文档库 最新最全的文档下载
当前位置:文档库 › 内毒素USP36

内毒素USP36

内毒素USP36
内毒素USP36

90?81? Antibiotics—Microbial Assays / Biological Tests USP 36

Table A2-1. Test for Outlier Measurements

In samples from a normal population, gaps equal to or larger than the following values of G1, G2, and G3 occur with a probability P = 0.01, when outlier measurements can occur only at one end; or with P = 0.02, when they may occur at either end.

N34567

G10.9870.8890.7810.6980.637

N8910

G20.6810.6340.597

N111213

G30.6740.6430.617

shows no reaction with the lysate employed, at the detec-?85? BACTERIAL ENDOTOXINS tion limit of the reagent.

Lysate TS—Dissolve Amoebocyte Lysate in Water for BET, TEST or in a buffer recommended by the lysate manufacturer, by

gentle stirring. Store the reconstituted lysate, refrigerated or

frozen, according to the specifications of the manufacturer. x Portions of this general chapter have been harmonized

with the corresponding texts of the European Pharmacopoeia PREPARATION OF SOLUTIONS

and/or the Japanese Pharmacopoeia. Those portions that are

not harmonized are marked with symbols (x x) to specify this

Standard Endotoxin Stock Solution—A Standard Endo-fact.x

toxin Stock Solution is prepared from a USP Endotoxin Refer-The Bacterial Endotoxins Test (BET) is a test to detect or

ence Standard that has been calibrated to the current WHO quantify endotoxins from Gram-negative bacteria using

International Standard for Endotoxin. Follow the specifica-amoebocyte lysate from the horseshoe crab (Limulus poly-

tions in the package leaflet and on the label for preparation phemus or Tachypleus tridentatus).

and storage of the Standard Endotoxin Stock Solution. Endo-There are three techniques for this test: the gel-clot tech-

toxin is expressed in Endotoxin Units (EU). [NOTE—One USP nique, which is based on gel formation; the turbidimetric

Endotoxin Unit (EU) is equal to one International Unit (IU) technique, based on the development of turbidity after

of endotoxin.]

cleavage of an endogenous substrate; and the chromogenic

technique, based on the development of color after cleav-Standard Endotoxin Solutions—After mixing the Stan-age of a synthetic peptide-chromogen complex. Proceed by dard Endotoxin Stock Solution vigorously, prepare appropriate any of the three techniques for the test. In the event of serial dilutions of Standard Endotoxin Solution, using Water doubt or dispute, the final decision is made based upon the for BET. Use dilutions as soon as possible to avoid loss of

gel-clot limit test unless otherwise indicated in the mono-activity by adsorption.

graph for the product being tested. The test is carried out in Sample Solutions—Prepare the Sample Solutions by dis-a manner that avoids endotoxin contamination.solving or diluting drugs using Water for BET. Some sub-

stances or preparations may be more appropriately dis-

solved, or diluted in other aqueous solutions. If necessary, APPARATUS adjust the pH of the solution to be examined (or dilution

thereof) so that the pH of the mixture of the lysate and Depyrogenate all glassware and other heat-stable materi-Sample Solution falls within the pH range specified by the als in a hot air oven using a validated process.x1x A com-lysate manufacturer, usually 6.0–8.0. The pH may be ad-monly used minimum time and temperature is 30 min at justed by use of an acid, base, or suitable buffer as recom-250°. If employing plastic apparatus, such as microplates mended by the lysate manufacturer. Acids and bases may and pipet tips for automatic pipetters, use apparatus that is be prepared from concentrates or solids with Water for BET shown to be free of detectable endotoxin and does not in-in containers free of detectable endotoxin. Buffers must be terfere in the test. [NOTE—In this chapter, the term “tube”validated to be free of detectable endotoxin and interfering includes any other receptacle such as a microtiter well.]factors.

REAGENTS AND TEST SOLUTIONS DETERMINATION OF MAXIMUM VALID

DILUTION (MVD) Amoebocyte Lysate—A lyophilized product obtained

from the lysate of amoebocytes (white blood cells) from the The maximum valid dilution is the maximum allowable horseshoe crab (Limulus polyphemus or Tachypleus dilution of a specimen at which the endotoxin limit can be tridentatus). This reagent refers only to a product manufac-determined. Determine the MVD from the following

tured in accordance with the regulations of the competent equation:

authority. [NOTE—Amoebocyte Lysate reacts to some β-glu-

cans in addition to endotoxins. Amoebocyte Lysate prepara-MVD = (endotoxin limit × concentration of Sample Solution)/ tions that do not react to glucans are available: they are(λ)

prepared by removing the G factor reacting to glucans from

Amoebocyte Lysate or by inhibiting the G factor reacting sys-Endotoxin Limit—The endotoxin limit for parenteral tem of Amoebocyte Lysate and may be used for endotoxin drugs, defined on the basis of dose, equals K/M

x2

testing in the presence of glucans.]

x, where K Water for Bacterial Endotoxins Test (BET)—Use Water

x2

intrathecal (for which K is 0.2 USP-EU/kg of body weight). For radiopharma-for Injection or water produced by other procedures that ceutical products not administered intrathecally, the endotoxin limit is calcu-

lated as 175 EU/V, where V is the maximum recommended dose in mL. For x1Dry-intrathecally administered radiopharmaceuticals, the endotoxin limit is ob-Heat Sterilization under Sterilization and Sterility Assurance of Compendial Arti-tained by the formula 14 EU/V. For formulations (usually anticancer products) cles ?1211?. Use Lysate TS having a sensitivity of not less than 0.15 Endotoxin administered on a per square meter of body surface, the formula is K/M,

Unit per mL.x where K = 100 EU/m

2 and M is the maximum dose/m2.x

USP 36

Biological Tests / ?85? Bacterial Endotoxins Test 91

is a threshold pyrogenic dose of endotoxin per kg of body turn directly from the incubator, and invert it through about weight, and M is equal to the maximum recommended bo-180° in one smooth motion. If a firm gel has formed that lus dose of product per kg of body weight. When the prod-remains in place upon inversion, record the result as posi-uct is to be injected at frequent intervals or infused continu-tive. A result is negative if an intact gel is not formed. The ously, M is the maximum total dose administered in a single test is considered valid when the lowest concentration of hour period. The endotoxin limit for parenteral drugs is the standard solutions shows a negative result in all replicate specified in the individual monograph in units such as EU/tests.

mL, EU/mg, EU/Unit of biological activity, etc.The endpoint is the smallest concentration in the series of decreasing concentrations of standard endotoxin that clots Concentration of Sample Solution—

the lysate. Determine the geometric mean endpoint by cal-mg/mL: in the case of endotoxin limit specified by weight culating the mean of the logarithms of the endpoint con-(EU/mg);

centrations of the four replicate series and then taking the Units/mL: in the case of endotoxin limit specified by unit antilogarithm of the mean value, as indicated in the follow-of biological activity (EU/Unit);

ing formula:

mL/mL: when the endotoxin limit is specified by volume (EU/mL).

geometric mean endpoint concentration = antilog (Σe /f )λ: the labeled sensitivity in the Gel-Clot Technique (EU/mL)or the lowest concentration used in the standard curve for where Σe is the sum of the log endpoint concentrations of the Turbidimetric Technique or Chromogenic Technique .

the dilution series used, and f is the number of replicate test tubes. The geometric mean endpoint concentration is the measured sensitivity of the lysate (in EU/mL). If this is not GEL-CLOT TECHNIQUE

less than 0.5λ and not more than 2λ, the labeled sensitivity is confirmed and is used in tests performed with this lysate.The gel-clot technique is used for detecting or quantifying endotoxins based on clotting of the lysate reagent in the Test for Interfering Factors—Usually prepare solutions presence of endotoxin. The minimum concentration of en-(A–D) as shown in Table 1, and perform the inhibition/en-dotoxin required to cause the lysate to clot under standard hancement test on the Sample Solutions at a dilution less conditions is the labeled sensitivity of the lysate reagent. To than the MVD, not containing any detectable endotoxins,ensure both the precision and validity of the test, perform operating as described for Test for Confirmation of Labeled the tests for confirming the labeled lysate sensitivity and for Lysate Sensitivity . The geometric mean endpoint concentra-interfering factors as described in Preparatory Testing , imme-tions of Solutions B and C are determined using the formula diately below.

described in the Test for Confirmation of Labeled Lysate Sensi-tivity . The test for interfering factors must be repeated when any condition changes that is likely to influence the result of Preparatory Testing

the test.

