Method for endotoxin detection of collagen solution

Abstract

The application discloses a method for detecting endotoxin of collagen solution, and relates to the technical field of collagen detection. The method comprises the following steps: after collagen solution is formed into collagen gel, endotoxin checking water is added to soak the collagen gel, the soaking solution is taken out and mixed with limulus reagent, the mixture is kept at a temperature of 35-38 DEG C, and then whether the mixture forms gel is determined to judge the content of endotoxin of the collagen solution. The method can reduce the probability of false positive of the detection result.

Description

Technical Field

[0001] This application relates to the field of collagen detection technology, and more specifically, to a method for detecting endotoxins in collagen liquid. Background Technology

[0002] Bacterial endotoxins are complexes of lipopolysaccharide (LPS) and protein found in the cell walls of Gram-negative bacteria. These endotoxins are released when bacteria die or undergo autolysis. Large amounts of endotoxin entering the bloodstream can cause a pyrogenic reaction, and endotoxins are closely associated with various infectious diseases; worsening of the condition is often accompanied by an increase in endotoxin levels.

[0003] Collagen liquid is extracted from animal tissues. When implanted into the human body as a medical device or cosmetic product, residual endotoxins can cause inflammation and affect the biosafety of the implant. Therefore, it is necessary to test the produced collagen for endotoxins to determine whether it has good biosafety.

[0004] Currently, endotoxin detection is generally performed using horseshoe crab (LCL) reagents. Detection methods based on LCL reagents include gelation, colorimetric methods, and turbidimetric methods. LCL reagents are reagents extracted from the blood of horseshoe crabs that can agglutinate bacterial endotoxins. Whether a gel forms indicates a positive endotoxin test result. Besides endotoxins, LCL reagents also react with certain β-glucans, which can produce false positive results. The inventors of this application discovered in their research that collagen solutions do not contain β-glucans, but false positive results frequently occur when using LCL reagents to detect collagen. Summary of the Invention

[0005] This application provides a method for detecting endotoxins in collagen liquid, which can reduce the probability of false positive results.

[0006] This application is implemented as follows:

[0007] This application provides a method for detecting endotoxins in collagen liquid, including:

[0008] After the collagen solution forms a collagen gel, it is soaked in endotoxin test water. The extract is then mixed with horseshoe crab reagent and kept at a temperature of 35-38°C. The endotoxin content of the collagen solution is then determined based on whether the mixture forms a gel.

[0009] Optionally, the heat preservation time is 50 to 70 minutes.

[0010] In one possible implementation, the step of forming a collagen gel from a collagen solution includes maintaining the collagen solution at a temperature of 35–38°C to form the collagen gel.

[0011] In one possible implementation, the detection method includes at least one of the following limitations:

[0012] First limitation: The pH value of the collagen solution is 6 to 8;

[0013] Second limitation: The collagen solution is obtained from animal tissue through a series of processes including defatting, protein removal, enzymatic hydrolysis, salting out, dialysis, and concentration.

[0014] The third limitation is that the concentration of collagen in the collagen solution is ≥5 mg / mL; optionally, the concentration of collagen in the collagen solution is 5 to 150 mg / mL.

[0015] In one possible implementation, the sensitivity of the horseshoe crab reagent is 0.03–0.5 EU / mL.

[0016] In one possible implementation, the endotoxin test is performed by soaking the sample in water for 1 to 30 hours.

[0017] In one possible implementation, the collagen solution contains a phosphate buffer solution in which the collagen is dissolved.

[0018] In one possible implementation, the volume ratio of the collagen solution to the water used for endotoxin testing is 1:2 to 20.

[0019] In one possible implementation, the step of mixing the extract with the horseshoe crab reagent includes: mixing the extract with at least two horseshoe crab reagents of different sensitivities respectively;

[0020] The step of determining the endotoxin content of the collagen based on whether the mixture forms a gel includes:

[0021] When the corresponding mixture forms a gel, the endotoxin content of the collagen solution is greater than or equal to the sensitivity value of the horseshoe crab reagent used.

[0022] If the corresponding mixture does not form a gel, the endotoxin content of the collagen solution is less than the sensitivity value of the horseshoe crab reagent used.

[0023] The range of endotoxin content in the collagen solution is determined based on the sensitivity values ​​of at least two different sensitivities of the horseshoe crab reagent added to the extract.

