CN102692494A - Endotoxin detection method for nano-particle size analyzer - Google Patents
Endotoxin detection method for nano-particle size analyzer Download PDFInfo
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- CN102692494A CN102692494A CN2012101966566A CN201210196656A CN102692494A CN 102692494 A CN102692494 A CN 102692494A CN 2012101966566 A CN2012101966566 A CN 2012101966566A CN 201210196656 A CN201210196656 A CN 201210196656A CN 102692494 A CN102692494 A CN 102692494A
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Abstract
The invention discloses an endotoxin detection method for a nano-particle size analyzer, and belongs to the field of bacterial endotoxin detection. According to the characteristic that the endotoxin can be agglomerated into nano-colloidal particles in a water solution, the method comprises the followings steps: preparing a series of endotoxin standard solutions with at least three dilution concentrations; detecting the particle sizes of the standard solutions by adopting the nano-particle size analyzer; performing regression analysis on the detected particle size data and the concentration of a corresponding standard solution, and establishing a standard curve; and finally calculating the content of the endotoxin of a sample according to the standard curve and the endotoxin particle size in the sample to be detected. The method has the advantages of quick detection speed, no consumption of detection reagent, low detection cost and high reliability.
Description
Technical field
The present invention relates to a kind of endotoxin detection method, relate in particular to a kind of nanometer particle size analyser and be used for the method that endotoxin detects, belong to the detection of bacterial endotoxin field.
Background technology
Bacterial endotoxin is a lipopolysaccharides, also is called as liposome, and it is the composition of Gram-negative bacteria mantle, extensively is present in occurring in nature, and this material gets into blood of human body can cause heating, is commonly called as pyrogen reaction.Because this type of material possibly cause the people and produce serious adverse effects, therefore in drug injection, need strict control.
Detection of bacterial endotoxin can be divided into two types of qualitative detection and detection by quantitative.Conventional detection method is the rabbit method, and the test sample vein is injected in the rabbit body, in official hour, observes the situation of change of body temperature.This method disturbing factor is many, and poor sensitivity particularly possibly also false negative can occur to the medicine of antipyretic effect or the injection of antipyretic and antidotal type.
Endotoxin in the pharmacopeia detects and all adopts limulus reagent test, and wherein: the TAL gel method is under suitable condition (temperature, pH value and noiseless material), and bacterial endotoxin activates the proclotting enzyme in the TAL, makes TAL produce agglutinating reaction and forms gel; The TAL nephelometry is linear with endotoxin concns according to the variation of the turbidity in TAL and the endotoxin course of reaction, thereby measures endotoxin content.These method preparatory stages are long, detect consuming timely, and cost is high, can not realize rapidly, continuously, online detection.
In addition the pilot study of some method is more, also fails to form and uses, and perhaps can not overcome needs to add reagent or use shortcoming such as expensive instrument, can't realize the direct detection to sample.
Summary of the invention
The present invention is directed to the defective of prior art, be used for the method that endotoxin detects and propose a kind of nanometer particle size analyser, to reduce cost and to improve detection speed.
This method comprises the steps:
Step 1: the endotoxin standard solution of preparation series concentration, adopt the nanometer particle size analyser that these endotoxin standard solution are carried out particle diameter and detect;
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done regretional analysis, set up typical curve ln (d)=kC+b, in the formula: d is a particle diameter, and C is an endotoxin concns, and k is a correction coefficient, and b is a correction factor;
Step 3: calculate the endotoxin content in the testing sample according to typical curve.
Series concentration in the said step 1 comprises at least three dilute concentrations; The curve evaluation coefficient of the typical curve in the said step 2 is greater than 0.98.
Technique effect:
1, the sense cycle of each sample has shortened the time that endotoxin detects greatly at 2～5 minutes, and detection speed is fast.
2, need not to consume detectable (TAL), can significantly reduce endotoxic detection cost.
3, detection method is easy, and testing result is comparatively accurate, and reliability is high, is applicable to that the endotoxin content in water for injection, injection semi-manufacture and the finished product is measured.