The test is considered valid when all replicates of Solu-Test for Confirmation of Labeled Lysate Sensitivity—tions A and D show no reaction and the result of Solution C Confirm in four replicates the labeled sensitivity, λ, ex-confirms the labeled sensitivity.

pressed in EU/mL of the lysate prior to use in the test. The If the sensitivity of the lysate determined in the presence test for confirmation of lysate sensitivity is to be carried out of Solution B is not less than 0.5λ and not greater than 2λ,when a new batch of lysate is used or when there is any the Sample Solution does not contain factors that interfere change in the test conditions that may affect the outcome under the experimental conditions used. Otherwise, the of the test. Prepare standard solutions having at least four Sample Solution to be examined interferes with the test.concentrations equivalent to 2λ, λ, 0.5λ, and 0.25λ by di-If the sample under test does not comply with the test at luting the USP Endotoxin RS with Water for BET .

a dilution less than the MVD, repeat the test using a greater Mix a volume of the Lysate TS with an equal volume dilution, not exceeding the MVD. The use of a more sensi-(such as 0.1-mL aliquots) of one of the Standard Endotoxin tive lysate permits a greater dilution of the sample to be Solutions in each test tube. When single test vials or ampuls examined, and this may contribute to the elimination of containing lyophilized lysate are used, add solutions directly interference.

to the vial or ampul. Incubate the reaction mixture for a Interference may be overcome by suitable treatment such constant period according to the directions of the lysate as filtration, neutralization, dialysis, or heating. To establish manufacturer (usually at 37±1° for 60±2 min), avoiding that the chosen treatment effectively eliminates interference vibration. To test the integrity of the gel, take each tube in

without loss of endotoxins, perform the assay described

Table 1. Preparation of Solutions for the Inhibition/Enhancement Test for Gel-Clot Techniques

Endotoxin Concentration/Solution to Which Endotoxin

Dilution Endotoxin Number of Solution

Is Added

Diluent Factor Concentration

Replicates

A a None/Sample Solution —

——4B b

2λ/Sample Solution

Sample Solution

1 2λ421λ440.5λ48

0.25λ4C c 2λ/Water for BET Water for BET 1

2λ221λ240.5λ28

0.25λ2D d None/Water for BET ———

2

a Solution A : A Sample Solution of the preparation under test that is free of detectable endotoxins.

b Solution B : Test for interference.

c Solution C : Control for labele

d lysat

e sensitivity.d Solution D : Negative control o

f Water for BET .

92?85? Bacterial Endotoxins Test / Biological Tests USP 36

above using the preparation to be examined to which Stan-be repeated using a greater dilution, not exceeding the dard Endotoxin has been added and which has then been MVD.

submitted to the chosen treatment.

Quantitative Test

Limit Test

Procedure—The test quantifies bacterial endotoxins in Procedure—Prepare Solutions A, B, C, and D as shown in Sample Solutions by titration to an endpoint. Prepare Solu-Table 2, and perform the test on these solutions following tions A, B, C, and D as shown in Table 3, and test these

the procedure above for Preparatory Testing, Test for Confir-solutions by following the procedure in Preparatory Testing, mation of Labeled Lysate Sensitivity.Test for Confirmation of Labeled Lysate Sensitivity.

Calculation and Interpretation—The test is considered

valid when the following three conditions are met: (1) Both Table 2. Preparation of Solutions for the Gel-Clot Limit Test

replicates of negative control Solution D are negative; (2) Endotoxin Concentration/Both replicates of positive product control Solution B are Solution to Which Number of positive; and (3) The geometric mean endpoint concentra-Solution*Endotoxin Is Added Replicates tion of Solution C is in the range of 0.5λ to 2λ.

A None/Diluted Sample Solution2To determine the endotoxin concentration of Solution A,

B2λ/Diluted Sample Solution2calculate the endpoint concentration for each replicate by

multiplying each endpoint dilution factor by λ. The endo-C2λ/Water for BET2

toxin concentration in the Sample Solution is the endpoint

D None/Water for BET2

concentration of the replicates. If the test is conducted with *Prepare Solution A and the positive product control Solution B using a diluted Sample Solution, calculate the concentration of en-a dilution not greater than the MVD and treatments as described for dotoxin in the original Sample Solution by multiplying by the the Test for Interfering Factors in Preparatory Testing. The positive con-dilution factor. If none of the dilutions of the Sample Solu-trol Solutions B and C contain the Standard Endotoxin Solution at a tion is positive in a valid assay, report the endotoxin concen-concentration corresponding to twice the labeled lysate sensitivity.tration as less than λ (if the diluted sample was tested, re-The negative control Solution D consists of Water for BET.port as less than λ times the lowest dilution factor of the

sample). If all dilutions are positive, the endotoxin concen-

tration is reported as equal to or greater than the greatest Interpretation—The test is considered valid when both dilution factor multiplied by λ (e.g., initial dilution factor replicates of Solutions B and C are positive and those of Solu-times eight times λ in Table 3).

tion D are negative. When a negative result is found for The preparation under test meets the requirements of the both replicates of Solution A, the preparation under test test if the concentration of endotoxin in both replicates is complies with the test. When a positive result is found for less than that specified in the individual monograph.

both replicates of Solution A, the preparation under test

does not comply with the test.

When a positive result is found for one replicate of Solu-PHOTOMETRIC QUANTITATIVE TECHNIQUES tion A and a negative result is found for the other, repeat

the test. In the repeat test, the preparation under test com-

plies with the test if a negative result is found for both repli-Turbidimetric Technique

cates of Solution A. The preparation does not comply with

the test if a positive result is found for one or both replicates

This technique is a photometric assay measuring increases of Solution A. However, if the preparation does not comply

in reactant turbidity. On the basis of the particular assay with the test at a dilution less than the MVD, the test may

principle employed, this technique may be classified as ei-

ther an endpoint-turbidimetric assay or a kinetic-turbidimet-

Table 3. Preparation of Solutions for the Gel-Clot Assay

Endotoxin Concentration/

Solution to Which Endotoxin Dilution Endotoxin Number of Solution Is Added Diluent Factor Concentration Replicates

A a None/Sample Solution Water for BET1 —2

2—2

4—2

8—2

B b2λ/Sample Solution—12λ2

C c2λ/Water for BET Water for BET1 2λ2

21λ2

4 0.5λ2

80.25λ2

D d None/Water for BET———2

a Solution A: Sample Solution under test at the dilution, not to exceed the MVD, with which the Test for Interfering Factors was completed. Subsequent dilution of the Sample Solution must not exceed the MVD. Use Water for BET to make a dilution series of four tubes containing the Sample Solution under test at concentrations of 1, 1/2, 1/4, and 1/8 relative to the concentration used in the Test for Interfering Factors. Other dilutions up to the MVD may be used as appropriate.

b Solution B: Solution A containing standard endotoxin at a concentration of 2λ (positive product control).

c Solution C: Two replicates of four tubes of Water for BET containing the standar

d endotoxin at concentrations of 2λ, λ, 0.5λ, and 0.25λ, respectively.

d Solution D: Water for BET (negativ

e control).

USP 36

Biological Tests / ?85? Bacterial Endotoxins Test 93

ric assay. The endpoint-turbidimetric assay is based on the cate, according to the instructions for the lysate employed,quantitative relationship between the concentration of en-for example, concerning volume of Sample Solution and Ly-dotoxins and the turbidity (absorbance or transmission) of sate TS, volume ratio of Sample Solution to Lysate TS, incu-the reaction mixture at the end of an incubation period.bation time, etc.

The kinetic-turbidimetric assay is a method to measure ei-The test is considered valid when the following conditions ther the time (onset time) needed to reach a predetermined are met.

absorbance or transmission of the reaction mixture, or the 1.The absolute value of the correlation coefficient of the rate of turbidity development. The test is carried out at the standard curve generated using Solution C is greater incubation temperature recommended by the lysate manu-than or equal to 0.980.

facturer (which is usually 37±1°).

2.The result with Solution D does not exceed the limit of the blank value required in the description of the lysate reagent employed, or it is less than the endo-Chromogenic Technique

toxin detection limit of the lysate reagent employed.Calculate the mean recovery of the added endotoxin by This technique is an assay to measure the chromophore subtracting the mean endotoxin concentration in the solu-released from a suitable chromogenic peptide by the reac-tion, if any (Solution A , Table 4), from that containing the tion of endotoxins with lysate. On the basis of the particular added endotoxin (Solution B , Table 4). In order to be consid-assay principle employed, this technique may be classified as ered free of factors that interfere with the assay under the either an endpoint-chromogenic assay or a kinetic-chromo-conditions of the test, the measured concentration of the genic assay. The endpoint-chromogenic assay is based on endotoxin added to the Sample Solution must be within the quantitative relationship between the concentration of 50%–200% of the known added endotoxin concentration endotoxins and the release of chromophore at the end of after subtraction of any endotoxin detected in the solution an incubation period. The kinetic-chromogenic assay is a without added endotoxin.

method to measure either the time (onset time) needed to When the endotoxin recovery is out of the specified

reach a predetermined absorbance of the reaction mixture,range, the Sample Solution under test is considered to con-or the rate of color development. The test is carried out at tain interfering factors. Then, repeat the test using a greater the incubation temperature recommended by the lysate dilution, not exceeding the MVD. Furthermore, interference manufacturer (which is usually 37±1°).

of the Sample Solution or diluted Sample Solution not to ex-ceed the MVD may be eliminated by suitable validated treatment such as filtration, neutralization, dialysis, or heat Preparatory Testing

treatment. To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform To assure the precision or validity of the turbidimetric and the assay described above, using the preparation to be ex-chromogenic techniques, preparatory tests are conducted to amined to which Standard Endotoxin has been added and verify that the criteria for the standard curve are valid and which has then been submitted to the chosen treatment.

that the sample solution does not interfere with the test.Validation for the test method is required when conditions that are likely to influence the test result change.

Test Procedure

Assurance of Criteria for the Standard Curve—The test must be carried out for each lot of lysate reagent. Using the Follow the procedure described for Test for Interfering Fac-Standard Endotoxin Solution, prepare at least three endotoxin tors under Preparatory Testing , immediately above.

concentrations within the range indicated by the lysate manufacturer to generate the standard curve. Perform the Calculation

assay using at least three replicates of each standard endo-toxin concentration according to the manufacturer’s instruc-Calculate the endotoxin concentration of each of the rep-tions for the lysate (volume ratios, incubation time, temper-licates of Solution A , using the standard curve generated by ature, pH, etc.). If the desired range is greater than two logs the positive control Solution C . The test is considered valid in the kinetic methods, additional standards should be in-when the following three requirements are met.

cluded to bracket each log increase in the range of the stan- 1.The results of the control Solution C comply with the dard curve. The absolute value of the correlation coeffi-requirements for validation defined for Assurance of cient, r , must be greater than or equal to 0.980 for the Criteria for the Standard Curve under Preparatory range of endotoxin concentrations set up.

