CN103760334A - Application of detection tool prepared by taking Acrp30 as marker and detection tool - Google Patents
Application of detection tool prepared by taking Acrp30 as marker and detection tool Download PDFInfo
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Abstract
The invention discloses application of a prepared tool for detecting corresponding diseases by using a detection marker of preeclampsia suffered by a pregnant woman, namely preeclampsia diseases, as well as corresponding detection tools obtained through the application mode and in particular relates to application by taking Acrp30 as a biomarker for preeclampsia during pregnancy. The application mode of taking the Acrp30 as the biomarker for preeclampsia diseases during pregnancy is adopted. The invention provides a novel biomarker application mode, and the biomarker can be more convenient in prediction, prognosis and diagnosis of the preeclampsia during pregnancy, so that a complicated blood collection step is avoided.
Description
Technical field
The present invention relates to can be used as preeclampsia that pregnant woman suffers from is also that the detection mark of pre-eclampsia disease is applied to prepare the testing tool of corresponding disease and the corresponding testing tool that obtains by this application mode etc.; Be specifically related to by Acrp30 as a token of thing prepare the application of testing tool preeclampsia and the testing tool obtaining.
Background technology
Hypertension is the modal medical care problem that pregnancy duration runs into, and makes the gestation of 2-3% complicated.The high blood pressure disease of pregnancy duration is divided into 4 classes: 1, chronic hypertension; 2, pre-eclampsia; 3, pre-eclampsia adds chronic hypertension and 4, the gestational period or P-AH.Before chronic hypertension is defined in gestation or before pregnant 20 weeks, blood pressure surpasses 140/90mmHg.During forepart pregnant women, first identified is to hypertension and pregnant less than in 20 weeks, blood pressure rising ordinary representation chronic hypertension.On the contrary, after pregnant 20 weeks, new blood pressure readings raises and requires consider and get rid of pre-eclampsia, is also preeclampsia.Preeclampsia with up to all gestation 5%, First pregnancy 10% and have the women's of chronic hypertension history the ratio of 20-25% to occur.High blood pressure disease during gestation may cause the incidence of disease of parent and fetus, and they remain the main source of maternal mortality rate.
Hypertension that gestation hypertension refers to appears at pregnant latter half (pregnant >20 week), without any the feature of other preeclampsias, and follow by postpartum blood pressure normalization.In originally showing the women of obvious gestation hypertension, approximately 1/3rd develop syndrome preeclampsia.Just because of this, progress that should these patient's this respects of close observation.The Pathological Physiology of gestation hypertension is unknown, but in the situation that there is no the feature of preeclampsia, the result of parent and fetus is generally normal.But gestation hypertension may be after hypertensive tendency in life.
Be the multisystem disease in gestation preeclampsia, mainly be new hypertension (systolic pressure and diastolic pressure difference >=140 and 90mmHg) and albuminuria (PE >=300mg in the urine collecting of 24 hours, or dipstick >=2+) that previous normotensive women developed after pregnant 20 weeks.Also can be divided into mild pre-eclampsia and serious preeclampsia (systolic pressure >=160mmHg or diastolic pressure >=110mmHg, albuminuria >5g/24 hour) preeclampsia, and can develop into eclampsia the most serious in the situation that.
Although clearly do not understand yet the definite pathophysiological mechanism of pre-eclampsia at present, it is mainly a kind of disorder of placental function disease, causes being attended by angiospastic endothelial dysfunction syndrome.
Although lack prevention and methods for the treatment of for preeclampsia, the Noninvasive biomarker of seeking the detection that can predict the development of this life-threatening disease of pregnancy or contribute to this disease remains most important.
The existing multiple biomarker that can be used for predicting this kind of disease preeclampsia in prior art, in number of patent application is the patent document of CN201180019135.7, CN201180030104.1, the multiple biomarker that can be used for diagnosis, prediction, prognosis and/or monitor this kind of disease is disclosed for example, also disclose simultaneously various biomarkers are prepared into the preeclampsia detected disease testing tool, kit for example.
But existing disclosed preeclampsia, the detected object of testing tool was mostly blood sample, most biological or chemical marks are all present in gravid woman's blood.By detecting the mode of the biological or chemical mark in blood sample, often relatively waste time and energy.
Summary of the invention
In view of this, the invention provides a kind of application mode, be specially using Acrp30 as detecting, the mark of diagnosis disease preeclampsia prepares the application of testing tool; This application mode provides a kind of new application mode of biomarker, and this biomarker is at the disease preeclampsia of prediction, prognosis, diagnosis gestation judicial convenience more, thereby avoids the loaded down with trivial details step of blood collection.
