CN106872687A - Lipoprotein associated phospholipase Lp PLA2Activity and total amount detection kit and preparation method thereof - Google Patents
Lipoprotein associated phospholipase Lp PLA2Activity and total amount detection kit and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of lipoprotein associated phospholipase Lp PLA2Activity and total amount detection kit and detection method, the detection kit includes that solid phase carrier, enzyme decompose substrate, buffer solution, enzyme label antibody reagent, cleaning solution, chromogenic substrate, terminate liquid, enzymatic activity calibration object and enzyme calibration-free product, and the ELISA Plate is coated with anti-Lp PLA2Special N-terminal non-inhibitory anti-body；It is the phosphatid ylcholine of platelet activating factor, platelet activating factor analog or its oxidative modification that the enzyme decomposes substrate；The enzyme label antibody reagent is the anti-Lp PLA of horseradish peroxidase or alkaline phosphatase or biotin labeling2Inhibiting antibody；The enzymatic activity calibration object includes the paranitrophenol standard items containing various concentration；The enzyme calibration-free product include containing Lp PLA2Multiple reference calibrations product of antigen.The present invention can avoid the interference of other materials in sample, and more accurately reaction Lp PLA2Situation in vivo, for clinic provides more accurately basis for estimation.
Description
Technical field
The present invention relates to in-vitro diagnosis field, more particularly to a kind of lipoprotein associated phospholipase Lp-PLA2Activity and total amount
Detection kit and preparation method thereof.
Background technology
Counted according to the World Health Organization, China dies from the people of cardiovascular and cerebrovascular disease up to more than 3,000,000 every year, just has within every 10 seconds
1 people dies from cardiovascular disease, China's cardiovascular and cerebrovascular disease morbidity and causes death toll to rank first, considerably beyond cancer etc. its
His disease.With the continuous aging of China human mortality, the crowd for suffering from cardiovascular and cerebrovascular disease increases year by year, and tends to becoming younger；I
Up to more than 60,000,000, annual that patients with stroke occurs up to 2,000,000, the incidence of disease is up to 1,20/,100,000 to the existing number of patients of state, annual soldier
Middle patient death 1,200,000, is often to send out a kind of common in elderly men more than 55 years old, women over-65s or postmenopausal women
Disease.The elderly, smoke and drink, hypertension, diabetes, obesity, dyslipidemia, to lack physical exertion and stress big et al.
Group is the Susceptible population of the serial angiocardiopathy such as coronary heart disease.
Plasma lipoprotein associated phospholipase A2(lipoprotein-associated phospholipase A2, Lp-
PLA2) it is phospholipase A2(Phospholipase A2, PLA2) a member in superfamily, it is made up of 441 amino acid residues, phase
It is 45.4kD to molecular weight, with the circulation of enzymatic activity form in blood.LP-PLA2It is the phosphatidase of serine dependence, its catalysis
Activity does not need calcium ion.LP-PLA2Enzymatic activity region is located at PROTEIN C end, S273, D296, H351 amino acid residue constitutive enzyme
Catalysis activity core site；189-239 amino acid residues form piece into structure, participate in the specificity of control zymolyte, and for low
Density lipoprotein (LDL) calmodulin binding domain CaM；367-370 amino acid residues participate in HDL (HDL) and combine.Lp-PLA2It is main
To be synthesized and be secreted by ripe macrophage and lymphocyte, and be adjusted by inflammatory mediator, the Lp-PLA in people's circulation2With
The form combined with hdl particle is present.
Lp-PLA2It is a kind of newly discovered biological marker with the effect of strong pro-inflammatory disease, is a kind of new
Specific vascular inflammation mark, its concentration or activity rising are the independent risk factors of cardiovascular event, with miocardial infarction
Onset risk and ishemic stroke (cerebral artery thrombosis) onset risk positive correlation, can be as coronary heart disease and ischemic cerebral apoplexy
The clinical advanced prediction index of the cardiovascular and cerebrovascular diseases such as atherosclerosis.Recently U.S. FDA ratifies it is used to predict coronary disease
Disease and risk of ischemic stroke.
