CN104165992A - Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia - Google Patents

Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia Download PDF

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CN104165992A
CN104165992A CN201410362427.6A CN201410362427A CN104165992A CN 104165992 A CN104165992 A CN 104165992A CN 201410362427 A CN201410362427 A CN 201410362427A CN 104165992 A CN104165992 A CN 104165992A
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apoa5
solution
monoclonal antibody
antibody
kit
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钱金宏
廖园园
廖伟
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Wuhan Jinghong Wanfangtang Medical Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

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Abstract

The invention relates to a kit for detecting an apolipoprotein A5 (ApoA5) for diagnosis of hypertriglyceridemia. The kit contains two unlabeled anti-ApoA5 monoclonal antibodies, an enzyme-labeled anti-ApoA5 monoclonal antibody, a blocking solution, an enzyme substrate solution, a coating buffer solution, a stop solution, a washing solution, and an ELISA plate; the two unlabeled anti-ApoA5 monoclonal antibodies coat the ELISA plate and are used as capture antibodies; the enzyme-labeled anti-ApoA5 monoclonal antibody is used as a detection antibody; the coating buffer solution is a carbonate buffer solution with the concentration of 0.05 mol/L and the pH of 9.6, wherein 1 L of the solution contains 1.59 g of Na2CO3 and 2.93 g of NaHCO3; the blocking solution is a bovine serum albumin with the mass concentration of 1%; the enzyme substrate solution is a phosphate buffer solution having the concentration of 0.1 mol/L and the pH of 6.0 and containing 3,3',5',5-tetramethylbenzidine with the concentration of 60 [mu]g/mL and H2O2 with the mass concentration of 0.05%; the stop solution is an H2SO4 solution with the concentration of 1 mol/L. The two monoclonal antibodies coat the ELISA plate to capture ApoA5 in human blood, the antibody capture ability is improved, the determination sensitivity and accuracy and linear range are improved, and test and clinical requirements are reached.

Description

A kind of ApoA5 that detects is with the kit of diagnosing high triglyceride
Technical field
The present invention relates to ApoA5 (ApoA5) as the biochemical indicator of hypertriglyceridemia (HTG) diagnosis, the right technology of preparing of anti-ApoA5 antibody, enzyme labeling colour developing immune response technology and utilize the antibody of ApoA5 to carry out the technical field of diagnosing high triglyceride (HTG), particularly a kind of quantitative detection ApoA5 is with the kit of diagnosing high triglyceride (HTG).
Background technology
The project that detects clinically blood fat 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 research shows, the hazards that high triglyceride, high cholesterol, low high-density lipoprotein (HDL)-C (HDL-C) and high low-density lipoprotein-C (LDL-C) have formed coronary heart disease jointly.Abnormal and the coronary heart disease of apolipoprotein (Apo) and apoplexy are closely related.Correctly diagnose as early as possible, be conducive to these serious diseases of early prevention and treatment.Apolipoprotein detects the exploitation of reagent, contributes to effective control of coronary heart disease and apoplexy.
Serum ApoAI can represent 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, under pathological state, HDL subclass and composition tend to change, thus ApoAI go up and down with different paces fixed and HDL-C variation is completely proportional.ApoB100 is nearly has 90% ratio to be distributed in LDL, so serum ApoB mainly represents 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, can there is high ApoB100 mass formed by blood stasis and the normal situation of LDL-C concentration, so these two kinds of apolipoprotein reliabilities and accuracy are very not satisfactory.
ApoA5 belongs to ApoAI/CIII/AIV gene group, at liver expression, relevant with HDL/VLDL.The single nucleotide variations of ApoA5 gene promoter (SNP) directly affects triglyceride levels.Experimental study shows, in the blood plasma of expressing the genetically modified mouse of mankind ApoA5, the concentration of triglyceride drops to 1/3rd of contrast mouse, and in lacking the mouse of this gene, has increased by 4 times.ApoA5 has very high susceptibility and specificity as the etiological diagnosis index of hypertriglyceridemia.
