CN110412194A - A kind of method of liquid chromatographic detection apolipoprotein concentration - Google Patents
A kind of method of liquid chromatographic detection apolipoprotein concentration Download PDFInfo
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- CN110412194A CN110412194A CN201810384177.4A CN201810384177A CN110412194A CN 110412194 A CN110412194 A CN 110412194A CN 201810384177 A CN201810384177 A CN 201810384177A CN 110412194 A CN110412194 A CN 110412194A
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- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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Abstract
The invention discloses a kind of methods of liquid chromatographic detection apolipoprotein concentration, and nano silica is added in plasma sample, mixes well, and are centrifuged after being stored at room temperature 20 ~ 50min, obtain solid sediment；Solid sediment is washed with sodium chloride solution, is centrifuged, is obtained sediment and be resuspended with eluent, is centrifuged, obtains supernatant；Chromatographic test strip part are as follows: chromatographic column: C18 chromatographic column, mobile phase: methanol-acetonitrile-aqueous solution, chromatographic column column temperature are 35 ~ 45 DEG C, Detection wavelength 220nm, and flow velocity is 1 ~ 3mL/min；APoA standard items are taken, various concentration is configured to, standard curve is drawn in measurement；Sample is measured, the concentration of apolipoprotein is calculated according to standard curve.By the simple process to plasma sample, eliminate influence of other complicated ingredients to testing result in blood plasma, treated sample can direct injected detection, detection sensitivity is high, and detection time is short, reproducible.
Description
Technical field
The invention belongs to Protein Detection fields, and in particular to a kind of method of liquid chromatographic detection apolipoprotein concentration.
Background technique
Apolipoprotein (apolipoprotein) be constitute plasma lipoprotein protein component, major function by: form
Lipoprotein and the stabilization and integrality, the activity of activation phosphatide cholesterol acyl transfer (LCAT), delivery lipid for maintaining its structure
Substance, activation hepatic lipase, the activation functions such as lipoprotein and identification receptor.Opposite pulmonary tuberculosis and lung cancer, aPoA is in silicosis
High expression in human serum prompts the albumen that may participate in silicotic fibrosis by adjusting the metabolic process of lipoprotein and GAP-associated protein GAP
Development.Have been reported that apolipoprotein (apo) C-I is lower than normal person's blood in the albumen of discovery serum of tuberculosis patients differential expression
Clearly, thus it is speculated that the albumen and blood lipid metabolism are closely related, it may be possible to which therefore one of potential marker of tuberculosis serum detects serum
Middle aPoA concentration has clinical meaning to respiratory diseases such as diagnosis silicosis, pulmonary tuberculosis and lung cancer.
Immuno-chemical method is almost all made of to the measurement of apolipoprotein at present, mainly there is radiation immunity diffusion method (RID)
, also known as rocket electrophoresis (" rocket "), enzyme linked immunosorbent assay (ELISA), fluorescence is exempted from for unidirectional immunoelectrophoresis measurement
Epidemic disease method (FIA), radioimmunology (RIA), Immune scatter turbidimetry (IN A) and Immunity transmission turbidity (IT A) etc..
Radiation immunity diffusion method is most common simple antigen quantitative technique.Antigen in agar plate well is according to diffusion
Principle generates precipitating in conjunction with the antibody of uniformly dispersing in agar plate, by measuring the diameter of precipitation ring or calculating the face of precipitation ring
Product carries out quantitative analysis to antigen, and measurement elapsed time is long (24~48 h), poor accuracy.Unidirectional immunoelectrophoresis measures
It establishes on the basis of RID, accelerates antigen to enter in homogeneous gel antibody-containing from well by voltage, calibration is bent
Line concentration range very little, therefore often sample need to be diluted or is concentrated.ELISA minute is short, and precision is in acceptable
In range, CV is usually within 10%.Although having the apo E LISA assay kit of commercialization, but still should not promote makes
With.FIA is belonged in immuno-labelling technique and develops earliest with fluorescent material labelled antibody and to the method that antigen is measured
It is a kind of.FIA has been used for the measurement of apoB, is better than ELIS A method in terms of the stability of reagent, analysis specificity and sensibility.
RIA is since sensibility is good in the detection for the radioactive isotope as marker, and accuracy is high, therefore also be used to carry rouge
Protein determination.But since isotope technology bring radioactive pollution easily causes certain harm to human body, and as label
The half-life period of the isotope of object is shorter, therefore to be not easy to be widely accepted and use as the kit technology of commercialization.IN
A is the scattering and refraction that can cause incident light according to immune complex caused by antigen-antibody reaction in the solution, is passed through
The Strength Changes of light scattering are quantitative determined.Immune complex is more, and the scattering light measured is stronger.Due to measurement pair
As that therefore the lipid turbidity in sample must be eliminated in advance and dissolve lipoprotein vulnerable to the disturbance caused for being rich in triglycerides.
