CN102305788A - Preparation of gram-negative bacterial lipopolysaccharide (endotoxin) colorimetric assay kit and using method for colorimetric assay kit - Google Patents
Preparation of gram-negative bacterial lipopolysaccharide (endotoxin) colorimetric assay kit and using method for colorimetric assay kit Download PDFInfo
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- 238000007398 colorimetric assay Methods 0.000 title abstract 4
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- YLKCHWCYYNKADS-UHFFFAOYSA-N 5-hydroxynaphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(O)=CC=CC2=C1S(O)(=O)=O YLKCHWCYYNKADS-UHFFFAOYSA-N 0.000 claims description 4
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- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 2
- 108010065152 Coagulase Proteins 0.000 claims description 2
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- JWAZRIHNYRIHIV-UHFFFAOYSA-N 2-naphthol Chemical compound C1=CC=CC2=CC(O)=CC=C21 JWAZRIHNYRIHIV-UHFFFAOYSA-N 0.000 claims 1
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- FUPMLXVNXBAYIE-UHFFFAOYSA-N n,n-bis(ethylamino)aniline Chemical compound CCNN(NCC)C1=CC=CC=C1 FUPMLXVNXBAYIE-UHFFFAOYSA-N 0.000 abstract 2
- 108010045487 coagulogen Proteins 0.000 abstract 1
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- IAAKNVCARVEIFS-UHFFFAOYSA-M sodium;4-hydroxynaphthalene-1-sulfonate Chemical compound [Na+].C1=CC=C2C(O)=CC=C(S([O-])(=O)=O)C2=C1 IAAKNVCARVEIFS-UHFFFAOYSA-M 0.000 abstract 1
- HWQLBKMVMXBGRC-UHFFFAOYSA-M sodium;5-hydroxynaphthalene-1-sulfonate Chemical group [Na+].C1=CC=C2C(O)=CC=CC2=C1S([O-])(=O)=O HWQLBKMVMXBGRC-UHFFFAOYSA-M 0.000 abstract 1
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- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 3
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical class OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
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Abstract
The invention relates to the preparation of a gram-negative bacterial lipopolysaccharide (endotoxin) colorimetric assay kit and a using method for the colorimetric assay kit. In the method, a C factor in a horseshoe crab hemocyte lysate is activated by using a trace amount of gram-negative bacterial endotoxin so as to hydrolyze a chromogenic matrix, namely Boc-Val-leu-Gly-Arg-DIAN(N,N-diethylaminoaniline), Boc-leu-Gly-Arg-DIAN or Boc-Thr-Gly-Arg-DIAN, and then a condensation reaction is performed so as to quantificationally detect the bacterial endotoxin. The method comprises the following steps that: the chromogenic matrix, namely the Boc-Val-leu-Gly-Arg-DIAN(N,N-diethylaminoaniline), the Boc-leu-Gly-Arg-DIAN or the Boc-Thr-Gly-Arg-DIAN is artificially synthesized, the C factor, proclotting enzyme and coagulogen in the horseshoe crab hemocyte lysate are specifically activated by using the trace amount of endotoxin, the activated proclotting enzyme hydrolyzes the chromogenic matrix so as to release a group DIAN, the DIAN and sodium 1-naphthol-4-sulfonate (sodium 1-naphthol-2-sulfonate or sodium 1-naphthol-5-sulfonate) form a blue condensate in the presence of sodium periodate, and the activated amount of the proclotting enzyme and the absorbance of the condensate are in linear relation within a certain range at the wavelength of between 630 and 680nm, so that the concentration of the endotoxin in a sample is quantificationally detected. The kit can be used for early diagnosis of the infection of gram-negative bacteria septicemia.
Description
Affiliated field:
The biological medicine category; The synthetic colour developing of the using artificial of more definitely saying so matrix; Utilize the gram-negative bacteria endotoxin to activate the C factor in the limulus blood cell lysate of east; Thereby the synthetic colour developing of hydrolysis matrix Boc-Val-leu-Gly-Arg-DIAN is (N; N-lignocaine aniline), Boc-leu-Gly-Arg-DIAN or Boc-Thr-Gly-Arg-DIAN (N, N-lignocaine aniline) so that condensation reaction takes place and detection by quantitative bacterial endotoxin method and related application in the product of clinical early diagnosis gram-negative bacterial infection.