For solving above technical matters, technical scheme of the present invention be adopt using Acrp30 as detecting, the mark of diagnosis disease preeclampsia prepares the application of testing tool.
Preferably, the urine that the detected object of described testing tool is gravid woman.
Preferably, described testing tool is qualitative detection instrument.
Preferably, described qualitative detection instrument is immune chromatography test paper.
Preferably, described immune chromatography test paper is colloidal gold immune chromatography test.
Preferably, described colloidal gold immune chromatography test is provided with detection line and control line, collaurum concentration is set to 0.5-2g/100ml, detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 0.5-3mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 0.2-1mg/ml.
Preferably, described testing tool is serious preeclampsia testing tool.
Preferably, described serious preeclampsia testing tool is 10-12ng/mL for the detectability concentration of Acrp30.
Preferably, described detectability concentration is 10ng/mL.
Testing tool preeclampsia that the present invention also provides aforementioned arbitrary application mode to prepare simultaneously.
Compared with prior art, it is described in detail as follows in the present invention:
The core technology scheme that the present invention adopts is: the Acrp30 of usining prepares the application of testing tool as the mark that detects, diagnoses disease preeclampsia
Acrp30, has another name called adiponectin, AdipoQ, OBG3, apm1 etc.; It is a kind of important Adipocyte Factor of discovered in recent years, by 244 amino acid, formed, comprise the globular domain (gAcrp30) that the signal peptide that is comprised of 1-17 amino acid, non-homologous district that 18-41 amino acids forms, collagenous region that 42-107 amino acids forms and 108-244 amino acids form.The inventor finds that after studying for a long period of time this biotic factor can be present in human urine, especially suffers from the gravid woman of preeclampsia and healthy gravid woman's urine, and the content difference of this biotic factor is larger.In gravid woman's the urine of suffering from preeclampsia, the content of Acrp30 can be up to 23.94 ± 15.69ng/ml, and in healthy gravid woman's urine, the content of Acrp30 is generally 1.06 ± 2.61ng/ml.This make Acrp30 as preeclampsia disease detection mark become possibility.
The inventor has also studied the content difference of suffering from Acrp30 in the gravid woman of preeclampsia and healthy gravid woman's blood; By studies show that, the content difference of suffering from Acrp30 in the gravid woman of preeclampsia and healthy gravid woman's blood is less; Therefore, adopt blood can not obtain well detecting effect as detected object.
Above-mentioned research by the inventor is known, Acrp30 as prediction, prognosis, diagnosis gestation preeclampsia disease application in, can preferably adopt this biomarker is applied as to the qualitative detection instrument of preeclampsia that is prepared into, also be fast detecting tool, this instrument can be realized patient and voluntarily this disease be predicted expediently, thereby can more effectively guarantee the safety of parent and fetus.
In addition, when using Acrp30 as biomarker, and for the preparation of the testing tool of pregnant preeclampsia of disease, when the detected object of described testing tool is the urine of biosome, due to the detected object urine that is biosome, gravid woman's urine particularly, thereby can realize the method detecting without wound; And this testing tool can be realized the qualitative detection to this disease, without large-scale medical treatment detection device, so a sample of its detection only needs 5-10 minute conventionally, simple to operate, pregnant woman can Self-operating, compares with existing clinical detection mode, and its detection speed increases substantially.
Further, also further research on aforesaid qualitative detection instrument of the present invention, to reach better detection effect.In existing qualitative detection instrument, the most conventional is immune chromatography test paper, more preferably can adopt colloidal gold immune chromatography test.When the preeclampsia of the qualitative detection gestation of application mode gained of the present invention, the testing tool of disease adopts following while preferably arranging: employing colloidal gold immune chromatography test, it is provided with detection line and control line, collaurum concentration is set to 0.5-2g/100ml, detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 0.5-3mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 0.2-1mg/ml.The testing tool of this concrete application mode gained arranging is detecting in effect, and sensitivity and specificity all can reach more than 80%.
The inventor has further studied Acrp30 content difference in mild pre-eclampsia and serious preeclampsia gravid woman patient's urine on this basis.By research, find, although the content difference of Acrp30 is not obvious especially in the Acrp30 content in mild pre-eclampsia gravid woman patient in urine and serious preeclampsia gravid woman patient, but by controlling testing tool in the application's application mode, for the detectability concentration of Acrp30, can reach better detection effect to serious preeclampsia gestational patients, its sensitivity and specificity can reach respectively more than 90% and 80%.Being specially and controlling testing tool is 10-12ng/mL for the detectability concentration of Acrp30, is particularly preferably 10ng/mL.
