CN116559456A - Endotoxin detection method of collagen liquid - Google Patents
Endotoxin detection method of collagen liquid Download PDFInfo
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- CN116559456A CN116559456A CN202211387146.7A CN202211387146A CN116559456A CN 116559456 A CN116559456 A CN 116559456A CN 202211387146 A CN202211387146 A CN 202211387146A CN 116559456 A CN116559456 A CN 116559456A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 167
- 108010035532 Collagen Proteins 0.000 title claims abstract description 167
- 229920001436 collagen Polymers 0.000 title claims abstract description 166
- 239000002158 endotoxin Substances 0.000 title claims abstract description 151
- 239000007788 liquid Substances 0.000 title claims abstract description 87
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 239000000243 solution Substances 0.000 claims abstract description 122
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 72
- 241000239218 Limulus Species 0.000 claims abstract description 71
- 238000012360 testing method Methods 0.000 claims abstract description 58
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 50
- 238000002156 mixing Methods 0.000 claims abstract description 43
- 238000002386 leaching Methods 0.000 claims abstract description 36
- 239000000512 collagen gel Substances 0.000 claims abstract description 20
- 239000011259 mixed solution Substances 0.000 claims abstract description 17
- 238000002791 soaking Methods 0.000 claims abstract description 13
- 239000000523 sample Substances 0.000 claims description 56
- 230000035945 sensitivity Effects 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 24
- 239000013642 negative control Substances 0.000 claims description 22
- 239000013641 positive control Substances 0.000 claims description 22
- 239000008055 phosphate buffer solution Substances 0.000 claims description 20
- 239000013068 control sample Substances 0.000 claims description 15
- 241001465754 Metazoa Species 0.000 claims description 7
- 238000005238 degreasing Methods 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 5
- 235000019799 monosodium phosphate Nutrition 0.000 description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 5
- 238000004321 preservation Methods 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 230000004520 agglutination Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229920006008 lipopolysaccharide Polymers 0.000 description 3
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- 229920002498 Beta-glucan Polymers 0.000 description 2
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
An endotoxin detection method of collagen liquid relates to the technical field of collagen detection. The endotoxin detection method of the collagen liquid comprises the following steps: after forming collagen gel from the collagen liquid, adding endotoxin test water for soaking, mixing the leaching solution with limulus reagent, preserving heat at 35-38 ℃, and judging the endotoxin content of the collagen liquid according to whether the mixed solution forms gel or not. Which can reduce the probability of false positives in the detection results.
Description
Technical Field
The application relates to the technical field of collagen detection, in particular to an endotoxin detection method of collagen liquid.
Background
Bacterial endotoxin is a complex of lipopolysaccharide (LipopolySaccharide, LPS) and proteins on the cell wall of gram-negative bacteria that are released when the bacteria die or autolyse. The massive entry of endotoxin into the blood causes a fever reaction, the "pyrogen reaction", and endotoxin is closely related to various infectious diseases, and the deterioration of the disease is often accompanied by an increase in endotoxin content.
Collagen liquid is extracted from animal tissues, and after the collagen liquid is used as a medical instrument and a medical product to be implanted into a human body, endotoxin residues can cause inflammation of the body and affect the biosafety of the implant. Thus, it is necessary to detect endotoxin in the produced collagen to determine whether it has good biosafety.
Currently, endotoxin is generally detected by using a limulus reagent, and detection methods based on the limulus reagent include a gel method, a chromogenic method and a turbidimetric method. The limulus reagent is a reagent extracted from the blood of limulus and can cause an agglutination reaction with bacterial endotoxin, and whether or not the detection result of endotoxin is positive is determined by whether or not a gel is formed. In addition to endotoxin, limulus reagents react with certain beta-glucans, producing false positive results. The inventors of the present application found that the collagen liquid does not contain β -glucan, but false positive results frequently occur when the collagen is detected by using a limulus reagent.
Disclosure of Invention
The application provides an endotoxin detection method of collagen liquid, which can reduce the probability of false positive of detection results.
