CN120594816A

CN120594816A - A reaction buffer for detecting endotoxins using a recombinant factor C method and its application

Info

Publication number: CN120594816A
Application number: CN202510832939.2A
Authority: CN
Inventors: 宇海慧; 秦凯涛; 付丽娜; 马静; 辛文
Original assignee: Beijing Quanshijin Biotechnology Co ltd
Current assignee: Beijing Quanshijin Biotechnology Co ltd
Priority date: 2025-06-20
Filing date: 2025-06-20
Publication date: 2025-09-05
Anticipated expiration: 2045-06-20
Also published as: CN120594816B

Abstract

The invention discloses a reaction buffer solution for detecting endotoxin by a recombinant factor C method and application thereof. The reaction buffer solution comprises the following components of 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. The reaction buffer solution for detecting endotoxin by the recombinant factor C method obviously improves the anti-interference capability on the basis of ensuring the response sensitivity of the endotoxin, and ensures the accuracy of endotoxin detection in an effective dilution range, thereby avoiding unreliable detection results caused by that the concentration of the endotoxin is lower than the detection limit due to excessive dilution.

Description

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

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a reaction buffer solution for detecting endotoxin by a recombinant factor C method and application thereof.

Background

Bacterial endotoxin is taken as lipopolysaccharide component of the cell wall of gram-negative bacteria, released into a circulatory system after the bacterial cells are cracked to trigger pyrogenic reaction, and when the concentration in blood exceeds the organism clearance threshold value, inflammatory factor cascade reaction is triggered, so that clinical risks such as fever, endotoxemia and even shock are caused. Because endotoxin has high thermal stability and strong pathogenicity, endotoxin detection is classified as a key quality control index of medicines, medical instruments and biological products in pharmacopoeias of various countries.

Traditional endotoxin detection techniques have undergone two major stages of development. The early rabbit pyrogen method evaluates whether the pyrogen limit meets the regulation by observing the temperature change of animals, but has the defects of low sensitivity (0.5 EU/mL), complicated operation, incapacity of meeting the ethical of animals and the like. The limulus reagent method (Limulus Amebocyte Lysate, LAL) found in 1964 is rapidly becoming an industry standard for its high sensitivity (0.005-0.03 EU/mL) and standardized protocols. The core mechanism is dependent on the unique coagulation cascade in the horseshoe crab blood, which is that endotoxin activates factor C, then activates factor B and clotting zymogens in turn, and finally cleaves clotting proteins to form gel. However, this technique has two inherent disadvantages, namely that the factor G in natural limulus blood cross-reacts with beta-1, 3-glucan to cause false positive results, especially significant interference to samples containing plant polysaccharide (such as cellulose purified monoclonal antibodies), and that the limulus numbers are drastically reduced due to environmental deterioration and overdrawing, and that the limulus and horseshoe crab species are listed as endangered species in the national important protection of wild animal directory (2021). As a secondary protective animal in China, the supply of limulus raw materials is strictly limited, the cost of reagents is greatly increased, and the batch-to-batch difference is difficult to control.

To break through the bottleneck, recombinant factor C (Recombinant Factor C, rFC) detection techniques have been developed. The technology obtains the C factor protein specifically combined with endotoxin through genetic engineering expression and purification, and the action mechanism thereof abandons the traditional cascade reaction, namely, after the endotoxin activates serine proteinase activity of recombinant C factor, a fluorescent substrate (such as Boc-Val-Pro-Arg-AMC) is directly cut to release a detectable signal. Compared with a limulus reagent method, the recombinant factor C technology has the advantages of (1) improving specificity, completely avoiding factor G interference, (2) standardizing production process, remarkably reducing inter-batch difference, and (3) getting rid of dependence on limulus blood resources, and conforming to animal protection principles. The method is fully accepted by the four general pharmacopoeias, japanese pharmacopoeia JP18 (2021) records recombinant protein detection guidelines, european pharmacopoeia EP11.5 (2024) sets up independent chapters, U.S. pharmacopoeia USP <86> (2025 takes effect) clear technical specifications, and annex-the recombinant factor C method is adopted in the guiding principle of China pharmacopoeia 2025. The current recombinant factor C technology covers the whole pharmaceutical industry chain (raw material screening, process monitoring and finished product release), and is particularly suitable for detecting complex samples containing glucan.