Qualitative detection instrument of the present invention, it obtains the colouring discrimination of product after can adopting mark Acrp30 to react with reaction substrate, or comes qualitative differentiation preeclampsia or healthy gravid woman crowd with the colouring discrimination of Binding Capacity afterproduct.
Detectability concentration for Acrp30 of the present invention is interpreted as, the testing tool that application process of the present invention obtains is when detection disease preeclampsia, when in detection sample, the concentration of Acrp30 is greater than or equal to this detectability concentration, qualitative or quantitative testing result is positive, suffers from preeclampsia; When in detecting sample, the concentration of Acrp30 is lower than this detectability concentration, qualitative or quantitative testing result is negative, does not suffer from preeclampsia.
Accompanying drawing explanation
Fig. 1 is cell factor/chemotactic factor (CF) antibody chip testing result figure of each two routine gravid woman's urine specimen in normal healthy controls group and preeclampsia group; Wherein, B, D are group preeclampsia, and A, C are normal healthy controls group.
Embodiment
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Following examples are the test specimen that the second hospital gathers in West China,
Embodiment 1
Preeclampsia group: 124 Cases with Preeclampsia gravid woman;
Normal healthy controls group: 135 routine healthy gravid woman;
Experimental subjects screening standard:
Preeclampsia systolic pressure >=the 140mmHg of group-(i);
(ii) diastolic pressure >=90mmHg;
(iii) PE >=300mg in the urine collecting of 24 hours, or dipstick >=2+.
The systolic pressure <140mmHg of normal healthy controls group-(i);
(ii) diastolic pressure <90mmHg;
(iii) PE <300mg in the urine collecting of 24 hours, or dipstick <2+.
Experimental subjects age distribution: 124 28 years old gravid woman's mean age of Cases with Preeclampsia group (the range of age 19-35 year), 30 years old normal healthy controls group mean age (the range of age 18-43 year).
Sample collection method: the urine specimen of getting each experimental group.
A, experimental technique: cell factor/chemotactic factor (CF) antibody chip qualitative detection
Experiment condition:
1) film (RayBiotech, the U.S.) that is marked with in advance 174 cytokine antibodies is put into and detected box, then add 2ml confining liquid, room temperature sealing 6 hours;
2) discard confining liquid, add 1 part, 1.2ml sample to be checked, 4 ℃ of night incubation;
3) discard sample, with 2ml lavation buffer solution, clean 5 times;
4) add the detection antibody of 1ml biotin coupling, incubated at room 2 hours;
5) discard antibody, with 2ml lavation buffer solution, clean 5 times;
6) add the streptomysin Avidin of 2ml horseradish peroxidase (HRP) coupling, incubated at room 2 hours;
7) discard liquid, with 2ml lavation buffer solution, clean 5 times;
8) on film, add chemical illuminating reagent 500 μ l, lucifuge effect 2 minutes, observations.
The sample to be tested of collecting group, normal healthy controls group from preeclampsia all adopts above-mentioned experiment condition to detect, and acquired results as shown in Figure 1.
Experimental result: the expression signal that the mark 1 in Fig. 1 is Acrp30, the expression signal that mark 2 is Adipsin, the expression signal that mark 3 is FLRG.
Fig. 1 is cell factor/chemotactic factor (CF) antibody chip testing result figure of each two routine gravid woman's urine specimen in normal healthy controls group and preeclampsia group; Wherein, B, D are group preeclampsia, and A, C are normal healthy controls group.
As can be seen from Figure 1, Acrp30, the Adipsin in preeclampsia group and the expression of FLRG are stronger, and in normal healthy controls group a little less than above-mentioned three's expression.
B, experimental technique: enzyme linked immunosorbent assay quantitatively detects
Experiment condition:
Experiment material: Acrp30ELISA detection kit (R & D company)
1. the preparation of reagent
Lavation buffer solution (Wash Buffer)
Substrate solution (Substrate Solution): before use, the developer A in kit and developer B equivalent, lucifuge are mixed 15 minutes, each hole needs the potpourri 200 μ l of two kinds of developers.
Acrp30 standard items: Acrp30 standard items are diluted with 1ml deionized water.Standard items original liquid concentration after dilution is 250ng/ml.For guaranteeing that standard items fully mix, before further diluting, standard items are put on shaking table and rock and mix more than at least 15 minutes gently.
