CN1556409A - Determination method of apoliprotein M - Google Patents
Determination method of apoliprotein M Download PDFInfo
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- CN1556409A CN1556409A CNA2004100138183A CN200410013818A CN1556409A CN 1556409 A CN1556409 A CN 1556409A CN A2004100138183 A CNA2004100138183 A CN A2004100138183A CN 200410013818 A CN200410013818 A CN 200410013818A CN 1556409 A CN1556409 A CN 1556409A
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Abstract
Using monoclonal antibody or polyclonal antibody of anti apolipoprotein M builds enzyme linked immunosorbent assay to measure content of apolipoprotein M in human blood plasma or blood serum. The method includes steps: coating monoclonal antibody or polyclonal antibody; preparing series of standard substances of apolipoprotein M; preparing sample to be tested; making series of standard substances and sample to be tested combine with coated antibody, then making labeled monoclonal antibody combine with them; stopping color development after development in ground substance liquid in 20-30 minutes; measuring absorbency of series of standard substances and sample to be tested; creating concentration and absorbency standard curve of series of standard substances, and looking up the curve to fine out content of apolipoprotein M of sample to be tested. The method is convenient and reliable.
Description
Technical field
The present invention relates to a kind of Apoliprotein M assay method of (Apoliprotein M is called for short ApoM), more specifically say, relate to ApoM Determination on content method in human plasma or the serum.
Background technology
Lipoprotein is the big molecular complex of the various lipids of transhipment in blood and other body fluid, participates in multiple physiology, pathologic process in the body, and especially generation, the development with body disorders of lipid metabolism and angiocardiopathy has extremely close getting in touch.Lipoprotein has dissimilar, classifies at present mainly the density classification during wherein most importantly according to ultracentrifugation according to physicochemical property.Can be divided into chylomicron (CM in view of the above, density＜1.006), very low density lipoprotein (VLDL) (VLDL, density＜1.006), intermediated-density lipoprotein (IDL, 1.006＜density＜1.019), low-density lipoprotein (LDL, 1.019＜density＜1.063) and high-density lipoprotein (HDL) (HDL, 1.063＜density＜1.210).Because of CM, VLDL, IDL are rich in triglyceride, so be referred to as rich triglyceride lipoprotein (TGRLP).Plasma lipoprotein is by lipid and protein component be combined into, and protein component wherein is called as apolipoprotein (Apos), has the character of some apolipoproteins clear, as ApoA-I, ApoA-II, ApoA-VI, and ApoB, ApoCs and ApoE.The function of apolipoprotein has: as the activity in conjunction with the catabolic regulatory factor of acceptor, lipoprotein and/or the adjusting lipid metabolism enzyme of aglucon.Some apolipoprotein is also closely related with senile dementia and adjusting organism immune response.
The inventor Xu Nin of this invention has found a kind of novel human Apoliprotein M (ApoM) in 1999, and first rear clone mouse, rat and people's ApoM cDNA, experiment finds that the ApoM gene is present in all mammals, and high homology is arranged.Preliminary research data discloses: ApoM may be relevant with body fat and lipoprotein metabolism, also may have necessarily with body disorders of lipid metabolism and body defense reaction and get in touch.Up-to-date research data further discloses the level of ApoM in the patients with coronary heart disease blood plasma apparently higher than the healthy volunteer.
Someone studies show that: growing up among the young people, (Maturity-onset diabetes of theyoung, MODY3) ApoM content obviously reduces morbidity type diabetes among the patients serum.
In sum, measure the content of ApoM in blood plasma or the serum, future clinically is widely used; Simultaneously because ApoM is a kind of albumen of finding recently, its function is still waiting further further investigation, under study for action, the method of the ApoM instrument that is absolutely necessary in high specificity, highly sensitive, good stability, simple and easy to do mensuration human plasma or the serum, but a kind of suitable assay method is not arranged so far yet.