High levels of triglycerides can make little dense low-density lipoprotein (sLDL) concentration raise, and has stronger atherogenicity effect.Triglyceride levels is higher, and the cholesteryl ester in low-density lipoprotein is more.People are verified, and the cholesterol in atherosclerotic plaque mainly comes from low-density lipoprotein, and low-density lipoprotein is maximum containing cholesterol in blood plasma, is also the lipoprotein that atherogenicity is the strongest.In addition, epidemiological study shows, high levels of triglycerides declines high-density lipoprotein concentration, and high-density lipoprotein concentration is subject to the impact of triglyceride levels, and both are negative correlation.Recently research shows, blood triglyceride levels is abnormal and the body intravascular coagulation factor is extremely closely related, plasma triglyceride level raises and can cause body intravascular coagulation factor level to raise, thereby promotion blood clotting, Bing Keshi tissue plasminogen activator inhibitor (PAI) is synthetic to be increased, 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 women is 76%.After proofreading and correct the impact of high-density lipoprotein (HDL), the level of significance male sex rises 14%, and women is 37%.These results show that it is the important risk factor of angiocardiopathy that serum triglyceride concentration raises.
Primary hypertriglyceridemiapatients patients often has hypertension and insulin resistance simultaneously.Insulin resistance is often referred to patient and tends to non-insulin dependence abnormal carbohydrate metabolism, and such patient often forms coronary artery disease earlier.Conventionally lipid triad, hypercoagulative state, hypertension, insulin resistance are called to metabolic syndrome.Confirmed that primary hypertriglyceridemiapatients patients is often associated with other in metabolic syndrome abnormal.The clinical meaning of hypertriglyceridemia is this.And seeming particularly important as the index ApoA5 that directly affects 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 by ApoA5 by ELISA experimental applications in clinical practice to reach the etiological diagnosis of hypertriglyceridemia, and the effective object of control of the angiocardiopathy such as coronary heart disease and apoplexy.
The enzyme linked immunological quantitative measurement of ApoA5, the overwhelming majority has been used two strain antibodies, if patent 200610018747.5, one strain monoclonal antibodies are capture antibody, another strain monoclonal antibody (or polyclonal antibody) is labelled antibody, forms enzyme connection sandwich method for determining ApoA5.But it is narrow that this method is measured the range of linearity of ApoA5, so it measures sensitivity and accuracy is all restricted.
Summary of the invention
The shortcoming that object of the present invention uses two strain antibodies to exist in order to overcome the enzyme linked immunological quantitative measurement of existing ApoA5, and provide the kit of a kind of ApoA5 of detection with diagnosing high triglyceride, the present invention has developed three anti-standard measures mensuration ApoA5 kits in conjunction with sandwich antibody technique and for the antibody of ApoA5 to development technique, by the strain monoclonal antibody that two strain monoclonal antibodies substitute in classic method, it is capture antibody, improved antibody capture ability, sensitivity and the accuracy measured have been improved, and the range of linearity, make quantitatively to detect ApoA5 in common microplate reader, reach check and clinical requirement.The present invention utilizes ApoA5 as the biochemical indicator of HTG diagnosis, the monoclonal antibody pair of preparing anti-ApoA5, and the concentration of utilizing ApoA5 in euzymelinked immunosorbent assay (ELISA) (ELISA) the quantitative measurement human serum improved, and hypertriglyceridemia is carried out to etiological diagnosis, thereby reach effective control of the angiocardiopathies such as coronary heart disease and apoplexy.