Respectively the standard items of 250ng/ml are configured to the Acrp30 examination criteria product that concentration is 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, 1.56ng/ml; RD5-7 calibration thinning agent is as blank (0ng/ml)
2. trace routine
In each micropore, add 100 μ l Acrp30Conjugate; The addition of standard items, tester, sample is 100 μ l, and joins in corresponding micropore.The adhesive tape that micropore is provided with kit is built.Microwell plate is placed on horizontal shaking table to 500 ± 50 revs/min at ambient temperature to be cultivated 1.5 hours.Wash plate; Every hole adds substrate solution (Substrate Solution) 200 μ l, under room temperature condition, lucifuge, keeps flat and cultivates 30 minutes; Every hole adds 50 μ l stop buffers (Stop Solution).In hole, the color of solution will become yellow from blueness.If solution colour is that green or change color are inconsistent in hole, pat gently flat board, to guarantee that solution fully mixes; In 30 minutes, survey 450nm light absorption value.
3. production standard curve, calculates sample content.
4. chart is added up and be depicted as to the content that calculates each sample to be tested of gained, and carry out statistical study.
The concentration that experimental result: Acrp30 organized in urine specimen in preeclampsia is the highest, on average can reach 23.94ng/mL; The concentration of Acrp30 in normal healthy controls group urine specimen is lower, average out to 1.06 ± 2.61ng/mL.
From the experimental result of embodiment 1, occur to contain a large amount of Acrp30 in gravid woman's the urine of preeclampsia, and Acrp30 content in the gravid woman's of normal healthy controls group urine is less.
And from the experimental result of embodiment 1, Acrp30 can be used as the application of biomarker for pregnant preeclampsia of disease.And preferred, can be using Acrp30 as biomarker, and for the preparation of the testing tool of pregnant preeclampsia of disease, the urine of the urine that the detected object of described testing tool is biosome, particularly gravid woman.
Be below to take Acrp30 in gravid woman's urine to detect target molecule, and according to different modes, prepare the reference examples of the qualitative or quantitative testing tool of gained, the preparation method of each reference examples is as follows:
Reference examples 1---quantitative testing tool 1
Adopt kit, this kit is provided with the 1. porous plate of anti-human Acrp30 monoclonal antibody, the every hole of porous plate is coated with mouse-anti human adiponectin monoclonal antibody 0.9 μ g～1 μ g, 2. biotin labeled mouse-anti human adiponectin monoclonal antibody detects liquid, mainly by biotin labeled mouse-anti human adiponectin monoclonal antibody, bovine serum albumin(BSA), glycerine and phosphate buffer form, the concentration of biotin labeled mouse-anti human adiponectin monoclonal antibody is 24mg/mL, the concentration of bovine serum albumin(BSA) is 1.5g～3g/100mL, the concentration of glycerine is the 3. Avidin-horseradish peroxidase of biotin labeled mouse-anti human adiponectin monoclonal antibody combination of 50mL/100mL, 4. chromogenic substrate 3 ', 3 ', 5, 5 '-tetramethyl benzidine and Acrp30 protein standard substance.Its detectability concentration for Acrp30 in gravid woman's urine is 20ng/mL.
Material source is as follows:
1, mouse-anti human adiponectin monoclonal antibody: purchased from R & D company;
2, Avidin-horseradish peroxidase: Shanghai Lei Hao Information technology company limited;
3, chromogenic substrate 3 ', 3 ', 5,5 '-tetramethyl benzidine and Acrp30 protein standard substance: U.S. R & D company.
Reference examples 2---quantitative testing tool 2
Adopt kit, this kit is provided with A, magnetic microsphere suspension: described magnetic microsphere is that hydroxylation magnetic microsphere and N-hydroxyl sulfo-succinimide, the carbodiimide of diameter 0.5 μ m-2 μ m is cross-linked gained jointly; In suspension, the concentration of magnetic microsphere is 75-85/μ L; The Streptavidin of B, phycoerythrin mark and biotin labeled antibody test liquid: described biotin is N-hydroxy succinic acid imines ester; Described antibody is mouse-anti human adiponectin monoclonal antibody; The concentration of biotin labeled antibody is 1.7-2mg/mL.C, Acrp30 protein standard substance and standard items dilution.
