CN100422743C - Kit for diagnosing high triglyceride - Google Patents
Kit for diagnosing high triglyceride Download PDFInfo
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- CN100422743C CN100422743C CNB2006100187475A CN200610018747A CN100422743C CN 100422743 C CN100422743 C CN 100422743C CN B2006100187475 A CNB2006100187475 A CN B2006100187475A CN 200610018747 A CN200610018747 A CN 200610018747A CN 100422743 C CN100422743 C CN 100422743C
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Abstract
The present invention relates to a kit used for diagnosing familial hypertriglyceridemia (FHTG). Apolipoprotein A5 (ApoA5) is used as a biochemical criterion for FHTG diagnosis to make prepare anti-ApoA5 monoclonal antibody pairs. Enzyme standard anti-ApoA5 monoclonal antibodies and unmarked anti-ApoA5 monoclonal antibodies, confining liquid, enzyme substrate solution, peridium buffer solution, termination solution, washing solution and an enzyme target are arranged in the kit. The kit uses the unmarked anti-ApoA5 monoclonal antibodies for coating the enzyme target so as to catch ApoA5 in a human body's blood and uses the enzyme standard anti-ApoA5 monoclonal antibodies for recognizing and detecting ApoA5 in a human body's blood for the etiological diagnosis of familial hypertriglyceridemia so as to effectively prevent and treat cardiovascular diseases, such as coronary heart disease, apoplexy, etc.
Description
Technical field
The present invention relates to the biochemical indicator of ApoA5 (ApoA5) as hypertriglyceridemia (FHTG) diagnosis, the right technology of preparing of anti-ApoA5 antibody, enzyme labeling colour developing immune response technology and the technical field of utilizing the antibody of ApoA5 to come diagnosing high triglyceride (FHTG), particularly a kind of kit and production method that is used for diagnosing high triglyceride (FHTG).
Background technology
Unusual and coronary heart disease of apolipoprotein and apoplexy are closely related.Correctly diagnose as early as possible, help these serious diseases of early prevention and treatment.The exploitation of apolipoprotein diagnostic kit helps effective control of coronary heart disease and apoplexy.ApoA5 belongs to the ApoAI/CIII/AIV gene group, and is at liver expression, relevant with HDL/VLDL.The density loss of triglyceride is 1/3rd of the contrast mouse in the blood plasma of expressing the genetically modified mouse of human ApoA5, and has increased by 4 times in lacking the mouse of this gene.The single nucleotide variations of ApoA5 gene promoter (SNP) directly influences triglyceride levels.Yet, at present and do not know that ApoA5 regulates the mechanism of action of triglyceride.
High triglyceride can make little dense low-density lipoprotein (sLDL) concentration raise, and has stronger atherogenicity effect.Triglyceride levels is high more, and the cholesteryl ester in the low-density lipoprotein is many more.People are verified, and the cholesterol in the atherosclerotic plaque mainly comes from low-density lipoprotein, and low-density lipoprotein is that to contain cholesterol in the blood plasma maximum, also is the strongest lipoprotein of atherogenicity.In addition, epidemiological study shows that high levels of triglycerides descends high-density lipoprotein concentration, and high-density lipoprotein concentration is subjected to the influence of triglyceride levels, and both are negative correlation.Recently studies show that, blood triglyceride levels is unusual and the body intravascular coagulation factor is closely related unusually, the plasma triglyceride level raises and can cause body intravascular coagulation factor level to raise, thereby promotion blood clotting, and can make the synthetic increase of tissue plasminogen activator's inhibitor (PAI), suppress fibrinolysis, form hypercoagulative state, promote the generation of coronary heart disease.There is experimental result to show, triglyceride concentration rising 1mmol/L, the cardiovascular accident incidence male sex rises 32%, and the women is 76%.After the influence of proofreading and correct high-density lipoprotein (HDL), the level of significance male sex rises 14%, and the women is 37%.These results show that it is the important risk factor of angiocardiopathy that serum triglyceride concentration raises.