The object of the invention is to be achieved through the following technical solutions:
Detect ApoA5 with a kit for diagnosing high triglyceride, in kit, include the monoclonal antibody of the unmarked anti-ApoA5 of two strains and the monoclonal antibody of the anti-ApoA5 of another strain enzyme mark, confining liquid, enzyme substrate solution, coated damping fluid, stop buffer, cleansing solution, ELISA Plate; It is characterized in that: the monoclonal antibody of the unmarked anti-ApoA5 of two strains of usining is evenly coated in the ELISA Plate of unit area as capture antibody, the monoclonal antibody of the anti-ApoA5 of another strain enzyme mark of take be to detect antibody, coated damping fluid is the carbonate buffer solution of 0.05mol/l, pH9.6, wherein in 1 liter of solution, contains 1.59gNa 2cO 3with 2.93g NaHCO 3, confining liquid is mass concentration 1% bovine serum albumin(BSA); Enzyme substrate solution is 3,3 ', 5 of 60 μ g/ml, and 5 ' tetramethyl is 0.05%H for biphenylamine (TMB) and mass concentration 2o 20.1mol/l, pH6.0 phosphate buffer; Stop buffer is 1mol/lH 2sO 4solution.
The described grand antibody of the unmarked anti-ApoA5 monoclonal antibody of two strains mixes by 1:0.1-9, is evenly coated on the ELISA Plate hole of unit area simultaneously.
The monoclonal antibody of the described unmarked anti-ApoA5 of two strains is designated as Ab1+Ab2; The monoclonal antibody of the anti-ApoA5 of another strain is designated as Ab3, and Ab3 mark horseradish peroxidase HRP postscript is Ab3-HRP; Thereby have Ab1+Ab2 to be combined with ApoA5, ApoA5 is combined with Ab3-HRP, successively form Ab1+Ab2～ApoA5～Ab3-HRP reaction chain, this is the core of the anti-standard measure mensuration of the present invention three ApoA5 kit.Ab1, Ab2, Ab3 require to act on the different loci of ApoA5 antigen, after Ab1+Ab2 and Ab3 match respectively, ApoA5 antigen is had to good adhesion and higher specificity, successively form Ab1+Ab2～ApoA5～Ab3-HRP reaction chain, HRP makes chromogenic substrate tetramethyl benzidine (TMB) colour developing, by microplate reader, measure OD value again, according to typical curve, calculate the content of trying to achieve testing sample ApoA5.
The antibody of described anti-ApoA5 is monoclonal antibody pair.
The ApoA5 immunity BALB/c mouse of purifying for described anti-ApoA5 monoclonal antibody, obtains the spleen cell of antigenic stimulus, 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 clonings secreting the cell line of the monoclonal antibody of high specific, preserve the cell line of the monoclonal antibody that the generation that screens is combined with ApoA5 high specific, by mouse ascites, produce the monoclonal antibody of anti-ApoA5.Recycling Protein A-SePharose affinity column is further purified the monoclonal antibody of anti-ApoA5 in ascites, and ELISA and Western-Blot identify the monoclonal antibody of separation and purification.
The right screening of the grand antibody of anti-ApoA5 monoclonal antibody, the main subclone that adopts inhibition method screening high-affinity, and then at cloning repeatedly and ELISA, be added on the basis of experimental result and 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 purifying obtain volume antibody, carry out individual plant mark, adopt ELISA direct method to carry out the working concentration titration of labelled antibody, then adopt ELISA A competitive inhibition method to carry out single labeling antibody epi-position mensuration, thereby filter out the sandwich antibody pair of efficient affinity.
What the monoclonal antibody of the anti-ApoA5 of enzyme mark adopted is sodium periodate method Horseradish Peroxidase Conjugates.With NaIO 4first the glycan molecule on HRP surface is oxidized to aldehyde radical, and then is that amino on immunoglobulin (Ig) combines with the monoclonal antibody of ApoA5, further use NaBH 4reduction generates stable enzymic-labelled antibody.Centrifugal removal precipitation after dialysis purifying, supernatant is enzyme-antibody conjugates.