Therefore a kind of new detection kit for cardiovascular and cerebrovascular disease advanced prediction is developed, being capable of look-ahead trouble
Person's state of an illness, in time treatment, reduces risk and death risk, mitigates burden on society, mitigates the pain of patient, simplifies
The flow for the treatment of, reduces the working strength of doctor, while can also substantially reduce the personal medical expense of country, hospital and patient
Expenditure, mitigates burden on society, with important, positive social effect.
In the prior art, with LP-PLA in ELISA measure blood2Content, LP-PLA in blood is shown indirectly2
Enzymatic activity, and artery sclerosis activity situation and LP-PLA in blood2Enzymatic activity is related, sometimes LP-PLA2Content and LP-PLA2It is living
Property and it is uncorrelated, such as LP-PLA2There is LP-PLA in genetic mutation crowd and blood2The crowd of mortifier；Directly determine blood LP-
PLA2Enzyme activity method is directly to add LP-PLA2Zymolyte in serum/plasma, wherein LP-PLA2Local behavior reaction substrate,
Generation color reaction product, then calculate LP-PLA2Enzymatic activity.The shortcoming of this scheme is that blood some compositions directly affect inspection
Survey LP-PLA2The result of enzymatic activity, such as ferroheme, bilirubin, metabolite, medicine etc.；There is LP- in blood or cell liquid
PLA2Similar PLA2Phosphatidase is present, and can also hydrolyze LP-PLA2Zymolyte, reduces detection blood LP-PLA2Enzymatic activity it is special
Property.
The content of the invention
Based on this, it is necessary to provide a kind of new lipoprotein associated phospholipase Lp-PLA2Detection mode removes sample kind
The interference of other impurities, improves Lp-PLA2The accuracy of measure.
According to the first aspect of the invention, the present invention provides a kind of lipoprotein associated phospholipase Lp-PLA2Activity and total amount
Detection kit, includes solid phase carrier, enzyme and decomposes substrate, buffer solution, enzyme label antibody reagent, cleaning solution, chromogenic substrate, termination
Liquid, enzymatic activity calibration object and/or enzyme calibration-free product, wherein：
The solid phase carrier is coated with anti-Lp-PLA2Special N-terminal non-inhibitory anti-body；
It is the phosphatide of platelet activating factor, platelet activating factor analog or its oxidative modification that the enzyme decomposes substrate
Phatidylcholine；
The enzyme label antibody reagent is the anti-Lp-PLA of horseradish peroxidase or alkaline phosphatase or biotin labeling2
Inhibiting antibody；
The enzymatic activity calibration object includes the paranitrophenol standard items containing various concentration；
The enzyme calibration-free product include containing Lp-PLA2Multiple reference calibrations product of antigen；
The chromogenic substrate includes the chromogenic substrate A liquid of the peroxide of 0.015-0.02% and comprising 0.25-0.32g/L
TMB chromogenic substrate B liquid.
According to the second aspect of the invention, the present invention provides a kind of lipoprotein associated phospholipase Lp-PLA2Detection side
Method, for carrying out activity and total amount inspection to the lipoprotein associated phospholipase in human serum or blood plasma using kit as described above
Survey, including：
The anti-Lp-PLA of 100ul are added in every hole of ELISA Plate2N-terminal antibody (5ug/ml) and 50mM carbonate buffer solutions,
Regulation pH value is 9.6, it is static 8-12 hour under the conditions of 4 DEG C after, removal buffer solution；
200 μ l confining liquids are added in every hole of ELISA Plate, after being closed 2 hours under the conditions of 4 DEG C, confining liquid is removed；
80ul sample diluting liquids and 20ul serum or plasma sample are added in every hole of ELISA Plate, 30 are reacted at room temperature
After minute, dilution is removed；
200ul buffer solutions and 50ul enzymes is added to decompose substrate, hybrid reaction determines OD values after 2 minutes；
Enzymatic activity-OD value standard curve is drawn, LP-PLA in testing sample is calculated to obtain2Enzymatic activity；
The μ l of enzyme label antibody reagent 100 are added, 37 DEG C is put and is continued to incubate 30 minutes；
Chromogenic substrate A liquid and each 50 μ l of chromogenic substrate B liquid is added to mix in every hole of ELISA Plate, 37 DEG C of lucifuge colour developings 15
After minute, the μ l of terminate liquid 50 are added per hole, after mixing in 10 minutes, determine the OD values of calibration object and measuring samples；
With calibration object antigen concentration as abscissa, the corresponding OD values of calibration object be that ordinate draws standard curve, and will treat
Test sample product OD values are substituted into standard curve, calculate to obtain LP-PLA in testing sample2Concentration.