Hypertriglyceridemia patient often has hypertension and insulin resistance simultaneously.Insulin resistance is often referred to the patient and tends to non-insulin dependence abnormal carbohydrate metabolism, and such patient often forms coronary artery disease earlier.Usually lipid triad, hypercoagulative state, hypertension, insulin resistance are called metabolic syndrome.It is unusual to have confirmed that the hypertriglyceridemia patient often is associated with in the metabolic syndrome other.The clinical meaning of hypertriglyceridemia is this.And seeming particularly important as the index ApoA5 that directly influences triglyceride levels, the single nucleotide polymorphism of ApoA5 is expected to the treatment target spot as risk indicateing arm and reduction serum lipid concentrations.The present invention be exactly with ApoA5 by the ELISA experimental applications in clinical practice reaching the etiological diagnosis of hypertriglyceridemia, and effective control of angiocardiopathies such as coronary heart disease and apoplexy.
The sandwich double-antibody technique is the newer back antibody technique of biological technical field that is based upon on the monoclonal antibody technique.Double antibody sandwich method is used antibody capture antigen is come in two sites of identification antigen, avoided antibody to combine with the competition in the same site of antigen, reduced the steric hindrance of antigenic determinant, so more general antigen trapping is more responsive, specificity is stronger, repeatability is better, is particularly useful for the detection of antigenic content sample seldom.The present invention is right in conjunction with the antibody at ApoA5 (ApoA5) of this technological development, and micro-biochemical indicator (antigen) more highlights its significance in the FHTG patient blood to detecting.
Summary of the invention
The purpose of this invention is to provide the biochemical indicator of a kind of ApoA5 of utilization as the FHTG diagnosis, the monoclonal antibody for preparing anti-ApoA5 is right, and a kind of easy kit of setting up based on euzymelinked immunosorbent assay (ELISA) (ELISA) principle that is easy to get, be used for measuring the concentration of human serum ApoA5 and hypertriglyceridemia is carried out etiological diagnosis, thereby reach effective control of angiocardiopathies such as coronary heart disease and apoplexy.
The object of the present invention is achieved like this: a kind of hypertriglyceridemia (FHTG) diagnostic kit comprises that enzyme marks the monoclonal antibody of anti-ApoA5 and the monoclonal antibody of unmarked anti-ApoA5; Confining liquid; Enzyme substrate solution; Bag is cushioned liquid; Stop buffer; Cleansing solution; ELISA Plate; Wherein the monoclonal antibody bag of unmarked anti-ApoA5 quilt is in ELISA Plate.The antibody of described anti-ApoA5 is that monoclonal antibody is right.
Described anti-ApoA5 monoclonal antibody obtains the spleen cell of antigenic stimulus with the ApoA5 immunity BALB/c mouse of purifying, merges the myeloma cell line SP2/0 of this spleen cell and mouse; Utilize HAT to select nutrient culture media to select hybridoma, and utilize ELISA method Screening and Identification to produce the cell of anti-ApoA5 antibody; Carry out obtaining after three time cloningizations secreting the cell line of the monoclonal antibody of high specific, preserve the cell line of the monoclonal antibody that the generation that screened combines with the ApoA5 high specific, produce the monoclonal antibody of anti-ApoA5 by mouse ascites.Utilize Protein A-SePharose affinity column to be further purified the monoclonal antibody of anti-ApoA5 in the ascites again, ELISA and Western-Blot identify the monoclonal antibody of separation and purification.
Anti-ApoA5 monoclonal antibody swells the right screening of antibody, the main subclone that adopts inhibition method screening high-affinity, and then on the basis of repeatedly cloning and ELISA addition experimental result, be at war with and suppress the mensuration of epitope analysis and affinity, obtain the monoclonal antibody cell line of different epi-positions, further production ascites and purifying obtain volume antibody, carry out the individual plant mark, adopt the ELISA direct method to carry out the working concentration titration of labelled antibody, adopt ELISA competition inhibition method to carry out single labeling antibody epi-position then and measure, thereby the sandwich antibody that filters out efficient affinity is right.