Background technology:
Through preliminary search, do not find with synthetic colour developing matrix Boc-Val-leu-Gly-Arg-DIAN (N, N-lignocaine aniline) and be prepared into colorimetric detection gram-negative bacteria endotoxin reagent patent.
Summary of the invention:
The present invention is with synthetic colour developing matrix Boc-Val-leu-Gly-Arg-DIAN (N; N-lignocaine aniline), Boc-leu-Gly-Arg-DIAN or Boc-Thr-Gly-Arg-DIAN (N; N-lignocaine aniline) utilize micro-bacterial endotoxin to activate the C factor, proclotting enzyme and the coagulagen that contains in the limulus blood cell lysate of east specifically, thereby bring out east limulus blood cell lysate cure reaction.The synthetic colour developing of the coagulase hydrolysis that is activated matrix discharges colour developing group DIAN (N; N-lignocaine aniline); DIAN and Neville acid sodium, (1-naphthols-2-sodium sulfonate or 1-naphthol-5-sulfonic acid sodium) generate blue condensation product in the presence of sodium metaperiodate; In the 630-680nm wavelength; The absorbance log of the condensation product of activation of zymogen amount and formation is linear dependence within the specific limits, thereby detection by quantitative goes out bacterial endotoxin concentration in the sample.This kit can be used for the clinical gram-negative bacteria endotoxin of early diagnosis and infect.With reference to Figure of description as follows with narration in the present invention:
One, synthetic colour developing matrix
Adopt biotechnology synthetic Boc-Val-leu-Gly-Arg-DIAN (N, N-lignocaine aniline), Boc-leu-Gly-Arg-DIAN or Boc-Thr-Gly-Arg-DIAN (N, N-lignocaine aniline) type of colour developing matrix.Concrete synthesis technique (seeing process route chart).
Two, limulus blood cell lysate in east extracts, separates
2.1 the preparation of haemocyte lysate: wherein composition should contain Na in the lysate
+, Mg
2+, Ca
2+Deng metallic ion, regulate behind the pH and carry out 121 ℃ and can use after the sterilization treatment in 90 minutes.
2.2 haemocyte cracking: sterile working; Get about 20g haemocyte; Add the haemocyte lysate according to the 1-5 ratio; And carry out 200000rpm/min with pulp grinder at a high speed all; 5-10 minute damaged cell; Histocyte liquid after the breakage is put into-30 ℃ of refrigerator snap frozen to be preserved 10 hours; Afterwards frozen cell liquid is taken out; Slowly dissolving at room temperature; After treating all dissolvings, carry out 200000rpm/min with high speed homogenate machine again, 5-10 minute damaged cell; Then the histocyte liquid after the breakage is put into once more-30 ℃ of refrigerator snap frozen and preserved 10 hours, repetition process last time gets final product for 2 times.
2.3 haemocyte separates, extracts: will carry out 3000rpm/min after the above-mentioned haemocyte lysate taking-up; 5-8 minute centrifugal; Get supernatant; Add chloroform according to the 1-2 ratio; Concuss 10 minutes; Be transferred to then and under 4 ℃, carry out 10000rpm/min in the apyrogeneity centrifugal precipition tube, separated in 5-10 minute, it is subsequent use carefully to take out supernatant.
Three, the preparation and the processing of colour developing synthetic substrate and condensation solution
1, colour developing synthetic substrate solution preparation
The synthetic substrate that will develop the color dissolves with sterilized water for injection, is mixed with 6mM concentration, uses the 0.22um membrane filtration, and 4 ℃ of preservations are subsequent use.
2, colour developing liquid A
Neville acid sodium solution (using of the borate buffer preparation of pH 8.5 concentration) for 6mM as 0.05mol.
3, colour developing liquid B
Be 2% sodium periodate solution (using the water for injection preparation).
4, colour developing liquid A and colour developing liquid B handle
After above-mentioned colour developing liquid A and colour developing liquid B preparation, divide to be filled to and carry out freeze drying in cillin bottle or the ampoule bottle then.