Material source is as follows:
1, Acrp30 protein standard substance: purchased from R & D company;
2, mouse-anti human adiponectin monoclonal antibody: purchased from R & D company;
3, N-hydroxyl sulfo-succinimide, N-hydroxy succinic acid imines ester, MES: purchased from Sigma company;
4, the Streptavidin of phycoerythrin mark: purchased from Molecular Probes company;
5, carbodiimide: purchased from Pierce company;
6, bovine serum albumin(BSA) (BSA): Roche company.
Kit is 15ng/mL for the detectability concentration of Acrp30 in gravid woman's urine.
Reference examples 3
Adopt the existing universal method of preparing colloidal gold immune chromatography test to prepare qualitative detection instrument preeclampsia of the present invention.
Described testing tool is provided with detection line and control line, collaurum concentration is set to 0.5g/100ml, detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 0.5mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 0.2mg/ml.
In this reference examples 3, testing tool is 13ng/mL for the detectability concentration of Acrp30 in gravid woman's urine.
Reference examples 4
Adopt the existing universal method of preparing colloidal gold immune chromatography test to prepare qualitative detection instrument preeclampsia of the present invention.
Described testing tool is provided with detection line and control line, and collaurum concentration is set to 2g/100ml, and detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 3mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 1mg/ml.
In this reference examples 4, testing tool is 12ng/mL for the detectability concentration of Acrp30 in gravid woman's urine.
Reference examples 5
Adopt the existing universal method of preparing colloidal gold immune chromatography test to prepare qualitative detection instrument preeclampsia of the present invention.
Described testing tool is provided with detection line and control line, collaurum concentration is set to 1.0g/100ml, detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 1.0mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 0.5mg/ml.
In this reference examples 5, testing tool is 10ng/mL for the detectability concentration of Acrp30 in gravid woman's urine.
Reference examples 6
Adopt the existing universal method of preparing colloidal gold immune chromatography test to prepare qualitative detection instrument preeclampsia of the present invention.
Described testing tool is provided with detection line and control line, collaurum concentration is set to 1.5g/100ml, detection line is provided with take mouse-anti human adiponectin antibody as detecting the testing liquid of antibody, and in described testing liquid, the concentration of mouse-anti human adiponectin antibody is 2.0mg/ml; Control line is provided with take sheep anti mouse polyclonal antibody IgG as controlling the control working fluid of antibody, and in described control working fluid, the concentration of sheep anti mouse polyclonal antibody IgG is 0.8mg/ml.
In this reference examples 6, testing tool is 11ng/mL for the detectability concentration of Acrp30 in gravid woman's urine.
Material source in above-mentioned reference examples 3-6 is as follows:
Mouse-anti human adiponectin antibody: RD company, the U.S.
Sheep anti mouse polyclonal antibody IgG: Wuhan doctor's moral company, China
Embodiment 2
The reference examples 1-6 qualitative detection preeclampsia instrument that distinct methods is obtained is respectively used to detect the clinical sample of the gravid woman's urine in embodiment 1, including preeclampsia organizes and normal healthy controls group, wherein 124 examples in group preeclampsia are divided into serious preeclampsia group (sPE according to systolic pressure and diastolic pressure size and 24 hours amounts of albuminuria, screening criteria: systolic pressure >=160mmHg, diastolic pressure >=110mmHg, albuminuria >5g/24 hour) 54 example and mild pre-eclampsia group (mPE, systolic pressure >=140mmHg, diastolic pressure >=90mmHg, PE >=300mg in the urine collecting of 24 hours) 70 examples.Normal healthy controls group is 135 examples.
Sample collection: get gravid woman's urine specimen.
Utilize reference examples 1-6 respectively 259 collected routine samples to be detected, the feminine gender of gained testing result and positive number of cases statistics are in Table one:
Table one
In upper table one, reference examples 1-2 preeclampsia of the present invention quantitative testing tool embodiment; Reference examples 3-6 is qualitative detection execution of instrument mode preeclampsia of the present invention, and special, reference examples 4-6 is the embodiment that is directed to the qualitative detection instrument of serious preeclampsia.
From embodiment 2, table one data can draw, application mode of the present invention can obtain directly carrying out the testing tool of qualitative detection preeclampsia, and preferred, after further improvement of the present invention, qualitative detection instrument preeclampsia obtaining can obviously improve it in the sensitivity and the specificity that detect placenta in preeclampsia, all can reach more than 80%; More particularly, after adjusting the detectability concentration of testing tool, the testing tool obtaining can further improve in the sensitivity and the specificity that detect serious preeclampsia disease, reaches respectively more than 90% and 85%.
Below be only the preferred embodiment of the present invention, it should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
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