Testing .

Test for Interfering Factors—Select an endotoxin con- 2.The endotoxin recovery, calculated from the concen-centration at or near the middle of the endotoxin standard tration found in Solution B after subtracting the con-curve. Prepare Solutions A, B, C, and D as shown in Table 4.centration of endotoxin found in Solution A , is within Perform the test on Solutions A, B, C, and D at least in dupli-

the range of 50%–200%.

Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques

Solution to Which

Solution

Endotoxin Concentration Endotoxin Is Added Number of Replicates

A a None Sample Solution Not less than 2

B b

Middle concentration of the standard curve Sample Solution Not less than 2At least three concentrations (lowest concentration is

C c designated λ)Water for BET Each not less than 2

D d None Water for BET Not less than 2a Solution A : The Sample Solution may be diluted not to exceed MVD.

b Solution B : The preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the standard curve.

c Solution C : The standar

d endotoxin at th

e concentrations used in the validation o

f the method described for Assurance of Criteria for the Standard Curve under Preparatory Testin

g (positive controls). d Solution D : Water for BET (negative control).

94?85? Bacterial Endotoxins Test / Biological Tests USP 36

3.The result of the negative control Solution D does not Apparatus—

exceed the limit of the blank value required in the Autoclave—Employ an autoclave capable of maintaining a description of the lysate employed, or it is less than temperature of 121±2°, equipped with a thermometer, a the endotoxin detection limit of the lysate reagent pressure gauge, a vent cock, a rack adequate to accommo-employed.date the test containers above the water level, and a water

cooling system that will allow for cooling of the test con-

tainers to about 20°, but not below 20°, immediately fol-Interpretation

lowing the heating cycle.

Oven—Use an oven, preferably a mechanical convection In photometric assays, the preparation under test com-

model, that will maintain operating temperatures in the plies with the test if the mean endotoxin concentration of

range of 50° to 70° within ±2°.

the replicates of Solution A, after correction for dilution and

concentration, is less than the endotoxin limit for the Incubator—Use an incubator capable of maintaining a product.temperature of 37±1° and a humidified atmosphere of

5±1% carbon dioxide in air.

Extraction Containers—Use only containers, such as am-

puls or screw-cap culture test tubes, or their equivalent, of

Type I glass. If used, culture test tubes, or their equivalent,

are closed with a screw cap having a suitable elastomeric

liner. The exposed surface of the elastomeric liner is com-?87? BIOLOGICAL REACTIVITY pletely protected with an inert solid disk 50 to 75 μm in

thickness. A suitable disk can be fabricated from polytef.

TESTS, IN VITRO Preparation of Apparatus—Cleanse all glassware thor-

oughly with chromic acid cleansing mixture and, if neces-

sary, with hot nitric acid followed by prolonged rinsing with

Sterile Water for Injection. Sterilize and dry by a suitable The following tests are designed to determine the biologi-

process containers and devices used for extraction, transfer, cal reactivity of mammalian cell cultures following contact

or administration of test material. If ethylene oxide is used with the elastomeric plastics and other polymeric materials

as the sterilizing agent, allow not less than 48hours for with direct or indirect patient contact or of specific extracts

complete degassing.

prepared from the materials under test. It is essential that

the tests be performed on the specified surface area. When Procedure—

the surface area of the specimen cannot be determined, use Preparation of Sample for Extracts—Prepare as directed in 0.1g of elastomer or 0.2g of plastic or other material for the Procedure under Biological Reactivity Tests, In Vivo ?88?. every mL of extraction fluid. Exercise care in the preparation Preparation of Extracts—Prepare as directed for Prepara-

of the materials to prevent contamination with microorgan-tion of Extracts in Biological Reactivity Tests, In Vivo ?88? using isms and other foreign matter.either Sodium Chloride Injection (0.9% NaCl) or serum-free Three tests are described (i.e., the Agar Diffusion Test, the mammalian cell culture media as Extraction Solvents. [N

OTE—Direct Contact Test, and the Elution Test).* The decision as to If extraction is done at 37° for 24hours in an incubator, use

which type of test or the number of tests to be performed cell culture media supplemented by serum. The extraction to assess the potential biological response of a specific sam-conditions should not in any instance cause physical

ple or extract depends upon the material, the final product,changes, such as fusion or melting of the material pieces, and its intended use. Other factors that may also affect the other than a slight adherence.]

suitability of sample for a specific use are the polymeric

composition; processing and cleaning procedures; contact-

ing media; inks; adhesives; absorption, adsorption, and per-Agar Diffusion Test

meability of preservatives; and conditions of storage. Evalua-

tion of such factors should be made by appropriate This test is designed for elastomeric closures in a variety additional specific tests before determining that a product of shapes. The agar layer acts as a cushion to protect the made from a specific material is suitable for its intended use.cells from mechanical damage while allowing the diffusion

of leachable chemicals from the polymeric specimens. Ex-USP Reference Standards ?11?—USP High-Density Poly-

tracts of materials that are to be tested are applied to a ethylene RS. USP Positive Bioreaction RS.

piece of filter paper.

Cell Culture Preparation—Prepare multiple cultures of L-

Sample Preparation—Use extracts prepared as directed or 929 (ATCC cell line CCL 1, NCTC clone 929) mammalian

use portions of the test specimens having flat surfaces not fibroblast cells in serum-supplemented minimum essential

less than 100mm2 in surface area.

medium having a seeding density of about 105 cells per mL.

Incubate the cultures at 37±1° in a humidified incubator Positive Control Preparation—Proceed as directed for Sam-for not less than 24hours in a 5±1% carbon dioxide at-ple Preparation.

mosphere until a monolayer, with greater than 80% conflu-Negative Control Preparation—Proceed as directed for ence, is obtained. Examine the prepared cultures under a Sample Preparation.

microscope to ensure uniform, near-confluent monolayers.Procedure—Using 7mL of cell suspension prepared as di-[N OTE—The reproducibility of the In Vitro Biological Reactivity rected under Cell Culture Preparation, prepare the monolay-Tests depends upon obtaining uniform cell culture density.]ers in plates having a 60-mm diameter. Following incuba-Extraction Solvents—Sodium Chloride Injection (see mon-tion, aspirate the culture medium from the monolayers, and ograph—use Sodium Chloride Injection containing 0.9% of replace it with serum-supplemented culture medium con-NaCl). Alternatively, serum-free mammalian cell culture me-taining not more than 2% of agar. [N OTE—The quality of dia or serum-supplemented mammalian cell culture media the agar must be adequate to support cell growth. The agar may be used. Serum supplementation is used when extrac-layer must be thin enough to permit diffusion of leached tion is done at 37° for 24hours.chemicals.] Place the flat surfaces of Sample Preparation,

Negative Control Preparation, and Positive Control Preparation *Further details are given in the following publications of the American Soci-

ety for Testing and Materials, 1916 Race St., Philadelphia, PA 19103: “Stan-or their extracts in an appropriate extracting medium, in dard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity,”duplicate cultures in contact with the solidified agar surface. ASTM Designation F 895-84; “Standard Practice for Direct Contact Cell Cul-Use no more than three specimens per prepared plate. Incu-ture Evaluation of Materials for Medical Devices,” ASTM Designation bate all cultures for not less than 24hours at 37±1°, prefer-F 813-83.