The beneficial effects of the invention are as follows：
Embodiments of the invention are by from LP-PLA2N ends non-inhibitory anti-body, energy specificity LP-PLA2N-terminal, does not do
Disturb LP-PLA2C-terminal enzymatic structural region enzymatic activity, carries out antibody and flutters to obtain LP-PLA2Enzyme assay is tested, and is flutterred and is obtained out blood
Middle LP-PLA2, eliminate the interference of other materials in sample, and foundation can be provided for the research and development of inhibitors of enzymes, enzyme total amount and
Enzymatic activity result is detected simultaneously can more accurately react Lp-PLA2 situations in vivo, make clinic and more accurately judge.Both
Traditional enzyme detection method is improved, enzyme linked immunosorbent detection, a sample, one-time detection can be continued to complete again, while obtaining enzyme activity
The detection of property and total amount.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is human lipoprotein associated phospholipase Lp-PLA of the invention2In activity and total quantity measuring method one embodiment
Lp-PLA2Content curve synoptic diagram.
Specific embodiment
The human lipoprotein associated phospholipase Lp-PLA of present invention offer is described below in detail2Activity and total amount detection kit
One embodiment；The present embodiment mainly includes solid phase carrier, enzyme and decomposes substrate, buffer solution, enzyme label antibody reagent, washing
Liquid, chromogenic substrate, terminate liquid, enzymatic activity calibration object and/or enzyme calibration-free product, wherein：
The solid phase carrier is coated with anti-Lp-PLA2Special N-terminal non-inhibitory anti-body；
It is the phosphatide of platelet activating factor, platelet activating factor analog or its oxidative modification that the enzyme decomposes substrate
Phatidylcholine；
The enzyme label antibody reagent is the anti-Lp-PLA of horseradish peroxidase or alkaline phosphatase or biotin labeling2
Inhibiting antibody；
The enzymatic activity calibration object includes the paranitrophenol standard items containing various concentration；
The enzyme calibration-free product include containing Lp-PLA2Multiple reference calibrations product of antigen；
The chromogenic substrate includes the chromogenic substrate A liquid of the peroxide of 0.015-0.02% and comprising 0.25-0.32g/L
TMB chromogenic substrate B liquid.
In a specific embodiment, the solid phase carrier can be described using the coating buffer coating containing following components
Anti- Lp-PLA2Special N-terminal antibody：
Na2CO3：1.59g/L；
NaHCO3：2.93g/L；
In a specific embodiment, the solid phase carrier can be described using the confining liquid closing containing following components
Anti- Lp-PLA2Special N-terminal antibody：
BSA：10.0g/L；
Sucrose：52.0g/L；
Casein：5.0g/L；
NBS：100ml/L；
Gelatin：5.0ml/L；
50mM TB 8.0：50ml/L；
Proclin300：1.0ml/L.
In a specific embodiment, the solid phase carrier can be described using the buffer solution buffering containing following components
Anti- Lp-PLA2Special N-terminal antibody：
Hepes：4.8g/L
NaCl：8.8g/L
EDTA：1.5g/L；
In a specific embodiment, the enzyme decomposes substrate solution and may include following components：
The phosphatid ylcholine of platelet activating factor (PAF), PAF analogs and its oxidative modification：1~3mmol/L；
Citric acid monohydrate：4.2g/L；
Ethanol：100ml/L.
In a specific embodiment, the enzyme label antibody reagent uses the dilution containing following components：
BSA：10.0g/L；
Sucrose：52.0g/L；
Casein：5.0g/L；
NBS：100ml/L；
Gelatin：5.0ml/L；
50mM TB 8.0：50ml/L；
Proclin300：1.0ml/L.