The application is realized in such a way that:
the application provides an endotoxin detection method of collagen liquid, which comprises the following steps:
after forming collagen gel from the collagen liquid, adding endotoxin test water for soaking, mixing the leaching solution with a limulus reagent, preserving heat at 35-38 ℃, and judging the endotoxin content of the collagen liquid according to whether the mixed solution forms gel or not.
Optionally, the time of heat preservation is 50-70 min.
In one possible embodiment, the step of forming the collagen liquid into a collagen gel comprises: the collagen liquid is kept at a temperature of 35-38 ℃ to form the collagen gel.
In one possible embodiment, the detection method comprises at least one of the following definitions:
first definition: the pH value of the collagen liquid is 6-8;
a second definition: the collagen liquid is obtained by degreasing animal tissues, removing impurity proteins, carrying out enzymolysis, salting out, dialyzing and concentrating.
Third definition: the concentration of collagen in the collagen liquid is more than or equal to 5mg/mL; optionally, the concentration of the collagen in the collagen liquid is 5-150 mg/mL.
In one possible embodiment, the sensitivity of the limulus reagent is 0.03 to 0.5EU/mL.
In one possible embodiment, the endotoxin test water is added for a period of time of 1 to 30 hours.
In one possible embodiment, the collagen solution contains a phosphate buffer solution in which the collagen is dissolved.
In one possible embodiment, the volume ratio of the collagen liquid to the endotoxin test water is 1:2 to 20.
In one possible embodiment, the step of mixing the leaching solution with a limulus reagent comprises: taking said leaching solution and mixing with at least two different sensitivity of said limulus reagents respectively;
the step of judging the endotoxin content of the collagen according to whether the mixed solution forms gel or not comprises the following steps:
when the corresponding mixed solution forms gel, the endotoxin content of the collagen solution is greater than or equal to the sensitivity value of the limulus reagent;
when the corresponding mixed solution does not form gel, the endotoxin content of the collagen solution is smaller than the sensitivity value of the limulus reagent;
judging the content range of endotoxin in the collagen solution according to the sensitivity values of at least two different sensitivities of the limulus reagent added into the leaching solution.
In one possible embodiment, the method further comprises providing a positive control sample, a positive test sample control sample and a negative control sample, and after mixing the positive control sample, the positive test sample control sample and the negative control sample with a limulus reagent respectively, determining whether the test is effective based on whether the positive control sample, the positive test sample control sample and the negative control sample form gel.
The endotoxin detection method of the collagen liquid has at least the following beneficial effects:
the inventors of the present application have found in the study that when endotoxin is detected in collagen using a limulus reagent, the collagen solution itself becomes gel due to the influence of temperature, and it is impossible to determine whether the collagen solution itself becomes gel or whether the endotoxin in the collagen solution has undergone an aggregation reaction with the limulus reagent. Therefore, the method comprises the steps of firstly forming the collagen liquid into gel, then adding endotoxin test water for soaking, mixing the leaching solution with the limulus reagent, and judging the content of endotoxin in the collagen liquid according to whether the mixed solution forms the gel or not, thereby eliminating the influence of the formation of the gel of the collagen liquid on the accuracy of the detection result of the endotoxin.
Drawings
It is to be understood that the following drawings illustrate only certain embodiments of the invention and are therefore not to be considered limiting of its scope, for the person of ordinary skill in the art may admit to other equally relevant drawings without inventive effort.
FIG. 1 is a graph of endotoxin test results of example 1 of the present application;
FIG. 2 is a graph of endotoxin test results of example 2 of the present application;
FIG. 3 is a graph of endotoxin test results of example 3 of the present application;
FIG. 4 is a graph of endotoxin test results of example 4 of the present application;
FIG. 5 is a graph of endotoxin test results of example 4 of the present application;
FIG. 6 is a graph showing endotoxin test results of comparative example 1 of the present application;
FIG. 7 is a graph showing endotoxin test results of comparative example 2 of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following specifically describes a method for detecting endotoxin in a collagen solution of the present application:
the application provides an endotoxin detection method of collagen liquid, which comprises the following steps:
after forming collagen gel from the collagen liquid, adding endotoxin test water for soaking, mixing the leaching solution with limulus reagent, preserving heat at 35-38 ℃, and judging the endotoxin content of the collagen liquid according to whether the mixed solution forms gel or not.