What the monoclonal antibody of anti-ApoA5 of marking enzyme adopted is sodium periodate method horseradish peroxidase-labeled antibody.With NaIO
4Earlier the glycan molecule with the HRP surface is oxidized to aldehyde radical, and then is that amino on the immunoglobulin (Ig) combines with the monoclonal antibody of ApoA5, further uses NaBH
4Reduction generates stable enzymic-labelled antibody.Centrifugal removal precipitation behind the dialysis purifying, supernatant is enzyme-antibody conjugates.
The grand antibody of anti-ApoA5 monoclonal antibody is evenly wrapped by on the ELISA Plate hole of unit area, and bag is cushioned the carbonate buffer solution that liquid is 0.05M pH9.6, promptly contains 1.59g Na in 1 liter of solution
2CO
3, 2.93gNaHCO
3Described confining liquid is 1% bovine serum albumin(BSA); Described enzyme substrate solution is that TMB uses liquid, i.e. 60 μ g/ml TMB and 0.05%H
2O
2In 0.1M pH6.0 phosphate buffer; Stop buffer is 1mol/LH
2SO
4Solution.
Hypertriglyceridemia diagnostic kit provided by the present invention is that biochemical indicator comes the diagnosing high triglyceride cause of disease with ApoA5.This comprises the right method of monoclonal antibody for preparing above-mentioned ApoA5, and utilizes the ELISA method to measure the concentration of ApoA5 in the human serum, and the cause of disease with diagnosis FHTG reaches the effective control to angiocardiopathies such as coronary heart disease and apoplexy.
Monoclonal antibody, ELISA Plate, confining liquid, enzyme substrate solution, bag that this kit is marked the monoclonal antibody of anti-ApoA5, unmarked anti-ApoA5 by enzyme are cushioned liquid, cleansing solution and stop buffer and form.
Compare with technology with existing index, the present invention has outstanding advantage and practicality:
1, detection specificity is strong.The project that detects blood fat clinically has a lot, and the elementary item of each hospital's general inspection has T-CHOL (TC), triglyceride (TG), apolipoprotein (Apo) B, Apo AI, lipoprotein (a) [Lp (a)] etc.Survey data studies show that high triglyceride, high cholesterol, low HDL-C and high LDL-C have constituted the hazards of coronary heart disease jointly.Serum ApoAI can represent the HDL level, is proportionate with HDL-C, and its clinical meaning is broadly similar also.But HDL is a series of grain sizes and form inhomogenous lipoprotein, and HDL subclass and composition tend to change under the pathological state, thus ApoAI go up and down with different paces calmly and the HDL-C variation proportional fully.ApoB100 is nearly to have 90% ratio to be distributed among the LDL, so serum ApoB mainly represents the LDL level, it and serum LDL-C level are obvious positive correlation, and the clinical meaning of ApoB level height is also similar to LDL-C.But, under a few cases, the normal situation of LDL-C concentration high ApoB100 mass formed by blood stasis can appear and, so these two kinds of apolipoprotein reliabilities and accuracy are very not satisfactory.ApoA5 has very high susceptibility and specificity as the etiological diagnosis index of hypertriglyceridemia.The single nucleotide variations of ApoA5 gene promoter (SNP) directly influences triglyceride levels.Experimental study shows that the density loss of triglyceride is 1/3rd of the contrast mouse in the blood plasma of expressing the genetically modified mouse of human ApoA5, and has increased by 4 times in lacking the mouse of this gene.
2, detection sensitivity height.The concentration of ApoA5 is very low in the human blood, on average is about 215ng/ml.The present invention adopts the ApoA5 in the double antibody sandwich method detection serum, makes the susceptibility of detection higher.
3, avoid the performance results merely of like product in the market, but can not offer some clarification on the shortcoming of the cause of disease.Hypertriglyceridemia is that ApoA5 gene mutation reason causes once finding, and can carry out corresponding treatment at the cause of disease, thereby fundamentally solve the prevention and the treatment of the relevant disease of hypertriglyceridemia and initiation thereof.
Experimental result of the present invention and analysis:
ELISA detects the concentration of ApoA5 in the different samples, utilizes the monoclonal antibody of prepared anti-ApoA5 right, adopts sandwich style ELISA method, measures the concentration of ApoA5 in the different samples.Result such as following table and Fig. 1.