抗菌药诱导的内毒素释放作用

论文关键词: 抗菌药内毒素青霉素结合蛋白 论文摘要: 一些实验研究及临床资料证明,有些抗菌药尤其是β-内酰胺类抗生素在治疗革兰氏阴性菌感染时,有诱导大量内毒素释放的副作用,在临床上可能引起或加重内毒素血症。不同类型抗菌药诱导内毒素释放的程度与其对不同类型青霉素结合蛋白(pbp)的亲和性有关,抗菌药与pbp3或pbp2结合所造成的内毒素释放的量较高。因此在抗菌药的研究设计中,除了要考虑其杀菌效力外,还应考虑可能诱导内毒素释放的问题。在败血症治疗中也应采取合理的治疗方案,以防止或减轻内毒素血症的发生。 内毒素是革兰氏阴性菌细胞壁外膜中的脂多糖成分,具有广泛的生物活性。内毒素对机体造成双重影响:少量内毒素刺激机体时,能增强特异性及非特异性免疫力,诱导产生干扰素等;而大量内毒素进入机体循环时,可造成广泛而强烈的病理反应,如弥漫性血管内凝血、多器官功能衰竭及休克等。内毒素在革兰氏阴性杆菌引起的感染性休克中起着关键作用[1-3]。对于革兰氏阴性菌败血症病人,有些广谱抗菌药能有效地控制菌血症,但是临床资料证明病人的死亡率仍然很高,其原因可能与抗菌药诱导细菌释放内毒素有关[4,5]。由于在抗菌药治疗的过程中,随着细菌的裂解,内毒素从细菌的细胞壁外膜释放后进入血液循环,因而可能加重内毒素血症,从而促进一系列的炎症反应,造成机体严重而广泛的病理损伤。 1 抗菌药诱导的内毒素释放 在体外细菌培养系统中,已发现多种抗菌药如β-内酰胺类[6~8]、氨基糖苷类[9]、氟喹诺酮类药物[10]等均能引起不同程度的内毒素释放,其中β-内酰胺类抗生素(如氨苄西林,头孢噻肟,头孢他啶,氨曲南等)能诱导多种细菌包括野生菌和突变菌(如大肠埃希氏菌,铜绿假单胞菌[6~8],肺炎克雷伯氏菌[11],流感嗜血菌[12]等)释放大量的内毒素,在这些抗菌药处理的细菌培养上清液中能检测到内毒素含量比无抗菌药处理者高几倍甚至几十倍。β-内酰胺类抗生素诱导的内毒素释放常常与其造成的细菌溶解伴随发生,并与细菌溶解程度有一定关系。内毒素-细胞因子级联反应所造成的全身炎症过程是发生内毒素休克的重要环节。抗菌药作用于细菌后,由于细菌裂解及伴随的内毒素释放到培养上清液中,其上清液可刺激巨噬细胞及血管内皮细胞等产生大量的肿瘤坏死因子(tnf)和白细胞介素-6(il-6)[13~15],这些炎性因子的大量产生可促进内毒素休克的发生。 有些动物体内实验可观察到与体外实验一致的结果。给大肠埃希氏菌性腹膜炎家兔注射庆大霉素后,动物血液中细菌数量急剧减少,但血浆中内毒素含量较未治疗者明显增高[9]。用氨苄西林治疗幼年大鼠流感嗜血菌性腹膜炎,造成较多的内毒素释放入血液循环[12]。烧伤后感染肺炎克雷伯氏菌的败血症小鼠,经头孢他啶、氨曲南、亚胺培南治疗后,小鼠血液中内毒素含量明显增高,同时内毒素激活巨噬细胞产生大量的il-6[16]。大肠埃希氏菌性脑膜炎家兔,经美罗培南和头孢噻肟治疗后,在脑脊液中可检测到高含量的内毒素,同时脑脊液中tnf 浓度也增高[17]。不同类型的抗菌药所诱导的内毒素释放水平有所不同,如拉氧头孢对菌血症的清除作用与庆大霉素相似,但其诱导内毒素释放的量比庆大霉素高20倍[18]。用d-半乳糖胺处理小鼠后,再造成大肠埃希氏菌性腹膜炎模型,发现用头孢他啶治疗比用亚胺培南治疗引起更多的小鼠死亡,推测头孢他啶诱导更多的内毒素释放[19]。 虽然动物体内的研究结果不能外推到人,但这些研究证据提示了抗菌药促进内毒素释放,加重内毒素血症的可能性。有些革兰氏阴性菌败血症患者在接受抗菌药治疗后,尽管血液中细菌数减少或消失,但大部分病人呈现血浆中游离及总内毒素含量增高(游离内毒素含量升高2~50倍)[5,20]。流感嗜血菌感染的脑膜炎患儿,在接受头孢他啶治疗后,所有患儿脑脊液中游 离内毒素含量均增高,同时脑脊液中乳酸盐及乳酸脱氢酶增高及葡萄糖下降,提示内毒素增高导致炎症加重[21]。用抗菌药治疗泌尿系感染后,尿液培养显示细菌数下降,但尿中内毒素含量却明显增高。内毒素释放的程度与所用的抗菌药种类有关,其中β-内酰胺类抗生素所

中国药典版《细菌内毒素检查法》.pdf

中国药典XXXX年版《细菌内毒素检查法》 ——凝胶法 凝胶法 凝胶法系通过鲎试剂与内毒素产生凝集反应的原理来检测或半定量内毒素的方法。 鲎试剂灵敏度复核试验在本检查法规定的条件下,使鲎试剂产生凝集的内毒素的最低浓度即为鲎试剂的标示灵敏度,用EU/ml表示。当使用新批号的鲎试剂或试验条件发生了任何可能影响检验结果的改变时,应进行鲎试剂灵敏度复核试验。 根据鲎试剂灵敏度的标示值(λ),将细菌内毒素国家标准品或细菌内毒素工作标准品用细菌内毒素检查用水溶解,在旋涡混合器上混匀15分钟,然后制成2λ、λ、0.5λ和0.25λ四个浓度的内毒素标准溶液,每稀释一步均应在旋涡混合器上混匀30秒钟。取分装有0.1ml鲎试剂溶液的10mm×75mm试管或复溶后的0.1ml/支规格的鲎试剂原安瓿18支,其中16管分别加入0.1ml不同浓度的内毒素标准溶液,每一个内毒素浓度平行做4管;另外2管加入0.1ml细菌内毒素检查用水作为阴性对照。将试管中溶液轻轻混匀后,封闭管口,垂直放入37℃±1℃恒温器中,保温60分钟±2分钟。 将试管从恒温器中轻轻取出,缓缓倒转180°,若管内形成凝胶,并且凝胶不变形、不从管壁滑脱者为阳性;未形成凝胶或形成的凝胶不坚实、变形并从管壁滑脱者为阴性。保温和拿取试管过程应避免受到振动造成假阴性结果。 当最大浓度2λ管均为阳性,最低浓度0.25λ管均为阴性,阴性对照管为阴性,试验方为有效。按下式计算反应终点浓度的几何平均值,即为鲎试剂灵敏度的测定值(λc). λc=1g-1(∑X/4)

式中 X为反应终点浓度的对数值(1g)。反应终点浓度是指系列递减的内毒素浓度中最后一个呈阳性结果的浓度。 当λc在0.5λ-2λ(包括0.5λ和2λ)时,方可用于细菌内毒素检查,并以标示灵敏度λ为该批鲎试剂的灵敏度。 干扰试验按表1制备溶液A、B、C和D,使用的供试品溶液应为未检验出内毒素且不超过最大有效稀释倍数(MVD)的溶液,按鲎试剂灵敏度复核试验项下操作。 只有当溶液A和阴性对照溶液D的所有平行管都为阴性,并且系列溶液C 的结果在鲎试剂灵敏度复核范围内时,试验方为有效。按下式计算系列溶液C和B的反应终点浓度的几何平均值(Es和Et)。 Es= 1g-1(∑Xs/4) Et= 1g-1(∑Xt/4) 式中,Xs、Xt分别为系列溶液C和溶液B的反应终点浓度的对数值(1g)。当Es在0.5λ—2λ(包括0.5λ和2λ)及Et在0.5Es—2Es (包括0.5Es 和2Es)时,认为供试品在该浓度下无干扰作用。若供试品溶液在小于MVD 的稀释倍数下对试验有干扰,应将供试品溶液进行不超过MVD的进一步稀释,再重复干扰试验。 表1 凝胶法干扰试验溶液的制备

内毒素和外毒素

内毒素和外毒素

1.外毒素产生菌主要是革兰阳性菌中的破伤风梭菌、肉毒梭菌、白喉杆菌、产气荚膜梭菌、A群链球菌、金黄色葡萄球菌等。某些革兰阴性菌中的痢疾志贺菌、鼠疫耶氏菌、霍乱弧菌、肠产毒素型大肠埃希菌、铜绿假单胞菌等也能产生外毒素。大多数外毒素是在菌细胞内合成后分泌至细胞外;也有存在于菌体内,待菌溶溃后才释放出来的,痢疾志贺菌和肠产毒素型大肠埃希菌的外毒素属此。 外毒素的毒性强。1mg肉毒毒素纯品能杀死2亿只小鼠,毒性比KCN大1万倍。不同细菌产生的外毒素,对机体的组织器官具有选择作用,各引起特殊的病变。例如肉毒毒素能阻断胆碱能神经末梢释放乙酰胆碱,使眼和咽肌等麻痹,引起眼睑下垂、复视、斜视、吞咽困难等,严重者可因呼吸麻痹而死。又如白喉毒素对外周神经末梢、心肌等有亲和性,通过抑制靶细胞蛋白质的合成而导致外周神经麻痹和心肌炎等。 多数外毒素不耐热。例如白喉外毒素在58—60℃经1—2h,破伤风外毒素在60℃经20min可被破坏。但葡萄球菌肠毒素是例外,能耐100℃30min。大多外毒素是蛋白质,具有良好的抗原性。在0.3%—0.4%甲醛液作用下,经一定时间,可以脱去毒性,但仍保有免疫原性,是为类毒素(toxoid)。类毒素注入机体后,可刺激机体产生具有中和外毒素作用的抗毒素抗体。类毒素和抗毒素在防治一些传染病中有实际意义,前者主要用于人工主动免疫,后者常用于治疗和紧急预防。 多数外毒素的分子结构为A-B模式,即由A和B两种亚单位组成。A亚单位是外毒素活性部分,决定其毒性效应。B亚单位无毒,能与宿主靶细胞表面的特殊受体结合,介导A亚单位进入靶细胞。A或B亚单独对宿主无致病作用,因而外毒素分子的完整性是致病的必要条件。利用B亚单位能与靶细胞受体结合后阻止受体再与完整外毒素分子结合,且B亚单位抗原性强;将B亚单位提纯制成疫苗,有可能预防相关的外毒素性疾病。 根据外毒素对宿主细胞的亲和性及作用方式等,可分成神经毒素、细胞毒

细菌内毒素检查标准操作规程..