The recombinant factor C is used for endotoxin detection, and is independent of horseshoe crab blood and the factor G is removed, so that the recombinant factor C has high specificity and good stability and accords with animal protection principles. These advantages will drive the gradual replacement of the conventional limulus reagent assay with recombinant factor C. When the endotoxin detection method is changed, in order to ensure the accuracy, reliability and compliance of the new method, the following key indexes such as specificity, sensitivity, linearity and range, accuracy, precision, equivalence, interference and stability are required to be systematically evaluated and verified. The interference experiment needs to verify the anti-interference capability of the factor C method in complex matrixes (such as medicines and biological products), and needs to verify the recovery rate of the sample under the maximum effective dilution, so that 50% -200% of the recovery rate needs to be met.

In the biological sample detection process, various components such as salt ions, surfactants, proteins, organic solvents, chelating agents and the like existing in the sample and the fluctuation of the pH value of the sample can have a significant interference effect on the detection result. These interfering factors may cause false positives or false negatives in the detection result by changing the ionic strength of the reaction system, affecting the enzyme activity, binding to endotoxin or competitive inhibition, and the like. For example, a high concentration of salt ions may interfere with the specific binding of the limulus reagent to endotoxin, a surfactant may destroy the aggregation state of endotoxin, a protein may nonspecifically adsorb endotoxin, an organic solvent may change the solubility of the reaction system, a chelating agent may affect the enzymatic reaction by chelating divalent cations, and an abnormal fluctuation in the pH of the sample may directly affect the enzymatic reaction efficiency of the limulus reagent.

Therefore, in order to ensure the accuracy and reliability of the detection result, a strong reaction buffer system must be established. The system needs to have the key characteristics that firstly, the system has excellent anti-interference capability, can effectively neutralize or shield the influence of various interference substances, secondly, the detection sensitivity needs to be kept under reasonable sample dilution, the endotoxin concentration is prevented from being lower than the detection limit due to excessive dilution, and finally, the system has broad-spectrum matrix applicability, and can accurately detect the real endotoxin content in samples from different sources (such as cell culture solution, injection, biological preparation and the like). By optimizing parameters such as ion composition, pH stability, chelating agent concentration and the like of the buffer solution, a robust detection system can be constructed, so that accurate quantification of endotoxin in complex biological samples is realized.

Disclosure of Invention

Aiming at the technical requirements, the first aim of the invention is to develop a reaction buffer solution for detecting endotoxin by a recombinant factor C method, which can obviously improve the anti-interference capability and ensure the accuracy of endotoxin detection in an effective dilution range on the basis of ensuring the response sensitivity of the endotoxin, thereby avoiding unreliable detection results caused by that the concentration of the endotoxin is lower than the detection limit due to excessive dilution.

The second object of the present invention is to provide a method for producing the reaction buffer.

A third object of the present invention is to provide the use of the above reaction buffer in endotoxin detection, which can be applied to endotoxin detection of various types of samples.

A fourth object of the present invention is to provide an endotoxin detection kit comprising the reaction buffer described above and use thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

In the first aspect, the reaction buffer for detecting endotoxin by a recombinant factor C method comprises a base buffer, magnesium chloride, trehalose and tween-20, wherein the base buffer is selected from one of MOPS, HEPES, TES, tris-HCl.

Further, the reaction buffer comprises 100-300mM, pH6.8-8.0 base buffer, 50-250mM magnesium chloride, 1-5% (w/v) trehalose, and 0.02-0.1% (w/v) Tween-20, based on the final concentration of each component.

Further, the reaction buffer comprises 150mM, pH 7.0 base buffer, 200mM magnesium chloride, 4% (w/v) trehalose, 0.04% (w/v) Tween-20, based on the final concentration of each component.

In a second aspect, the invention provides a preparation method of the reaction buffer solution for detecting endotoxin by the recombinant factor C method, which comprises the steps of dissolving all components in water according to the concentration of a formula, preparing, fully mixing, and filtering. The preparation process needs to ensure aseptic operation, and the whole process is carried out in an ultra clean bench. According to the specific embodiment of the invention, the preparation process of the reaction buffer solution with the volume of 100mL comprises the following steps of taking a proper volume of sterilized injection water, sequentially adding each component, namely TES buffer solution with the final concentration of 100-300mM and pH of 6.8-8.0, 50-250mM magnesium chloride, 1-5% (w/v) trehalose and 0.02-0.1% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume to 100 mL.

