in Example 6, the test sample was serially diluted, and 100 μl was added to an endotoxin-free ELISA plate. A spiked sample well was also prepared; 10 μl of the test sample was added to a well with 10 μl of 5 EU / ml endotoxin, and then added to the ELISA plate. The plate was preheated at 37°C. Then, 100 μl of reaction solution was added to both the test sample and the spiked sample, and the zero-point fluorescence value was immediately read. The ELISA plate was then incubated at 37°C for one hour, and the value was read again by the ELISA reader. The zero-hour reading was subtracted from the one-hour reading, and then the difference was subtracted from the 0 EU / ml difference in the standard curve to obtain ΔRFU. The endotoxin content of the test sample and the spiked sample was then calculated based on the standard curve. The recovery rate was calculated as (spike sample - test sample) / spiked endotoxin content × 100%. When the recovery rate was between 50% and 200%, the sample was considered to have no interference with the endotoxin reaction, and the results were reliable. The endotoxin content of the original sample solution is equal to the endotoxin content measured in the diluted sample solution multiplied by the dilution factor. Comparing the recovery rate and the corresponding dilution factor of different reaction buffers when detecting the endotoxin content of the sample demonstrates the anti-interference capability.

[0058] Experimental Example 1: Determining 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 identical. The effects of different reaction buffers on the sensitivity of the endotoxin reaction were compared, as shown in Table 1.

[0060] Table 1. Effect of different reaction buffers on the detection sensitivity of recombinant factor C endotoxin

[0061] ΔRFU 0.005 EU / ml 0.05 EU / ml 0.5 EU / ml 5 EU / ml [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] The results above show that magnesium chloride, trehalose, and Tween-20 all significantly improve the sensitivity of the reaction and the linearity of the standard curve. The detection limit of the reaction buffer in Examples 1-5 is lower than that in Comparative Example 1, reaching 0.005 EU / ml, and the reaction buffer reading in Example 2 shows the best linearity.

[0063] Experiment 2: Detection of the anti-interference ability of different reaction buffers on the recombinant factor C endotoxin response.

[0064] Endotoxin reactions were performed using the reaction buffers 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 identical. The effects of different reaction buffers on the anti-interference ability of the endotoxin reaction in PBS solution were compared, as shown in Table 2.

[0065] Table 2. Effects of different reaction buffers on interference resistance in the recombinant factor C method for endotoxin detection.

[0066] PBS solution Dilution fold Recovery rate Endotoxin content EU / ml LAL turbidity 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, the reaction buffers 1-5 of the present invention can perform recombinant factor C endotoxin reaction without diluting the sample when detecting PBS solution, with a recovery rate between 50-200%, and the detected endotoxin content in PBS solution is accurate (the accuracy of endotoxin detection in the present invention is based on the recovery rate being qualified, and the difference between the result and the result detected by the Limulus amebocyte lysate (LAL) reagent is less than 2 times, which is considered a reliable result), indicating that it has excellent anti-interference ability. In contrast, Comparative Example 1 requires at least 5 times dilution to achieve a qualified recovery rate, and its anti-interference ability is poor.

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

[0069] Following the method described in Example 7, the reaction buffer prepared in Example 2 and Comparative Example 1 was used to perform the endotoxin reaction. The sample was serially diluted. When the recovery rate first met the requirements within the effective dilution factor, i.e., between 50-200%, 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 in samples using recombinant factor C endotoxin reaction buffer.

[0071]

[0072]

[0073] As shown in Table 3, Comparative Example 1 has poor anti-interference ability, and the recovery rate of serum, serum substitutes and albumin injection is not up to standard within the effective dilution factor, so the endotoxin content cannot be detected. However, Example 2 can achieve a recovery rate of 50-200% with a lower dilution factor, has excellent anti-interference ability, and can accurately measure the endotoxin content of various samples.

[0074] The reaction buffers prepared in other embodiments also have the same excellent properties as the reaction buffer in Example 2, and will not be listed here.

[0075] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. An application of a reaction buffer solution for endotoxin detection using the recombinant C factor method in endotoxin detection, wherein the application is a non-diagnostic application, characterized in that... Based on the final concentration of each component, the reaction buffer consists of the following components: 100-300 mM, pH 6.8-8.0 basal buffer, 50-250 mM magnesium chloride, 1-5% (w / v) trehalose, and 0.02-0.1% (w / v) Tween-20. The base buffer is selected from one of MOPS, HEPES, TES, and Tris-HCl.

2. The application according to claim 1, characterized in that, Based on the final concentration of each component, the reaction buffer consists of the following components: 150 mM, pH 7.0 basal buffer, 200 mM magnesium chloride, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20.

3. The application according to claim 1, characterized in that, The preparation method of the reaction buffer includes: dissolving all components in water according to the formula concentration, mixing thoroughly, and then filtering to obtain the solution.

4. The application according to claim 3, characterized in that, Filtration was performed using a 0.1 μm filter membrane; The endotoxin level in the reaction buffer was controlled to be below 0.005 EU / ml.

5. The application according to claim 1, characterized in that, The application includes the following steps: Prepare a series of endotoxin standard solutions of different concentrations; A reagent for detecting endotoxins is prepared, comprising a fluorescent substrate, the reaction buffer, and a recombinant factor C protein solution; The reagents for detecting endotoxins were mixed with endotoxin standard solutions of each concentration and incubated at 37°C. The microplate reader readings were taken at 0h and 1h. The logarithm of the fluorescence intensity change after calibration of the negative control well was plotted as the ordinate, and the logarithm of the endotoxin standard solution concentration was plotted as the abscissa to establish a standard curve. The sample to be tested was diluted to prepare a test sample. At the same time, a spiked test sample was prepared by adding a certain concentration of endotoxin standard solution. The reagent for detecting endotoxin was then mixed with the test sample and the spiked test sample, respectively, and incubated at 37°C. The ELISA reader readings were taken at 0h and 1h. The endotoxin content of the test sample was calculated using the established standard curve. The endotoxin content of the initial undiluted test sample was calculated based on the dilution factor.

6. The application according to claim 5, characterized in that, In the reagent for detecting endotoxins, the volume ratio of the fluorescent substrate, the reaction buffer, and the recombinant factor C protein solution is 5:4:

1. The concentration of the recombinant factor C protein solution was 20 μg / ml; The volume ratio of the reagent for detecting endotoxins to the endotoxin standard solution is 1:

1. The volume ratio of the reagent for detecting endotoxins to the sample to be tested or the spiked sample to be tested is 1:

1.

7. The application of a reaction buffer solution for detecting endotoxins using the recombinant factor C method in the preparation of an endotoxin detection kit, characterized in that, Based on the final concentration of each component, the reaction buffer consists of the following components: 100-300 mM, pH 6.8-8.0 basal buffer, 50-250 mM magnesium chloride, 1-5% (w / v) trehalose, and 0.02-0.1% (w / v) Tween-20. The base buffer is selected from one of MOPS, HEPES, TES, and Tris-HCl.

8. The application according to claim 7, characterized in that, Based on the final concentration of each component, the reaction buffer consists of the following components: 150 mM, pH 7.0 basal buffer, 200 mM magnesium chloride, 4% (w / v) trehalose, and 0.04% (w / v) Tween-20.

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