细菌内毒素检查标准操作规程 1 简述 1.1 本规范适用于中国药典2005年版附录中细菌内毒素检查法一凝胶法和光度测定法。后者包括浊度法和显色基质法。供试品检测时,可使用其中任何一种方法进行实验。当 测定结果有争议时,除另有规定外,以凝胶法结果为准。 1.2 供试品细菌毒素限值的确定。 (一)药典中有规定的,按供试品各论中规定限值; (二)尚无标准规定的,按以下公式确定供试品内毒素限值: L=K/M 式中 L为供试品的细菌内毒素限值,以EU/ml、EU/mg、EU/U表示。 K为按规定的给药途径,人用每公斤体重每小时最大可接受的内毒素剂量,以EU/kg/h表示。其中注射剂,K=5EU/kg/h;放射性药品注射剂,K=2.5EU/kg/h;鞘内用注射剂, K=0.2EU/kg/h。 M为人用每公斤体重每小时的最大供试品剂量,以ml/kg/h、ml/kg/h、U/kg/h表示。药品人用最大剂量可参阅国家批准的药品说明书和《临床用药须知》等权威著作,中国人 均体重按60kg计算,注射时间小于1小时的按1小时计。按人用剂量计算限值时,如遇特殊情况,可根据生产和临床用实际情况做必要调整,但需说明理由。 1.3 供试品最大有效稀释倍数的确定 供试品的最大有效稀释倍数(MV D)按下式计算: MV D=C?L/λ L为供试品的细菌内毒素限值;C为供试品溶液的浓度。当L以EU/ml表示时,C等于1.0ml/ml;当L的单位以EU/mg或EU/U表示时,C为供试品制备成溶液后的浓度,单位为mg/ml 或U/ml。如供试品为注射用无菌粉末或原料药,则MV D取1,可计算供试品的最小有效稀释浓度C: λ/L。

外毒素和内毒素

外毒素和内毒素是细茵产生的两大类毒素物质。外毒素是病原菌在代谢过程中分泌到菌体外的物质。产生外毒素的细菌主要是一些革兰氏阳性细菌,例如金黄色葡萄球菌、白喉杆菌、破伤风杆菌等。少数革兰氏阴性菌如霍乱弧菌和产毒性大肠杆菌等也能产生外毒素。我们把产生外毒素的细菌接种到液体培养基中培养,经过滤除培养液中的细菌,即可得到外毒素。 外毒素的化学成分是蛋白质。毒性极不稳定,对热和某些化学物质敏感,容易受到破坏。用3%~4%的甲醛溶液处理,其毒性完全消失,可制成类毒素。外毒素的抗原性较强,能刺激机体产生抗毒素。 外毒素毒性很强,例如纯化的肉毒杆菌外毒素,1毫克可以杀死2000万只小鼠,对人的最小致死量为0.1微克,其毒性比氰化钾强1万倍。 细菌产生的外毒素对组织的毒性作用有高度的选择性,各自引起特殊的临床症状。如白喉杆菌产生的白喉外毒素,能抑制人体细胞蛋白质的合成,使细胞变性死亡,导致心肌炎、肾上腺出血和神经麻痹;破伤风杆菌产生的是破伤风外毒素,作用到脊髓和脑,引起肌肉的痉挛和强直;霍乱杆菌产生的肠毒素作用到小肠粘膜,使粘膜细胞分泌功能加强,引起严重的呕吐和腹泻。 内毒素是革兰氏阴性细菌细胞壁的组成成分、细菌在生活时不能释放出来,当细胞死亡而溶解或用人工方法破坏菌体时才释放出来,因而称为内毒素。常用超声波处理细菌或反复冻融细菌的方法制备内毒素。 内毒素化学成分比较复杂,它是磷酸-多糖-蛋白质的复合物。主要成分为脂多糖。其性质较稳定、耐热、毒性比外毒素低、其作用没有组织器官选择性,不同病原菌所产生的内毒素引起的症状大致相同,都能引起机体体温升高、腹泻和出现出血性休克和其他组织损伤现象。 外毒素和内毒素的区别如下表:

内毒素和外毒素

For personal use only in study and research; not for commercial use 内毒素和外毒素

1.外毒素产生菌主要是革兰阳性菌中的破伤风梭菌、肉毒梭菌、白喉杆菌、产气荚膜梭菌、A群链球菌、金黄色葡萄球菌等。某些革兰阴性菌中的痢疾志贺菌、鼠疫耶氏菌、霍乱弧菌、肠产毒素型大肠埃希菌、铜绿假单胞菌等也能产生外毒素。大多数外毒素是在菌细胞内合成后分泌至细胞外;也有存在于菌体内,待菌溶溃后才释放出来的,痢疾志贺菌和肠产毒素型大肠埃希菌的外毒素属此。 外毒素的毒性强。1mg肉毒毒素纯品能杀死2亿只小鼠,毒性比KCN大1万倍。不同细菌产生的外毒素,对机体的组织器官具有选择作用,各引起特殊的病变。例如肉毒毒素能阻断胆碱能神经末梢释放乙酰胆碱,使眼和咽肌等麻痹,引起眼睑下垂、复视、斜视、吞咽困难等,严重者可因呼吸麻痹而死。又如白喉毒素对外周神经末梢、心肌等有亲和性,通过抑制靶细胞蛋白质的合成而导致外周神经麻痹和心肌炎等。多数外毒素不耐热。例如白喉外毒素在58—60℃经1—2h,破伤风外毒素在60℃经20min可被破坏。但葡萄球菌肠毒素是例外,能耐100℃30min。大多外毒素是蛋白质,具有良好的抗原性。在0.3%—0.4%甲醛液作用下,经一定时间,可以脱去毒性,但仍保有免疫原性,是为类毒素(toxoid)。类毒素注入机体后,可刺激机体产生具有中和外毒素作用的抗毒素抗体。类毒素和抗毒素在防治一些传染病中有实际意义,前者主要用于人工主动免疫,后者常用于治疗和紧急预防。多数外毒素的分子结构为A-B模式,即由A和B两种亚单位组成。A亚单位是外毒素活性部分,决定其毒性效应。B 亚单位无毒,能与宿主靶细胞表面的特殊受体结合,介导A亚单位进入靶细胞。A或B亚单独对宿主无致病作用,因而外毒素分子的完整性是致病的必要条件。利用B亚单位能与靶细胞受体结合后阻止受体再与完整外毒素分子结合,且B 亚单位抗原性强;将B亚单位提纯制成疫苗,有可能预防相关的外毒素性疾病。 根据外毒素对宿主细胞的亲和性及作用方式等,可分成神经毒素、细胞毒素和肠毒素三大类。细菌的外毒素多数为A-B型分子结构,这类外毒素的作用机制不完全相同,又可分为几种类型:(1)具腺苷二磷酸核糖基转

细菌内毒素的检测

细菌内毒素的检测 摘要:本文研究了注射用头孢哌酮钠他唑巴坦钠细菌内毒素的检测。 本文是在通过调查大量的科技文献的基础上,按照中国药典2000版二部附录XI E细菌内毒素检查法所规定的试验方法而进行的。实验方案是根据注射用头孢哌酮钠他唑巴坦钠的细菌内毒素限度,并结合现有的实验条件而确定的。注射用头孢哌酮钠他唑巴坦钠(Cefoperazone Sodium and Tazuobatanna)为头孢类抗生素头孢哌酮与β-内酰胺酶抑制剂他唑巴坦钠组成的复方注射用无菌粉针,在临床上用于治疗下呼吸道感染、泌尿生殖系统感染、腹腔盆腔感染、以及其他感染。质量标准[国家食品药品监督管理局标准(试行)YBH0538 2003]中控制热原的方法仍为家兔热原法,鉴于家兔热原法有影响因素复杂、操作繁琐、检验成本高等缺点,而用细菌内毒素检查法来控制产品中的热原具有操作简便快捷、检验灵敏度高等优点[1]。为了在大范围内开展本品的细菌内毒素检查,我们对本品进行了细菌内毒素检查凝胶法的研究。本品细菌内毒素限值为0.15Eu/ml,在1.667至0.400mg/ml的浓度范围内,本品对细菌类毒素与鲎试剂的反应无干扰。我们对3批样品进行了检验,并与家兔热原法进行对比,结果一致都为阴性,认为本品可用细菌内毒素检查凝胶法代替家兔热原法。

关键词:注射用头孢哌酮钠他唑巴坦钠细菌内毒素鲎试剂灵敏度复核试验干扰预试验干扰实验热原检查 0引言 细菌内毒素是革兰氏阴性菌细胞壁上的一种脂多糖(Lipoply Saccharide)和微量蛋白(Protein)的复合物,它的特殊性不是细菌或细菌的代谢产物,而是细菌死亡或解体后才释放出来的一种具有内毒素生物活性的物质。其化学成分广泛分布于革兰氏阴性菌(如大肠杆菌、布氏杆菌、伤寒杆菌、变形杆菌、沙门氏菌等)及其它微生物(如衣原体、立克次氏体、螺旋体等)的细胞壁层的脂多糖,其化学成份主要是由O-特异性链、核心多糖、类脂A三部分组成。(附图1) 附图1 内毒素脂多糖结构示意图 <一>、O—特异性链:位于脂多糖分子最外层的多糖链,是由3—5个单糖(一般不多于25个)连成为一个多糖链。其单糖包括戊糖、氨基戊糖、已糖、氨基已糖、脱氧已糖等,单糖的种类、位置和排列顺序和空间构型,因菌种不同而异。因此,它决定菌体热原的特异性。