Further, the reaction buffer was filtered using a 0.1 μm filter, and the endotoxin level was controlled to be 0.005EU/ml or less.

In a third aspect, the invention provides an application of the reaction buffer solution for detecting endotoxin by the recombinant factor C method in endotoxin detection.

Further, the application comprises the following steps:

Preparing endotoxin standard solution with serial concentration;

Preparing a reagent for detecting endotoxin, which comprises a fluorogenic substrate, the reaction buffer solution and a recombinant factor C protein solution;

Mixing the reagent for detecting endotoxin with endotoxin standard substance solutions with each concentration respectively, incubating at 37 ℃, reading by an enzyme-labeling instrument at 0h and 1h, taking the logarithm of the change value of the fluorescence intensity after the calibration of a negative control hole as an ordinate, taking the logarithm of the endotoxin standard substance solution concentration as an abscissa, and establishing a standard curve;

Diluting a sample to be detected, preparing a labeled sample to be detected, adding an endotoxin standard substance solution with a certain concentration, mixing the endotoxin detection reagent with the sample to be detected and the labeled sample to be detected respectively, incubating at 37 ℃, reading by an enzyme-labeled instrument at 0h and 1h, calculating the endotoxin content of the sample to be detected by using the established standard curve, and calculating according to the dilution multiple to obtain the endotoxin content of the sample to be detected which is not diluted initially.

In the reagent for detecting endotoxin, the volume ratio of the fluorogenic substrate (such as Boc-Val-Pro-Arg-AMC), the reaction buffer solution and the recombinant factor C protein solution is 5:4:1.

Further, the concentration of the recombinant factor C protein solution is 20 mug/ml.

Further, the volume ratio of the reagent for detecting endotoxin to the endotoxin standard solution is 1:1.

Further, the volume ratio of the reagent for detecting endotoxin to the sample to be detected or the sample to be detected by adding the label is 1:1.

In a fourth aspect, the present invention provides an endotoxin detection kit comprising the reaction buffer described above.

Furthermore, the invention also claims the application of the endotoxin detection kit in endotoxin detection.

The beneficial effects of the invention are as follows:

1. when the reaction buffer of the present invention is used for detecting endotoxin by the recombinant factor C method, endotoxin can be stably detected at a minimum of 0.005EU/ml.

2. When the reaction buffer solution is used for detecting endotoxin by a recombinant factor C method, the interference capability of other substances (including proteins, metal ions and the like) in a sample on the reaction can be effectively reduced, the actual content of the endotoxin in the sample can be accurately detected within an effective dilution range without excessive dilution, and the sample labeling recovery rate is 50-200%.

3. The reaction buffer solution can be used for accurately detecting the endotoxin content in biological samples such as glucose injection, dextran injection, albumin injection, compound amino acid injection, eimerizumab, cell culture medium (DMEM, RPMI 1640), cell cryopreservation solution, serum substitutes and the like, and the required dilution factor is far smaller than that of the prior art, including but not limited to the method disclosed in U.S. Pat. No. 6645724B 1.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

In the examples described below, reagents and consumables were purchased from conventional biochemical reagent manufacturers, and experimental methods were conventional in the art, unless otherwise specified.

Example 1

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 100mM TES buffer solution pH 6.8,250mM magnesium chloride solution, 5% (w/v) trehalose, 0.02% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a reaction buffer solution 1.

Example 2

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 150mM TES buffer solution pH 7.0,200mM magnesium chloride solution, 4% (w/v) trehalose, 0.04% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a reaction buffer solution 2.

Example 3

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 200mM TES buffer solution pH 7.5,150mM magnesium chloride solution, 3% (w/v) trehalose, 0.06% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a reaction buffer solution 3.

Example 4

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 250mM TES buffer solution pH 7.8,100mM magnesium chloride solution, 2% (w/v) trehalose, 0.08% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a reaction buffer solution 4.