In a specific embodiment, the calibration object includes Lp-PLA2Antigenic content is respectively 1000ng/
At least one calibration object of ml, 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml and 0ng/ml；And the calibration object is adopted
With the calibration object dilution containing following components：
Na2HPO4·12H2O：53.7g/L；
NaH2PO4·2H2O：7.8g/L；
NBS：200ml/L；
Glycerine：50ml/L；
Tween-20：1ml/L；
Proclin300：1ml/L.
In a specific embodiment, the chromogenic substrate A liquid includes following components：
Sodium acetate trihydrate：27.2g/L；
Citric acid：3.2g/L；
30%H2O2：0.6ml/L；
And/or, the chromogenic substrate B liquid includes following components：
Disodium ethylene diamine tetraacetate 0.4g/L；
Citric acid；1.9g/L；
Glycerine；100ml/L；
TMB；0.3g/L；
DMSO：6ml.
In a specific embodiment, the concentrated cleaning solution includes following components：
Na2HPO4·12H2O：71.6g/L；
KH2PO4：4.8g/L；
NaCl：160g/L；
KCl：4g/L；
Tween-20：20ml/L.
In a specific embodiment, the concentrated cleaning solution (20X)：0.01M PBS, containing being not less than 1.5%
Surfactant；The buffer solution contains the ethylenediamine tetra-acetic acid (EDTA) less than 50mmol/L；The terminate liquid contains dense
Degree is not higher than the sulfuric acid of 2mol/L.
In one embodiment of the invention, the solid phase carrier can be ELISA Plate or flat based tubes, or
Other can be used for coated solid phase carrier etc..
The phosphatid ylcholine of the platelet activating factor (PAF), PAF analogs or its oxidative modification can be：
1- myristoyls -2- (4- nitrobenzophenones succinyl group)-sn- glyceryl -3- phosphocholines；1- myristoyl bases -2-
4- p-nitrophenol succinic anhydride -3- phosphatidyl-ethanolamines；1- myristoyl base -2-4- p-nitrophenol succinic anhydride -3- phosphatide
Acyl glycerine；1- myristoyl base -2-4- p-nitrophenol succinic anhydride -3- phosphatidyl courage fat；1- myristoyl base -2-4- p-nitrophenyls
Oxyphenisatin dicarboxylic anhydride -3- phosphoric acid etc..
Embodiments of the invention are used with the phosphorus of the platelet activating factor (PAF), PAF analogs or its oxidative modification
Phosphatidylcholine carrys out Lp-PLA in determination sample for the detection method and DASELISA immunization of substrate2Activity and total amount.
After adding sample, the pre-coated anti-Lp-PLA on ELISA Plate2N-terminal specific antibody can be with the Lp-PLA in sample2With reference to addition
1- myristoyls -2- (4- nitrobenzophenones succinyl group)-sn- glyceryl -3- phosphocholines, Lp-PLA2Hydrolysis substrate 1- Pork and beans
The sn sites of cool acyl -2- (4- nitrobenzophenones succinyl group)-sn- glyceryl -3- phosphocholines, generate 4- nitrobenzophenone succinyls
Base, its paranitrophenol generated after degrading in aqueous can be monitored by optical system, by extinction under specific wavelength
The change of degree can detect Lp-PLA2Activity.Further add the anti-Lp-PLA of horseradish peroxidase (HRP) mark2It is anti-
Body is incubated.When there is Lp-PLA in sample2When, " coated antibody-antigen-enzyme labelled antibody " compound will be formed.Compound
The HRP catalyzed colorations agent reaction of upper connection, generates blue product, and yellow is changed into after terminating reaction.If without Lp-PLA in sample2
When, do not develop the color.The colour developing depth and Lp-PLA in sample2Content it is proportional.Absorbance (OD values) is detected by ELIASA, with school
Quasi- product concentration value is abscissa, calibration object OD values for ordinate does standard curve, Lp-PLA in calculating sample2Content.