Antibody: anti-ApoA5,0.1 μ g/ml, 100 μ l/ holes; Confining liquid: 1%BSA.By measurement result as seen, sandwich style ELISA method is that sensitivity is about 150ng/ml to the detection lower limit of ApoA5, is lower than the mean concentration (215ng/ml) of ApoA5 in the human blood.
Description of drawings
Fig. 1 is the curve map of ApoA5 concentration and absorbance.
Embodiment
The production method of hypertriglyceridemia (FHTG) diagnostic kit:
1, the right preparation of the monoclonal antibody of ApoA5
ApoA5 with purifying distinguishes immune BALB/c mouse as antigen, immunity in 21 days for the second time at interval, the immunity in 10 days of later every interval once, immune 3-4 time altogether, take out the spleen cell of immune mouse then, with PEG the myeloma cell line SP2/0 of this spleen cell and mouse is merged, utilize the HAT selective medium on 96 porocyte culture plates, to screen hybridoma, identify the cell line of anti-ApoA5 with the ELISA method, carry out obtaining after three time cloningizations cell line of stably excreting highly specific monoclonal antibody, adopt ELISA to suppress method at the same antigen bag on by plate, be to add cells and supernatant in the reference group, add cells and supernatant and antigen mixture in the inhibition group, thereby compare two groups of different subclones that filter out high-affinity of OD value difference.Experimentally carry out the additive process epitope analysis at indirect ELISA then, promptly add two individual plant cells and supernatant in the reference group respectively, add two strain cells and supernatant in the mensuration group simultaneously, compare two groups of OD values (A1, A2 and A1+2), calculate by following formula:
On the basis of comparing result of calculation, carry out the mensuration of epitope analysis and affinity, the AI value is the addition feminine gender less than 50%, the epi-position of the antibody recognition antigen of i.e. two cell lines secretion is identical, the AI value is the addition positive greater than 50%, represent between two cell lines uncontested, two different epi-positions of the antibody recognition antigen of i.e. two cell lines secretion, thus monoclonal antibody cell line tentatively obtained at the different epi-positions of antigen.Further the monoclonal antibody cell line that obtains is injected into mouse peritoneal respectively and carries out ascites production, utilize ProteinA-Sepharose affinity column purifying ascites to obtain the volume monoclonal antibody, with the HRP enzyme antibody purified is carried out the individual plant mark, carry out the working concentration titration of labelled antibody with the ELISA direct method, in reference group, add enzyme labelled antibody with ELISA competition inhibition method then, in the mensuration group, add enzyme labelled antibody and unmarked mixtures of antibodies to be measured, compare two groups of OD values, reference group OD value exists competition to suppress greater than the expression of mensuration group OD value, and the epi-position that is considered as two antibody recognition antigens is identical.Otherwise two antibody are considered as the epi-position difference of two antibody recognition antigens when not existing competition to suppress, thereby the sandwich antibody that final screening obtains efficient affinity is right.
2, the anti-ApoA5 monoclonal antibody method of enzyme labeling
What the monoclonal antibody of anti-ApoA5 of marking enzyme adopted is sodium periodate method horseradish peroxidase-labeled antibody.Take by weighing in the HRP dissolving distilled water.Add the 0.1M NaIO that newly joins
4Solution, lucifuge stirred 20 minutes under the room temperature.NaIO
4Glycan molecule with the HRP surface is oxidized to aldehyde radical earlier.Above-mentioned solution is packed in the bag filter, adds the sodium-acetate buffer dialysis of 1mM PH4.4, and 4 ℃ are spent the night.Add 0.2M PH9.5 carbonate buffer solution, make the pH of above hydroformylation HRP be elevated to 9.0～9.5, add the monoclonal antibody of ApoA5 then immediately, in the 0.01M carbonate buffer solution, the room temperature lucifuge stirred 2 hours gently.The HRP that treats hydroformylation and the monoclonal antibody of ApoA5 are after the amino on the immunoglobulin (Ig) combines, to add the 4mg/ml NaBH that newly joins again
4Liquid, put 4 ℃ 2 hours, NaBH
4Its reduction is generated stable enzymic-labelled antibody.Above-mentioned liquid is packed in the bag filter, and in 0.15M pH7.4PBS dialysis, 4 ℃ are spent the night.Centrifugal removal precipitation behind the dialysis purifying, supernatant is the enzyme antibody bond.