内毒素外毒素区别

细菌外毒素和内毒素 外毒素和内毒素是细茵产生的两大类毒素物质。外毒素是病原菌在代谢过程中分泌到菌体外的物质。产生外毒素的细菌主要是一些革兰氏阳性细菌,例如金黄色葡萄球菌、白喉杆菌、破伤风杆菌等。少数革兰氏阴性菌如霍乱弧菌和产毒性大肠杆菌等也能产生外毒素。我们把产生外毒素的细菌接种到液体培养基中培养,经过滤除培养液中的细菌,即可得到外毒素。 外毒素的化学成分是蛋白质。毒性极不稳定,对热和某些化学物质敏感,容易受到破坏。用3%~4%的甲醛溶液处理,其毒性完全消失。外毒素的抗原性较强,能刺激机体产生抗毒素。 的毒性很强,例如纯化的肉毒杆菌外毒素,1毫克可以杀死2000万只小鼠,对人的最小致死量为0.1微克,其毒性比氰化钾强1万倍。 细菌产生的外毒素对组织的毒性作用有高度的选择性,各自引起特殊的临床症状。如白喉杆菌产生的白喉外毒素,能抑制人体细胞蛋白质的合成,使细胞变性死亡,导致心肌炎、肾上腺出血和神经麻痹;破伤风杆菌产生的是破伤风外毒素,作用到脊髓和脑,引起肌肉的痉挛和强直;霍乱杆菌产生的肠毒素作用到小肠粘膜,使粘膜细胞分泌功能加强,引起严重的呕吐和腹泻。 内毒素是革兰氏阴性细菌细胞壁的组成成分、细菌在生活时不能释放出来,当细胞死亡而溶解或用人工方法破坏菌体时才释放出来,因而称为内毒素。常用超声波处理细菌或反复冻融细菌的方法制备内毒素。 1 / 3下载文档可编辑

内毒素化学成分比较复杂,它是磷酸一多糖一蛋白质的复合物。主要成分为脂多糖。其性质较稳定、耐热、毒性比外毒素低、其作用没有组织器官选择性,不同病原菌所产生的内毒素引起的症状大致相同,都能引起机体体温升高、腹泻和出现出血性休克和其他组织损伤现象。 外毒素和内毒素的区别如下表: 2 / 3下载文档可编辑

QIAGEN 中文版去内毒素大提试剂盒

纯化高转染级别的质粒DNA 【工作台流程】BENCH PROTOCOL 准备工作: 1.将RNase A 溶液加入P1缓冲液(Buffer P1) 2.加40ml96-100%乙醇(ethanol)于试剂盒的去内毒素水中(endotoxin-fre water) 3.可选:加 LyseBlue 试剂于 Buffer P1 4.检查 Buffer P2是否有SDS沉淀precipitation 有沉淀可在37摄氏度下暖化溶解 5.Buffer P3 置于4摄氏度中预冷pre-chill 6.细菌扩增培养 补充: 1.准备4-6个50ml新的或灭菌的离心管用于冷冻离心机下离心过夜菌液,2个用于接下来 的菌块重悬和与buffer混合 2.1个无内毒素的30ml聚丙烯(不推荐聚碳酸酯,因为不耐乙醇)管子(最好用圆底的离 心管以利于高速离心)用于收集洗提的质粒DNA 3.5个1.5ml的EP管用于收集抽提过程各步骤的产物用于实验后分析和质量控制 4.准备1个冰浴盒用于步骤6 5.准备室温下的异丙醇10.5ml 6.4摄氏度冰冻离心机和分光光度仪,琼脂糖凝胶电泳 步骤:peocedure A.细菌培养、收获和裂解bacterial culture,harvest & lysis 1.100ml高拷贝high-copy或250ml低拷贝low-copy过夜overnight LB培养菌液于冰冻离心机4摄氏度;6000×g;15min 从新鲜的抗生素平板上挑取一个单菌落,2-5ml抗生素LB液体初始培养基30摄氏度孵化约8小时(振荡300rpm,注意孵化容器容积至少4倍于培养液) 抗生素LB液体培养基稀释初始培养液到1:500-1000。(高拷贝质粒100-200ul初始培养液加于100ml培养基;低拷贝质粒250-500ul初始培养液加于250ml培养基)37摄氏度300rpm振荡12-16小时。(孵化容器容积至少4倍于培养液,培养至细菌密度约3-4×109/ml,即离心后菌块湿重约3g/L培养基) 培养基配方:10g蛋白胨+5g酵母+10g氯化钠溶于800ml蒸馏水,用1 N NaOH 调pH值至7.0,用蒸馏水调整至1L。 可将离心后菌块-20摄氏度下冰冻保存暂时终止实验。

如何去除内毒素

3.2.1原本法用于去除实验用玻璃器皿上的内毒素。将玻璃器皿置于烤箱中,加热到250℃,持续30min,即可去除内毒素。 3.2.2方法与步骤1、内毒素去除效率的评估(1)制备浓度分别为1000和1000EU/mL 的CSE溶液。按内毒素测量方法检测这些溶液。(2)向所有待去除内毒素的玻璃器皿滴入1mL的CSE溶液。(每种类型的玻璃器皿至少有3EU的CSE)。(3)将玻璃器皿置放烤箱中,在60℃下,干烤持续30min,烘干,然后在烤箱中,250℃下,干烤30min。(4)加入适量的LAL水,剧烈振荡5min,以清洗玻璃器皿,然后测量清洗后的LAL水中的内毒素的浓度。(5)比较原来的内毒素含量和现存的内毒素浓度。当内毒素含量至少减少了3—log时,该方法有效。必要时,重复操作,去除内毒素。用鲎度验法计算内毒素去除的百分比。2、去除实验玻璃器皿上的内毒素确定可使内毒素玻璃器皿上的内毒素对于用来配置无同一内毒素溶液的玻璃器皿,重复操作,直至内毒素含量下降3—log。(例如,1000 IEU和10000 10EU)数个加热循环后,用前述的鲎试验法,测定内毒素含量。3、对照阴性对照:用只盛有LAL水的玻璃器皿,在250℃下,干烤30min并用LAL法测量内毒素的含量。阳性对照:干燥(60℃,30min)每种玻璃器皿中的内毒素溶液,不经过干烤(250℃,30min)去内毒素的操作,然后测定其内毒素质量。 3.2.3质控与提示(1)保烤箱散热均匀,为此,在操作过程中,可将玻璃器皿放置在烤箱中的每一层上,当温度在250℃后,温度的波动不要超过±15℃。(2)用含100EU的内毒素过行的检测,可用来确定去内毒素的效率。用含10000EU的内毒素进行的检测,可用来确定对内毒素含量较高的器皿去除内毒素的效率。(3)如果如果不理想,可增加每一次操作的时间和操作次数。(4)其他技术适用于培养基的内毒素,例如,超滤,反向渗透,但比较昂贵。选取有效的滤过膜,确保去内毒素后,培养基的营养成分不丢失并且内毒素含量下降3—log。

内毒素LAL检测使用说明(中文版)

Page 1 前协议 定量检测协议 ToxinSensor?显色 LAL 内毒素检测试剂盒 执行所有的测试步骤,在室温下。操作前请使用认证的内毒素免费产品。 样品制备 所有材料或稀释剂用于标本收集和准备,测试试剂必须无内毒素。在任何时候都使用无菌技术。待测样品必须存储在所有细菌活动受阻的方式中。 例如,样品之前使用,24 小时内可以存储在 2-8 ° C,但长期使用需要冻结。 pH 溶解或稀释试样用 LAL 试剂水。 由于 LAL 内毒素反应是依赖 ph的,故样品的 pH 值应为 6-8,确保良好的线性度。因此,如有需要,我们推荐使用氢氧化钠 (0.1 N,溶解在LAL试剂水中) 或盐酸 (0.1 N,LAL 试剂水稀释)调pH 值。 I.试剂制备、 鲎试剂溶解物 (LAL) 再造冻干裂解液加 1.7 毫升 LAL 试剂水。轻轻打旋每个组成30 秒,避免起泡。再造裂解液如果存储在-20 ° C,可以保持稳定一周,不推荐tonger 存储。

避免反复冻融。 Page 2 继续ToxinSensor? 显色 LAL 内毒素检测试剂盒 显色底物 重建衬底加入1.7 毫升 LAL 试剂水浓度至~ 2 mM。一旦重建,底物溶液存储在2-8 ° c可以保持稳定一个月。防止底物溶液长时间直接接触光。 终止液 重组颜色稳定剂 #1 (终止液)用 10 毫升的缓冲液。重组终止液如果存储在 2-8° c可以保持稳定一周。 重组颜色稳定剂 #2 和 #3 : 每个添加 10 ml LAL 水。每个重组液在 2-8 ° c下可以保持稳定一周。 标准内毒素的溶液 此试剂盒中提供的冻干的内毒素标准量应参考内毒素标准液瓶上的标签。 加 2ml的 LAL 试剂水溶解冻干的内毒素。 涡漩充分混匀 15 分钟以获得内毒素贮备液。重组后的内毒素贮备液若存储在 2-8 ° C可保持稳定一周 请不要冻结内毒素贮备液。 Page3 II。试剂制备,继续准备 1 EU/ml 内毒素溶液,使标准系列稀释。