Wherein, the principle of detecting the endotoxin content in the collagen liquid by the gel method is that when the endotoxin in the collagen liquid and the limulus reagent are subjected to agglutination reaction, gel is formed, whether the endotoxin in the collagen liquid exceeds the limit is judged by whether the endotoxin forms the gel, if the endotoxin forms the gel, the detection result is judged to be positive, and the endotoxin content exceeds the sensitivity value of the limulus reagent; if gel is not formed, the detection result of endotoxin is judged to be negative, and the content of endotoxin does not exceed the sensitivity value of the limulus reagent used.
However, the inventors of the present application have found in the study that, when endotoxin is detected in a collagen liquid, false positive results often exist in a plurality of parallel samples, or the detection results are inconsistent, and that some samples are negative and some samples are positive, and the results cannot be determined. The inventors of the present application considered that it is necessary to incubate a sample with a limulus reagent at a temperature of 35 to 38℃and then determine the detection result of endotoxin based on whether the sample forms a gel. Since the detection of endotoxin in the collagen liquid is aimed at, if the collagen liquid is directly used as a sample, the collagen liquid may self-assemble to form collagen gel due to the influence of temperature, and thus it cannot be judged whether the collagen liquid itself becomes gel or whether the endotoxin in the collagen liquid and the limulus reagent undergo an agglutination reaction, thereby causing false positive results. Further, the inventors of the present application considered that a leaching solution of a collagen solution was used as a sample, and that a collagen solution was mixed with bacterial endotoxin for a certain period of time and then the leaching solution was mixed with a limulus reagent, and the temperature was kept at 35 to 38℃to determine whether the endotoxin test result was negative or positive depending on whether the mixed solution formed a gel. However, after the bacterial endotoxin is detected, the collagen liquid is soaked in water, the mixed liquid is in a suspension state, the residual collagen exists in the leaching liquid after the leaching liquid is extracted, and the detection result of the bacterial endotoxin is easy to be false positive. In addition, the inventors of the present application have attempted to select a limulus reagent having a higher sensitivity, mix the limulus reagent with a collagen solution, dilute the mixture, and reduce the influence of collagen itself by increasing the dilution factor, but as a result, false positives often occur. This approach is also unable to get rid of false positives caused by the influence of the collagen itself.
In the endotoxin detection method of the collagen liquid, the collagen liquid is firstly formed into gel, then endotoxin inspection water is added for soaking, the leaching solution is mixed with the limulus reagent, and after the temperature is kept at 35-38 ℃, the content of the endotoxin in the collagen liquid is judged according to whether the mixed solution forms the gel or not, so that the accuracy that the endotoxin detection result is influenced by the gel formed by the collagen liquid per se is eliminated. Alternatively, the incubation temperature is 35 ℃, 36 ℃, 37 ℃ or 38 ℃. Illustratively, the incubation time is 50 to 70 minutes, for example, a value of any one or any two of 50 minutes, 60 minutes, and 70 minutes. It should be noted that, in the process of performing heat preservation and judging endotoxin detection results, false negative results caused by vibration of the sample should be avoided.
Preferably, the pH value of the collagen liquid is 6-8, and after the collagen liquid forms gel, the pH of the mixed liquid can be basically controlled to be 6-8 after the leaching liquid is mixed with the limulus reagent, and no acid, alkali or buffer solution is needed to be added for adjusting the pH. If during the procedure an acid, base or buffer is also added to adjust the pH, the bacterial endotoxin needs to be formulated with water in the endotoxin-removed container, increasing the procedure and increasing the risk of false positives.