Summary of the invention
The objective of the invention is to, a kind of assay method that can easy, accurately measure the Apoliprotein M of novel Apoliprotein M content in human plasma or the serum is provided, this method is not subjected to the influence of blood plasma or serum medium, accuracy and favorable reproducibility.
Realize the technical scheme of the object of the invention: a kind of assay method of Apoliprotein M, utilize the monoclonal antibody or the polyclonal antibody of anti-Apoliprotein M, set up enzyme linked immunosorbent assay, measure the content of Apoliprotein M in human plasma or the serum, the monoclonal of above-mentioned anti-Apoliprotein M or polyclonal antibody, have the only specificity of anti-Apoliprotein M, concrete determination step is: bag is by the monoclonal of anti-Apoliprotein M or polyclonal antibody; Prepare serial Apoliprotein M standard items; Prepare sample to be checked; Make series standard product and sample to be checked simultaneously respectively with the antibodies of wrapping quilt, make the monoclonal antibody combination with it respectively simultaneously of mark again, with matrix liquid colour developing color development stopping after 20～30 minutes, measure above-mentioned series standard product and the reacted absorbance of sample to be checked after 10 minutes; Set up the typical curve of the concentration of series standard product and absorbance and find the content of the Apoliprotein M of sample to be checked from this curve.
In the said determination method, described bag is to be 9.6 coating buffer with pH by the monoclonal of anti-Apoliprotein M or polyclonal antibody, the monoclonal antibody or the polyclonal antibody of anti-Apoliprotein M are fully combined with 96 hole microwell plates, with pH is after 7.4 washing lotion is cleaned micropore, with remaining the site in the confining liquid blind hole that contains the protein except that Apoliprotein M, after above-mentioned washing lotion cleaning, microwell plate is deposited in 4 ℃, 6 months operating periods.
In the said determination method, the monoclonal antibody of described mark is directly to use enzyme, is different from the monoclonal antibody that the monoclonal antibody that is coated on the 96 hole microwell plates makes mark with binding site; Or use biotin earlier, and be different from the monoclonal antibody that is coated on the 96 hole microwell plates with binding site and make biotin labeled monoclonal antibody, before with the colour developing of matrix liquid, fully combine with enzyme again.
In the said determination method, used coating buffer is by 1.56gNa
2CO
3, 2.93gNaHCO
3And 0.20gNaN
3Add deionized water and make to 1000ml, its pH is 9.6; Used washing lotion is by KH
2P0
40.2g, Na
2HPO
412H
2O2.9g, NaCl8g, Tween20 0.5ml, adding distil water is made to 1000ml, and its pH is 7.4; Described sealer is 1% bovine serum albumin(BSA), 5% sucrose and 0.05%NaN
3Phosphate buffer form.
In the said determination method, described matrix liquid is H
2O
21: 1 potpourri with tetramethyl benzidine; Color development stopping is to use 1M H
2SO
4Make the stop buffer color development stopping; The dilution that described dilution sample to be checked and preparation series standard product use is formed by add 3% bovine serum albumin(BSA) in washing lotion.
In the said determination method, described serial Apoliprotein M standard items initial concentration is 2.60g/L, does dilution in 1: 1000 with dilution earlier, remakes doubling dilution during use.
In the said determination method, described sample to be checked is people's fasting plasma, serum or pooled plasma, uses the 50mM NaH of pH7.4 during detection earlier
2PO
4Damping fluid is done dilution in 1: 100, does dilution in 1: 10 with dilution again.
In the said determination method, described monoclonal antibody and polyclonal antibody are rabbit source or mouse source, and these antibody also can come from other mammal.
The measurement range of assay method of the present invention is that Apoliprotein M content is at 0.25g/L～2.60g/L.The coefficient of variation scope of assay method is 5.45～9.38%.