去内毒素实验方法介绍

去内毒素质粒提取Protocol 内毒性也称为脂多糖或LPS,是革兰氏阴性(如大肠杆菌,E.coli)胞膜上一种成分。细菌外膜的外部脂质成分完全由内毒素分子组成。一个E.coli包含约2百万个LPS分子,每个LPS 分子又由疏水性的脂质A、复杂的多糖链以及带负电荷的磷酸基团组成。因此每个内毒素既包含疏水区域也包含了亲水和带电区域,从而赋予其与其它分子相互作用的独特性质。 图1. 内毒素结构图。 细菌在其活跃生长时表面的内毒素成分较少,而一旦其死亡则会释放大量内毒素。在质粒提取的裂解过程中,内毒素会从细菌的外膜释放到裂解液中。内毒素的存在会严重的影响质粒转染细胞的效率,此外会激活造血细胞(如B细胞、巨噬细胞等)的非特异免疫反应,造成实验的假阳性,所以转染级质粒的提取纯化必须去除内毒素。 内毒素如何测定? 历史上,内毒素测定主要是基于内毒素与鲎(一种海洋节支动物,也称马蹄型蟹)血液中的变形细胞冻融后的溶解物(鲎试剂)接触时可发生凝血反应。鲎试剂与细菌内毒素产生凝胶反应的机理是:鲎的血液及淋巴液中有一种有核的变形细胞,胞浆内有大量的致密颗粒,内含凝固酶及凝固蛋白原。当内毒素与鲎变形细胞冻融后的溶解物(鲎试剂)接触时,可激活凝固酶原,继而使可溶性的凝固蛋白原变成凝固蛋白而使鲎变形细胞冻融物呈凝胶状态。现在已经发展出更加灵敏的光度计测试方法,该方法主要基于鲎试剂以及一种人工合成的可产生颜色反应的底物。 LPS污染通常用内毒素单位(Endotoxin Units, EU)表示,通常情况下,1ng LPS对应于1到10个EU。

Protocol 常用的试剂盒选择包括Qiagen、Sigma都挺好用的。或者omega、天根等。在选择取出内毒素的质粒提取试剂盒的时候有一个小窍门,就是有的盒子上写着“Endofree”字样,这种试剂盒是专门用来提取用于细胞转染实验的质粒的。一般这种质粒提取试剂盒采用独特的硅胶膜吸附技术,高效专一地结合质粒DNA。同时采用特殊的溶液P4和过滤柱CS,可以有效的去除内毒素、蛋白等杂质。没有这个字样的试剂盒,对于常规的分子克隆实验,如,PCR,酶切,克隆等完全够用了。 如果不是特别的试验,建议采用手工方法,丁香园战友推荐以下的protocol提的质粒免疫动物没有问题,曾用于制备DNA疫苗,可进行大量的去内毒素质粒提取。 1)挑取一个新鲜菌落接种于含5ml LB及相应抗生素的试管,37℃培养8-12小时。 2)用上述新鲜培养物小提质粒鉴定后,剩余的菌液接种于含相应抗生素的400ml LB培养基中,37℃培养12-16小时。 3)用500ml离心筒收集菌液,5000rpm离心10分钟,弃上清。 4)用40ml预冷的STE重悬菌体,转移至2支30ml离心管中,6000rpm 5分钟,弃上清。 5)用3ml预冷的溶液I重悬菌体,再加入6ml新鲜配制的溶液II,轻轻颠倒混匀;再加入4.5ml预冷的溶液III,轻轻颠倒混匀。 6)4℃12000rpm离心10分钟,将上清转移至另外的30ml管中。 7)加等体积的异丙醇,颠倒混匀,置于-20℃20分钟以上。 8)4℃12000rpm离心15分钟,弃上清,70%乙醇洗涤沉淀,37℃蒸发至沉淀边缘透明。 9)加入5ml TE使DNA溶解后,加入5mol/L的LiCl溶液5ml,轻轻混匀,-20℃放置5-10分钟。 10)4℃12000rpm离心15分钟,转移上清,加入等体积的异丙醇,-20℃放置20分钟以上。 11)4℃12000rpm离心15分钟,弃上清,用70%的乙醇洗涤沉淀,倒置控干后,37℃蒸发至沉淀基本透明。 12)用2ml TE溶解沉淀,并加入50μl/管的RNase(5mg/ml),37℃消化过夜。

去内毒素的方法及检查方法

去内毒素的方法及检查方法 注:这部分内容为内毒素的检测方法及去除方法。 热原(pyrogen)是微生物产生的内毒素,是由磷脂、脂多糖和蛋白质组成的复合物,微量即可引起恒温动物体温异常升高。其中脂多糖具有很强的热原活性。由革兰氏阴性杆菌产生的热原致热能力最强,真菌、病毒也可以产生热原。 【相关链接】热原的危害 一、热原的性质及除去热原的方法 1.高温法 热原的耐热性能良好,60℃加热1h不被分解破坏,100℃不降解,但180℃3~4h、200℃60min或250℃30~45min可使热原彻底破坏。因此耐热物品如玻璃制品、金属制品、生产过程中所用的容器和其它用具以及注射时使用的注射器等,均可采用此法破坏热原。但在通常使用的注射剂热压灭菌条件下不足以破坏热原。 2.吸附法 热原在水溶液中可被活性炭、石棉、白陶土等吸附而除去。由于活性炭性质稳定、吸附性强兼具助滤和脱色作用,故广泛用于注射剂生产,但应注意吸附药液所造成的主药的损失。 3.超滤法 热原分子量为1×106左右,体积较小,约1~5nm,可以通过一般滤器和微孔滤膜,但采用超滤法如用 3.0~15nm超滤膜可将其除去。

4.蒸馏法 热原能溶于水但不挥发,但可随水蒸气的雾滴进入注射用水中,因此制备注射用水时,原水中的热原可经蒸馏除去,但需多次蒸馏,,并加有隔沫装置,单次蒸馏往往效果不理想。 5.酸碱法 热原能被强酸、强碱、强氧化剂破坏。玻璃容器及用具如配液用玻璃器皿、输液瓶等可用重铬酸钾硫酸清洁液或稀氢氧化钠处理,破坏热原。 6.其它 包括离子交换法、凝胶滤过法、反渗透法等。 二、热原的检查方法 《中国药典》2005年版规定热原检查采用家兔法,细菌内毒素检查采用鲎试剂法。 1.热原检查法 由于家兔对热原的反应与人基本相似,目前家兔法仍为各国药典规定的检查热原的法定方法。 《中国药典》2005年版规定的热原检查法系将一定剂量的供试品,静脉注入家兔体内,在规定时间内,观察家兔体温升高的情况,以判定供试品中所含热原的限度是否符合规定。检查结果的准确性和一致性取决于试验动物的状况、试验室条件和操作的规范性。家兔法检测内毒素的灵敏度为0.001μg/ml,试验结果接近人体真实情况,但操作繁琐费时,不能用于注射剂生产过程中的质量监控,且不适用

内毒素测定方法

2005年版中国药典《细菌内毒素检查法》 来源--中华人民共和国药典2005年版附录Ⅺ E 本法系利用鲎试剂来检测或量化由革兰阴性菌产生的细菌内毒素,以判断供试品中细菌内毒素的限量是否符合规定的一种方法。 细菌内毒素检查包括两种方法,即凝胶法和光度测定法,后者包括浊度法和显色基质法。供试品检测时,可使用其中任何一种方法进行试验。当测定结果有争议时,除另有规定外,以凝胶法结果为准。 细菌内毒素的量用内毒素单位(EU)表示。 细菌内毒素国家标准品系自大肠杆菌提取精制而成,用于标定、复核、仲裁鲎试剂灵敏度和标定细菌内毒素工作标准品的效价。 细菌内毒素工作标准品系以细菌内毒素国家标准品为基准标定其效价,用于试验中鲎试剂灵敏度复核、干扰试验及设置的各种阳性对照。 凝胶法细菌内毒素检查用水系指内毒素含量小于0.015EU/ml的灭菌注射用水。定量测定用的细菌内毒素检查用水,其内毒素的含量应小于0.005EU/ml。 试验所用的器皿需经处理,以去除可能存在的外源性内毒素。常用的方法是在250℃干烤至少30分钟,也可用其他确证不干扰细菌内毒素检查的适宜方法。若使用塑料器械,如微孔板和与微量加样器配套的吸头等,应选用标明无内毒素并且对试验无干扰的器械。试验操作过程应防止微生物的污染。 供试品溶液的制备某些供试品需进行复溶、稀释或在水性溶液中浸提制成供试品溶液。一般要求供试品溶液的PH值在6.0-8.0的范围内。对于过酸、过碱或本身有缓冲能力的供试品,需调节被测溶液(或其稀释液)的PH值,可使用酸、碱溶液或鲎试剂生产厂家推荐的适宜的缓冲剂调节PH值。酸或碱溶液须用检查用水在已去除内毒素的容器中进行配制。缓冲液必须经过验证不含内毒素和干扰因子。 内毒素限值的建立药品、生物制品的细菌内毒素限值(L)一般按以下公式确定: L=K/M 式中L为供试品的细菌内毒素限值,以EU/ml、EU/mg或EU/U(活性单位)表示; K为人每公斤体重每小时最大可接受的内毒素剂量,以EU/(kg?h)表示,注射剂K=5E U/(kg?h),放射性药品注射剂K=2.5 EU/(kg?h),鞘内用注射剂K=0.2 EU/(kg?h)。 M为人用每公斤体重每小时最大剂量,以ml(kg?h)、mg (kg?h)或U/ (kg?h)表示,人均体重按60kg计算,注射时间若不足1小时,按1小时计算。 按人用剂量计算限值时,如遇特殊情况,可根据生产和临床用药实际情况做必要调整,但需说明理由。 确定最大有效稀释倍数(MVD)最大有效稀释倍数是指供试品溶液被允许稀释的最大倍数,在不超过此稀释倍数下可进行内毒素限值的检测。用以下公式来确定MVD: MVD=CL/λ 式中L为供试品的细菌内毒素限值; C为供试品溶液的浓度,当L以EU/ml表示时,则C等于1.0ml/ml,当L以EU/mg或EU/U表示时,C的单位需为mg/ml或U /ml。如供试品为注射用无菌粉末或原料药,则MVD 取1,计算供试品的最小有效浓度C=λ/L。 λ为在凝胶法中鲎试剂的标示灵敏度(EU/ml),或是在光度测定法中所使用的标准曲线上最低的内毒素浓度。 方法1 凝胶法