3, the compound method of various damping fluids and reagent
1. bag is cushioned liquid: the Na of 0.05M pH9.6
2CO
3-NaHCO
3: Na
2CO
3(MW 105.99) 1.59g and NaHCO
3(MW 84.01) 2.93g, adding distil water is to 1000ml.
2. confining liquid: 1% bovine serum albumin(BSA) (BSA) solution: bovine serum albumin(BSA) 1g; Add phosphate buffer (PBS) (being formulated as follows) to 100ml.
0.01M, the PBS solution of pH7.4: NaCI (MW 58.44) 8.0g, KCl (MW 74.55) 0.2g, KH
2PO
4(MW 136.09) 0.24g, Na
2HPO
412H
2O (MW 358.14) 3.6g, adding distil water is to 1000ml.
3. PBS one Tween-20 of cleansing solution: pH7.4 at 1000ml 0.01M, adds 0.5ml Tween-20 (Tween-20) and gets final product in the PBS solution of pH7.4.
4. zymolyte 3,3,5,5 tetramethyl benzidines (TMB) solution:
I) four times substrate buffer solution: take by weighing 35.8g Na
2HPO
4.12H
2O; Take by weighing the 21.1g citric acid; Respectively add ultrapure water and promptly obtain A mother liquor B mother liquor respectively to 500ml, get A liquid 300ml, transfer pH value to 5.2 promptly with B liquid, four times of dilutions during use.
Ii) TMB concentrate: accurately weighing pulvis TMB400mg adds in the 10ml dimethyl sulfoxide (DMSO) (DMSO), promptly gets fortyfold tmb substrate concentrate after making it to dissolve fully.
Iii) working fluid preparation: the hydrogen peroxide of 4 μ l 30% gets final product behind the complete mixing of 10ml substrate buffer solution (1 *) middle adding 0.025ml TMB concentrate, faces with preceding and now joins.
5. stop buffer: 1mol/LH
2SO
4Solution: the concentrated sulphuric acid (95-98%) 54.3ml, adding distil water gets final product to 1000ml, and timing slowly splashes into the concentrated sulphuric acid in the distilled water, limit edged mixing.
Hypertriglyceridemia (FHTG) diagnostic kit operation steps:
1, formulates typical curve
The monoclonal anti liquid solution of the unmarked anti-ApoA5 of purifying is diluted to 0.1 μ g/ml with coating buffer, joins in the 96 hole ELISA Plate, every hole 100 μ l, 37 ℃ of bags were spent the night by 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds the 1%BSA sealing, every hole 200 μ l, and 37 ℃ were sealed 1 hour; Do not wash, dry, add ApoA5 (0-406ng/ml) standard antigen of variable concentrations, every hole 100 μ l were hatched 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds the anti-ApoA5 monoclonal antibody solution (being diluted to 0.1 μ g/ml with cleansing solution) of enzyme labeling, and every hole 100 μ l were hatched 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds enzyme substrate solution, every hole 100 μ l, and lucifuge colour developing 5 minutes adds stop buffer, every hole 50 μ l.Utilizing microplate reader to measure absorbance value (A450nm) at 450nm, is horizontal ordinate with concentration (ng/ml), and A450nm is an ordinate, the drawing standard curve.
2, the ApoA5 concentration in the mensuration serum
The monoclonal anti liquid solution of the unmarked anti-ApoA5 of purifying is diluted to 0.1 μ g/ml with coating buffer, joins in the 96 hole ELISA Plate, every hole 100 μ l, 37 ℃ of bags were spent the night by 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds the 1%BSA sealing, every hole 200 μ l, and 37 ℃ were sealed 1 hour; Do not wash, dry, add the serum to be detected of suitable multiple dilution, every hole 100 μ l are hatched for 37 ℃ and were spent the night in 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds the anti-ApoA5 monoclonal antibody solution of enzyme labeling, is diluted to 0.1 μ g/ml with cleansing solution, and every hole 100 μ l were hatched 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds enzyme substrate solution, every hole 100 μ l, and lucifuge colour developing 5 minutes adds stop buffer, every hole 50 μ l.From utilizing microplate reader to measure absorbance value A450nm,,, try to achieve the concentration value of ApoA5 in the serum by measured A450nm according to typical curve at 450nm.