The grand antibody of the unmarked anti-ApoA5 monoclonal antibody of two strains is evenly coated in by 1:0.1-9 on the ELISA Plate hole of unit area, and coated damping fluid is the carbonate buffer solution of 0.05mol/l, pH9.6, in 1 liter of solution, contains 1.59g Na 2cO 3with 2.93g NaHCO 3.Confining liquid is mass concentration 1% bovine serum albumin(BSA); Enzyme substrate solution is 3,3 ', 5 of 60 μ g/ml, and 5 ' tetramethyl is 0.05%H for biphenylamine (TMB) and mass concentration 2o 20.1mol/l, pH6.0 phosphate buffer; Stop buffer is 1mol/lH 2sO 4solution.
Compare with technology and product with existing detection index, the present invention has outstanding advantage and practicality:
1, detection specificity is strong and practical.ApoA5 belongs to ApoAI/CIII/AIV gene group, at liver expression, relevant with HDL/VLDL.The single nucleotide variations of ApoA5 gene promoter (SNP) directly affects triglyceride levels.ApoA5 has very high susceptibility and specificity as the etiological diagnosis index of hypertriglyceridemia.The present invention has avoided the performance results merely of like product in the market, but can not clearly state the shortcoming of the cause of disease.Hypertriglyceridemia is that ApoA5 gene mutation reason causes once finding, and can carry out corresponding treatment for the cause of disease, thereby fundamentally solve prevention and the treatment of the relevant disease of hypertriglyceridemia and initiation thereof.
2, detection sensitivity and accuracy are high, and the range of linearity is large.In human blood, the concentration range of ApoA5 alters a great deal, and is about 20-410ng/ml very low, is on average about 215ng/ml.The present invention has developed three anti-standard measures mensuration ApoA5 kits in conjunction with sandwich double-antibody technique and for the antibody of ApoA5 to development technique, by the strain monoclonal antibody that two strain monoclonal antibodies substitute in classic method, it is capture antibody, improved antibody capture ability, sensitivity and the accuracy measured have been improved, and the range of linearity, make quantitatively to detect ApoA5 and reach check and clinical requirement in common microplate reader.
Experimental result of the present invention and analysis:
ELISA detects the concentration of ApoA5 in different samples, utilizes the monoclonal antibody pair of prepared anti-ApoA5, adopts three anti-ELISA methods, measures the concentration of ApoA5 in different samples.Result is as following table and Fig. 1.
Antibody: anti-ApoA5,0.1 μ g/ml, 100 μ l/ holes; Confining liquid: 1%BSA.From measurement result, three anti-ELISA methods are that sensitivity is 30ng/ml left and right to the detection lower limit of ApoA5, approach the minimum (24ng/ml) of the ApoA5 concentration of Caucasia white man's serum, far below the minimum (91ng/ml) of Asian ApoA5 concentration, and well below the mean concentration (215ng/ml) of ApoA5 in human blood.
Accompanying drawing explanation
Fig. 1 is the curve map that ApoA5 concentration of the present invention and different two resists dilution absorbances.
Embodiment
The production method of hypertriglyceridemia (HTG) diagnostic kit:
1, the right preparation of the monoclonal antibody of ApoA5
With the ApoA5 of purifying, as antigen, distinguish immune BALB/c mouse, interval immunity in 21 days for the second time, once at interval of immunity in 10 days later, immune 3-4 time altogether, then take out the spleen cell of immune mouse, with PEG, the myeloma cell line SP2/0 of this spleen cell and mouse is merged, utilize HAT selective medium to screen hybridoma on 96 porocyte culture plates, by ELISA method, identify the cell line of anti-ApoA5, carry out obtaining after three time clonings the cell line of stably excreting highly specific monoclonal antibody, on the coated plate of same antigen, adopt ELISA to suppress method, be in reference group, to add cells and supernatant, in inhibition group, add cells and supernatant and antigen mixture, thereby compare two groups of different subclones that filter out high-affinity of OD value difference.Then at indirect ELISA, experimentally carry out additive process epitope analysis, be in reference group, to add respectively two individual plant cells and supernatant, in mensuration group, add two strain cells and supernatant simultaneously, two groups of OD values (A1, A2 and A1+2) relatively, by formula below, calculate:
