CN109030486A - A kind of Test for Bacterial Endotoxins of hemoglobin samples - Google Patents
A kind of Test for Bacterial Endotoxins of hemoglobin samples Download PDFInfo
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- CN109030486A CN109030486A CN201811135808.5A CN201811135808A CN109030486A CN 109030486 A CN109030486 A CN 109030486A CN 201811135808 A CN201811135808 A CN 201811135808A CN 109030486 A CN109030486 A CN 109030486A
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- test
- hemoglobin samples
- blocking agent
- hemoglobin
- bacterial endotoxins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
Abstract
The invention discloses a kind of Test for Bacterial Endotoxins of hemoglobin samples, include the following steps: that a G-factor blocking agent dissolves reagents dry powder；Hemoglobin samples are diluted 20-40 times by b；It after reactant obtained in step a and b is sufficiently mixed uniformly by c, is incubated in 37 DEG C, monitors the absorbance at 660nm, carry out detection of bacterial endotoxin.The G-factor blocking agent used in step a makes Aprotinin containing 5ug-15ug and 10mg-20mg pachymaran in every milliliter of blocking agent solution for Aprotinin and pachymaran are added in purified water.The Test for Bacterial Endotoxins of hemoglobin samples of the invention, detection limit is low, and accuracy is high, easy to operate, can be realized the accurate detection to hemoglobin samples bacterial endotoxin.
Description
Technical field
The invention belongs to drug measurement techniques fields, and in particular to a kind of detection of bacterial endotoxin side of hemoglobin samples
Method.
Background technique
Endotoxin is the general name of toxicant present in the thallus of Gram-negative bacteria, and main component is that phosphatide is more
Sugar can cause to generate heat, therefore also referred to as ' heat source '.
Endotoxin is the potential pollutant of biological products, for big infusion products, needs a kind of reliable, effective method
Induced by endotoxin residual is quantified.
Endotoxic detection method can be divided into gel method and photometry.Gel method is produced by reagents and endotoxin
The principle of agglutinating reaction is given birth to carry out qualitative detection or half-quantitative detection.Photometry includes nephelometry and chromogenic substrate method,
It is further divided into terminal nephelometry, dynamic turbidimetric, terminal development process and dynamic color method.Terminal nephelometry is mixed according to reaction
Existing quantitative relationship between the endotoxin concns in object and its turbidity (absorbance or light transmittance) when being incubated for termination is closed to come
The method for measuring endotoxin content.Dynamic turbidimetric is to detect a certain preset absorbance of turbidity arrival of reaction mixture
Or the reaction time needed for light transmittance, or the method that detection turbidity is increased speed.Terminal development process is according to reaction mixture
Existing quantitative relationship measures endotoxin between middle endotoxin concns and the amount of its colour generation released when being incubated for and terminating group
The method of content.Dynamic color method is that the absorbance for detecting reaction mixture or light transmittance reach a certain preset detected value
The method that required reaction time or detected value are increased speed.
Hemoglobin is to be responsible for unique non-memebrane protein in the protein and red blood cell of delivery oxygen in higher organism body.
Hemoglobin is a kind of iron-containing compound allosteric protein, is made of ferroheme and globin.By a series of separation purifying techniques
The hemoglobin samples of preparation have particularity, and the hemoglobin released from red blood cell exists with dissolved state
In solution, since hemoglobin samples have very deep color, strong interference can be generated to the detection of bacterial endotoxin, at present also
The report that bacterial endotoxin of hemoglobin samples is not detected.Chinese patent CN102901726 discloses a kind of blood
The preparation and application of liquid detection of bacterial endotoxin kit are carrying out blood detection of bacterial endotoxin using the kit of the invention
When need first to blood sample carry out centrifugal treating, the red blood cell in blood is eliminated after centrifugal treating, to eliminate
Influence of the hemoglobin to detection of bacterial endotoxin can not carry out endotoxin detection to hemoglobin samples using the kit.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of suitable for detecting bacterial endotoxin hemoglobin samples
Method realizes the accurate detection to hemoglobin samples bacterial endotoxin, to make up the blank in this field.