5, colour developing synthetic substrate solution-treated
The synthetic substrate solution that will develop the color divides to be filled to and carries out freeze drying in cillin bottle or the ampoule bottle then.
Four, the preparation and the demarcation of reaction host
1, gets the supernatant of the east limulus blood cell of extraction, separator well, be added to and contain in the 1.0M magnesium chloride 0.5M calcium chloride solution, divide to be filled to and carry out freeze drying in cillin bottle or the ampoule bottle and be reaction host.
2, get the good reaction host of above-mentioned freeze drying, add lysate according to the sign value it is dissolved fully, doubly dilute with sterilized water for injection 1-10 then, use UV spectrophotometer measuring, should absorption peak be arranged at 270 ± 3nm place.
3, the bacterial endotoxin standard items are used lysate dissolve and be diluted to ultimate density and be 50pg/ml solution,, anti-S type detected peaks should occur with the reaction of reaction host.
Five, the performance of gram-negative bacteria lipopolysaccharides (endotoxin) colorimetric detection kit
1, sensitivity: the bacterial endotoxin standard items are diluted to 100pg/ml, 50pg/ml, 25pg/ml successively with lysate; 12.5pg/ml, 6.25pg/ml, the standard series of 3.125pg/ml; Reference reagent box operation instructions detect, and measure absorbance separately; With each concentration of standard solution is X-axis, is the Y-axis mapping analysis with the absorbance, and its linear correlation coefficient r should be greater than 0.980.
2, specificity: in the blank plasma of known bacterial endotoxin concentration, add a certain amount of bacterial endotoxin standard solution, it detects the recovery should be between 80-120%.
3, repeatability: same sample is carried out 4 times detect, the CV value of its absorbance should be in 10%.
4, measurement range: between the 3.125-100pg/ml.
Six, the composition of gram-negative bacteria lipopolysaccharides (endotoxin) colorimetric detection kit
1, composition is as shown in the table
| Form | Specification, quantity |
| (1) reaction host | 0.1ml*20 |
| (2) sample preparation liquid | 0.9ml*20 |
| (3) reaction host lysate | 0.6ml*4 |
| (4) colour developing synthetic substrate | 0.6ml*4 |
| (5) colour developing synthetic substrate lysate | 0.8ml*4 |
| (6) colour developing liquid A | 0.6ml*2 |
| (7) colour developing liquid A lysate | 0.8ml*2 |
| (8) colour developing liquid B | 0.6ml*4 |
| (9) colour developing liquid B lysate | 0.8ml*4 |
2, quality control method: use the bacterial endotoxin standard items, its standard items kit is formed as follows.
| Form | Specification, quantity |
| (1) bacterial endotoxin standard items | 100pg/vial*2 |
| (2) standard items lysate | 5ml*2 |
Seven, detection kit method of operating
(1) sample preparation liquid test sample treating fluid packing has crack-free or liquid to overflow, and uses emery wheel to carry out cut at the ampoule neck, uses 75% alcohol swab disinfection then, places ampoule to be opened in 10 minutes to get final product again.
(2) vacuum test tube aseptic, that apyrogeneity contains heparin class anti-coagulants is used in specimen preparation sterile working, collects 2-4ml venous blood; Mix fair gently; Carry out the quick centrifuging of 3000rpm/min 1min then, must contain the platelet rich plasma sample, directly get supernatant and get final product.
(3) sample pre-treatments sterile working is got the adding of 0.1ml supernatant and is equipped with in the 0.9ml sample preparation liquid, and mixing is put into 70 ℃ of insulations of T01 heated at constant temperature appearance 10 minutes at once gently, takes out and prevents at once cooling off 10 minutes in the ice-water bath, is testing sample.
