阅读说明：本技术 一种检测含有正电荷聚合物细菌内毒素的方法 (Method for detecting bacterial endotoxin containing positive charge polymer ) 是由 张锐 鲁建国 王姣 于 2021-07-30 设计创作，主要内容包括：本发明公开了一种检测含有正电荷聚合物细菌内毒素的方法,包括如下步骤：步骤一,器皿前处理；步骤二,树脂前处理；步骤三,制备细菌内毒素阳性对照液；步骤四,制备供试品阳性液；步骤五,准备鲎试剂；步骤六,取供试品溶液、检查用水、供试品阳性液、阳性对照液,分别加入已复溶好的鲎试剂中,观察结果；采用本发明的检测方法能够排除正电荷聚合物对细菌内毒素检测过程的干扰,同时不引入外源性内毒素。(The invention discloses a method for detecting bacterial endotoxin containing a positively charged polymer, which comprises the following steps: firstly, pretreating a vessel; step two, resin pretreatment; step three, preparing a bacterial endotoxin positive control solution; step four, preparing positive liquid of the test sample; step five, preparing a limulus reagent; taking the test solution, the examination water, the test positive solution and the positive control solution, respectively adding the test solution, the examination water, the test positive solution and the positive control solution into the redissolved limulus reagent, and observing the result; the detection method can eliminate the interference of the positive charge polymer on the detection process of the bacterial endotoxin, and does not introduce exogenous endotoxin.)

1. A method for detecting bacterial endotoxin containing positively charged polymers, comprising the steps of:

firstly, pretreating a vessel, and removing exogenous endotoxin;

step two, resin pretreatment: soaking anion exchange resin and cation exchange resin with strong base and strong acid respectively, and cleaning the anion exchange resin and the cation exchange resin with water until the cleaning solution is neutral when detected by test paper; placing cation exchange resin below the filter screen, placing anion exchange resin above the filter screen, adding pyrogen-free examination water until the anion exchange resin is submerged, shaking, taking out, pouring out the anion exchange resin, cleaning the upper layer of the filter screen with the pyrogen-free examination water, and sealing and storing the cation exchange resin for later use;

step three, preparing a bacterial endotoxin positive control solution;

step four, preparing a test sample solution and a test sample positive solution;

step five, preparing a limulus reagent;

and step six, adding the test solution, the test water, the test positive solution and the positive control solution into the redissolved limulus reagent, placing the redissolved limulus reagent in a thermostat after the sample is added, preserving the heat for 60 +/-2 minutes to observe results, wherein the test is effective when the negative control tubes are negative, the positive control tubes are positive, and the test positive control tubes are positive.

2. The method for detecting bacterial endotoxin of a polymer containing positive charges in claim 1, wherein the second step comprises the steps of soaking the anion exchange resin and the cation exchange resin in strong alkali and strong acid respectively: adding 1mol/L HCl solution into cation exchange resin, soaking for not less than 1 hour, and adding 1mol/L NaOH solution into anion exchange resin, soaking for not less than 1 hour.

3. The method for detecting bacterial endotoxin containing positively charged polymer as claimed in claim 1, wherein the ratio of the anion exchange resin to the cation exchange resin is 1:1 to 5: 1.

4. The method for detecting bacterial endotoxin in polymers containing positive charges according to claim 1, wherein the method for preparing the bacterial endotoxin positive control solution comprises the following steps: taking a bacterial endotoxin working standard substance, adding detection water after opening, sealing a sealing film, placing on a vortex oscillator, mixing uniformly, adding the detection water, and gradually diluting to 0.5EU/ml and 0.25EU/ml, wherein 0.25EU/ml is a positive control solution.