Embodiment
Be described further in the face of the present invention down.
Because endotoxic structure one end is hydrophobic fat chain, an end is hydrophilic sugar chain, the hydrophobic side of surfactant-like and water-wet side.Bacterial endotoxin exists with the form of reuniting in the WS, and the reunion molecular weight is in several thousand to 1,000,000 scope, and according to the characteristic of nano colloidal particles of solution, its size increases with the increase of concentration.The nanometer particle size analyser is the instrument of the detection particle diameter used always, and the particle diameter of bacterial endotoxin molecular group can be detected by particle size analyzer be that we are serendipitous.
The inventive method specifically comprises the steps:
Step 1: adopt normative reference, international standard, national standard or working stamndard endotoxin as the standard endotoxin; Confirm required standard endotoxin series concentration; This series concentration comprises at least three dilute concentrations; The endotoxin standard solution of preparation series concentration adopts the nanometer particle size analyser that these endotoxin standard solution are carried out particle diameter and detects.
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done regretional analysis; Set up typical curve ln (d)=kC+b; The curve evaluation coefficient of this typical curve needs greater than 0.98, in the formula: d is that (unit: nm), C is an endotoxin concns (unit: EU/ml) to particle diameter; K is a correction coefficient, and b is a correction factor.
Step 3: detect the endotoxin particle diameter in the testing sample solution under the same conditions, calculate the endotoxin content in the testing sample according to typical curve.
One embodiment of the present of invention are provided below:
The foundation of I, bacterial endotoxin typical curve
Equipment and materials: a, Ma Erwen Nano ZS ZEW3600 type laser particle size analyzer; B, bacterial endotoxin working standard (lot number: 150601-201070, specification: 140EUAmp
-1, Nat'l Pharmaceutical & Biological Products Control Institute); C, bacterial endotoxin inspection water (lot number: 100130, Zhanjiang Bo Kang sea life company limited).
Method step: get the working stamndard endotoxin; Use the inspection water to be mixed with the endotoxin standard solution of concentration as 100EU/ml; Re-use the endotoxin standard solution that the inspection water progressively is diluted to series concentration, series concentration is respectively 50EU/ml, 10EU/ml, 1EU/ml, 0.25EU/ml.
Open the particle size analyzer power supply; More than the preheating 30min; Serial endotoxin standard solution joined in the detection cell to high concentration by low concentration successively detect;, peak area strong to the peak among the measured size distribution figure and particle size distribution range carry out the data model computing, obtain the logarithm ln (d) of endotoxin particle diameter.As ordinate Y, carry out regretional analysis with endotoxin concns as horizontal ordinate X with ln (d), linear equation is: ln (d)=0.061C+0.846, R
2=0.996, the curve evaluation coefficient is greater than 0.98, and typical curve is set up, and the typical curve result is as shown in table 1.
Table 1
II, measure the endotoxin content of red sage roo drip liquid intermedium respectively with this method and dynamic turbidimetric
Equipment and materials: a, Ma Erwen Nano ZS ZEW3600 type laser particle size analyzer; B, BET-16M bacteria endotoxin detector; C, dynamic turbidimetric TAL (lot number: 0711010, λ=0.03EUmL
-1, specification: 0.6mLAmp
-1, Zhanjiang Bo Kang sea life company limited); D, bacterial endotoxin working standard (lot number: 2007-1, specification: 150EUAmp
-1, Nat'l Pharmaceutical & Biological Products Control Institute); E, bacterial endotoxin inspection water (lot number: 070130, specification: 5mLAmp
-1, Zhanjiang Bo Kang sea life company limited); F, bacterial endotoxin indicator (lot number: 071114, specification: 3000EUAmp
-1, Zhanjiang Bo Kang sea life company limited); G, red sage roo drip liquid intermedium (lot number: 05110209, Shanghai Worldbest & Anhui Jinhui Pharmaceutical Co., Ltd.).
Method: get the red sage roo drip liquid intermedium, adopt this method (calculating by the typical curve in the part I) and TAL nephelometry to measure endotoxin content respectively, the result is as shown in table 2.