3, the result judges
According to cutoff value (the cut off value) 215ng/ml of ApoA5 as hypertriglyceridemia (FHTG) etiological diagnosis index, the decidable that concentration is lower than this value is that ApoA5 lacks, and the danger of hypertriglyceridemia is arranged; Or decidable ApoA5 gene mutation be hypertriglyceridemia main diseases because of.
Claims (1)
1. kit that is used for diagnosing high triglyceride, include enzyme in the kit and mark the monoclonal antibody of anti-ApoA5 and the monoclonal antibody of unmarked anti-ApoA5, confining liquid, enzyme substrate solution, bag is cushioned liquid, stop buffer, cleansing solution, ELISA Plate, it is characterized in that: the monoclonal antibody bag quilt of unmarked anti-ApoA5 is in ELISA Plate, and will resist the grand antibody of ApoA5 monoclonal antibody evenly to wrap by on the ELISA Plate hole of unit area, bag is cushioned the carbonate buffer solution that liquid is 0.05M pH9.6, and wherein carbonate buffer solution is to contain 1.59g Na in 1 liter of solution
2CO
3And 2.93gNaHCO
3, confining liquid is 1% bovine serum albumin(BSA); Enzyme substrate solution is 3,3,5,5 tetramethyl benzidine and the 0.05%H of 60 μ g/ml
2O
2In 0.1M pH6.0 phosphate buffer; Stop buffer is 1mol/LH
2SO
4Solution.
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| CN100422743C true CN100422743C (en) | 2008-10-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018020477A1 (en) * | 2016-07-28 | 2018-02-01 | The Regents Of The University Of California | Methods and compositions for treating hypertriglyceridemia |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559850B (en) * | 2010-12-16 | 2014-03-05 | 益善生物技术股份有限公司 | ApoA5 genic mutation detection specific primer and liquid phase chip |
| CN102586404B (en) * | 2011-01-07 | 2013-10-16 | 同济大学 | Kit for specifically detecting genotype of ApoE4 and application of kit |
| CN102279262A (en) * | 2011-06-30 | 2011-12-14 | 南开大学 | Enzyme-linked immuno-detection assay kit of sunset yellow |
| CN104165992A (en) * | 2014-07-28 | 2014-11-26 | 武汉璟泓万方堂医药科技股份有限公司 | Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia |
| CN104597248B (en) * | 2015-01-05 | 2016-11-23 | 中南大学湘雅二医院 | The method of a kind of non-diagnostic purpose detection ApoA5 biologic activity and test kit |
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2006
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Patent Citations (5)
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|---|---|---|---|---|
| WO2003094852A2 (en) * | 2002-05-11 | 2003-11-20 | Ilex Products, Inc. | Hydroxyphosphonates and phosphonophosphates as apolipoprotein e modulators |
| WO2003105899A1 (en) * | 2002-06-13 | 2003-12-24 | Neuropro Technologies, Inc. | Apparatus and methods for detecting cerebrospinal fluid |
| WO2004108071A2 (en) * | 2003-06-05 | 2004-12-16 | Salk Institute For Biological Studies | Targeting polypeptides to the central nervous system |
| CN1603824A (en) * | 2003-09-29 | 2005-04-06 | 杜凤鸣 | Quantitative enzyme linked immunosorbent assay kit for Apo AI protein in human urine and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018020477A1 (en) * | 2016-07-28 | 2018-02-01 | The Regents Of The University Of California | Methods and compositions for treating hypertriglyceridemia |
| US11090310B2 (en) | 2016-07-28 | 2021-08-17 | The Regents Of The University Of California | Methods and compositions for treating hypertriglyceridemia |
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| CN1834654A (en) | 2006-09-20 |
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