In order to solve the above technical problems, the present invention provides a kind of Test for Bacterial Endotoxins of hemoglobin samples,
It includes the following steps:
A G-factor blocking agent dissolves reagents dry powder；
Hemoglobin samples are diluted 20-40 times by b；
C will be obtained in step a and b reactant be sufficiently mixed uniformly after, in 37 DEG C be incubated for, monitor 660nm at absorbance,
Carry out endotoxin detection.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein in the step a G-factor blocking agent be
Aprotinin is added in purified water, makes Aprotinin containing 5ug-15ug in every milliliter of blocking agent solution.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein in the step a G-factor blocking agent be
Pachymaran is added in purified water, makes pachymaran containing 10mg-20mg in every milliliter of blocking agent solution.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein in the step a G-factor blocking agent be
Aprotinin and pachymaran are added in purified water, makes Aprotinin containing 5ug-15ug and 10mg-20mg in every milliliter of blocking agent solution
Pachymaran.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein G-factor blocking agent described in the step a
The G-factor blocking agent that proportion with reagents is 1ml dissolves 1-3ug reagents powder.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein in the step c, what the step a was obtained
The addition volume ratio for the hemoglobin samples that reagents solution and the step b are obtained is 1:4-5.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein with tiny electrolytic cell water in the step b
Dilute hemoglobin samples.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein with tiny electrolytic cell water in the step b
Hemoglobin samples are diluted 20 times.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein incubation time is 3 hours in the step c.
The Test for Bacterial Endotoxins of above-mentioned hemoglobin samples, wherein using in the progress of reagents dynamic turbidimetric
Mycotoxin identification.
The Test for Bacterial Endotoxins of hemoglobin samples of the invention has the following beneficial effects:
1, hemoglobin samples are diluted 20-40 times in the present invention, can utmostly reduced that may be present in sample
Various interference to detection of bacterial endotoxin, and method of the invention is not necessarily to pre-process hemoglobin samples, it can
Realize that method is simple and easy, and high sensitivity, minimum detection limit reaches to the quantitative detection of hemoglobin samples bacterial endotoxin
0.001EU/ml；
2, using the compound G-factor blocking agent being made of Aprotinin and pachymaran in detection method of the invention, sufficiently
Structure in hemoglobin samples is inhibited to avoid the appearance of false positive similar to the interference of callose substance, use is additional
The endotoxic method of known concentration carries out accuracy test, and the rate of recovery reaches 90-130%, significantly larger than international standard 50-
200% requirement, testing result are more accurate.
3, reagents kinetic turbidimetric assay is used in the present invention, is measured the absorption value at 660nm, is utmostly reduced blood red
The interference of albumen, high sensitivity realize accurate detection endotoxic to hemoglobin samples.
4, standard curve, positive control, negative control, test sample have been detected every time in the present invention, one-time detection 3
A sample more than 20 can be detected in hour simultaneously, and 3, each sample parallel, detection method quickly, accurately, greatly mentions
High detection efficiency.
Detailed description of the invention
Fig. 1 is endotoxin standard curve graph.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples.
Embodiment 1
One, endotoxin titer is prepared with tiny electrolytic cell water.
1. taking one bottle of endotoxin, metal cover is removed, bottle stopper is slowly opened, tiny electrolytic cell is added with 1000ul pipettor
With water 5.0ml, bottle stopper is covered, is sealed with sealing film, is mixed in vortex oscillator, primary, each 1min is mixed every 5min,
Total 60-120min.Concentration is 1000EU/ml.
2. tiny electrolytic cell water 1800ul is added in pyrogen-free test tube, step 1. sample 200ul is added, in whirlpool
About 1min is mixed on the oscillator of whirlpool.Concentration is 100EU/ml.
3. tiny electrolytic cell water 1800ul is added in pyrogen-free test tube, step 2. sample 200ul is added, in whirlpool
About 1min is mixed on the oscillator of whirlpool.Concentration is 10EU/ml.
4. tiny electrolytic cell water 1800ul is added in pyrogen-free test tube, step 3. sample 200ul is added, in whirlpool
About 1min is mixed on the oscillator of whirlpool.Concentration is 1EU/ml.
5. tiny electrolytic cell water 360ul is added in pyrogen-free test tube, step 4. sample 640ul is added, in whirlpool
About 1min is mixed on oscillator.Concentration is 0.64EU/ml.
6. standard curve sample preparation, endotoxin standard curve is shown in Fig. 1.
It is separately added into endotoxin standard in test tube by table 1, and mixes 1min in vortex oscillator.
Table 1
Endotoxin standard accuracy in detection is shown in Table 2, and each endotoxin standard contains 3 Duplicate Samples.
Table 2
Two, reagents dry powder is dissolved with G-factor blocking agent
1, it prepares G-factor blocking agent: Aprotinin being added in purified water, make to press down peptide containing 10ug in every milliliter of blocking agent solution
Enzyme；
2, configured G-factor blocking agent 5.0ml is added in 10ug reagents dry powder, mixes gently, using preceding static
At least 5min.