AI = [ 2 A 1 + 2 A 1 + A 2 - 1 ] × 100 %
On the basis of comparing result of calculation, carry out the mensuration of epitope analysis and affinity, AI value is less than 50% for being added feminine gender, the epi-position of the antibody recognition antigen of two cell line secretions is identical, AI value is greater than 50% for being added the positive, represent between two cell lines uncontested, two different epi-positions of the antibody recognition antigen that two cell lines are secreted, thus the monoclonal antibody cell line for the different epi-positions of antigen tentatively obtained.Further the monoclonal antibody cell line obtaining is injected into respectively to mouse peritoneal and carries out ascites production, utilize Protein A-Sepharose affinity column purifying ascites to obtain volume monoclonal antibody, with HRP enzyme, the antibody of purifying is carried out to individual plant mark, by ELISA direct method, carry out the working concentration titration of labelled antibody, then with ELISA A competitive inhibition method, in reference group, add enzyme labelled antibody, in mensuration group, add enzyme labelled antibody and unmarked mixtures of antibodies to be measured, compare two groups of OD values, the expression that reference group OD value is greater than mensuration group OD value exists competition to suppress, the epi-position that is considered as two antibody recognition antigens is identical.Otherwise two antibody are considered as two antibody recognition antigens epi-position while not existing competition to suppress is different, thereby final screening obtains the sandwich antibody pair of efficient affinity.
2, the method for the anti-ApoA5 monoclonal antibody of enzyme labeling
What the monoclonal antibody of the anti-ApoA5 of enzyme mark adopted is sodium periodate method Horseradish Peroxidase Conjugates.Taking HRP dissolves in distilled water.Add the 0.1mol/l, the NaIO that newly join 4solution, under room temperature, lucifuge stirs 20 minutes.NaIO 4first the glycan molecule on HRP surface is oxidized to aldehyde radical.Above-mentioned solution packs in bag filter, adds the sodium-acetate buffer dialysis of 1m mol/l, PH4.4, and 4 ℃ are spent the night.Add 0.2mol/l, PH9.5 carbonate buffer solution, make the pH of above hydroformylation HRP be elevated to 9.0～9.5, then add immediately the monoclonal antibody of ApoA5, in 0.01mol/l carbonate buffer solution, room temperature lucifuge stirs 2 hours gently.Until the HRP of hydroformylation and the monoclonal antibody of ApoA5, be after amino on immunoglobulin (Ig) combines, then add the 4mg/ml NaBH newly joining 4liquid, put 4 ℃ 2 hours, NaBH4 is reduced generates stable enzymic-labelled antibody.Above-mentioned liquid packs in bag filter, and in 0.15mol/l, pH7.4PBS dialysis, 4 ℃ are spent the night.Centrifugal removal precipitation after dialysis purifying, supernatant is enzyme--antibody conjugates (Ab3-HRP).
3, the compound method of various damping fluids and reagent
1. be coated with the Na of damping fluid: 0.05mol/l, pH9.6 2cO 3-NaHCO 3: Na 2cO 3(MW105.99) 1.59g and NaHCO 3(MW84.01) 2.93g, adding distil water is to 1OO0ml.
2. confining liquid: 1% bovine serum albumin(BSA) (BSA) solution: bovine serum albumin(BSA) 1g; Add phosphate buffer (PBS) (being formulated as follows) to 10Oml.
The PBS solution of 0.01mol/l, pH7.4: NaCI (MW58.44) 8.0g, KCl (MW74.55) 0.2g, KH 2pO 4(MW136.09) 0.24g, Na 2hPO 412H 2o (MW358.14) 3.6g, adding distil water is to 1OO0ml.
3. PBS mono-Tween-20 of cleansing solution: pH7.4 adds 0.5ml Tween-20 (Tween-20) in the PBS of 1OO0ml0.01mol/l, pH7.4 solution.