5. The method of claim 1, wherein the method for preparing the test solution and the test positive solution comprises the steps of:

(1) if the sample to be detected is liquid, the sample can be directly diluted to obtain the liquid to be detected for use; if the sample to be detected is solid, the sample needs to be leached, and the leaching liquor is the liquid to be detected for later use;

(2) mixing the solution to be tested with the cation exchange resin preserved in a sealed manner in the step two for later use in a pyrogen-free test tube according to the volume mass ratio of 1:1-10:1, oscillating in a shaking table for not less than 2 hours, and finally standing for 20-60min at the temperature of 20-35 ℃;

(3) diluting the treated sample solution with water for examination to prepare a test solution under an effective dilution factor MVD (CL/lambda), wherein L is the limit value of bacterial endotoxin of the test solution, C is the concentration of the test solution, C is equal to 1.0ml/ml when L is expressed by EU/ml, the unit of C is required to be mg/ml or U/ml when L is expressed by EU/mg or EU/U, and lambda is the marking sensitivity EU/ml of limulus reagent;

(4) diluting the treated sample solution by using a bacterial endotoxin working standard solution to prepare a test article positive solution containing 2 lambda endotoxin and MVD under an effective dilution factor.

6. The method for detecting bacterial endotoxin of bacteria containing positively charged polymers as claimed in claim 1, wherein the limulus reagent is prepared by the method comprising: 0.1 ml/branch limulus reagent 5 times the amount of the test sample is added to the test water according to the labeled amount for re-dissolution and standby.

7. The method of claim 1, wherein said cation exchange resin comprises: a strongly acidic cation exchange resin or a weakly acidic cation exchange resin; the anion exchange resin is hydroxide type anion exchange resin.

8. The method of claim 1, wherein said positively charged polymer comprises: quaternary ammonium salts, biguanides, chlorhexidine, alcohols, phenols, organic amines, pyridines or isothiazolinones.

Technical Field

The invention relates to the field of pharmaceutical analysis, in particular to a method for detecting bacterial endotoxin containing a positively charged polymer.

Background

Bacterial endotoxins are cell wall components of gram-negative bacteria that release endotoxins when they die or autolyze. Thus, bacterial endotoxins are widely found in nature. The damage of endotoxins is very severe and causes: 1. the fever response, the body is extremely sensitive to bacterial endotoxins. The extremely trace (1-5 ng/kg) of endotoxin can cause the body temperature to rise; 2. leukocyte reaction, after bacterial endotoxin enters a host body, the quantity of neutrophils accounting for 60-70% of the total number of leukocytes in blood flow is rapidly reduced because cells move and adhere to tissue capillaries; 3. endotoxin shock, the action of a large amount of endotoxin on macrophages, neutrophils, a complement system, a coagulation system and the like of a body, causes microcirculatory disturbance, and causes microcirculatory failure, hypotension, hypoxia, acidosis and the like, thereby causing shock of a patient. Therefore, preparations such as biological products, injection medicines, chemical medicines, radiopharmaceuticals, antibiotics, vaccines, dialyzates and medical instruments (such as disposable syringes and implantable biomaterials) can be used after being qualified by bacterial endotoxin detection tests.

The main problems existing in the prior art are as follows: the most commonly used test method for bacterial endotoxin is the limulus reagent test, and in 1964, the American scholars Levin and Bang found that trace gram-negative bacterial endotoxin can also cause gel reaction, so that an in vitro analysis method (limulus reagent test) for qualitatively or quantitatively detecting trace bacterial endotoxin was established in 1968. The detection principle is as follows: the limulus body has a deformed cell, and the hemocyanin in the cell can activate protein coagulogen once contacting with the endotoxin of bacteria, so that the agglutination phenomenon can be caused. The limulus reagent assay is widely used because of its simple operation, but the limulus reagent assay requires that the interference of other substances in a sample with the assay be eliminated to prevent the deviation of the assay result. In particular, the polymer having a positive charge adsorbs and binds to endotoxin having a negative charge, so that endotoxin in the test solution cannot be coagulated with limulus reagent.