The lipoprotein associated phospholipase Lp-PLA that the present invention is provided is described in detail below with reference to Fig. 12One of detection method
Embodiment；The present embodiment is used for using the kit described in previous embodiment to the lipoprotein correlation phosphorus in human serum or blood plasma
Lipase is detected that the sample containing anti-coagulants such as EDTA, sodium citrate or heparin can be detected with this reagent.The present embodiment is main
Including：
The anti-Lp-PLA of 100ul are added in every hole of ELISA Plate2N-terminal antibody (5ug/ml) and 50mM carbonate buffer solutions,
Regulation pH value is 9.6, it is static 8-12 hour under the conditions of 4 DEG C after, removal buffer solution；
200 μ l confining liquids are added in every hole of ELISA Plate, after being closed 2 hours under the conditions of 4 DEG C, confining liquid is removed；
80ul sample diluting liquids and 20ul serum or plasma sample are added in every hole of ELISA Plate, 30 are reacted at room temperature
After minute, dilution is removed；
Add 200ul buffer solutions (200mM Hepes pH7.6,150mM NaCl, 5mM EDTA) and 50ul enzyme bottom explodeds
Thing (1~3mmol/L zymolyte 1- myristoyls -2- (4- nitrobenzophenones succinyl group)-sn- glyceryl -3- phosphocholines,
10%Ethanol, 20mM Citric acid monohydrate pH4.5), hybrid reaction determines OD values, specifically after 2 minutes
When realizing, 405nm sections of measure OD value of wavelength is can select；
With 0,50,100,200,400 and 800UI Lp-PLA2Enzymatic activity-OD value standard curves are drawn, testing sample is calculated to obtain
Middle LP-PLA2Enzymatic activity.
In addition, in the present embodiment, the LP-PLA in blood sample is calculated2Also include after enzymatic activity：
The μ l of enzyme label antibody reagent 100 are added, 37 DEG C is put and is continued to incubate 30 minutes；
Chromogenic substrate A liquid and each 50 μ l of chromogenic substrate B liquid is added to mix in every hole of ELISA Plate, 37 DEG C of lucifuge colour developings 15
After minute, the μ l of terminate liquid 50 are added per hole, after mixing in 10 minutes, determine the OD values of calibration object and measuring samples；It is (now blue vertical
Turn yellow).Blank well returns to zero, each hole absorbance of 450nm wavelength measurements (OD values).
With calibration object antigen concentration as abscissa, the corresponding OD values of calibration object be that ordinate draws standard curve, and will treat
Test sample product OD values are substituted into standard curve, calculate to obtain LP-PLA in testing sample2Concentration.
It is described to calculate LP-PLA in blood sample in a specific embodiment2Enzymatic activity includes：
It is standard items with pure paranitrophenol, the paranitrophenol mark of 0,10,20,40,80 and 120nmol/ml is prepared respectively
Quasi- product, determine the OD values of various concentrations paranitrophenol standard items, draw out paranitrophenol standard concentration and OD value relation marks
Directrix curve, calculates the slope of curve (K)：
K=(paranitrophenol 80nmol-40nmol/ml)/(80nmol/ml OD value -40nmol/ml OD values)；
50th, 100,200 and 400ng/ml concentration LP-PLA2With zymolyte reaction, paranitrophenol is produced, in color reaction,
Differential responses time (2,3,4,5,6,7,8min) OD values are determined, LP-PLA2 units of enzyme activity are calculated according to below equation：
LP-PLA2Unit of enzyme activity (IU, nmol/ml/min)=K (OD3min-OD2min)/0.02ml.
It is as follows：
If OD value=0.9780 of a sample, standard curve is substituted into, calculate to obtain Lp-PLA2Concentration ≈ 244.61ng/ml.
(this calibration object OD values and the merely illustrative explanation of standard curve, without actual quantification effect；Experiment is with the actual OD of calibration object every time
Value is defined).
In a specific embodiment, Warm adds detergent washed once before educating, and Warm adds detergent after the completion of educating
Washing 3 times.
Following each table shows in detail concrete component, content and the system of the various solution used in embodiments of the invention
Standby step.
Table 1, coating formula of liquid
| Raw material title | Prepare 1000ml consumptions |
| 1.59g | |
| 2.93g |
During preparation, 900ml purified waters are first added, after after total material dissolving in table 1, adjustment pH value is 9.6 ± 0.1, then
It is settled to 1000ml.
Table 2, closing formula of liquid
During preparation, 800ml purified waters are first added, after total material in table 2 dissolves, then be settled to 1000ml.