内毒素外毒素区别

内毒素外毒素区别 This model paper was revised by the Standardization Office on December 10, 2020

细菌外毒素和内毒素 外毒素和内毒素是细茵产生的两大类毒素物质。外毒素是病原菌在代谢过程中分泌到菌体外的物质。产生外毒素的细菌主要是一些革兰氏阳性细菌,例如金黄色葡萄球菌、白喉杆菌、破伤风杆菌等。少数革兰氏阴性菌如霍乱弧菌和产毒性大肠杆菌等也能产生外毒素。我们把产生外毒素的细菌接种到液体培养基中培养,经过滤除培养液中的细菌,即可得到外毒素。 外毒素的化学成分是蛋白质。毒性极不稳定,对热和某些化学物质敏感,容易受到破坏。用3%~4%的甲醛溶液处理,其毒性完全消失。外毒素的抗原性较强,能刺激机体产生抗毒素。 的毒性很强,例如纯化的肉毒杆菌外毒素,1毫克可以杀死2000万只小鼠,对人的最小致死量为0.1微克,其毒性比氰化钾强1万倍。 细菌产生的外毒素对组织的毒性作用有高度的选择性,各自引起特殊的临床症状。如白喉杆菌产生的白喉外毒素,能抑制人体细胞蛋白质的合成,使细胞变性死亡,导致心肌炎、肾上腺出血和神经麻痹;破伤风杆菌产生的是破伤风外毒素,作用到脊髓和脑,引起肌肉的痉挛和强直;霍乱杆菌产生的肠毒素作用到小肠粘膜,使粘膜细胞分泌功能加强,引起严重的呕吐和腹泻。 内毒素是革兰氏阴性细菌细胞壁的组成成分、细菌在生活时不能释放出来,当细胞死亡而溶解或用人工方法破坏菌体时才释放出来,因而称为内毒素。常用超声波处理细菌或反复冻融细菌的方法制备内毒素。 内毒素化学成分比较复杂,它是磷酸一多糖一蛋白质的复合物。主要成分为脂多糖。其性质较稳定、耐热、毒性比外毒素低、其作用没有组织器官选择性,不同病原菌所产生

内毒素检测

说明书1份1份 封板膜2片(48)2片(96) 密封袋1个1个 酶标包被板1×481×962-8℃保存标准品:0.9Eu/L0.5ml×1瓶0.5ml×1瓶2-8℃保存标准品稀释液 1.5ml×1瓶 1.5ml×1瓶2-8℃保存酶标试剂 3 ml×1瓶 6 ml×1瓶2-8℃保存样品稀释液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂A液 3 ml×1瓶 6 ml×1瓶2-8℃保存显色剂B液 3 ml×1瓶 6 ml×1瓶2-8℃保存终止液3ml×1瓶6ml×1瓶2-8℃保存浓缩洗涤液(20ml×20倍)×1瓶(20ml×30倍)×1瓶 样本处理及要求: 1.血清:室温血液自然凝固10-20分钟,离心20分钟左右(2000-3000转/分)。仔细收集上 清,保存过程中如出现沉淀,应再次离心。 2.血浆:应根据标本的要求选择EDTA或柠檬酸钠作为抗凝剂,混合10-20分钟后,离心 20分钟左右(2000-3000转/分)。仔细收集上清,保存过程中如有沉淀形成,应该再次离心。 3.尿液:用无菌管收集,离心20分钟左右(2000-3000转/分)。仔细收集上清,保存过程 中如有沉淀形成,应再次离心。胸腹水、脑脊液参照实行。 4.细胞培养上清:检测分泌性的成份时,用无菌管收集。离心20分钟左右(2000-3000转/ 分)。仔细收集上清。检测细胞内的成份时,用PBS(PH7.2-7.4)稀释细胞悬液,细胞

RD Drug Names Generic Name:Mouse endotoxin(ET)ELISA Kit. Purpose This kit allows for the determination of ET concentrations in Mouse serum, blood plasma, and other biological fluids. Principle of the assay T he kit assay Mouse ET level in the sample,use Purified Mouse ET antibody to coat microtiter plate wells, make solid-phase antibody, then add ET to wells,Combined antibody which With HRP labeled goat anti-mouse become antibody - antigen - enzyme-antibody complex, after washing Completely,Add TMB substrate solution,TMB substrate becomes blue color At HRP enzyme-catalyzed,reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of ET in the samples is then determined by comparing the O.D. of the samples to the standard curve.

内毒素是革兰阴性菌细胞壁的脂多糖(LPS)成分,脂多糖的类脂A …汇编

细菌内毒素的研究进展 目录 中文摘要及关键词 (1) 英文摘要及关键词 (2) 前言 (3) 1 细菌内毒素的化学组成 (3) 2 细菌内毒素的生物学活性 (4) 3 内毒素的病理生理作用机制 (5) 4 细菌内毒素受体的研究进展 (5) 4.1 CD14 (6) 4.2 TOLL样受体 (7) 4.3 清道夫受体 (8) 4.4 LBP (9) 5 细菌内毒素的检测方法 (10) 5.1 鲎氏实验 (10) 5.1.1 半定量测定-凝胶法 (10) 5.1.2 定量测定 (10) 5.1.2.1 浊度法(比浊法) (10) 5.1.2.2 显色基质法(比色法) (11) 5.1.2.3 酶联免疫吸附测定法(ELISA) (11) 5.2 免疫学方法 (11) 5.3 生物学方法 (11) 5.4 化学发光法 (11)

5.5 流式细胞术 (11) 6 内毒素的制备 (11) 7 内毒素抗体的保护作用 (12) 参考文献 (13) 致谢 (14) 摘要 本文从细菌内毒素的化学组成、生物学活性、致病机理、内毒素的制备与检测及内毒素受体的作用等方面,对细菌内毒素的研究进展进行了综述,并对本领域的研究方向及前景进行了讨论。

关键词: 内毒素;生物学活性;致病机理;检测方法 Abstract This article from the bacterial endotoxin in chemical composition, biological activity, the pathogenic mechanism, within the endotoxin and the preparation and testing, endotoxin receptor in the role of bacterial endotoxins of progress were reviewed, and this field of study direction and prospects were discussed. Key words:endotoxin; biological activity; pathogenic mechanism; examination method

内毒素清除剂使用说明

内毒素清除剂使用说明 货号:E1040 规格:20ml/100ml 保存:4℃半年有效,-20℃长期储存。 产品简介: 内毒素清除剂主要用于清除DNA、蛋白质或其他液体样品中的内毒素。在特定的pH值、盐浓度和温度条件下,内毒素清除剂能与DNA、重组蛋白以及液体样品中的内毒素特异性结合,经过室温高速离心后,DNA或蛋白质等保留在水相,而内毒素则被浓缩到极小体积的下层相而被清除。经过3次以上重复抽提后可将活性为5000~50000EU/ml的内毒素水平降低到5~0.5EU/ml以下,即降低1000~10000倍。 操作步骤: 提取前请将内毒素清除剂放在冰上冰浴5min,期间翻转瓶子数次使试剂均匀预冷。 1、a)已纯化的质粒DNA内毒素清除:吸取500μl DNA溶液于微型离心管,加入1/10体积3M NaAc pH5.2或1/20体积5M NaCl溶液,冰浴5min。 b)在提取质粒DNA过程中清除内毒素:以碱裂解法提取质粒为例,在加入裂解液和中和液并离心去除碎片之后,吸取含质粒DNA的上清于新离心管中,冰浴5min。 2、加入1/5体积预冷的内毒素清除剂,振荡混匀,溶液变浑浊。

3、冰浴5min,溶液应变清亮。 4、37℃水浴5min,不时振荡,溶液又变浑浊。 5、12000rpm室温离心5min,溶液应分为两相,上层水相含DNA,下层油状相含内毒素。 6、将含DNA的上层水相转移到新管,弃油状相,重复抽提三次,即重复步骤2-6三次。 7、加入 2.5体积无水乙醇,-20℃沉淀30min或过夜;12000rpm离心10min,弃上清;加入70%乙醇洗涤沉淀,12000rpm离心5min弃上清;空气干燥沉淀,加入100~200μl无内毒素的高纯水或TE溶解沉淀。 8、用内毒素检测试剂测定样品中内毒素活性,并与初始样品比较。 注意事项: 1、DNA浓度>1mg/ml时清除内毒素效率降低。由于DNA和蛋白质本身的性质,清除程序可导致10-20%的DNA丢失,所幸的是与清除内毒素的艰难相比DNA更容易提取制备。 2、所有溶液应用无内毒素的高纯水配制,所有器械材料均应不含内毒素,玻璃器皿可高温烘烤,非挥发性水溶液可高压120℃高温处理。 相关试剂: D1140无内毒素质粒小量提取试剂盒 D1150无内毒素质粒大量提取试剂盒 12100DMEM(H)

相关文档