(4) standard operation method
Sterile working will be reacted the host ampoule and broken into two with one's hands, add reaction host lysate 0.1ml, and mixing dissolving gently adds each 0.1ml of standard items dilution series, negative control and testing sample then respectively, and 37 ℃ are incubated 60 minutes; Add the synthetic colour developing of 0.1ml matrix afterwards respectively, 37 ℃ are incubated 15 minutes; (or use colour developing matrix with lysate 0.6ml dissolving colour developing matrix mixing, and then solubilizing reaction host; Take out 0.1ml; Add each 0.1ml of standard items dilution series, negative control and testing sample respectively; 37 ℃ of insulations 60-120 minute) it is even to take out jolting; Add developer A50ul then; Mixing adds 0.1ml developer B, mixing once more; Room temperature was placed 5 minutes, detected absorbance at wavelength 630-680nm.Computing method as a result
According to the bacterial endotoxin concentration in the formula calculation sample:
Csp1＝(Asp1-Ab1)*Cstd*10/(Astd-Ab1)
Wherein: Csp1 is the bacterial endotoxin concentration in the sample;
Cstd is the bacterial endotoxin concentration in the titer;
The absorbance of Asp1 sample
The absorbance of Ab1 blank
Astd is the absorbance of titer
Measure result's judgement:
With reference to Cut off value is 10pg/ml.
Eight, operational points for attention
Detect sample
Blood plasma: use aseptic, the pyrogen-free vacuum test tube that contains heparin class anti-coagulants;
Serum: use aseptic, pyrogen-free vacuum test tube;
Bronchoalveolar lavage fluid: use aseptic, pyrogen-free vacuum test tube.
No matter sterile working still in the transfer process, must avoid the pollution of bacterial endotoxin in blood sampling.
Interfering material
The pollution of the bacterial endotoxin that contains in the influence of external environment and the institute's using appliance in the mensuration process can exert an influence to measured value; Especially operation must be avoided the pollution of microorganism and bacterial endotoxin before chromogenic reagent added.
Description of drawings:
Fig. 1 is the reaction mechanism figure of gram-negative bacteria lipopolysaccharides (endotoxin) colorimetric detection kit;
Fig. 2 is extraction, the separation process figure of reaction host in the chromogenic reagent box;
Fig. 3 is N, N-lignocaine aniline (Boc-Val-leu-Gly-Arg-DIAN) nuclear-magnetism figure.
Claims (8)
1. the preparation and the method for application thereof of gram-negative bacteria lipopolysaccharides (endotoxin) colorimetric detection kit; With synthetic colour developing matrix Boc-Val-leu-Gly-Arg-DIAN (N; N-lignocaine aniline); Boc-leu-Gly-Arg-DIAN or Boc-Thr-Gly-Arg-DIAN (N; N-lignocaine aniline); Utilize micro-bacterial endotoxin to activate the C factor that contains in the limulus blood cell lysate of east specifically; Proclotting enzyme and coagulagen; Thereby bring out east limulus blood cell lysate cure reaction. the synthetic colour developing of the coagulase hydrolysis that is activated matrix discharges colour developing group DIAN (N; N-lignocaine aniline); DIAN and developer A (Neville acid sodium; 1-naphthols-2-sodium sulfonate or 1-naphthol-5-sulfonic acid sodium) the blue condensation product of generation in the presence of sodium metaperiodate developer B; In the 630-680nm wavelength; The absorbance log of the condensation product of activation of zymogen amount and formation is linear dependence within the specific limits, thereby detection by quantitative goes out bacterial endotoxin concentration in the sample; Process of the test should be operated in strict accordance with concrete operation method on the operation instructions, will note the points for attention in the certain operations process simultaneously; The clinical gram-negative bacteria septicemia of early diagnosis that can be used for this kit infects, as one of reference of clinical diagnosis.
2. synthetic as claimed in claim 1 colour developing matrix comprises three kinds of Boc-Val-leu-Gly-Arg-DIAN (N, N-lignocaine aniline), Boc-leu-Gly-Arg-DIAN and Boc-Thr-Gly-Arg-DIAN (N, N-lignocaine aniline).
3. developer A as claimed in claim 1 comprises Neville acid sodium, 1-naphthols-2-sodium sulfonate and 1-naphthol-5-sulfonic acid sodium and developer B (sodium metaperiodate) etc.
4. the blue condensation product that generates as claimed in claim 1, in the 630-680nm wavelength, the absorbance of the condensation product of activation of zymogen amount and formation is linear dependence within the specific limits.
5. the concentration of synthetic colour developing matrix as claimed in claim 2 is between the 1-10Mol, and preparation back packing freeze drying or solution keep in Dark Place at 2-8 ℃, and Boc-Val-leu-Gly-Arg-DIAN (N, N-lignocaine aniline) nuclear-magnetism figure sees Figure of description 3.