Table 3, buffer formulation
| Raw material title | Prepare 1000ml consumptions |
| Hepes | 4.8g |
| NaCl | 8.8g |
| EDTA | 1.5g |
During preparation, 800ml purified waters are first added, after after total material dissolving in table 3, adjust pH to 7.6, then be settled to
1000ml.(200mM Hepes pH7.6,150mM NaCl, 5mM EDTA)
Table 4, enzyme decomposes substrate solution formula
| Raw material title | Prepare 1000ml consumptions |
| Citric acid monohydrate | 4.2g |
| Ethanol | 100ml |
During preparation, 800ml purified waters are first added, after after total material dissolving in table 4, adjust pH to 2.7, then be settled to
1000ml。
Zymolyte 1- myristoyls -2- (4- nitrobenzophenones succinyl group)-sn- the third three is added in enzyme decomposes substrate solution
Base -3- phosphocholines, make its concentration be 1~3mmol/L.
Table 5, enzyme labelled antibody dilutes formula of liquid
During preparation, 800ml purified waters are first added, after total material in table 5 dissolves, then be settled to 1000ml.
After enzyme labelled antibody diluent preparing is good, HRP labelled antibodies are added, fully mixed, that is, obtain enzyme label antibody reagent.
Table 6, calibration object dilutes formula of liquid
| Raw material title | Prepare 1000ml consumptions |
| 53.7g | |
| 7.8g | |
| NBS | 200ml |
| Glycerine | 50ml |
| Tween-20 | 1ml |
| Proclin300 | 1ml |
During preparation, 600ml purified waters are first added, after total material in table 6 dissolves, then be settled to 1000ml.
Table 7,20X thickening and washing formula of liquid
| Raw material title | Prepare 1000ml consumptions |
| 71.6g | |
| 4.8g | |
| NaCl | 160g |
| KCl | 4g |
| Tween-20 | 20ml |
During preparation, 700ml purified waters are first added, after total material in table 7 dissolves, then be settled to 1000ml.
Table 8, TMB chromogenic substrate A liquid, B formula of liquid
Chromogenic substrate A liquid is prepared：800ml purified waters are first added, after after whole components dissolving of chromogenic substrate A liquid then fixed
Hold to 1000ml.
Chromogenic substrate B liquid is prepared：(1) first load weighted TMB is dissolved in DMSO, is completely dissolved, mixes, added before treating constant volume
Enter；(2) 800ml purified waters are first added, after after whole components dissolving of chromogenic substrate B liquid, the TMB- in step (1) is added
DMSO solution and glycerine, are settled to 1000ml.
Table 9, termination formula of liquid
| Raw material title | Prepare 1000ml consumptions |
| The concentrated sulfuric acid | 108ml |
Take 800ml purified waters to be placed in beaker, be slowly added to the concentrated sulfuric acid, last constant volume is 1000ml.
Above content is to combine specific preferred embodiment further description made for the present invention, it is impossible to assert
Specific implementation of the invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of not departing from present inventive concept, some simple deduction or replace can also be made, should be all considered as belonging to of the invention
Protection domain.
Claims (10)
1. a kind of lipoprotein associated phospholipase Lp-PLA2Activity and total amount detection kit, it is characterised in that include solid phase load
Body, buffer solution, enzyme decompose substrate, enzyme label antibody reagent, cleaning solution, chromogenic substrate, terminate liquid, enzymatic activity calibration object and enzyme and exempt from school
Quasi- product, wherein：
The solid phase carrier is coated with anti-Lp-PLA2Special N-terminal non-inhibitory anti-body；
It is the phosphatidyl courage of platelet activating factor, platelet activating factor analog or its oxidative modification that the enzyme decomposes substrate
Alkali；
The enzyme label antibody reagent is the anti-Lp-PLA of horseradish peroxidase or alkaline phosphatase or biotin labeling2Inhibition
Antibody；
The enzymatic activity calibration object includes the paranitrophenol standard items containing various concentration；
The enzyme calibration-free product include containing Lp-PLA2Multiple reference calibrations product of antigen；
The chromogenic substrate includes the chromogenic substrate A liquid of the peroxide of 0.015-0.02% and comprising 0.25-0.32g/L's
The chromogenic substrate B liquid of TMB.