In the work of detecting endotoxin in bacteria, if impurity interference is encountered, a dilution method or a method for adjusting the pH value of a liquid to be detected is generally adopted so as to reduce the interference encountered in detection. However, when some medical devices, drugs or their leaching solutions contain positively charged polymers, these substances are adsorbed and bound to endotoxin after dissolution. The phenomenon of false negative during detection is usually caused by a dilution multiple method or the adjustment of the pH value of a liquid to be detected; the market needs a detection method which can eliminate the interference of a positively charged polymer on the detection process of bacterial endotoxin and does not introduce exogenous endotoxin, and the invention solves the problem.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for detecting bacterial endotoxin containing a positively charged polymer, which can eliminate the interference of the positively charged polymer on the detection process of the bacterial endotoxin and simultaneously does not introduce exogenous endotoxin.

In order to achieve the above object, the present invention adopts the following technical solutions:

a method for detecting bacterial endotoxin containing positively charged polymers comprising the steps of:

firstly, pretreating a vessel, and removing exogenous endotoxin;

step two, resin pretreatment: soaking anion exchange resin and cation exchange resin with strong base and strong acid respectively, and cleaning the anion exchange resin and the cation exchange resin with water until the cleaning solution is neutral when detected by test paper; placing cation exchange resin below the filter screen, placing anion exchange resin above the filter screen, adding pyrogen-free examination water until the anion exchange resin is submerged, shaking, taking out, pouring out the anion exchange resin, cleaning the upper layer of the filter screen with the pyrogen-free examination water, and sealing and storing the cation exchange resin for later use;

step three, preparing a bacterial endotoxin positive control solution;

step four, preparing a test sample solution and a test sample positive solution;

step five, preparing a limulus reagent;

and step six, adding the test solution, the test water, the test positive solution and the positive control solution into the redissolved limulus reagent, placing the redissolved limulus reagent in a thermostat after the sample is added, preserving the heat for 60 +/-2 minutes to observe results, wherein the test is effective when the negative control tubes are negative, the positive control tubes are positive, and the test positive control tubes are positive.

In the second step, the specific method for soaking the anion exchange resin and the cation exchange resin in strong base and strong acid respectively is as follows: adding 1mol/L HCl solution into cation exchange resin, soaking for not less than 1 hour, and adding 1mol/L NaOH solution into anion exchange resin, soaking for not less than 1 hour.

In the method for detecting bacterial endotoxin containing the positively charged polymer, the part ratio of the anion exchange resin to the cation exchange resin is 1:1-5: 1.

In the method for detecting bacterial endotoxin containing a positively charged polymer, the method for preparing the bacterial endotoxin positive control solution comprises the following steps: taking a bacterial endotoxin working standard substance, adding detection water after opening, sealing a sealing film, placing on a vortex oscillator, mixing uniformly, adding the detection water, and gradually diluting to 0.5EU/ml and 0.25EU/ml, wherein 0.25EU/ml is a positive control solution.

In the method for detecting bacterial endotoxin containing a positively charged polymer, the method for preparing the test solution and the test positive solution comprises the following steps:

(1) if the sample to be detected is liquid, the sample can be directly diluted to obtain the liquid to be detected for use; if the sample to be detected is solid, the sample needs to be leached, and the leaching liquor is the liquid to be detected for later use;

(2) mixing the solution to be tested with the cation exchange resin preserved in a sealed manner in the step two for later use in a pyrogen-free test tube according to the volume mass ratio of 1:1-10:1, oscillating in a shaking table for not less than 2 hours, and finally standing for 20-60min at the temperature of 20-35 ℃;

(3) diluting the treated sample solution with water for examination to prepare a test solution under an effective dilution factor MVD (CL/lambda), wherein L is the limit value of bacterial endotoxin of the test solution, C is the concentration of the test solution, C is equal to 1.0ml/ml when L is expressed by EU/ml, the unit of C is required to be mg/ml or U/ml when L is expressed by EU/mg or EU/U, and lambda is the marking sensitivity EU/ml of limulus reagent;

(4) diluting the treated sample solution by using a bacterial endotoxin working standard solution to prepare a test article positive solution containing 2 lambda endotoxin and MVD under an effective dilution factor.

The method for detecting bacterial endotoxin containing the positively charged polymer comprises the following steps: 0.1 ml/branch limulus reagent 5 times the amount of the test sample is added to the test water according to the labeled amount for re-dissolution and standby.