4. zymolyte 3, and 3 ', 5,5 ' tetramethyl replaces biphenylamine (TMB) solution:
I) substrate buffer solution of four times: take 35.8g Na 2hPO 4.12H 2o; Take 21.1g citric acid; Respectively add ultrapure water and obtain respectively A mother liquor B mother liquor to 500ml, get A liquid 300ml, with B liquid, adjust pH value to 5.2 and get final product four times of dilutions during use.
Ii) TMB concentrate: accurately weigh pulvis TMB400mg and add in 10ml dimethyl sulfoxide (DMSO) (DMSO), obtain fortyfold tmb substrate concentrate after making it to dissolve completely.
Iii) working fluid preparation: the hydrogen peroxide of 4 μ l30% after adding 0.025ml TMB concentrate to mix completely in 10ml substrate buffer solution, now join before use.
5. stop buffer: 1mol/l H 2sO 4solution: the concentrated sulphuric acid (95-98%) 54.3ml, adding distil water is to 1000ml, and timing slowly splashes into the concentrated sulphuric acid in distilled water, and limit edged mixes.
The operation steps of hypertriglyceridemia (HTG) diagnostic kit:
1, formulate typical curve
Unmarked anti-ApoA5 monoclonal antibody solution Ab1 and the Ab2 of two strain purifying are mixed in 1:1 ratio, then with coated damping fluid, be diluted to 0.l μ g/ml, join in 96 hole ELISA Plate, every hole 100 μ l, 37 ℃ of coated spending the night for 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds l%BSA sealing, every hole 200 μ l, and 37 ℃ are sealed 1 hour; Do not wash, dry, add respectively 0,50,100,200,400, the ApoA5 standard antigen of 800ng/ml, every hole 100 μ l, hatch 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds anti-ApoA5 monoclonal antibody (Ab3-HRP) solution (being diluted to 0.1 μ g/ml with cleansing solution) of enzyme labeling, and every hole 100 μ l, hatch 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds zymolyte (TMB) working fluid, every hole 100 μ l, and lucifuge colour developing 5 minutes, adds stop buffer, every hole 50 μ l.Utilize microplate reader to measure absorbance value (A450nm) at 45Onm wavelength, the concentration (ng/ml) of take is horizontal ordinate, and A450nm is ordinate, drawing standard curve.
2, measure the ApoA5 concentration in serum
Monoclonal antibody solution Ab1 and the Ab2 of the unmarked anti-ApoA5 of two strains of purifying are mixed in 1:1 ratio, monoclonal antibody mixed liquor (Ab1+Ab2) is diluted to 0.l μ g/ml with coated damping fluid, join in 96 hole ELISA Plate every hole 100 μ l, 37 ℃ of coated spending the night for 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds l%BSA sealing, every hole 200 μ l, and 37 ℃ are sealed 1 hour; Do not wash, dry, add the serum to be detected of suitable multiple dilution, every hole 100 μ l, hatch for 37 ℃ and spend the night for 1 hour or 4 ℃; Cleansing solution is washed plate 3 times, dries, and adds the anti-ApoA5 monoclonal antibody solution (Ab3-HRP) of enzyme labeling, with cleansing solution, is diluted to 0.1 μ g/ml, and every hole 100 μ l, hatch 1 hour for 37 ℃; Cleansing solution is washed plate 3 times, dries, and adds zymolyte (TMB) working fluid, every hole 100 μ l, and lucifuge colour developing 5 minutes, adds stop buffer, every hole 50 μ l.Utilize microplate reader to measure absorbance value (A450nm) at 450nm wavelength, according to typical curve, by measured A450nm value, calculate the concentration of trying to achieve ApoA5 in serum.
3, result is judged
Cutoff value (cut off value) 215ng/ml according to ApoA5 as hypertriglyceridemia (HTG) etiological diagnosis index, concentration lacks lower than the be judged to be ApoA5 of this value, has the danger of hypertriglyceridemia; Maybe can judge the Etiological that ApoA5 gene mutation is hypertriglyceridemia.