[0024] In one possible implementation, the method further includes providing a positive control sample, a positive test sample control sample, and a negative control sample. After mixing the positive control sample, the positive test sample control sample, and the negative control sample with the horseshoe crab reagent respectively, the validity of the experiment is determined by whether the positive control sample, the positive test sample control sample, and the negative control sample form a gel.

[0025] The endotoxin detection method for collagen liquid in this application has at least the following beneficial effects:

[0026] The inventors of this application discovered in their research that when using Limulus Amebocyte Lysate (LAL) reagent to detect endotoxins in collagen, the collagen solution itself can gel due to temperature variations, making it impossible to determine whether the gel formation is due to the collagen solution itself or to agglomeration of endotoxins within the collagen solution with the LAL reagent. Therefore, this invention eliminates the possibility that the collagen solution itself forming a gel, which could affect the accuracy of endotoxin detection. Attached Figure Description

[0027] It should be understood that the following figures only illustrate certain embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related figures can be obtained from these figures without creative effort.

[0028] Figure 1 This is a graph showing the endotoxin test results of Example 1 of this application;

[0029] Figure 2 This is a graph showing the endotoxin test results of Example 2 of this application;

[0030] Figure 3 This is a graph showing the endotoxin test results of Example 3 of this application;

[0031] Figure 4 This is a graph showing the endotoxin test results of Example 4 of this application;

[0032] Figure 5 This is a graph showing the endotoxin test results of Example 4 of this application;

[0033] Figure 6 This is a graph showing the endotoxin test results of Comparative Example 1 of this application;

[0034] Figure 7 The figure shows the endotoxin test results of Comparative Example 2 of this application. Detailed Implementation

[0035] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0036] The following is a detailed description of the endotoxin detection method for collagen liquid in this application:

[0037] This application provides a method for detecting endotoxins in collagen solution, comprising:

[0038] After the collagen solution forms a collagen gel, it is soaked in water for endotoxin testing. The extract is then mixed with horseshoe crab reagent and kept at 35-38°C. The endotoxin content of the collagen solution is then determined based on whether the mixture forms a gel.

[0039] The principle behind using the gelation method to detect endotoxin content in collagen solution is that when endotoxins in the collagen solution react with the horseshoe crab reagent, a gel-like substance is formed. Whether or not a gel forms determines whether the endotoxin content in the collagen solution exceeds the limit. If a gel forms, the endotoxin test result is positive, indicating that the endotoxin content exceeds the sensitivity value of the horseshoe crab reagent used. If no gel forms, the endotoxin test result is negative, indicating that the endotoxin content does not exceed the sensitivity value of the horseshoe crab reagent used.

[0040] However, the inventors of this application discovered in their research that when detecting endotoxins in collagen liquid, false positive results or inconsistent results frequently occur among multiple parallel samples, with some samples showing negative results and others positive, making it impossible to determine the outcome. The inventors of this application considered that after adding the horseshoe crab reagent to the sample, it needs to be kept at a temperature of 35–38°C, and then the endotoxin detection result is determined based on whether the sample forms a gel. Since the target is endotoxin detection in collagen liquid, if collagen liquid is used directly as the sample, the collagen liquid may self-assemble to form a collagen gel due to temperature effects, making it impossible to determine whether the collagen liquid itself has become a gel or whether the endotoxins in the collagen liquid have undergone a coagulation reaction with the horseshoe crab reagent, thus leading to false positive results. Furthermore, the inventors of this application considered using a collagen extract as a sample. The collagen extract was mixed with bacterial endotoxin and soaked in water for a period of time. The extract was then mixed with a horseshoe crab reagent and incubated at 35–38°C. The endotoxin test result was then determined to be negative or positive based on whether the mixture formed a gel. However, when using water to soak the collagen extract for bacterial endotoxin detection, the mixture became a suspension. Even after taking the extract, collagen residue remained, making false positives in the bacterial endotoxin test highly likely. Additionally, the inventors also tried using a more sensitive horseshoe crab reagent, mixing it with the collagen extract and then diluting it to reduce the influence of collagen itself. However, false positives still frequently occurred. This method still cannot eliminate false positives caused by the influence of collagen itself.

