CN1111201C - Low-density liporotein acceptor testing reagent and method - Google Patents
Low-density liporotein acceptor testing reagent and method Download PDFInfo
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- CN1111201C CN1111201C CN 99121274 CN99121274A CN1111201C CN 1111201 C CN1111201 C CN 1111201C CN 99121274 CN99121274 CN 99121274 CN 99121274 A CN99121274 A CN 99121274A CN 1111201 C CN1111201 C CN 1111201C
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- density lipoprotein
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Abstract
The present invention relates to a reagent and a method for testing low-density lipoprotein acceptors. The reagent of the present invention is composed of low-density lipoprotein, amphoteric adhesives, cell separation liquid, composite agents for fixing cells and preventing composition from moving inward, reaction system stop and color developing agents, and system quantization standard references. The test method comprises: a standard reaction pool is prepared; the cell amphoteric adhesives are placed for 2 hours; cells in blood are strictly separated by the cell separation liquid; a certain amount of the cells are placed in the standard reaction pool; the composite agents are added in the standard reaction pool; a reaction pool and an empty reaction pool are prepared; the low-density lipoprotein is added; low-density lipoprotein antibodies labeled by enzyme are added; the reaction system stop and color developing agents are added. The method can directly and quantitatively analyze low-density lipoprotein acceptors of which the cell surfaces have biological functions, and is an ideal test method for diagnosing familial hyperlipemia.
Description
The present invention relates to a kind of low-density liporotein acceptor testing reagent and method, particularly diagnose detection reagent and the method for familial hypercholesterolemia pair cell LDL-R.
The low-density lipoprotein metabolism is the low density lipoprotein receptor that function is arranged by cell surface, the LDL-R of round-robin LDL and cell surface is mutually combined, carried out katabolism by cytophagy, lipophorin resolves into amino acid, lipid is participated in lipid metabolism, prevent that the LDL level increases in the blood circulation, too much LDL is engulfed by phagocytic cell and artery substrate smooth muscle cell, forms foam cell and assembles and cause atherosclerosis.Familial hypercholesterolemia is because LDL-R gene and other factors cause cell surface LDL-R dysfunction, can not combine with round-robin LDL or in conjunction with after LDL can not be swallowed cell and causes LDL to increase, nonage just forms arteriosclerosis, xanthoma, other factors also can make LDL increase with the exception of this, how to diagnose the patient whether the proteic defective of cell surface LDL-R is arranged, be that whether the LDL level increases and come Indirect evaluation from blood plasma the earliest, its weak point is: this method can not determine that the high-caliber LDL of blood plasma is owing to the LDL-R dysfunction causes.Then foreign study utilizes the method for the anti-LDL antibody of labelled with radioisotope to check free LDL-R in the blood plasma, but free LDL-R and the LDL-R of cell surface are in line relevantly fully, therefore detect the LDL-R protein function that free LDL-R in the blood plasma can not estimate cell surface fully.To the domestic and international 2,300,000 pieces of literature searches of 1994-1999, not seeing has the pertinent literature report through Institute of Medical Information of the Chinese Academy of Medical Sciences.Detect industry for this reason and thirst for having the cell LDL-R detection method that to diagnose familial hypercholesterolemia.
The object of the present invention is to provide a kind of detection reagent, with having the low density lipoprotein receptor LDL-R of biological function to carry out quantitative analysis by the direct pair cell of certain operation steps surface, come the low-density liporotein acceptor testing reagent and the method for diagnostic detection familial hypercholesterolemia with this reagent.
The object of the present invention is achieved like this: preparation reagent: the component of reagent has: low-density lipoprotein; The anti-low-density lipoprotein both sexes tackiness agent of horseradish peroxidase-labeled; Cellular segregation liquid; Cell fixation and prevent to move recombiner in the receptor-ligand binding substances; Reaction system stops and the reaction solution agent; System quantitative criterion object of reference.Detect step: it is identical to get the reaction tank floorage, the standard reaction pond that the bottom absorbancy is identical; Get cell both sexes tackiness agent, under 20 ℃-50 ℃ temperature, placed 2 hours; With the cell in the strict separating blood of cellular segregation liquid, get certain cell and be positioned in the reaction tank 20-60 minute, add in the cell fixation acceptor and move recombiner, 2-18 minute; System quantitative criterion object of reference reaction tank and blank reaction tank are set; Add low-density lipoprotein, 20-50 ℃ was reacted one hour; Add the anti-low-density lipoprotein antibody of enzyme labelling, reacted 5-50 minute; Add reaction system and stop and reaction solution liquid, stablized 5-40 minute; Detect with enzyme micro-plate reader.
Advantage of the present invention is:
1, directly the pair cell surface has the low density lipoprotein receptor (LDL-R) of biological function to carry out quantitative analysis, solved at present and can not carry out the direct quantitative analysis by pair cell Surface L DL-R, can only measure the low density lipoprotein cholesterol (LDL-C) in the blood circulation or check what problem of free LDL-R protein level.