Table 2
The result shows that the result that this method and TAL nephelometry record is comparatively approaching, and testing result is comparatively accurate, and this has also explained the reliability of this method.
Claims (3)
1. a nanometer particle size analyser is used for the method that endotoxin detects, and it is characterized in that:
This method comprises the steps:
Step 1: the endotoxin standard solution of preparation series concentration, adopt the nanometer particle size analyser that these endotoxin standard solution are carried out particle diameter and detect;
Step 2: the particle size data of detection gained and the concentration of corresponding endotoxin standard solution are done regretional analysis, set up typical curve ln (d)=kC+b, in the formula: d is a particle diameter, and C is an endotoxin concns, and k is a correction coefficient, and b is a correction factor;
Step 3: calculate the endotoxin content in the testing sample according to typical curve.
2. nanometer particle size analyser according to claim 1 is used for the method that endotoxin detects, and it is characterized in that: the series concentration in the said step 1 comprises at least three dilute concentrations.
3. nanometer particle size analyser according to claim 1 is used for the method that endotoxin detects, and it is characterized in that: the curve evaluation coefficient of the typical curve in the said step 2 is greater than 0.98.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077307A (en) * | 2018-10-21 | 2020-04-28 | 厦门鲎试剂生物科技股份有限公司 | Novel method for rapidly detecting sepsis by using gram-negative bacterial infection |
| CN112505279A (en) * | 2020-12-04 | 2021-03-16 | 北京师范大学 | Method for detecting endotoxin concentration in biochemical tail water by using nanotube membrane pressure difference |
| CN113804593A (en) * | 2020-06-11 | 2021-12-17 | 北京科兴生物制品有限公司 | Detection method and application of split vaccine splitting effect |
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| US4221865A (en) * | 1978-10-06 | 1980-09-09 | Baxter Travenol Laboratories, Inc. | Method for determining endotoxin concentration |
| CN101903776A (en) * | 2007-12-19 | 2010-12-01 | 兴和株式会社 | Method for measuring endotoxin and reagent kit for measuring endotoxin |
| EP2397857A1 (en) * | 2009-02-13 | 2011-12-21 | Kowa Company, Ltd. | Method for assaying physiological substance of biological origin and assay device therefor |
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| US4221865A (en) * | 1978-10-06 | 1980-09-09 | Baxter Travenol Laboratories, Inc. | Method for determining endotoxin concentration |
| CN101903776A (en) * | 2007-12-19 | 2010-12-01 | 兴和株式会社 | Method for measuring endotoxin and reagent kit for measuring endotoxin |
| EP2397857A1 (en) * | 2009-02-13 | 2011-12-21 | Kowa Company, Ltd. | Method for assaying physiological substance of biological origin and assay device therefor |
Non-Patent Citations (2)
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| NUNO C. SANTOS ET AL: "Evaluation of Lipopolysaccharide Aggregation by Light Scattering Spectroscopy", 《CHEMBIOCHEM》, no. 4, 3 January 2003 (2003-01-03), pages 1 - 5 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111077307A (en) * | 2018-10-21 | 2020-04-28 | 厦门鲎试剂生物科技股份有限公司 | Novel method for rapidly detecting sepsis by using gram-negative bacterial infection |
| WO2020083261A1 (en) * | 2018-10-21 | 2020-04-30 | 厦门鲎试剂生物科技股份有限公司 | Novel method for rapid test of sepsis with gram-negative bacterial infections |
| CN111077307B (en) * | 2018-10-21 | 2022-08-23 | 厦门鲎试剂生物科技股份有限公司 | Novel method for rapidly detecting sepsis by using gram-negative bacterial infection |
| CN113804593A (en) * | 2020-06-11 | 2021-12-17 | 北京科兴生物制品有限公司 | Detection method and application of split vaccine splitting effect |
| CN112505279A (en) * | 2020-12-04 | 2021-03-16 | 北京师范大学 | Method for detecting endotoxin concentration in biochemical tail water by using nanotube membrane pressure difference |
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