In some embodiments, the collagen solution is obtained from animal tissue by sequentially degreasing, deproteinizing, enzymatic hydrolysis, salting out, dialysis, and concentrating. The animal tissue may be skin tissue, organ tissue, cartilage tissue, etc. as long as collagen can be extracted, and the animal may be pig, cow, sheep, fish, silkworm, donkey, etc. For example, animal tissue may be selected from pigskin, sheep embryo skin, cow hide, tendon, fish scale, fish skin, chicken skin, etc. The high-concentration collagen solution is obtained by concentration. In addition, the specific preparation process of the collagen liquid is not particularly limited, and the method of the present application may be used to reduce the occurrence of false positive results, as long as the collagen gel can be formed and endotoxin is detected.
In some embodiments, the step of forming the collagen liquid into a collagen gel comprises: the collagen liquid is maintained at a temperature of 35 to 38 ℃ to form a collagen gel. The temperature was kept at 35 to 38℃during the immersion in the endotoxin test water. For example, the collagen solution is maintained at a temperature of 35 ℃, 36 ℃, 37 ℃ or 38 ℃.
Alternatively, the time for soaking in the endotoxin test water is 1 to 30 hours, for example, a value of any one or any two of 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.
Alternatively, the sensitivity of the limulus reagent is 0.03 to 0.5EU/mL, for example, a value of any one or any two of 0.03EU/mL, 0.05EU/mL, 0.10EU/mL, 0.15EU/mL, 0.20EU/mL, 0.25EU/mL, 0.30EU/mL, 0.40EU/mL and 0.5EU/mL.
Optionally, the concentration of collagen in the collagen liquid is more than or equal to 5mg/mL. The inventor of the application found in the research that when the concentration of the collagen in the collagen liquid is too low, the collagen liquid is not easy to form gel at the temperature of 35-38 ℃, and when the concentration of the collagen liquid is more than or equal to 5mg/mL, the inventor of the application found that the collagen gel can be formed through the test of the application. Alternatively, the concentration of collagen in the collagen liquid is 5 to 150mg/mL, for example, a value of any one or any two of 5mg/mL, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 100mg/mL, 120mg/mL, 140mg/mL and 150mg/mL.
Optionally, the collagen solution contains a phosphate buffer solution, and the collagen is dissolved in the phosphate buffer solution. Among them, the phosphate buffer solution contains disodium hydrogen phosphate and sodium dihydrogen phosphate, and the inventors of the present application have found in the study that collagen forms a whole collagen gel more easily when it is dissolved in the phosphate buffer solution. It will be appreciated that the phosphate buffer solution in the collagen solution according to embodiments of the present application may be introduced during the production process steps of the collagen solution, for example, during ultrafiltration concentration; the high-concentration collagen solution and the phosphate buffer solution can be compounded so as to bring the phosphate buffer solution in. The procedure and the way in which the phosphate buffer solution is introduced into the collagen solution are not particularly limited in this application.
In addition, when NaCl and/or KCl are/is also contained in the phosphate buffer solution, and the concentration of salt ions is not less than 3g/L, collagen is partially separated out to generate layering phenomenon, only partial collagen forms gel at the temperature of 35-38 ℃, bacterial endotoxin is added to soak the solution with water, and then leaching solution is extracted, and collagen can be also generated in the leaching solution, so that the result can be false positive.
In some embodiments, the step of mixing the leaching solution with the limulus reagent comprises: the leaching solution is mixed with at least two limulus reagents with different sensitivities. For example, a limulus reagent having a sensitivity of A, B, C may be separately prepared, wherein A > B > C, and three identical amounts of the leaching solutions may be taken and mixed with a limulus reagent having a sensitivity of A, B, C. When the corresponding mixed solution forms gel in the result determination, the endotoxin content of the collagen solution is greater than or equal to the sensitivity of the limulus reagent used; when the corresponding mixed solution does not form gel, the endotoxin content of the collagen solution is smaller than the sensitivity value of the limulus reagent; the range of endotoxin content in the collagen solution can be judged based on the sensitivity of at least two different sensitivity limulus reagents added to the leaching solution. For example, when the mixed solution corresponding to the limulus reagent added with the sensitivity A does not form a gel, and the mixed solution corresponding to the limulus reagent added with the sensitivity B and the limulus reagent added with the sensitivity C both form a gel, it can be judged that the endotoxin content in the collagen solution is between B and A.