Example 5

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 300mM TES buffer solution pH 8.0,50mM magnesium chloride solution, 1% (w/v) trehalose, 0.1% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a reaction buffer solution 5.

Comparative example 1

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of each component is 50mM, the pH value is 8.0Tris-HCl,0.1M sodium chloride and 50mM calcium chloride, fully dissolving and uniformly mixing, fixing the volume, and filtering by using a 0.1 mu M filter membrane to obtain a comparative reaction buffer solution 1 (prepared by referring to the formula of the reaction buffer solution disclosed in U.S. Pat. No. 3, 6645724B 1).

Comparative example 2

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 100mM TES buffer solution pH 7.5,50mM magnesium chloride solution, 1% (w/v) trehalose, fully dissolving and uniformly mixing, fixing the volume, and filtering with a 0.1 mu m filter membrane to obtain a comparative reaction buffer solution 2.

Comparative example 3

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 100mM TES buffer solution pH 7.5,50mM magnesium chloride solution, 0.02% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain the comparative reaction buffer solution 3.

Comparative example 4

Taking a proper volume of sterilized water for injection, sequentially adding the components, wherein the final concentration of the components is 100mM TES buffer pH 7.5,1% (w/v) trehalose and 0.02% (w/v) Tween-20, fully dissolving and uniformly mixing, and then fixing the volume, and filtering by using a 0.1 mu m filter membrane to obtain a comparison reaction buffer 4.

Example 6 preparation of endotoxin Standard solution, preparation of reagent for detecting endotoxin and drawing of Standard Curve

After dissolving endotoxin standard substances to 20EU/ml, gradient dilution (5, 0.5, 0.05, 0.005 EU/ml) was continued to obtain endotoxin standard substance solutions of various concentrations. 100 μl of endotoxin standard substance and endotoxin-free water with each concentration are respectively added into the endotoxin-free ELISA plate, each reaction is 2-3 multiple holes, and the reaction plates are placed in a 37 ℃ incubator for preheating. Then, fluorogenic substrate (Boc-Val-Pro-Arg-AMC), reaction buffer prepared in examples 1 to 5 of the present invention, and recombinant factor C protein solution (concentration of recombinant factor C protein solution: 20. Mu.g/ml) were mixed at a volume ratio of 5:4:1 to prepare a reagent for detecting endotoxin. After 100. Mu.l of endotoxin detection reagent is added into endotoxin standard solution or endotoxin-free water with different concentrations, the zero fluorescence value is immediately read, and then the ELISA plate is placed in a 37 ℃ incubator for incubation for one hour, and the value is read again by an ELISA reader. After subtracting the zero hour reading from the one hour reading, the 0EU/ml difference is subtracted from the 5, 0.5, 0.05, 0.005EU/ml reading to obtain the final ΔRFU. And drawing a standard curve after taking logarithm of the result, counting a correlation coefficient R ², and comparing the sensitivity and the linear difference of different reaction buffers in endotoxin detection reaction.

Example 7 dilution of sample to be tested and labeling experiments, detection of recovery

On the basis of the standard detection in example 6, the sample to be detected is subjected to gradient dilution, 100 mu l of the sample to be detected is added into an endotoxin-free ELISA plate, and a standard sample adding hole is arranged at the same time, namely, 10 mu l of 5EU/ml endotoxin is added into another 100 mu l of the sample to be detected, the ELISA plate is added, and the sample is placed in a 37 ℃ incubator for preheating. Then 100 μl of reaction solution is added into the sample to be detected and the labeled sample, the zero fluorescence value is immediately read, then the ELISA plate is placed in a 37 ℃ incubator for incubation for one hour, and the value is read again by the ELISA plate. After subtracting the reading value from the reading value for one hour by the reading value for zero hour, subtracting the 0EU/ml difference value in the standard curve from the obtained difference value to obtain the delta RFU. And then calculating the endotoxin contents of the sample to be detected and the labeled sample according to the standard curve, wherein the recovery rate is (labeled sample-sample to be detected)/the endotoxin content of the labeled sample is multiplied by 100 percent, and when the recovery rate is between 50 and 200 percent, the sample can be considered to have no interference on endotoxin reaction, and the result is reliable. The endotoxin content of the stock solution of the sample to be detected is the endotoxin content multiplied by dilution of the diluent of the sample to be detected. And comparing the recovery rate of the different reaction buffers when the endotoxin content of the sample to be detected is detected with the dilution multiple of the corresponding sample, namely the anti-interference capability.