[0041] The endotoxin detection method for collagen solution in this application involves first forming a gel from the collagen solution, then soaking it in endotoxin test water. The extract is mixed with a horseshoe crab reagent, and the mixture is incubated at 35–38°C. The endotoxin content of the collagen solution is determined based on whether the mixture forms a gel, thus eliminating the possibility that the collagen solution itself forming a gel could affect the accuracy of the endotoxin detection results. Optionally, the incubation temperature is 35°C, 36°C, 37°C, or 38°C. For example, the incubation time is 50–70 minutes, such as any one of 50 minutes, 60 minutes, and 70 minutes, or any value between two of them. It should be noted that during the incubation process and the determination of the endotoxin detection results, sample vibration should be avoided to prevent false negative results.

[0042] Preferably, the pH of the collagen solution is 6-8. After the collagen solution forms a gel, the pH of the mixture after mixing with the horseshoe crab reagent can be maintained at approximately 6-8, eliminating the need for adding acids, alkalis, or buffer solutions to adjust the pH. If acid, alkali, or buffer solutions need to be added during the operation to adjust the pH, it requires preparing the solution with bacterial endotoxin in water in a container where the endotoxin has been removed, which increases the number of steps and the risk of false positives.

[0043] In some embodiments, the collagen solution is obtained from animal tissue through a series of processes including defatting, protein removal, enzymatic hydrolysis, salting out, dialysis, and concentration. The animal tissue can be skin tissue, organ tissue, cartilage tissue, etc., as long as collagen can be extracted from it. Suitable animals include pigs, cattle, sheep, fish, silkworms, and donkeys. For example, suitable animal tissues include pigskin, sheep placenta, cowhide, tendons, fish scales, fish skin, and chicken skin. It should be noted that high-concentration collagen solutions require concentration. Furthermore, this application does not specify a particular preparation process for the collagen solution; as long as it can form a collagen gel, the method described in this application can be used to reduce false positive results when detecting endotoxins.

[0044] In some embodiments, the step of forming a collagen gel from a collagen solution includes maintaining the collagen solution at a temperature of 35–38°C to form a collagen gel. It should be noted that the temperature is also maintained at 35–38°C throughout the immersion process using water for endotoxin testing. For example, the collagen solution is maintained at temperatures of 35°C, 36°C, 37°C, or 38°C.

[0045] Optionally, the immersion time in water for endotoxin testing is 1 to 30 hours, for example, any one or any two of the following: 1 hour, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 22 hours, 24 hours, 25 hours, 26 hours, 28 hours, and 30 hours.

[0046] Optionally, the sensitivity of the horseshoe crab reagent is 0.03 to 0.5 EU / mL, for example, any one or any two of 0.03 EU / mL, 0.05 EU / mL, 0.10 EU / mL, 0.15 EU / mL, 0.20 EU / mL, 0.25 EU / mL, 0.30 EU / mL, 0.40 EU / mL and 0.5 EU / mL.

[0047] Optionally, the concentration of collagen in the collagen solution is ≥5 mg / mL. The inventors of this application have discovered in their research that when the collagen concentration in the collagen solution is too low, the collagen solution does not easily form a gel at temperatures of 35–38°C. However, when the concentration of the collagen solution is greater than or equal to 5 mg / mL, the inventors of this application have found that collagen gel can be formed. Optionally, the concentration of collagen in the collagen solution is 5–150 mg / mL, for example, any one or any combination of 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 100 mg / mL, 120 mg / mL, 140 mg / mL, and 150 mg / mL.

[0048] Optionally, the collagen solution contains a phosphate buffer solution in which the collagen is dissolved. The phosphate buffer solution contains disodium hydrogen phosphate and sodium dihydrogen phosphate. The inventors of this application have discovered that when collagen is dissolved in a phosphate buffer solution, it more easily forms a monolithic collagen gel. It is understood that the phosphate buffer solution in the collagen solution in this application's embodiments may be introduced during the collagen solution production process, for example, during ultrafiltration concentration; or it may be introduced by compounding a high-concentration collagen solution with a phosphate buffer solution. This application does not specify in which step or how the phosphate buffer solution is introduced into the collagen solution.

[0049] Additionally, it should be noted that when the phosphate buffer solution also contains NaCl and / or KCl and the salt ion concentration is ≥3g / L, collagen may partially separate and exhibit stratification. Only a portion of it will form a gel at a temperature of 35–38°C. After adding bacterial endotoxin and soaking in water, the extract may also contain collagen, which could lead to false positive results.