Further, in order to improve the accuracy of endotoxin detection results, the present application further includes providing a positive control sample, a positive test sample control sample and a negative control sample, mixing the positive control sample, the positive test sample control sample and the negative control sample with a limulus reagent, respectively, and determining whether the experiment is effective or not in an auxiliary manner according to whether the positive control sample, the positive test sample control sample and the negative control sample form gel or not, wherein the gel is positive if formed, and the gel is negative if not formed. When the positive control and the positive test sample are positive and the negative control sample is negative, the whole experiment is effective, so that the accuracy of the endotoxin detection result of the collagen liquid can be improved.
The negative control sample is water for bacterial endotoxin, the positive control sample is a 2 lambda bacterial endotoxin standard substance solution and water for bacterial endotoxin, and the positive test sample is a leaching solution of collagen gel formed by the collagen protein liquid and the 2 lambda bacterial endotoxin standard substance solution. Wherein lambda is a value indicative of the sensitivity of the limulus reagent used.
The method for detecting endotoxin in the collagen liquid of the present application is described in further detail below with reference to examples.
Example 1
The embodiment provides an endotoxin detection method of collagen liquid, which comprises the following steps:
(1) Providing collagen liquid obtained by degreasing, removing impurity protein, carrying out enzymolysis, salting out, dialyzing and concentrating the pigskin serving as a raw material; wherein the concentration of collagen in the collagen solution is 5.25mg/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 7.23.
(2) Taking 0.1mL of the collagen liquid obtained in the step (1), keeping the temperature at 37+/-1 ℃ to enable collagen to form collagen gel, adding 1.67mL of bacterial endotoxin detection water, and soaking for 24 hours under the water bath heat preservation condition at 37+/-1 ℃.
(3) Adding 1mL of bacterial endotoxin inspection water into the bacterial endotoxin standard substance, and carrying out vortex shaking for 5 minutes to uniformly mix to obtain an E10 solution; uniformly mixing the E10 solution with bacterial endotoxin inspection water to obtain an E1 solution (the bacterial endotoxin concentration is 0.5 EU/mL); mixing 0.2mL of E1 solution with 0.2mL of bacterial endotoxin water to prepare a positive control sample solution; mixing 0.2mL of E1 solution with 0.2mL of the leaching solution obtained in the step (2) to prepare a positive test sample control sample; taking 0.2mL of bacterial endotoxin water as a negative control sample;
(4) A limulus reagent solution was obtained by dissolving 0.1mL of a limulus reagent having a sensitivity lambda of 0.25EU/mL with 0.1mL of bacterial endotoxin with water;
(5) Mixing 0.1mL of the leaching solution in the step (2) with 0.1mL of the limulus reagent solution in the step (4), respectively mixing 0.1mL of the positive control sample solution, the positive test sample solution and the negative control sample with 0.1mL of the limulus reagent solution, sealing a pipe orifice by using a sealing film after mixing, vertically placing into a drying oven at 37+/-1 ℃ and preserving heat for 60+/-2 minutes.
(6) Taking the test tube out of the drying box gently, and slowly reversing for 180 degrees, if gel is formed in the tube, and the gel is not deformed, the test tube does not slip off the tube wall, and the test tube is positive and recorded as (+); the gel not formed or formed was not firm, deformed and negative for slipping off the tube wall, recorded as (-s).
Example 2
The embodiment provides an endotoxin detection method of collagen liquid, which comprises the following steps:
(1) Providing collagen liquid obtained by degreasing, removing impurity protein, carrying out enzymolysis, salting out, dialyzing and concentrating the pigskin serving as a raw material; wherein the concentration of the collagen solution is 10.62mg/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 7.15.
(2) Taking 0.1mL of the collagen liquid obtained in the step (1), preserving the temperature at 37+/-1 ℃ to enable collagen to form collagen gel, then adding 0.2mL of bacterial endotoxin detection water, and soaking for 24 hours under the water bath preserving condition at 37+/-1 ℃.