Three, the preparation of hemoglobin samples
1. the preparation of hemoglobin samples: in pyrogen-free test tube, tiny electrolytic cell water 1950ul is added, blood is added
Lactoferrin sample to be tested 50ul, mixes 1min in vortex oscillator, and hemoglobin samples extension rate is 40 times；
2. the preparation of hemoglobin samples positive control: in pyrogen-free test tube, tiny electrolytic cell water is added
1925ul is added hemoglobin sample to be tested 50ul, the endotoxin standard 25ul of 0.64EU/ml is added, in vortex oscillation
1min is mixed on device, hemoglobin samples positive control extension rate is 40 times；
3. the preparation of negative control sample: in pyrogen-free test tube, tiny electrolytic cell water 2ml is added.
Four, it detects
1. opening instrument, 30min is preheated, keeps temperature constant at 37 DEG C, measurement wavelength is 660nm；
2. connect network analysis software, be arranged instrument parameter, reaction time 180min, fill in standard curve, sample and
Positive control title and extension rate fill in the lot number of reagents and tiny electrolytic cell water；
3. negative control: taking tiny electrolytic cell to be set in reaction tubule with water 200ul, do 3 in parallel；
4. standard curve: each dilution gradient (from low to high) takes 200ul to set in reaction tubule, and each gradient each 3 flat
Row；
5. sample: taking each 200ul of the sample diluted to set in reaction tubule, each 3 parallel；
6. Sample Positive compares: taking each 200ul of the control sample diluted to set in reaction tubule, each 3 parallel；
7. 50ul reagents is respectively added in each reaction tubule, 2s is mixed, the reacting hole of tubule placement instrument will be reacted
In detected, i.e. reagents and sample to be tested volume ratio are as follows: 1:4；
8. analysis software analyzes data, and testing result is shown in Table 3 when detection terminates.
Embodiment 2-9, comparative example 1-2 are identical as the detection method of embodiment 1, the difference is that using different G because
Sub- blocking agent, testing result are shown in Table 3.
Table 3
It is to reduce similar (1 → 3)-β-D- of all structures in hemoglobin samples with factor blocking agent dissolution reagents
Glucan substance induced by endotoxin interferes caused by detecting, and (1 → 3)-callose can activate the glucan in reagents quick
Feel the factor, keeps test result higher or false positive occur.From table 3 it is observed that if not using any G in detection process
Factor blocking agent, is shown in comparative example 1, and the endotoxic positive rate of recovery is very high, illustrate to have in hemoglobin samples structure similar to 1 →
3)-callose substance, has activated G access, causes the enhancing of endotoxin testing result.Aprotinin is used in embodiment 1-3
As G-factor blocking agent, G access can either be effectively blocked using the Aprotinin of very low concentrations, make endotoxic positive recycling
Rate is substantially reduced, and the positive rate of recovery reaches 140% or less.Using pachymaran as G-factor blocking agent, energy in embodiment 4-6
The interference of (1 → 3)-callose is enough effectively reduced, the positive rate of recovery is 140-150%, is far below international standard 50-
200%.Embodiment 7-9 uses compound G-factor blocking agent, and compound G-factor blocking agent is made of Aprotinin and pachymaran, together
Embodiment 1-6 is compared, and the positive rate of recovery is substantially reduced, and basically reaches 130% or less, it is seen then that uses compound G-factor of the invention
Blocking agent can block (1 → 3)-callose analogue in hemoglobin samples comprehensively, improve endotoxin inspection
The accuracy of survey.
Comparative example 3-7, embodiment 10 are identical as the detection method of embodiment 8, the difference is that hemoglobin samples
Extension rate is different, and positive control sample also dilutes in the same fashion, and each dilution does three in parallel, and testing result is shown in
Table 4.
Table 4
By table 4 it is found that when sample dilutes 10 times, since the color of hemoglobin itself is interfered, determined by system software
Test invalidation；Extension rate is from 1:20 to 1:140 without inhibition or enhancement effect.Extension rate in 1:20 between 1:40,
The rate of recovery is 130% hereinafter, interfering minimum, accuracy highest.When extension rate is more than 40 times, due to exceeding the detection of system
Limit, system can not provide accurate data, use 20-40 times of extension rate in the present invention, can be effectively reduced hemoglobin sheet
The detection to bacterial endotoxin in hemoglobin samples is realized in the interference of body color.