[0050] In some implementations, the step of mixing the extract with the horseshoe crab reagent includes: mixing the extract with at least two horseshoe crab reagents of different sensitivities separately. For example, horseshoe crab reagents with sensitivities A, B, and C can be prepared separately, where A>B>C. Then, three equal amounts of the extract are taken and mixed with horseshoe crab reagents of sensitivities A, B, and C, respectively. When determining the results, if the corresponding mixture forms a gel, the endotoxin content of the collagen solution is greater than or equal to the sensitivity of the used horseshoe crab reagent; if the corresponding mixture does not form a gel, the endotoxin content of the collagen solution is less than the sensitivity value of the used horseshoe crab reagent. Based on the sensitivity of the at least two horseshoe crab reagents of different sensitivities added to the extract, the range of endotoxin content in the collagen solution can be determined. For example, if the mixture corresponding to the addition of horseshoe crab reagent with sensitivity A does not form a gel, but the mixtures corresponding to the addition of horseshoe crab reagents with sensitivities B and C both form gels, then the endotoxin content in the collagen solution can be determined to be between B and A.

[0051] Furthermore, to improve the accuracy of endotoxin detection results, this application also includes providing a positive control sample, a positive test sample control sample, and a negative control sample. These samples are mixed separately with horseshoe crab reagent. The validity of the experiment is determined by whether the positive control sample, positive test sample control sample, and negative control sample form a gel; gel formation indicates a positive result, while non-gel formation indicates a negative result. Therefore, when both the positive control sample and the positive test sample control sample are positive, and the negative control sample is negative, the entire experiment is valid, thereby improving the accuracy of endotoxin detection results for collagen solution.

[0052] The negative control was bacterial endotoxin-infused water; the positive control was 2λ bacterial endotoxin standard solution + bacterial endotoxin-infused water; and the positive test sample control was an extract of collagen gel formed from the collagen solution of this application + 2λ bacterial endotoxin standard solution. Here, λ represents the labeled sensitivity value of the Limulus Amebocyte Lysate (LAL) reagent used.

[0053] The following detailed description of the endotoxin detection method for collagen liquid of this application is provided in conjunction with the embodiments.

[0054] Example 1

[0055] This embodiment provides a method for detecting endotoxins in collagen solution, which includes the following steps:

[0056] (1) Provide a collagen solution obtained from pig skin as raw material through defatting, removing impurities and proteins, enzymatic hydrolysis, salting out, dialysis and concentration; wherein the concentration of collagen in the collagen solution is 5.25 mg / mL, the collagen solution contains phosphate buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate), the collagen in the collagen solution is dissolved in phosphate buffer solution, and the pH of the collagen solution is 7.23.

[0057] (2) Take 0.1 mL of collagen solution from step (1) and keep it at a temperature of 37±1℃ to allow the collagen to form a collagen gel. Then add 1.67 mL of water for bacterial endotoxin testing and soak it in a water bath at 37±1℃ for 24 hours.

[0058] (3) Add 1 mL of bacterial endotoxin test water to the bacterial endotoxin standard, vortex for 5 minutes to mix evenly, and obtain E10 solution; mix E10 solution with bacterial endotoxin test water evenly to obtain E1 solution (bacterial endotoxin concentration is 0.5 EU / mL); mix 0.2 mL of E1 solution with 0.2 mL of bacterial endotoxin water to prepare a positive control sample solution; mix 0.2 mL of E1 solution with 0.2 mL of the extract in step (2) to prepare a positive test sample control sample; take 0.2 mL of bacterial endotoxin water as a negative control sample;

[0059] (4) Dissolve 0.1 mL of Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.25 EU / mL in water with 0.1 mL of bacterial endotoxin to obtain LAL reagent solution;

[0060] (5) Take 0.1 mL of the extract from step (2) and mix it with 0.1 mL of the horseshoe crab reagent solution from step (4). Also, take 0.1 mL of the positive control sample solution, the positive test sample control sample and the negative control sample and mix them with 0.1 mL of the horseshoe crab reagent solution. All mixing is done in test tubes. After mixing, seal the tube opening with sealing film and place it vertically in a drying oven at 37℃±1℃ for 60 minutes±2 minutes.