The technology of the present invention effect: the monoclonal of used anti-Apoliprotein M or polyclonal antibody have the only specificity of anti-Apoliprotein M in the assay method of the present invention, thereby have got rid of the influence of other materials to measurement result; The utensil that is adopted is as 96 hole microwell plates and instrument (microplate reader) etc.; Used main agents all belongs to market as coating buffer, washing lotion, dilution, matrix liquid, stop buffer, confining liquid etc. and easily buys.The used Apoliprotein M standard items and the monoclonal antibody of anti-Apoliprotein M and polyclonal antibody invention can provide per capita, the mensuration process is easy to operate and control, therefore, the present invention has set up a kind of feasible assay method for newfound Apoliprotein M, and Apoliprotein M is provided clinical directive function and the function of further studying Apoliprotein M may.In concrete mensuration process, no matter be with antibody sandwich on 96 hole microwell plates, still with sealer sealing residue site, labeled monoclonal antibody, serial Apoliprotein M standard items and sample to be checked combine with coated antibody, all adopt and hatch certain hour guaranteeing various reactions or in conjunction with reaching fully, and all want the clear liquid several subsequently, guarantee that unreacted matters or excessive reagent washes off fully, make the result precision that detects improve favorable reproducibility like this.Determine the typical curve that sample to be checked is used, all carry out simultaneously at every turn that it is minimum that environment or other human factors are reduced to, and improved method and accuracy and reappearance with sample to be checked.
Embodiment
Below in conjunction with embodiment the present invention is further described in detail, but is not limited to this.
One, measures with reagent and equipment
Removing refers else is the medical agent of market purchasing.
1, coating buffer: get 1.56gNa
2CO
3, 2.93gNaHCO
3And 0.20gNaN
3With deionized water dissolving and be diluted to 1000ml, its pH is 9.6;
2, washing lotion: get 0.2gKH
2PO
4, 2.9gNa
2HPO
412H
2O, 8gNaCl, 0.5ml Tween20 add deionized water to 1000ml, and its pH is 7.4;
3, dilution: the washing lotion that contains 3% bovine serum albumin(BSA);
4, matrix liquid: H
2O
2With 1: 1 potpourri of tetramethyl benzidine, available from R﹠amp; D Systems, catalog number (Cat.No.): DY999;
5, stop buffer: 1M H
2SO
4
6, confining liquid: the phosphate buffer by 1% bovine serum albumin(BSA), 5% sucrose and 0.05% mixes;
7, the polyclonal antibody of Apoliprotein M standard items, anti-Apoliprotein M and monoclonal antibody, the monoclonal antibody of the anti-Apoliprotein M different with the monoclonal antibody binding site with anti-Apoliprotein M polyclone, being inventor's self-control also can provide;
8, Streptavidin-horseradish peroxidase is available from R﹠amp; D Systems, catalog number (Cat.No.): DY998;
9, the microplate reader model is Benchmark, BIO-RAD company;
10,96 hole microwell plates: binding ability is arranged with protein.
Two, the mensuration of human plasma Apoliprotein M
In following examples, Apoliprotein M all is called for short ApoM.
Embodiment 1
1, bag is by the polyclonal antibody of anti-Apoliprotein M
1. use the polyclonal antibody (concentration is 6.6mg/ml) of 1: 1 anti-ApoM of dilution of coating buffer, in each hole of 96 hole microwell plates, add 100 μ l, about 6.6 μ g in each hole, incubated at room is spent the night, guaranteeing that antibody interacts with microwell plate fully combines, and inhales with washing lotion subsequently and washes microwell plate 3 times;
2. add 300 μ l confining liquids in every hole again, seal residue site in every hole, incubated at room 1 hour is guaranteed complete closed, inhales with washing lotion and washes microwell plate 2 times;
3. microwell plate is deposited in 4 ℃, standby, 6 months operating periods.