Claims (3)

1. detect ApoA5 (ApoA5) with a kit for diagnosing high triglyceride, in kit, include the monoclonal antibody of the unmarked anti-ApoA5 of two strains and the monoclonal antibody of the anti-ApoA5 of another strain enzyme mark, confining liquid, enzyme substrate solution, coated damping fluid, stop buffer, cleansing solution, ELISA Plate; It is characterized in that: the monoclonal antibody of the unmarked anti-ApoA5 of two strains of usining is evenly coated in the ELISA Plate of unit area as capture antibody, the monoclonal antibody of the anti-ApoA5 of another strain enzyme mark of take be to detect antibody, coated damping fluid is the carbonate buffer solution of 0.05mol/l, pH9.6, wherein in 1 liter of solution, contains 1.59g Na 2cO 3with 2.93g NaHCO 3, confining liquid is mass concentration 1% bovine serum albumin(BSA); Enzyme substrate solution is 3,3 ', 5 of 60 μ g/ml, and 5 ' tetramethyl is 0.05%H for biphenylamine (TMB) and mass concentration 2o 20.1mol/l, pH6.0 phosphate buffer; Stop buffer is 1mol/lH 2sO 4solution.
2. detect according to claim 1 ApoA5 with the kit of diagnosing high triglyceride, it is characterized in that: the described grand antibody of the unmarked anti-ApoA5 monoclonal antibody of two strains mixes by 1:0.1-9, be evenly coated on the ELISA Plate hole of unit area simultaneously.
3. detect according to claim 1 ApoA5 with the kit of diagnosing high triglyceride, it is characterized in that: the monoclonal antibody of the described unmarked anti-ApoA5 of two strains is designated as Ab1+Ab2; The monoclonal antibody of the anti-ApoA5 of another strain is designated as Ab3, and Ab3 mark horseradish peroxidase HRP postscript is Ab3-HRP; Thereby have Ab1+Ab2 to be combined with ApoA5, ApoA5 is combined with Ab3-HRP, successively form Ab1+Ab2～ApoA5～Ab3-HRP reaction chain, this is the core of the anti-standard measure mensuration of the present invention three ApoA5 kit.
CN201410362427.6A 2014-07-28 2014-07-28 Kit for detecting apolipoprotein A5 for diagnosis of hypertriglyceridemia Pending CN104165992A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104076158A (en) * 2014-07-10 2014-10-01 深圳市新产业生物医学工程股份有限公司 Kit for measuring high-density lipoprotein cholesterol
CN104498586A (en) * 2014-11-28 2015-04-08 山东博科生物产业有限公司 Single reagent serum triglyceride detection reagent with strong stability
CN104597248A (en) * 2015-01-05 2015-05-06 中南大学湘雅二医院 Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically
CN114703230A (en) * 2022-05-19 2022-07-05 北京大学 Construction method of ApoA5 gene knockout hamster model

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CN1834654A (en) * 2006-04-13 2006-09-20 廖伟 Kit for diagnosing high triglyceride

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CN1834654A (en) * 2006-04-13 2006-09-20 廖伟 Kit for diagnosing high triglyceride

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104076158A (en) * 2014-07-10 2014-10-01 深圳市新产业生物医学工程股份有限公司 Kit for measuring high-density lipoprotein cholesterol
CN104076158B (en) * 2014-07-10 2016-08-17 深圳市新产业生物医学工程股份有限公司 A kind of test kit for measuring HDL-C
CN104498586A (en) * 2014-11-28 2015-04-08 山东博科生物产业有限公司 Single reagent serum triglyceride detection reagent with strong stability
CN104498586B (en) * 2014-11-28 2016-06-22 山东博科生物产业有限公司 The single reagent serum triglycerides detectable that a kind of stability is strong
CN104597248A (en) * 2015-01-05 2015-05-06 中南大学湘雅二医院 Method and kit for detecting biological activity of apolipoprotein ApoA5 non-diagnostically
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
CN114703230A (en) * 2022-05-19 2022-07-05 北京大学 Construction method of ApoA5 gene knockout hamster model

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