[0061] (6) When the test tube is gently removed from the drying oven and slowly inverted 180°, if a gel forms inside the tube and the gel does not deform or slip off the tube wall, it is positive and recorded as (+); if no gel forms or the formed gel is not firm, deformed and slips off the tube wall, it is negative and recorded as (-).

[0062] Example 2

[0063] This embodiment provides a method for detecting endotoxins in collagen solution, which includes the following steps:

[0064] (1) Provide a collagen solution obtained by defatting, removing impurities and proteins from pig skin, enzymatic hydrolysis, salting out, dialysis and concentration; wherein the concentration of the collagen solution is 10.62 mg / mL, the collagen solution contains a phosphate buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate), the collagen in the collagen solution is dissolved in the phosphate buffer solution, and the pH of the collagen solution is 7.15.

[0065] (2) Take 0.1 mL of collagen solution from step (1) and keep it warm at 37±1℃ to form collagen gel. Then add 0.2 mL of water for bacterial endotoxin testing and soak it in a water bath at 37±1℃ for 24 hours.

[0066] (3) Add 1 mL of bacterial endotoxin test water to the bacterial endotoxin standard, vortex for 5 minutes to mix evenly, and obtain E10 solution; take E10 solution and add bacterial endotoxin test water to mix evenly to obtain E1 solution (bacterial endotoxin concentration is 0.5 EU / mL); take 0.2 mL of E1 solution and 0.2 mL of bacterial endotoxin water to prepare a positive control sample solution; take 0.2 mL of E1 solution and 0.2 mL of the extract in step (2) to prepare a positive test sample control sample; take 0.2 mL of bacterial endotoxin water as a negative control sample;

[0067] (4) Dissolve 0.1 mL of Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.25 EU / mL in water with 0.1 mL of bacterial endotoxin to obtain LAL reagent solution;

[0068] (5) Take 0.1 mL of the extract from step (2) and mix it with 0.1 mL of the horseshoe crab reagent solution from step (4). Also, take 0.1 mL of the positive control sample solution, the positive test sample control sample and the negative control sample and mix them with 0.1 mL of the horseshoe crab reagent solution. All mixing is done in test tubes. After mixing, seal the tube opening with sealing film and place it vertically in a drying oven at 37℃±1℃ for 60 minutes±2 minutes.

[0069] (6) When the test tube is gently removed from the drying oven and slowly inverted 180°, if a gel forms inside the tube and the gel does not deform or slip off the tube wall, it is positive and recorded as (+); if no gel forms or the formed gel is not firm, deformed and slips off the tube wall, it is negative and recorded as (-).

[0070] Example 3

[0071] This embodiment provides a method for detecting endotoxins in collagen solution, which includes the following steps:

[0072] (1) Provide a collagen solution obtained by defatting, removing impurities and proteins, enzymatic hydrolysis, salting out, dialysis, filtration and sterilization and concentration of pig skin as raw material; wherein the concentration of the collagen solution is 35.12 mg / mL, the collagen solution contains phosphate buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate), the collagen in the collagen solution is dissolved in the phosphate buffer solution, and the pH of the collagen solution is 7.46.

[0073] (2) Take 0.1 mL of collagen solution from step (1) and keep it warm at 37±1℃ to form collagen gel. Then add 0.2 mL of water for bacterial endotoxin testing and soak it in a water bath at 37±1℃ for 24 hours.

[0074] (3) Add 1 mL of bacterial endotoxin test water to the bacterial endotoxin standard, vortex for 5 minutes to mix evenly, and obtain E10 solution; take E10 solution and add bacterial endotoxin test water to mix evenly to obtain E1 solution (bacterial endotoxin concentration is 0.5 EU / mL); take 0.2 mL of E1 solution and 0.2 mL of bacterial endotoxin water to prepare a positive control sample solution; take 0.2 mL of E1 solution and 0.2 mL of the extract in step (2) to prepare a positive test sample control sample; take 0.2 mL of bacterial endotoxin water as a negative control sample;

[0075] (4) Dissolve 0.1 mL of Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.25 EU / mL in water with 0.1 mL of bacterial endotoxin to obtain LAL reagent solution;

[0076] (5) Take 0.1 mL of the extract from step (2) and mix it with 0.1 mL of the horseshoe crab reagent solution from step (4). Also, take 0.1 mL of the positive control sample solution, the positive test sample control sample and the negative control sample and mix them with 0.1 mL of the horseshoe crab reagent solution. All mixing is done in test tubes. After mixing, seal the tube opening with sealing film and place it vertically in a drying oven at 37℃±1℃ for 60 minutes±2 minutes.