2, prepare series A poM standard items
With initial concentration is 2.60g/l, and standard items ApoM does dilution in 1: 1000 with dilution, further makes 1: 1,1: 2,1: 4,1: 8 doubling dilution again, and the series A poM standard items that acquisition is made up of the ApoM standard items of 4 kinds of variable concentrations are standby;
3, prepare sample to be checked
With human plasma 50mMNaH to be checked
2PO
4Damping fluid is done dilution in 1: 100, does dilution in 1: 10 with dilution again, is sample to be checked;
4, with above-mentioned series A poM standard items and sample to be checked, in each hole of the microwell plate that wraps quilt, respectively add 100 μ l respectively, blank well adds dilution, and incubated at room 2 hours makes it fully to combine with coated antibody, inhales with washing lotion and washes microwell plate 3 times;
5, make biotin labeled ApoM monoclonal antibody (catalog number (Cat.No.) 21450 of this kit) with the plain labelling kit of EZ-Link NHS-PEO solid phase biological earlier, therefrom getting 100 μ l is added to respectively in each hole of microwell plate, incubated at room 1 hour is inhaled with washing lotion and is washed microwell plate 5 times; Add 100 μ l Streptavidin-horseradish peroxidases in every hole, combine, enzyme is combined with sample to be checked and ApoM standard items respectively, inhale with washing lotion and wash microwell plate 5 times with biotin;
6, add 100 μ l matrix liquid in every hole, matrix liquid colour developing under the catalysis of enzyme, incubated at room 30 minutes adds 50 μ l stop buffers then, does not measure sample to be checked and the series standard product absorbance at the 450nm place with the luminosity score after 10 minutes;
7, set up the typical curve of the concentration of standard items and absorbance and find the ApoM content of sample to be checked from this curve.
Measurement range is at 0.25g/l～2.60g/l ApoM, and the scope of the coefficient of variation is 5.45～9.38%.
Embodiment 2
The personnel selection pooled plasma replaces the single blood plasma of embodiment 1 as sample to be checked, and all the other are operated with embodiment 1, and the coefficient of variation scope of testing result is identical with embodiment 1.
Claims (8)
1, a kind of assay method of Apoliprotein M, it is characterized in that: the monoclonal antibody or the polyclonal antibody that utilize anti-Apoliprotein M, set up enzyme linked immunosorbent assay, measure the content of Apoliprotein M in human plasma or the serum, the monoclonal of above-mentioned anti-Apoliprotein M or polyclonal antibody, have the only specificity of anti-Apoliprotein M, concrete determination step is: bag is by the monoclonal of anti-Apoliprotein M or polyclonal antibody; Prepare serial Apoliprotein M standard items; Prepare sample to be checked; Make series standard product and sample to be checked simultaneously respectively with the antibodies of wrapping quilt, make the monoclonal antibody combination with it respectively simultaneously of mark again, with matrix liquid colour developing color development stopping after 20～30 minutes, measure above-mentioned series standard product and the reacted absorbance of sample to be checked after 10 minutes; Set up the typical curve of the concentration of series standard product and absorbance and find the content of the Apoliprotein M of sample to be checked from this curve.
2, assay method according to claim 1, it is characterized in that: described bag is to be 9.6 coating buffer with pH by the monoclonal of anti-Apoliprotein M or polyclonal antibody, the monoclonal antibody or the polyclonal antibody of anti-Apoliprotein M are fully combined with 96 hole microwell plates, with pH is after 7.4 washing lotion is cleaned micropore, with remaining the site in the confining liquid blind hole that contains the protein except that Apoliprotein M, after above-mentioned washing lotion cleaning, microwell plate is deposited in 4 ℃, 6 months operating periods.
3, assay method according to claim 1 is characterized in that: the monoclonal antibody of described mark is directly to use enzyme, is different from the monoclonal antibody that the monoclonal antibody that is coated on the 96 hole microwell plates makes mark with binding site; Or use biotin earlier, and be different from the monoclonal antibody that is coated on the 96 hole microwell plates with binding site and make biotin labeled monoclonal antibody, before with the colour developing of matrix liquid, fully combine with enzyme again.