2. kit as claimed in claim 1, it is characterised in that the solid phase carrier uses the coating buffer containing following components
It is coated with the anti-Lp-PLA2Special N-terminal non-inhibitory anti-body：
Na2CO3：1.59g/L；
NaHCO3：2.93g/L；
And/or, the solid phase carrier closes the anti-Lp-PLA using the confining liquid containing following components2The non-suppression of special N-terminal
Property antibody processed：
BSA：10.0g/L；
Sucrose：52.0g/L；
Casein：5.0g/L；
NBS：100ml/L；
Gelatin：5.0ml/L；
50mM TB 8.0：50ml/L；
Proclin300：1.0ml/L.
3. kit as claimed in claim 1, it is characterised in that the solid phase carrier uses the buffer solution containing following components
Buffer the anti-Lp-PLA2Special N-terminal non-inhibitory anti-body：
Hepes：4.8g/L
NaCl：8.8g/L
EDTA：1.5g/L；
The enzyme decomposes substrate solution and includes following components：
The phosphatid ylcholine of platelet activating factor, platelet activating factor analog or its oxidative modification：1~3mmol/L；
Citric acid monohydrate：4.2g/L；
Ethanol：100ml/L.
4. kit as claimed in claim 1, it is characterised in that the enzyme label antibody reagent is using containing the dilute of following components
Release liquid：
BSA：10.0g/L；
Sucrose：52.0g/L；
Casein：5.0g/L；
NBS：100ml/L；
Gelatin：5.0ml/L；
50mM TB 8.0：50ml/L；
Proclin300：1.0ml/L.
5. kit as claimed in claim 1, it is characterised in that the calibration object includes Lp-PLA2Antigenic content is respectively
At least one calibration object of 1000ng/ml, 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml and 0ng/ml；And the school
Quasi- product use the calibration object dilution containing following components：
Na2HPO4·12H2O：53.7g/L；
NaH2PO4·2H2O：7.8g/L；
NBS：200ml/L；
Glycerine：50ml/L；
Tween-20：1ml/L；
Proclin300：1ml/L.
6. kit as claimed in claim 1, it is characterised in that the chromogenic substrate A liquid includes following components：
Sodium acetate trihydrate：27.2g/L；
Citric acid：3.2g/L；
30%H2O2：0.6ml/L；
And/or, the chromogenic substrate B liquid includes following components：
Disodium ethylene diamine tetraacetate 0.4g/L；
Citric acid；1.9g/L；
Glycerine；100ml/L；
TMB；0.3g/L；
DMSO：6ml.
7. kit as claimed in claim 1, it is characterised in that the concentrated cleaning solution includes following components：
Na2HPO4·12H2O：71.6g/L；
KH2PO4：4.8g/L；
NaCl：160g/L；
KCl：4g/L；
Tween-20：20ml/L.
8. the method as any one of claim 1-7, it is characterised in that：
The solid phase carrier is ELISA Plate or flat based tubes；
The phosphatid ylcholine of the platelet activating factor, platelet activating factor analog or its oxidative modification is 1- nutmegs
Acyl -2- (4- nitrobenzophenones succinyl group)-sn- glyceryl -3- phosphocholines；1- myristoyl base -2-4- p-nitrophenyls oxyphenisatin two
Acid anhydrides -3- phosphatidyl-ethanolamines；1- myristoyl base -2-4- p-nitrophenol succinic anhydride -3- phosphatidyl glycerols；1- myristoyls
Base -2-4- p-nitrophenol succinic anhydride -3- phosphatidyl courage fat；Or 1- myristoyl base -2-4- p-nitrophenols succinic anhydride -
3- phosphoric acid.