Test example 1 sensitivity of different reaction buffers to recombinant factor C endotoxin

Endotoxin reactions were carried out using the reaction buffers prepared in examples 1 to 5 and comparative examples 1 to 4. Following the procedure described in example 6, and ensuring consistency of the remaining components except for the reaction buffer. The effect of different response buffers on endotoxin response sensitivity was compared and is shown in table 1.

TABLE 1 Effect of different reaction buffers on the detection sensitivity of recombinant factor C endotoxins

ΔRFU	0.005EU/ml	0.05EU/ml	0.5EU/ml	5EU/ml	R²
						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

From the above results, it was found that magnesium chloride, trehalose and Tween-20 all had a remarkable improvement in the sensitivity of the reaction and the linearity of the standard curve, the reaction buffers of examples 1 to 5 had a lower minimum detection limit than that of comparative example 1, and the reaction buffer of example 2 had the best reading and linearity at 0.005 EU/ml.

Test example 2 detection of the anti-interference ability of different reaction buffers against the recombinant factor C endotoxin reaction

Endotoxin reactions were carried out using the reaction buffers prepared in comparative example 1, examples 1 to 5. Following the procedure described in example 7, and ensuring consistency of the remaining components except for the reaction buffer. The effect of different reaction buffers on the anti-interference ability of endotoxin reactions in PBS solutions was compared and is shown in table 2.

TABLE 2 influence of different reaction buffers on the anti-interference detection of recombinant factor C endotoxin

PBS solution	Dilution factor	Recovery rate	Endotoxin content EU/ml
				Limulus reagent 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

As shown in Table 2, the reaction buffers 1 to 5 of the present invention can perform the recombinant factor C endotoxin reaction without diluting the sample when detecting PBS solution, the recovery rate is between 50 and 200%, and the endotoxin content of the detected PBS solution is accurate (the detection endotoxin accuracy of the present invention is based on that the recovery rate is qualified, and the detection result difference of the limulus reagent is less than 2 times is considered as credible), which indicates that the anti-interference ability is excellent, whereas the comparative example 1 requires at least 5 times dilution and the recovery rate is qualified, and the anti-interference ability is poor.

Test example 3 detection of the anti-interference Capacity of optimal reaction buffer to different samples

The endotoxin reaction was performed using the reaction buffer prepared in comparative example 1 according to the method described in example 7, and the samples were subjected to stepwise gradient dilution, and the corresponding dilution and endotoxin content of the samples were recorded when the recovery rate was satisfactory for the first time, i.e., between 50 and 200%, within the effective dilution ratio, as shown in table 3.

TABLE 3 results of detection of endotoxin content in samples by recombinant factor C endotoxin reaction buffer

As shown in table 3, comparative example 1 has poor anti-interference ability, and the recovery rate of serum, serum substitute and albumin injection is not qualified in the effective dilution, the endotoxin content cannot be detected, whereas example 2 has low dilution, which can make the recovery rate between 50-200%, has excellent anti-interference ability, and can accurately measure the endotoxin content of various samples.

The reaction buffers prepared in the other examples also have the same excellent properties as those of the reaction buffer of example 2, which are not exemplified herein.

It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that other and different forms of changes and modifications may be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments, and all obvious changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

1. A reaction buffer for detecting endotoxins using a recombinant Factor C method, wherein the reaction buffer comprises 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.

2. The reaction buffer according to claim 1, characterized in that, based on the final concentration of each component, the reaction buffer comprises 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.

3. The reaction buffer according to claim 1, characterized in that, 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.

4. The method for preparing a reaction buffer for detecting endotoxins using the recombinant Factor C method according to any one of claims 1 to 3, characterized in that the preparation method comprises: dissolving all components in water according to the formulated concentrations for preparation, thoroughly mixing, and then filtering to obtain the reaction buffer.

5. The preparation method according to claim 4, characterized in that filtration is performed 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 application 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, wherein the volume ratio of the fluorescent substrate, the reaction buffer, and the recombinant Factor C protein solution in the endotoxin detection reagent 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 by comprising the 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.