Claims (10)
1. a kind of Test for Bacterial Endotoxins of hemoglobin samples comprising following steps:
A G-factor blocking agent dissolves reagents dry powder；
Hemoglobin samples are diluted 20-40 times by b；
It after reactant obtained in step a and b is sufficiently mixed uniformly by c, is incubated in 37 DEG C, monitors the absorbance at 660nm, into
Row detection of bacterial endotoxin.
2. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1, wherein G-factor in the step a
Blocking agent is that Aprotinin is added in purified water, makes Aprotinin containing 5ug-15ug in every milliliter of blocking agent solution.
3. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1, wherein G-factor in the step a
Blocking agent is that pachymaran is added in purified water, makes pachymaran containing 10mg-20mg in every milliliter of blocking agent solution.
4. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1, wherein G-factor in the step a
Blocking agent is that Aprotinin and pachymaran are added in purified water, make in every milliliter of blockings agent solution Aprotinin containing 5ug-15ug with
10mg-20mg pachymaran.
5. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1 or 4, wherein institute in the step a
It states the proportion of G-factor blocking agent and reagents and dissolves 1-3ug reagents powder for the G-factor blocking agent of 1ml.
6. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1 or 4, wherein in the step c,
The addition volume ratio for the hemoglobin samples that the reagents solution and the step b that the step a is obtained obtain is 1:4-5.
7. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1 or 4, wherein used in the step b
Hemoglobin samples are diluted with water in tiny electrolytic cell.
8. the Test for Bacterial Endotoxins of hemoglobin samples as claimed in claim 7, wherein with interior in the step b
Hemoglobin samples are diluted 20 times by toxin inspection water.
9. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1 or 4, wherein incubated in the step c
Educating the time is 3 hours.
10. the Test for Bacterial Endotoxins of hemoglobin samples as described in claim 1 or 4, wherein dynamic using reagents
State nephelometry carries out endotoxin detection.
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| CN201811135808.5A CN109030486A (en) | 2018-09-28 | 2018-09-28 | A kind of Test for Bacterial Endotoxins of hemoglobin samples |
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| CN201811135808.5A CN109030486A (en) | 2018-09-28 | 2018-09-28 | A kind of Test for Bacterial Endotoxins of hemoglobin samples |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007253A (en) * | 2019-12-26 | 2020-04-14 | 常州千红生化制药股份有限公司 | Method for detecting bacterial endotoxin in heparin |
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|---|---|---|---|---|
| US5318893A (en) * | 1988-02-27 | 1994-06-07 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin |
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
| CN102901726A (en) * | 2012-09-14 | 2013-01-30 | 莫水晶 | Preparation and application of detection kit for blood bacterial endotoxin |
| CN107478477A (en) * | 2017-08-04 | 2017-12-15 | 福州新北生化工业有限公司 | A kind of jaundice, haemolysis, the serum processing method of piarhemia |
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2018
- 2018-09-28 CN CN201811135808.5A patent/CN109030486A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5318893A (en) * | 1988-02-27 | 1994-06-07 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin |
| CN1105757A (en) * | 1993-02-26 | 1995-07-26 | 生化学工业株式会社 | Reagent for endotoxin assay and method for endotoxin assay using the same |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
| CN102901726A (en) * | 2012-09-14 | 2013-01-30 | 莫水晶 | Preparation and application of detection kit for blood bacterial endotoxin |
| CN107478477A (en) * | 2017-08-04 | 2017-12-15 | 福州新北生化工业有限公司 | A kind of jaundice, haemolysis, the serum processing method of piarhemia |
Non-Patent Citations (1)
| Title |
|---|
| 万丽卿等: "血浆中细菌内毒素定量测定方法的研究", 《药物分析杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007253A (en) * | 2019-12-26 | 2020-04-14 | 常州千红生化制药股份有限公司 | Method for detecting bacterial endotoxin in heparin |
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Effective date of registration: 20220318 Address after: 101111 1602, building 3, yard 88, Kechuang 6th Street, economic and Technological Development Zone, Daxing District, Beijing Applicant after: REDPHARM (BEIJING) BIOMEDICAL RESEARCH INSTITUTE Co.,Ltd. Applicant after: You Kewei Address before: 101111 room 1602, building 3, No.88 courtyard, Kechuang Sixth Street, Daxing Economic and Technological Development Zone, Beijing Applicant before: REDPHARM (BEIJING) BIOMEDICAL RESEARCH INSTITUTE Co.,Ltd. |
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Application publication date: 20181218 |