(3) Adding 1mL of bacterial endotoxin inspection water into the bacterial endotoxin standard substance, and carrying out vortex shaking for 5 minutes to uniformly mix to obtain an E10 solution; adding the E10 solution into water for bacterial endotoxin detection, and uniformly mixing to obtain an E1 solution (the bacterial endotoxin concentration is 0.5 EU/mL); mixing 0.2mL of E1 solution with 0.2mL of bacterial endotoxin water to prepare a positive control sample solution; mixing 0.2mL of E1 solution with 0.2mL of the leaching solution obtained in the step (2) to prepare a positive test sample control sample; taking 0.2mL of bacterial endotoxin water as a negative control sample;
(4) A limulus reagent solution was obtained by dissolving 0.1mL of a limulus reagent having a sensitivity lambda of 0.25EU/mL with 0.1mL of bacterial endotoxin with water;
(5) Mixing 0.1mL of the leaching solution in the step (2) with 0.1mL of the limulus reagent solution in the step (4), respectively mixing 0.1mL of the positive control sample solution, the positive test sample solution and the negative control sample with 0.1mL of the limulus reagent solution, sealing a pipe orifice by using a sealing film after mixing, vertically placing into a drying oven at 37+/-1 ℃ and preserving heat for 60+/-2 minutes.
(6) Taking the test tube out of the drying box gently, and slowly reversing for 180 degrees, if gel is formed in the tube, and the gel is not deformed, the test tube does not slip off the tube wall, and the test tube is positive and recorded as (+); the gel not formed or formed was not firm, deformed and negative for slipping off the tube wall, recorded as (-s).
Example 3
The embodiment provides an endotoxin detection method of collagen liquid, which comprises the following steps:
(1) Providing collagen liquid obtained by degreasing, removing protein, enzymolysis, salting out, dialysis, filtering, sterilizing and concentrating pigskin serving as a raw material; wherein the concentration of the collagen liquid is 35.12mg/mL, the collagen liquid contains phosphate buffer solution (containing disodium hydrogen phosphate and sodium dihydrogen phosphate), the collagen in the collagen liquid is dissolved in the phosphate buffer solution, and the pH of the collagen liquid is 7.46.
(2) Taking 0.1mL of the collagen liquid obtained in the step (1), preserving the temperature at 37+/-1 ℃ to enable collagen to form collagen gel, then adding 0.2mL of bacterial endotoxin detection water, and soaking for 24 hours under the water bath preserving condition at 37+/-1 ℃.
(3) Adding 1mL of bacterial endotoxin inspection water into the bacterial endotoxin standard substance, and carrying out vortex shaking for 5 minutes to uniformly mix to obtain an E10 solution; adding the E10 solution into water for bacterial endotoxin detection, and uniformly mixing to obtain an E1 solution (the bacterial endotoxin concentration is 0.5 EU/mL); mixing 0.2mL of E1 solution with 0.2mL of bacterial endotoxin water to prepare a positive control sample solution; mixing 0.2mL of E1 solution with 0.2mL of the leaching solution obtained in the step (2) to prepare a positive test sample control sample; taking 0.2mL of bacterial endotoxin water as a negative control sample;
(4) A limulus reagent solution was obtained by dissolving 0.1mL of a limulus reagent having a sensitivity lambda of 0.25EU/mL with 0.1mL of bacterial endotoxin with water;
(5) Mixing 0.1mL of the leaching solution in the step (2) with 0.1mL of the limulus reagent solution in the step (4), respectively mixing 0.1mL of the positive control sample solution, the positive test sample solution and the negative control sample with 0.1mL of the limulus reagent solution, sealing a pipe orifice by using a sealing film after mixing, vertically placing into a drying oven at 37+/-1 ℃ and preserving heat for 60+/-2 minutes.
(6) Taking the test tube out of the drying box gently, and slowly reversing for 180 degrees, if gel is formed in the tube, and the gel is not deformed, the test tube does not slip off the tube wall, and the test tube is positive and recorded as (+); the gel not formed or formed was not firm, deformed and negative for slipping off the tube wall, recorded as (-s).