9. a kind of lipoprotein associated phospholipase Lp-PLA2Detection method, for using the examination any one of claim 1-8
Agent box is detected to the activity and total amount of the lipoprotein associated phospholipase in human serum or blood plasma, it is characterised in that including：
The anti-Lp-PLA of 100ul are added in every hole of ELISA Plate2N-terminal non-inhibitory anti-body (5ug/ml) and 50mM carbonate buffers
Liquid, regulation pH value is 9.6, it is static 8-12 hour under the conditions of 4 DEG C after, removal buffer solution；
200 μ l confining liquids are added in every hole of ELISA Plate, after being closed 2 hours under the conditions of 4 DEG C, confining liquid is removed；
80ul sample diluting liquids and 20ul serum or plasma sample are added in every hole of ELISA Plate, is reacted 30 minutes at room temperature
Afterwards, dilution is removed；
200ul buffer solutions and 50ul enzymes is added to decompose substrate, hybrid reaction determines OD values after 2 minutes；
Enzymatic activity-OD value standard curve is drawn, LP-PLA in testing sample is calculated to obtain2Enzymatic activity；
The μ l of enzyme label antibody reagent 100 are added, 37 DEG C is put and is continued to incubate 30 minutes；
Chromogenic substrate A liquid and each 50 μ l of chromogenic substrate B liquid is added to mix in every hole of ELISA Plate, 37 DEG C of lucifuges develop the color 15 minutes
Afterwards, add the μ l of terminate liquid 50 per hole, after mixing in 10 minutes, determine the OD values of calibration object and measuring samples；
With calibration object antigen concentration as abscissa, the corresponding OD values of calibration object be that ordinate draws standard curve, and test sample will be treated
Product OD values are substituted into standard curve, calculate to obtain LP-PLA in testing sample2Concentration.
10. method as claimed in claim 9, it is characterised in that described to calculate LP-PLA in blood sample2Enzymatic activity includes：
It is standard items with pure paranitrophenol, the paranitrophenol standard items of 0,10,20,40,80 and 120nmol/ml is prepared respectively,
The OD values of various concentrations paranitrophenol standard items are determined, paranitrophenol standard concentration is drawn out bent with OD values affinity criterions
Line, calculates the slope of curve (K)：
K=(paranitrophenol 80nmol-40nmol/ml)/(80nmol/ml OD value -40nmol/ml OD values)；
50th, 100,200 and 400ng/ml concentration LP-PLA2With zymolyte reaction, paranitrophenol is produced, in color reaction, determined not
Same reaction time (2,3,4,5,6,7,8min) OD values, LP-PLA2 units of enzyme activity are calculated according to below equation：
LP-PLA2Unit of enzyme activity (IU, nmol/ml/min)=K (OD3min-OD2min)/0.02ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175242A (en) * | 2020-03-17 | 2020-05-19 | 湖南新大陆生物技术有限公司 | Lipoprotein phospholipase A2 detection kit and application thereof |
| CN112415196A (en) * | 2020-11-25 | 2021-02-26 | 刘世杰 | System and application for analyzing molecular activity |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101441219A (en) * | 2007-11-20 | 2009-05-27 | 山东泰邦生物制品有限公司 | Coronary heart disease and palsy risk diagnosis reagent kit and manufacturing method thereof |
| CN104133062A (en) * | 2014-08-20 | 2014-11-05 | 方磊 | Activity detection kit for lipoprotein phospholipase A2 in plasma and detection method thereof |
| CN104730241A (en) * | 2015-03-02 | 2015-06-24 | 深圳市第二人民医院 | Kit used for detecting enzymatic activity of cardiovascnlar and cerebrovascular diseases |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101441219A (en) * | 2007-11-20 | 2009-05-27 | 山东泰邦生物制品有限公司 | Coronary heart disease and palsy risk diagnosis reagent kit and manufacturing method thereof |
| CN104133062A (en) * | 2014-08-20 | 2014-11-05 | 方磊 | Activity detection kit for lipoprotein phospholipase A2 in plasma and detection method thereof |
| CN104730241A (en) * | 2015-03-02 | 2015-06-24 | 深圳市第二人民医院 | Kit used for detecting enzymatic activity of cardiovascnlar and cerebrovascular diseases |
| CN105219750A (en) * | 2015-08-25 | 2016-01-06 | 长春恒晓生物科技有限责任公司 | Nature enzymic activity platelet-activating factor acetylhydro-lase and preparation process |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175242A (en) * | 2020-03-17 | 2020-05-19 | 湖南新大陆生物技术有限公司 | Lipoprotein phospholipase A2 detection kit and application thereof |
| CN112415196A (en) * | 2020-11-25 | 2021-02-26 | 刘世杰 | System and application for analyzing molecular activity |
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