In the method for detecting bacterial endotoxin containing a positively charged polymer, the cation exchange resin comprises: a strongly acidic cation exchange resin or a weakly acidic cation exchange resin; the anion exchange resin is hydroxide type anion exchange resin.

One of the foregoing methods for detecting bacterial endotoxin containing a positively charged polymer, the positively charged polymer comprising: quaternary ammonium salts, biguanides, chlorhexidine, alcohols, phenols, organic amines, pyridines or isothiazolinones.

The invention has the advantages that:

the detection method can detect the bacterial endotoxin of the polymer sample containing positive charges, and the polymer containing positive charges in the liquid to be detected can be effectively reduced by treating the liquid to be detected through the cation exchange resin, so that a false negative detection result can be effectively avoided;

the invention has simple operation;

the invention provides a method for preparing pyrogen-free cation exchange resin.

Drawings

FIG. 1 is a schematic structural diagram of one embodiment of a detection apparatus of the present invention;

FIG. 2 is a schematic structural view of an embodiment of the inspection apparatus for resin pretreatment according to the present invention;

the meaning of the reference symbols in the figures:

1 vessel, 2 screens, 3 resins, 301 anion exchange resins, 302 cation exchange resins.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

The following examples are used to demonstrate that the present invention is effective in avoiding false negatives.

Example 1:

taking a certain care solution containing a positively charged polymer, namely polyhexamethylene biguanide hydrochloride, as a sample to be detected. The limit of endotoxin detection in the nursing solution is 0.5EU/ml, and the sensitivity of limulus reagent is 0.125 EU/ml.

The bacterial endotoxin test method was as follows:

1. pretreatment of the vessel 1: the glassware 1 and the metal tool used in the test need to be subjected to high-temperature heat treatment to remove possible exogenous endotoxin;

2. resin 3 pretreatment: respectively taking 10g of strong-acid cation exchange resin 302 (styrene-divinylbenzene sulfonic acid group cation exchange resin) and strong-base anion exchange resin 301 (styrene quaternary ammonium group anion exchange resin), putting the resins into the device shown in the figure 1, and respectively soaking the cation exchange resin and the anion exchange resin with 1mol/L HCl solution and 1mol/L NaOH solution for 2 hours to inactivate microorganisms contained in the resins and simultaneously remove bacterial endotoxin in partial resins. Then, the HCl solution and the NaOH solution are respectively poured off, and the resin is washed by water for detection for a plurality of times until the detection washing liquid of the test paper is neutral. Then, the anion exchange resin which is rinsed to be neutral is filled into the tube shown in the figure 1, the anion exchange resin is positioned on the filter screen 2, the part ratio of the anion exchange resin to the cation exchange resin is 1:1, and then a proper amount of pyrogen-free detection water is added to at least submerge the anion exchange resin, as shown in the figure 2. The tube containing the resin was placed in a shaker, shaken for 2 hours, removed and the anion exchange resin decanted, and the upper layer of the filter screen 2 was cleaned with pyrogen-free test water. And sealing and storing the cationic resin for later use.

3. Preparation of bacterial endotoxin positive control solution:

taking 1 count of bacterial endotoxin working standard substance, adding 1ml of detection water after opening, placing on a vortex oscillator after sealing a sealing film, uniformly mixing for 15 minutes, and gradually diluting to 0.5EU/ml and 0.25EU/ml by using the detection water, wherein 0.25EU/ml is a positive control solution.

4. Preparing a test solution:

and (3) mixing the care solution with the resin treated in the step (2) according to a volume-to-mass ratio of 1:1, shaking the mixture on a shaking table for 3 hours, and standing the mixture for 30min under the condition of water bath at the temperature of 30 ℃. Test solution preparation: 0.2ml of the shaking-treated liquid and 0.2ml of the test water were mixed, respectively.

Positive liquid of the test sample: 0.2ml of the concussion-treated solution was mixed with 0.2ml of a 0.5EU/ml endotoxin solution.

5. Preparation of limulus reagent:

according to the amount of the test sample, several 0.1ml limulus reagents are taken, and the test water is added according to the marked amount for re-dissolution for standby.