Example 4
The embodiment provides an endotoxin detection method of collagen liquid, which comprises the following steps:
(1) Providing collagen liquid obtained by degreasing, removing impurity protein, carrying out enzymolysis, salting out, dialyzing and concentrating the pigskin serving as a raw material; wherein the concentration of collagen in the collagen solution is 15.96mg/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 7.32.
(2) Taking 0.1mL of the collagen liquid obtained in the step (1), keeping the temperature at 37+/-1 ℃ to enable collagen to form collagen gel, adding 1mL of bacterial endotoxin detection water, and soaking for 24 hours under the water bath heat preservation condition at 37+/-1 ℃.
(3) Adding 1mL of bacterial endotoxin inspection water into the bacterial endotoxin standard substance, and carrying out vortex shaking for 5 minutes to uniformly mix to obtain an E10 solution; uniformly mixing the E10 solution with bacterial endotoxin inspection water to obtain E11 solution (bacterial endotoxin concentration is 0.5 EU/mL); mixing 0.2mL of E11 solution with 0.2mL of water for bacterial endotoxin to prepare a first positive control sample solution; mixing 0.2mL of E11 solution with 0.2mL of the leaching solution obtained in the step (2) to prepare a first positive test sample control sample; taking 0.2mL of bacterial endotoxin water as a first negative control sample;
(4) Uniformly mixing the E10 solution with bacterial endotoxin inspection water to obtain an E12 solution (the bacterial endotoxin concentration is 0.06 EU/mL); mixing 0.2mL of E12 solution with 0.2mL of bacterial endotoxin with water to prepare a second positive control sample solution; mixing 0.2mL of E12 solution with 0.2mL of the leaching solution obtained in the step (2) to prepare a second positive test sample control sample; taking 0.2mL of bacterial endotoxin water as a second negative control sample;
(5) Dissolving 0.1mL of a limulus reagent having a sensitivity lambda of 0.25EU/mL with 0.1mL of bacterial endotoxin with water to obtain a first limulus reagent solution;
(6) Dissolving 0.1mL of a limulus reagent having a sensitivity lambda of 0.03EU/mL with 0.1mL of bacterial endotoxin to obtain a second limulus reagent solution;
(7) Mixing 0.1mL of the leaching solution in the step (2) with 0.1mL of the first limulus reagent solution in the step (5), respectively mixing 0.1mL of the first positive control sample solution, the first positive test sample solution and the first negative control sample with 0.1mL of the first limulus reagent solution, sealing a pipe orifice with a sealing film after mixing, vertically placing into a drying box at 37+/-1 ℃, and preserving heat for 60 minutes+/-2 minutes;
(8) Mixing 0.1mL of the leaching solution in the step (2) with 0.1mL of the second limulus reagent solution in the step (6), respectively mixing 0.1mL of the second positive control sample solution, the second positive test sample solution and the second negative control sample with 0.1mL of the second limulus reagent solution, sealing a pipe orifice with a sealing film after mixing, vertically placing into a drying box at 37+/-1 ℃, and preserving heat for 60 minutes+/-2 minutes;
(9) Taking the test tube out of the drying box gently, and slowly reversing for 180 degrees, if gel is formed in the tube, and the gel is not deformed, the test tube does not slip off the tube wall, and the test tube is positive and recorded as (+); the gel not formed or formed was not firm, deformed and negative for slipping off the tube wall, recorded as (-s).
Comparative example 1
This comparative example provides a method for detecting endotoxin in collagen, which differs from example 1 only in that the leaching solutions in step (3) and step (5) are replaced with collagen solutions (provided by step (1)).
Comparative example 2
This comparative example provides a method for detecting endotoxin of collagen, which is different in step (2) from example 1, and the added leaching solution in step (3) and step (5) is the leaching solution of step (2) of this comparative example.
The step (2) of this comparative example is: taking 0.1mL of the collagen solution obtained in the step (1), adding 1.67mL of bacterial endotoxin inspection water at room temperature (25+/-1 ℃), and soaking for 24 hours at room temperature.