ITA as apolipoprotein common detection methods and be widely adopted.Above-mentioned detection method detection time is long, is not suitable for quickly
Detection and batch detection.
Summary of the invention
It is an object of the invention to: a kind of method of liquid chromatographic detection apolipoprotein concentration is provided, detection time is short, spirit
Sensitivity is high.
In order to achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of method of liquid chromatographic detection apolipoprotein concentration, includes the following steps:
1) nano silica is added in plasma sample, mixes well, is centrifuged after being stored at room temperature 20 ~ 50min, it is heavy to obtain solid
Starch；
2) solid sediment of step 1) is washed with sodium chloride solution, is centrifuged, obtains sediment and be resuspended with eluent, be centrifuged,
Obtain supernatant；
3) chromatographic test strip part are as follows: chromatographic column: C18 chromatographic column, mobile phase: methanol-acetonitrile-aqueous solution, chromatographic column column temperature be 35 ~
45 DEG C, Detection wavelength 220nm, flow velocity is 1 ~ 3mL/min；
4) production of standard curve: taking aPoA standard items, be configured to various concentration, is surveyed using the condition of step 3)
It is fixed, draw standard curve；
5) sample of step 2 is measured using the condition of step 3), the dense of apolipoprotein is calculated according to standard curve
Degree.
Nano silica additional amount is 1 ~ 5mg/mL plasma sample in step 1).
Concentration of sodium chloride solution described in step 2 is 0.5 ~ 1.5mol/L.
Eluent described in step 2 is containing 10 ~ 15%(wt) the Tris-HCl eluent or phosphate buffer of ethyl alcohol.
The volume ratio of methanol, acetonitrile and aqueous solution in mobile phase described in step 3) are as follows: 50: 5:(30 ~ 60).
Chromatographic column described in step 3) is ZORBAX 80A Extend-C18.
Taking aPoA standard items to be configured to concentration in step 4) is respectively 0 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ
G/mL, 120 μ g/mL, 160 μ g/mL, 200 μ g/mL, 400 μ g/mL, 600 μ g/mL, are measured using the condition of step 3), with
Peak area Y carries out linear regression to concentration X, draws standard curve.
The utility model has the advantages that
The present invention uses the concentration of liquid chromatographic detection apolipoprotein for the first time, by the simple process to plasma sample, eliminates
Influence of other complicated ingredients to testing result in blood plasma, treated sample can direct injected detection, detection sensitivity is high, inspection
The survey time is short, reproducible.
Detailed description of the invention
Fig. 1: the chromatograms of apolipoprotein, No. 1 peak are aPoA.
Specific embodiment
APoA (Apo A) standard items are purchased from Shanghai Jun Rui Bioisystech Co., Ltd;1100 efficient liquid phase of HP
Chromatograph (Hewlett Packard, Germany);Beckman L8-55 centrifuge (Beckman Coulter, the U.S.)；
Chromatographic column is that ZORBAX 80A Extend-C18 is purchased from Agilent Technologies (China) Co., Ltd.
Embodiment 1
A kind of method of liquid chromatographic detection apolipoprotein concentration, includes the following steps:
1) 2 mg nano silicas are added in 1mL plasma sample, mix well, are centrifuged after being stored at room temperature 30min, 8000r/
M is centrifuged 10 min, obtains solid sediment；
2) solid sediment of step 1) is washed with 1.5mol/L sodium chloride solution, is centrifuged, 8000r/m is centrifuged 10 min, obtains
It is resuspended to sediment with eluent, is centrifuged, obtains supernatant；The eluent is containing 10%(wt) the Tris-HCl eluent of ethyl alcohol,
7.0 20mM of Tris-HCl eluent pH.
3) chromatographic test strip part are as follows: chromatographic column: ZORBAX 80A Extend-C18, mobile phase: methanol-acetonitrile-is water-soluble
Liquid, chromatographic column column temperature are 35 ~ 45 DEG C, Detection wavelength 220nm, flow velocity 2mL/min；Methanol in mobile phase, acetonitrile with it is water-soluble
The volume ratio of liquid are as follows: 10: 1:12.
4) production of standard curve: taking aPoA standard items, and being configured to concentration is respectively 0 μ g/mL, 20 μ g/mL, 40
μ g/mL, 80 μ g/mL, 120 μ g/mL, 160 μ g/mL, 200 μ g/mL, 400 μ g/mL, 600 μ g/mL, using the condition of step 3)
Be measured, measure the chromatographic peak area of each standard solution, each concentration replication 3 times, with peak area Y to concentration X into
Standard curve, standard curve are as follows: Y=328.24x+1837.1, correlation coefficient r=0.9999 are drawn in row linear regression;Detection limit
It determines and uses Noise Method, the corresponding detection limit concentration of 3 times of noises is 8 μ g/mL.
5) sample of step 2 is measured using the condition of step 3), apolipoprotein is calculated according to standard curve
Concentration.