6. the concentration of developer Neville acid sodium as claimed in claim 3,1-naphthols-2-sodium sulfonate and 1-naphthol-5-sulfonic acid sodium is between the 0.2-10Mol; And use the 0.05-5Mol borate buffer to prepare (pH 8.5-8.9); The concentration of its developer sodium metaperiodate is between the 0.2-20%; Use sterilized water for injection preparation is divided in the brown bottle freeze drying or solution and keeps in Dark Place at 2-8 ℃.
7. kit concrete operation method as claimed in claim 1 is: will react host and break into two with one's hands; Add reaction host lysate 0.1ml; Mixing dissolving gently adds each 0.1ml of standard items dilution series, negative control and testing sample respectively, and 37 ℃ are incubated 60-120 minute; Add the synthetic colour developing of 0.1ml matrix afterwards respectively, 37 ℃ are incubated 15 minutes; (or use colour developing matrix with lysate 0.6ml dissolving colour developing matrix mixing, and then solubilizing reaction host; Take out 0.1ml; Add each 0.1ml of standard items dilution series, negative control and testing sample respectively; 37 ℃ of insulations 60-120 minute) it is even to take out jolting; Add developer A 50ul mixing then; Add 0.1ml developer B mixing afterwards; Room temperature was placed 5-20 minute, detected absorbance at wavelength 630-680nm at last, calculated content.
8. the pollution of the bacterial endotoxin that contains in the influence of attention external environment and the institute's using appliance in the kit use as claimed in claim 1 can exert an influence to measured value; Especially operation must be avoided the pollution of microorganism and bacterial endotoxin before chromogenic reagent added.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN103454235B (en) * | 2013-09-13 | 2016-04-20 | 广州康盛生物科技有限公司 | A kind of ultrasonic wave added measures the method for bacteria endotoxin content in blood plasma |
| CN103454235A (en) * | 2013-09-13 | 2013-12-18 | 广州康盛生物科技有限公司 | Method for measuring content of bacterial endotoxin in blood plasma under assistance of ultrasonic waves |
| CN105021817B (en) * | 2014-04-24 | 2017-02-15 | 天津汇滨生物科技有限公司 | Developing-process fungus 1,3-beta-D-glucan detection kit for human body fluid |
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| EP3418730A4 (en) * | 2016-02-16 | 2019-10-30 | Seikagaku Corporation | ELECTROCHEMICAL MEASUREMENT USING PHENYLENEDIAMINE DERIVATIVE |
| US11906521B2 (en) | 2016-08-03 | 2024-02-20 | Lonza Walkersville, Inc. | Method of detecting an endotoxin using limulus amebocyte lysate substantially free of coagulogen |
| US11352656B2 (en) | 2017-01-11 | 2022-06-07 | Lonza Walkersville, Inc. | Coagulogen-free clarified limulus amebocyte lysate and chromogenic assay of endotoxin |
| CN110192110A (en) * | 2017-01-11 | 2019-08-30 | 隆萨沃克斯维尔股份有限公司 | Clarified Limulus amoeba-like cell lysate without coagulogen |
| US12359241B2 (en) | 2017-01-11 | 2025-07-15 | Lonza Walkersville, Inc. | Coagulogen-free clarified limulus amebocyte lysate |
| CN109323996B (en) * | 2018-08-24 | 2021-09-07 | 宁波市奉化区人民医院 | Fungus detection kit |
| CN109323996A (en) * | 2018-08-24 | 2019-02-12 | 宁波市奉化区人民医院 | A kind of fungal detection kit |
| CN113607903A (en) * | 2021-07-30 | 2021-11-05 | 振德医疗用品股份有限公司 | Method for detecting bacterial endotoxin containing positive charge polymer |
| CN113607903B (en) * | 2021-07-30 | 2024-03-15 | 振德医疗用品股份有限公司 | Method for detecting bacterial endotoxin containing positive charge polymer |
| CN116609521A (en) * | 2023-01-17 | 2023-08-18 | 河北省药品医疗器械检验研究院(河北省化妆品检验研究中心) | Recombinant Hepatitis B Vaccine Bacterial Endotoxin Rapid Detection Kit and Application Method |
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