The experimental results of examples 1 to 4 and comparative examples 1 to 2 are shown in fig. 1 to 7, respectively. It should be noted that 2 parallel samples were tested for each sample.
As can be seen from the results of fig. 1 to 7, in examples 1 to 4 and comparative examples 1 to 2, the endotoxin detection results of both the positive control sample and the positive test sample control sample were positive, and the endotoxin detection results of the negative control sample were negative, indicating that the endotoxin detection experiments of examples 1 to 4 and comparative examples 1 to 2 were effective. As can be seen from the results of fig. 1 to 3, in examples 1 to 3 of the present application, the endotoxin detection results were negative, indicating that the endotoxin content in the collagen of examples 1 to 3 was less than 0.25EU/mL. The results according to FIGS. 6 and 7 show that the final endotoxin test results were positive after the extract was mixed with the limulus reagent by soaking in the endotoxin test water after the collagen solution was not formed into a collagen gel, indicating that the endotoxin test methods of comparative examples 1 and 2 easily caused false positive results.
From the results shown in FIGS. 4 and 5, it was found that the endotoxin content in the collagen liquid of example 4 was greater than 0.03EU/mL and less than 0.25EU/mL.
The foregoing is merely a specific embodiment of the present application and is not intended to limit the present application, and various modifications and variations may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (9)
1. The method for detecting endotoxin in a collagen solution is characterized by comprising the steps of:
after forming collagen gel from the collagen liquid, adding endotoxin test water for soaking, mixing the leaching solution with a limulus reagent, preserving heat at 35-38 ℃, and judging the endotoxin content of the collagen liquid according to whether the mixed solution forms gel or not.
2. The method of claim 1, wherein the step of forming the collagen gel from the collagen solution comprises: the collagen liquid is kept at a temperature of 35-38 ℃ to form the collagen gel.
3. The method of claim 2, comprising at least one of the following limitations:
first definition: the pH value of the collagen liquid is 6-8;
a second definition: the collagen liquid is obtained by degreasing animal tissues, removing impurity proteins, carrying out enzymolysis, salting out, dialyzing and concentrating;
third definition: the concentration of collagen in the collagen liquid is more than or equal to 5mg/mL; optionally, the concentration of the collagen in the collagen liquid is 5-150 mg/mL.
4. The method for detecting endotoxin in a collagen solution according to any one of claims 1 to 3, wherein the sensitivity of the limulus reagent is 0.03 to 0.5EU/mL.
5. The method for detecting endotoxin in a collagen liquid according to any one of claims 1 to 3, wherein the time for immersing in the endotoxin test water is 1 to 30 hours.
6. The method for endotoxin detection of a collagen solution according to any one of claims 1 to 3, wherein said collagen solution comprises a phosphate buffer solution, and said collagen is dissolved in said phosphate buffer solution.
7. The method for detecting endotoxin in a collagen liquid according to any one of claims 1 to 3, wherein the volume ratio of said collagen liquid to said endotoxin test water is 1:2 to 20.
8. The method for detecting endotoxin in a collagen solution according to any one of claims 1 to 3, wherein the step of mixing the extract with a limulus reagent comprises: taking said leaching solution and mixing with at least two different sensitivity of said limulus reagents respectively;
the step of judging the endotoxin content of the collagen according to whether the mixed solution forms gel or not comprises the following steps:
when the corresponding mixed solution forms gel, the endotoxin content of the collagen solution is greater than or equal to the sensitivity value of the limulus reagent;
when the corresponding mixed solution does not form gel, the endotoxin content of the collagen solution is smaller than the sensitivity value of the limulus reagent;
judging the content range of endotoxin in the collagen solution according to the sensitivity values of at least two different sensitivities of the limulus reagent added into the leaching solution.
9. The method according to any one of claims 1 to 3, further comprising providing a positive control sample, a positive test sample control sample and a negative control sample, mixing the positive control sample, the positive test sample control sample and the negative control sample with a limulus reagent, and determining whether the test is effective based on whether the positive control sample, the positive test sample control sample and the negative control sample form a gel.
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