4, according to claim 1 or 2 or 3 described assay methods, it is characterized in that: used coating buffer is by 1.56gNa
2CO
3, 2.93gNaHCO
3And 0.20gNaN
3Add deionized water and make to 1000ml, its pH is 9.6; Used washing lotion is by KH
2PO
40.2g, Na
2HPO
412H
2O2.9g, NaCl8g, Tween20 0.5ml, adding distil water is made to 1000ml, and its pH is 7.4; Described sealer is 1% bovine serum albumin(BSA), 5% sucrose and 0.05%NaN
3Phosphate buffer form.
5, assay method according to claim 1 is characterized in that: described matrix liquid is H
2O
21: 1 potpourri with tetramethyl benzidine; Color development stopping is to use 1M H
2SO
4Make the stop buffer color development stopping; The dilution that described dilution sample to be checked and preparation series standard product use is formed by add 3% bovine serum albumin(BSA) in washing lotion.
6, assay method according to claim 1 is characterized in that: described serial Apoliprotein M standard items initial concentration is 2.60g/L, does dilution in 1: 1000 with dilution earlier, remakes doubling dilution during use.
7, assay method according to claim 1 is characterized in that: described sample to be checked is people's fasting plasma, serum or pooled plasma, uses the 50mM NaH of pH7.4 during detection earlier
2PO
4Damping fluid is done dilution in 1: 100, does dilution in 1: 10 with dilution again.
8, assay method according to claim 1 is characterized in that: described monoclonal antibody and polyclonal antibody are rabbit source or mouse source.
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|---|---|---|---|
| CNA2004100138183A CN1556409A (en) | 2004-01-02 | 2004-01-02 | Determination method of apoliprotein M |
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|---|---|---|---|
| CNA2004100138183A CN1556409A (en) | 2004-01-02 | 2004-01-02 | Determination method of apoliprotein M |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007058588A1 (en) * | 2005-11-18 | 2007-05-24 | T.A.C Thrombosis And Coagulation Aktiebolag | Monoclonal antibodies against apolipoprotein m |
| CN102680710A (en) * | 2012-05-22 | 2012-09-19 | 成都华神生物技术有限责任公司 | Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application |
| CN102707068A (en) * | 2012-05-31 | 2012-10-03 | 北京大学 | Application of complement factor H (CFH) to genetic expression products of methamphetamine (METH) addiction patient |
| CN110325201A (en) * | 2016-08-15 | 2019-10-11 | 儿童医学中心公司 | ApoM-Fc fusion protein, its method with the compound of sphingol L- phosphoric acid (SIP) and for treating blood vessel and non-vascular disease |
-
2004
- 2004-01-02 CN CNA2004100138183A patent/CN1556409A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007058588A1 (en) * | 2005-11-18 | 2007-05-24 | T.A.C Thrombosis And Coagulation Aktiebolag | Monoclonal antibodies against apolipoprotein m |
| CN102680710A (en) * | 2012-05-22 | 2012-09-19 | 成都华神生物技术有限责任公司 | Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application |
| CN102680710B (en) * | 2012-05-22 | 2014-06-11 | 成都华神生物技术有限责任公司 | Enzyme-linked immunosorbent assay kit for human oxidized low-density lipoprotein, and using method and application |
| CN102707068A (en) * | 2012-05-31 | 2012-10-03 | 北京大学 | Application of complement factor H (CFH) to genetic expression products of methamphetamine (METH) addiction patient |
| CN102707068B (en) * | 2012-05-31 | 2015-03-18 | 北京大学 | Application of complement factor H (CFH) to genetic expression products of methamphetamine (METH) addiction patient |
| CN110325201A (en) * | 2016-08-15 | 2019-10-11 | 儿童医学中心公司 | ApoM-Fc fusion protein, its method with the compound of sphingol L- phosphoric acid (SIP) and for treating blood vessel and non-vascular disease |
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