[0077] (6) When the test tube is gently removed from the drying oven and slowly inverted 180°, if a gel forms inside the tube and the gel does not deform or slip off the tube wall, it is positive and recorded as (+); if no gel forms or the formed gel is not firm, deformed and slips off the tube wall, it is negative and recorded as (-).

[0078] Example 4

[0079] This embodiment provides a method for detecting endotoxins in collagen solution, which includes the following steps:

[0080] (1) Provide a collagen solution obtained from pig skin as raw material through defatting, removing impurities and proteins, enzymatic hydrolysis, salting out, dialysis and concentration; wherein the concentration of collagen in the collagen solution is 15.96 mg / mL, the collagen solution contains phosphate buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate), the collagen in the collagen solution is dissolved in phosphate buffer solution, and the pH of the collagen solution is 7.32.

[0081] (2) Take 0.1 mL of collagen solution from step (1) and keep it at a temperature of 37±1℃ to allow the collagen to form a collagen gel. Then add 1 mL of water for bacterial endotoxin testing and soak it in a water bath at 37±1℃ for 24 hours.

[0082] (3) Add 1 mL of bacterial endotoxin test water to the bacterial endotoxin standard, vortex for 5 minutes to mix evenly, and obtain E10 solution; take E10 solution and mix evenly with bacterial endotoxin test water to obtain E11 solution (bacterial endotoxin concentration is 0.5 EU / mL); take 0.2 mL of E11 solution and 0.2 mL of bacterial endotoxin water to prepare the first positive control sample solution; take 0.2 mL of E11 solution and 0.2 mL of the extract in step (2) to prepare the first positive test sample control sample; take 0.2 mL of bacterial endotoxin water as the first negative control sample;

[0083] (4) Mix E10 solution with water for bacterial endotoxin testing to obtain E12 solution (bacterial endotoxin concentration is 0.06 EU / mL); mix 0.2 mL of E12 solution with 0.2 mL of bacterial endotoxin water to prepare a second positive control sample solution; mix 0.2 mL of E12 solution with 0.2 mL of the extract from step (2) to prepare a second positive test sample control sample; use 0.2 mL of bacterial endotoxin water as a second negative control sample.

[0084] (5) Dissolve 0.1 mL of Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.25 EU / mL in water with 0.1 mL of bacterial endotoxin to obtain the first LAL reagent solution;

[0085] (6) Dissolve 0.1 mL of Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.03 EU / mL in water with 0.1 mL of bacterial endotoxin to obtain a second LAL reagent solution;

[0086] (7) Take 0.1 mL of the extract from step (2) and mix it with 0.1 mL of the first horseshoe crab reagent solution from step (5). Also, take 0.1 mL of the first positive control sample solution, the first positive test sample control sample and the first negative control sample and mix them with 0.1 mL of the first horseshoe crab reagent solution. All mixing is done in test tubes. After mixing, seal the tube opening with sealing film and place it vertically in a drying oven at 37℃±1℃ for 60 minutes±2 minutes.

[0087] (8) Take 0.1 mL of the extract from step (2) and mix it with 0.1 mL of the second horseshoe crab reagent solution from step (6). Also, take 0.1 mL of the second positive control sample solution, the second positive test sample control sample, and the second negative control sample and mix them with 0.1 mL of the second horseshoe crab reagent solution. All mixing is done in test tubes. After mixing, seal the tube opening with sealing film and place it vertically in a drying oven at 37℃±1℃ for 60 minutes±2 minutes.

[0088] (9) When the test tube is gently removed from the drying oven and slowly inverted 180°, if a gel forms inside the tube and the gel does not deform or slip off the tube wall, it is positive and recorded as (+); if no gel forms or the formed gel is not firm, deformed and slips off the tube wall, it is negative and recorded as (-).

[0089] Comparative Example 1

[0090] This comparative example provides a method for detecting endotoxins in collagen. The steps are the same as those in Example 1, except that the extract in steps (3) and (5) is replaced with collagen solution (provided by step (1)).

[0091] Comparative Example 2

[0092] This comparative example provides a method for detecting endotoxins in collagen. The steps are different from those in Example 1, except that step (2) is different, and the extract added in steps (3) and (5) is the extract from step (2) of this comparative example.