2 Precision Experiment of embodiment
8 same processing and sample introduction will be divided into a sample (sample containing 208 μ g/mL carrier protein A accurately configured), surveyed
Determine as a result, the results are shown in Table 1.
Table 1
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Concentration μ g/mL | 208.6 | 208.1 | 207.9 | 207.7 | 208.4 | 208.5 | 208.6 | 207.9 |
The experiment of 3 rate of recovery of embodiment
The spiked plasma sample of low middle high three kinds of concentration, 40,200,500 μ g/mL in preparation standard curve range, every kind 6 parts of concentration,
It is measured by sample pre-treatments and analysis method, METHOD FOR CONTINUOUS DETERMINATION 3d, calculates average recovery rate and precision.As a result, high, medium and low dense
The recovery of standard addition for spending sample is 95.43%~102.96%, and withinrun precision is 1.8%~4.5%, and betweenrun precision is
2.8%~5.4%, it the results are shown in Table 2.Meet the requirement of " Development Criteria of biological material analysis method ".
The average recovery rate of 2 method of table and relative standard deviation (n=6), %
| 40 μ g/mL of mark-on | 200 μ g/mL of mark-on | 500 μ g/mL of mark-on | |
| Recovery | 95. 43 | 98. 29 | 102.96 |
| In RSD(batches) | 3.7 | 1.8 | 4.5 |
| Between RSD(batches) | 4.6 | 2.8 | 5.4 |
Claims (7)
1. a kind of method of liquid chromatographic detection apolipoprotein concentration, which comprises the steps of:
1) nano silica is added in plasma sample, mixes well, is centrifuged after being stored at room temperature 20 ~ 50min, it is heavy to obtain solid
Starch；
2) solid sediment of step 1) is washed with sodium chloride solution, is centrifuged, obtains sediment and be resuspended with eluent, be centrifuged,
Obtain supernatant；
3) chromatographic test strip part are as follows: chromatographic column: C18 chromatographic column, mobile phase: methanol-acetonitrile-aqueous solution, chromatographic column column temperature be 35 ~
45 DEG C, Detection wavelength 220nm, flow velocity is 1 ~ 3mL/min；
4) production of standard curve: taking aPoA standard items, be configured to various concentration, is surveyed using the condition of step 3)
It is fixed, draw standard curve；
5) sample of step 2 is measured using the condition of step 3), the dense of apolipoprotein is calculated according to standard curve
Degree.
2. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that in step 1)
Nano silica additional amount is 1 ~ 5mg/mL plasma sample.
3. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that in step 2
The concentration of sodium chloride solution is 0.5 ~ 1.5mol/L.
4. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that in step 2
The eluent is containing 10 ~ 15%(wt) the Tris-HCl eluent or phosphate buffer of ethyl alcohol.
5. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that in step 3)
The volume ratio of methanol, acetonitrile and aqueous solution in the mobile phase are as follows: 50: 5:(30 ~ 60).
6. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that in step 3)
The chromatographic column is ZORBAX 80A Extend-C18.
7. a kind of method of liquid chromatographic detection apolipoprotein concentration according to claim 1, which is characterized in that step 4)
In take aPoA standard items be configured to concentration be respectively 0 μ g/mL, 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 120 μ g/mL,
160 μ g/mL, 200 μ g/mL, 400 μ g/mL, 600 μ g/mL, are measured, with peak area Y to concentration X using the condition of step 3)
Linear regression is carried out, standard curve is drawn.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114414819A (en) * | 2022-03-28 | 2022-04-29 | 中元伯瑞生物科技(珠海横琴)有限公司 | Biomarker for diagnosing pneumoconiosis and application thereof |
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| CN105527449A (en) * | 2015-12-24 | 2016-04-27 | 山东博科生物产业有限公司 | Apolipoprotein A1 immunoturbidimetry detection kit |
| CN105646699A (en) * | 2016-01-15 | 2016-06-08 | 新乡医学院 | Method for extracting apolipoprotein A-1 from human plasma by means of fumed silica |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671722A (en) * | 2009-09-28 | 2010-03-17 | 华东理工大学 | Evaluation method of cell biology safety of silicon dioxide nanoparticle |
| CN105527449A (en) * | 2015-12-24 | 2016-04-27 | 山东博科生物产业有限公司 | Apolipoprotein A1 immunoturbidimetry detection kit |
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Non-Patent Citations (2)
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| RICHARD WEINBERG ET AL: "Analytic and preparative separation of apolipoproteins A-Ⅰ，A-Ⅱ，and A-Ⅳ by reverse phase high pressure liquid chromatography", 《JOURNAL OF LIPID RESEARCH》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114414819A (en) * | 2022-03-28 | 2022-04-29 | 中元伯瑞生物科技(珠海横琴)有限公司 | Biomarker for diagnosing pneumoconiosis and application thereof |
| CN114414819B (en) * | 2022-03-28 | 2022-06-21 | 中元伯瑞生物科技(珠海横琴)有限公司 | Biomarker for diagnosing pneumoconiosis and application thereof |
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