6. Detection of

Taking 0.1ml of each of the test solution, the test water, the test positive solution and the positive control solution, respectively adding the test solution, the test water, the test positive solution and the positive control solution into the reconstituted limulus reagent obtained in the step 5, placing the limulus reagent in a thermostat after the sample is added, keeping the temperature for 60 +/-2 minutes, observing the result, and when the negative control tubes are negative, the positive control tubes are positive, and the test positive control tubes are positive, the test is effective.

Example 2:

taking a wet dressing containing a positively charged polymer-biguanide polymer as a sample to be tested. The limit of endotoxin detection of the dressing is 20EU/ml, and the sensitivity of the limulus reagent is 0.25 EU/ml.

The steps of the method for checking the bacterial endotoxin are the same as those of the embodiment 1, and the part ratio of the anion-cation exchange resin to the cation-anion exchange resin in the step 2 is 5: 1; mixing the wet dressing leaching liquor in the step 4 with the resin treated in the step 2 according to a volume-to-mass ratio of 5: 1;

example 3:

taking a certain liquid dressing containing a positively charged polymer-chlorhexidine as a sample to be tested. The liquid dressing is used for endotoxin detection, the limit value is 20EU/ml, and the sensitivity of the limulus reagent is 0.5 EU/ml.

The steps of the method for checking the bacterial endotoxin are the same as those of the embodiment 1, and the part ratio of the anion-cation exchange resin to the cation-anion exchange resin in the step 2 is 3: 1; step 4, mixing the liquid dressing with the resin treated in the step 2 according to a volume mass ratio of 10: 1;

comparative example 1: the same as example 1 except for the absence of resin pretreatment.

Taking a certain care solution containing a positively charged polymer, namely polyhexamethylene biguanide hydrochloride, as a sample to be detected. The limit of endotoxin detection in the nursing solution is 0.5EU/ml, and the sensitivity of limulus reagent is 0.125 EU/ml.

The bacterial endotoxin test method was as follows:

1. pretreatment of the vessel 1: the glassware and metal tools used in the test need to be subjected to high-temperature heat treatment to remove possible exogenous endotoxin;

3. preparation of bacterial endotoxin positive control solution:

taking 1 count of bacterial endotoxin working standard substance, adding 1ml of detection water after opening, placing on a vortex oscillator after sealing a sealing film, uniformly mixing for 15 minutes, and gradually diluting to 0.5EU/ml and 0.25EU/ml by using the detection water, wherein 0.25EU/ml is a positive control solution.

4. Preparing a test solution:

mixing the care solution and cation exchange resin at a volume-to-mass ratio of 1:1, shaking on a shaking table for 3 hours, and standing in a water bath at 30 deg.C for 30 min. Test solution preparation: 0.2ml of the shaking-treated liquid and 0.2ml of the test water were mixed, respectively.

Positive liquid of the test sample: 0.2ml of the concussion-treated solution was mixed with 0.2ml of a 0.5EU/ml endotoxin solution.

5. Preparation of limulus reagent:

according to the amount of the test sample, several 0.1ml limulus reagents are taken, and the test water is added according to the marked amount for re-dissolution for standby.

6. Detection of

Taking 0.1ml of each of the test solution, the test water, the test positive solution and the positive control solution, respectively adding into the reconstituted limulus reagent obtained in the step 5, placing in a thermostat after the sample addition is finished, preserving the temperature for 60 +/-2 minutes, and observing the result, wherein the results are shown in table 1:

TABLE 1

	Positive control	Negative control	Test solution	Positive liquid for test article
					Example 1	+	—	—	+
Example 2	+	—	—	+
					Example 3	+	—	—	+
Comparative example 1	+	—	—	—

From the above experiments, it can be seen that: the positive test solution of comparative example 1 without pretreatment has a negative result, resulting in an invalid test result. The invention can effectively reduce the polymer containing positive charges in the liquid to be detected and effectively avoid false negative detection results by treating the liquid to be detected by the cation exchange resin.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.