[0093] Step (2) of this comparative example is as follows: Take 0.1 mL of collagen solution from step (1) and add 1.67 mL of bacterial endotoxin test water at room temperature (25±1℃), and soak for 24 h at room temperature.

[0094] The experimental results of Examples 1 to 4 and Comparative Examples 1 to 2 are as follows: Figures 1 to 7 As shown in the figure. It should be noted that each sample was tested in duplicate.

[0095] from Figures 1 to 7 The results show that in Examples 1-4 and Comparative Examples 1-2, the endotoxin detection results of the positive control samples and positive test sample control samples were all positive, while the endotoxin detection results of the negative control samples were all negative, indicating that the endotoxin detection experiments in Examples 1-4 and Comparative Examples 1-2 were all effective. Furthermore, according to... Figures 1-3 The results show that the endotoxin test results in Examples 1 to 3 of this application were all negative, indicating that the endotoxin content in the collagen of Examples 1 to 3 was less than 0.25 EU / mL. According to... Figure 6and Figure 7 The results showed that without forming a collagen gel from the collagen solution, the final endotoxin test result was positive after mixing the extract with the horseshoe crab reagent in water for endotoxin testing. This indicates that the endotoxin detection methods in Comparative Examples 1 and 2 are prone to false positive results.

[0096] In addition, according to Figure 4 and Figure 5 The results showed that the endotoxin content in the collagen solution of Example 4 was greater than 0.03 EU / mL and less than 0.25 EU / mL.

[0097] The above description is merely a specific embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting endotoxins in collagen solution, characterized in that, include: After the collagen solution forms a collagen gel, it is soaked in endotoxin test water. The extract is then mixed with horseshoe crab reagent and kept at a temperature of 35-38°C. The endotoxin content of the collagen solution is then determined based on whether the mixture forms a gel. The step of forming collagen gel from collagen liquid includes: maintaining the collagen liquid at a temperature of 35~38°C to form collagen gel; The collagen solution has a pH of 6-8; the collagen concentration in the collagen solution is ≥5 mg / mL; the collagen solution contains a phosphate buffer solution, and the collagen is dissolved in the phosphate buffer solution.

2. The method for detecting endotoxins in collagen solution according to claim 1, characterized in that, The collagen solution is obtained by sequentially defatting, removing impurities and proteins from animal tissues, followed by enzymatic hydrolysis, salting out, dialysis, and concentration.

3. The method for detecting endotoxins in collagen solution according to claim 1, characterized in that, The concentration of collagen in the collagen solution is 5~150 mg / mL.

4. The method for detecting endotoxins in collagen solution according to any one of claims 1 to 3, characterized in that, The sensitivity of the Limulus amebocyte lysate (LAL) reagent is 0.03~0.5 EU / mL.

5. The method for detecting endotoxins in collagen solution according to any one of claims 1 to 3, characterized in that, The endotoxin test involves soaking the sample in water for 1 to 30 hours.

6. The method for detecting endotoxins in collagen solution according to any one of claims 1 to 3, characterized in that, The volume ratio of the collagen solution to the water used for endotoxin testing is 1:2~20.

7. The method for detecting endotoxins in collagen solution according to any one of claims 1 to 3, characterized in that, The step of mixing the extract with the horseshoe crab reagent includes: mixing the extract with at least two horseshoe crab reagents of different sensitivities respectively; The step of determining the endotoxin content of the collagen based on whether the mixture forms a gel includes: When the corresponding mixture forms a gel, the endotoxin content of the collagen solution is greater than or equal to the sensitivity value of the horseshoe crab reagent used. If the corresponding mixture does not form a gel, the endotoxin content of the collagen solution is less than the sensitivity value of the horseshoe crab reagent used. The range of endotoxin content in the collagen solution is determined based on the sensitivity values ​​of at least two different sensitivities of the horseshoe crab reagent added to the extract.

8. The method for detecting endotoxins in collagen solution according to any one of claims 1 to 3, characterized in that, It also includes providing a positive control sample, a positive test sample control sample, and a negative control sample. After mixing the positive control sample, the positive test sample control sample, and the negative control sample with the horseshoe crab reagent respectively, the validity of the experiment is determined by whether the positive control sample, the positive test sample control sample, and the negative control sample form a gel.

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