2, make medical diagnosis on disease be transformed into to measure bioactive mcroorganism macromole is arranged, can more direct inspection analyze the biological function material of body by the present simple macromolecular substance of measuring.
3, can effectively detect the familial high-cholesterol disease.
Below in conjunction with embodiment in detail the present invention is described in detail.
Technical scheme of the present invention is: diagnosis employed reagent of familial hypercholesterolemia and detection method.It comprises two big steps, the one, and preparation reagent, the 2nd, operation steps.Reagent is the product that uses in the testing process, and its component is: 1, low-density lipoprotein, content 0.8-10.0mmol/L; 2, the anti-low-density lipoprotein both sexes tackiness agent 20-30W/V of horseradish peroxidase-labeled, the main component of anti-low-density lipoprotein both sexes tackiness agent is N1 N-dioctadeylsuccinamic a ciddioctadecyl ylutdmate HCL salt, chemical molecular formula;
Its making method is, the high molecular bonding thing is applied on the Sptting plate, forms film, and cell can firmly adsorb above it.3, cellular segregation liquid 10-40W/V, NaHCO30.1-1.5g; 4, cell fixation and prevent to move recombiner in the receptor-ligand binding substances, ldl receptor and LDL part on the cytolemma, cell fixation before combination, dehydration does not exist in the receptor-ligand binding substances and moves problem, and fixedly dewatering agent is a methyl alcohol; Reaction system stops and the reaction solution agent, and the main component of reaction solution agent is: 33 '-Diaminobenzidinetetrhydrochlorido, C
12H
18CL
4N
4Utilize normal people's mixed lymphocytes as system quantitative criterion object of reference.
Detection method is undertaken by following step: it is identical 1, to get the reaction tank floorage, the standard reaction pond that the bottom absorbancy is identical; 2, get cell both sexes tackiness agent 20-25ul in the part, placed 2 hours for 20 ℃-50 ℃; 3, with cellular segregation liquid (FICOLL) 10-40w/v in the part, the cell in the strict separating blood of NaHCO30.1-1.5g is got certain cell and is positioned in the reaction tank, and 20 ℃-80 ℃, 20-60 minute; 4, move recombiner 4-30UL, 2-18 minute in the cell fixation acceptor in the adding part; 5, system quantitative criterion object of reference reaction tank and blank reaction tank are set; 6, add low-density lipoprotein 5-150ul, 20-50 ℃ was reacted one hour; 7, the anti-low-density lipoprotein antibody that adds enzyme labelling, certain temperature (according to the practical situation fixed temperature) reaction 5-50 minute; 8, add reaction system and stop and reaction solution liquid 5-30ul, stablized 5-40 minute; Detect with enzyme micro-plate reader.9, determine whether to be hypercholesterolemia according to detected result.
Claims (2)
1, a kind of low-density liporotein acceptor testing reagent is characterized in that the component of reagent has: low-density lipoprotein; The anti-low-density lipoprotein both sexes tackiness agent of horseradish peroxidase-labeled; Cellular segregation liquid; Cell fixation and prevent to move recombiner in the receptor-ligand binding substances; Reaction system stops and the reaction solution agent; System quantitative criterion object of reference.
2, a kind of detection method with claim 1 pair low density lipoprotein receptor is characterized in that it detects by following detection step:
A, to get the reaction tank floorage identical, the standard reaction pond that the bottom absorbancy is identical;
B, get cell both sexes tackiness agent, under 20 ℃-50 ℃ temperature, placed 2 hours;
C, with the cell in the cellular segregation liquid strict separating blood, get certain cell and be positioned in the reaction tank 20-60 minute, add in the cell fixation acceptor and move recombiner, 2-18 minute;
D, system quantitative criterion object of reference reaction tank and blank reaction tank are set; Add low-density lipoprotein, 20-50 ℃ was reacted one hour;
The anti-low-density lipoprotein antibody of e, adding enzyme labelling reacted 5-50 minute;
F, adding reaction system stop and reaction solution liquid, stablize 5-40 minute;
G, detect with enzyme micro-plate reader.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99121274 CN1111201C (en) | 1999-11-04 | 1999-11-04 | Low-density liporotein acceptor testing reagent and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99121274 CN1111201C (en) | 1999-11-04 | 1999-11-04 | Low-density liporotein acceptor testing reagent and method |
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| Publication Number | Publication Date |
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| CN1295132A CN1295132A (en) | 2001-05-16 |
| CN1111201C true CN1111201C (en) | 2003-06-11 |
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| CN 99121274 Expired - Fee Related CN1111201C (en) | 1999-11-04 | 1999-11-04 | Low-density liporotein acceptor testing reagent and method |
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| JP4677545B2 (en) * | 2003-10-15 | 2011-04-27 | 積水メディカル株式会社 | Method for fractional determination of multimeric adiponectin |
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