CN103207274B - Screening kit for seventh chromosome abnormality diseases of fetus - Google Patents
Screening kit for seventh chromosome abnormality diseases of fetus Download PDFInfo
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- CN103207274B CN103207274B CN201310156706.2A CN201310156706A CN103207274B CN 103207274 B CN103207274 B CN 103207274B CN 201310156706 A CN201310156706 A CN 201310156706A CN 103207274 B CN103207274 B CN 103207274B
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Abstract
The invention discloses a screening kit for the seventh chromosome abnormality diseases of a fetus. The screening kit comprises a reagent for detecting the PLGF (placental growth factor) level of the serum or urine of a pregnant woman. The invention further discloses an application for the screening kit for the seventh chromosome abnormality diseases of a fetus in detection for the PLGF level of the serum or urine of a pregnant woman. The kit disclosed by the invention can be used for evaluating whether the seventh chromosome of the fetus is normal or not, and is good in clinical application prospect.
Description
Technical field
The present invention relates to a kind of kit, particularly the kit for screening of a kind of fetus No. 7 chromosome diseases.
background technology
No. 7 chromosomes of the mankind be 23 to chromosomal one of them, under normal condition, each cell has two, and this chromosome, containing 1000 to 1400 genes of having an appointment, accounts for 5% to 5.5% of all DNA in cell.
No. 7 chromosomal exceptions, as pericentric inversion, can produce unbalanced gamete, usually can cause miscarriage or monster clinically.
Therefore, detect at early pregnancy, determine that whether No. 7 chromosomes of fetus are abnormal, to the timely treatment after fetal birth, there is very great importance.
summary of the invention
In order to solve the problem, the invention provides the kit for screening of a kind of fetus No. 7 chromosome diseases, and detect the purposes of reagent in preparation fetus No. 7 chromosome disease screening agents of pregnancy serum or urine PLGF level.
The kit for screening of fetus of the present invention No. 7 chromosome diseases, it comprises the reagent detecting pregnancy serum or urine PLGF level.
Wherein, described reagent is ELISA detection reagent.
Described ELISA detection reagent is ELISA direct method, ELISA indirect method, ELISA double antibody sandwich method or ELISA competition law detection reagent.
Described ELISA double antibody sandwich method detection reagent comprises anti-human PLGF antibody, people PLGF standard items, ELISA detection enzyme and ELISA detection substrate.
Preferably, described anti-human PLGF antibody comprises anti-human PLGF monoclonal antibody and biotin labeled anti-human PLGF polyclonal antibody; Described ELISA detection enzyme is horseradish peroxidase-labeled Streptavidin; Described ELISA detection substrate is TMB.
Present invention also offers the purposes of reagent in preparation fetus No. 7 chromosome disease screening agents detecting pregnancy serum or urine PLGF level.
Wherein, the reagent of described detection pregnancy serum or urine PLGF level is ELISA detection reagent.
Described ELISA detection reagent is ELISA direct method, ELISA indirect method, ELISA double antibody sandwich method or ELISA competition law detection reagent.
Described ELISA double antibody sandwich method detection reagent comprises anti-human PLGF antibody, people PLGF standard items, ELISA detection enzyme and ELISA detection substrate.
Preferably, described anti-human PLGF antibody comprises anti-human PLGF monoclonal antibody and biotin labeled anti-human PLGF polyclonal antibody; Described ELISA detection enzyme is horseradish peroxidase-labeled Streptavidin; Described ELISA detection substrate is TMB.
Kit for screening of the present invention and method can No. 7 chromosomes of effective examination fetus whether abnormal, and simple to operate, only need to carry out blood or uroscopy to pregnant woman, patient compliance is high, has a good application prospect.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
Embodiment 1 sample collection
1, acquisition target: in April ,-2012 in October, 2011 is in pregnant early stage pregnant woman totally 35 examples of West China No.2 Hospital, Sichuan University's outpatient service and affiliated hospital of Chengdu University of Traditional Chinese Medicine second outpatient clinic.
2, diagnosis of early gestation standard: the method recorded with reference to national higher medical education teaching material " gynecotokology " the 7th edition (comprises 1. menelipsis history; 2. HCG is positive; 3. uterine pregnancy (ultrasonic the seeing in uterine cavity of Type B has gestation sac) is diagnosed as.
3, inclusive criteria: 1. meet above-mentioned diagnostic criteria person; 2. pregnant week: 5 weeks-13+6 weeks; 3. informed consent, volunteers tested.
4, exclusion standard: have cardiovascular, liver, the complication such as kidney and hemopoietic system, or other primary diseases and mental patient.
5, come off standard: 1. include rear discovery in and do not meet inclusive criteria or rejected with the case of exclusion standard; 2. because of case that other reasons is lost to follow-up; 3. patient exits voluntarily; 4. include that rear discovery cannot obtain truly from experimenter, comprehensive clinical data in.
6, sample population characteristic: include pregnant woman age scope in 21 years old-43 years old, 31.96 ± 6.1 years old mean age.
7, fetus No. 7 dyeing method for detecting abnormality: after amniocentesis, the aobvious band of G is analyzed.Testing result: 34 examples are normal, and 1 example is No. 7 chromosome pericentric inversions.
8, sample collection
(1) blood sample: adopt the test tube containing EDTA to gather pregnant woman venous blood (on an empty stomach) 4ml, by its supernatant after 4 DEG C of low-temperature centrifugations (4000rpm, 10min), it is to be measured that numbering is stored in-80 DEG C of refrigerator freezings.
(2) urine specimen: leave and take experimenter's midstream urine 6ml, by its supernatant after 4 DEG C of low-temperature centrifugations (4000rpm, 10min), it is to be measured that numbering is stored in-80 DEG C of refrigerator freezings.
The detection method of embodiment 2PLGF level
1, key instrument
(1) microplate reader: Molecular Devices company, Spectra Max5
(2) refrigerated centrifuge: BECKMAN company, AllegraTM64R
(3)-20 DEG C of refrigerators: Electrolux company
(4) ultra low temperature freezer: Thermo company, SK501
(5) electro-heating standing-temperature cultivator: Shanghai is gloomy reliablely tests Instrument Ltd., DRP-9162
2, experiment reagent and material
(1) Capture Antibody: mouse-anti people PLGF monoclonal antibody Human PLGF Antibody (Monoclonal Mouse IgG1), purchased from R & D company, MAB264;
(2) Detection Antibody: biotin labeled goat-anti people PLGF polyclonal antibody HumanPLGF Biotinylated Antibody (Ployclonal Goat IgG), purchased from R & D company, BAF264;
(3) PLGF standard items: recombined human PLGF Recombinant Human PLGF, purchased from R & D company, 264-PG-10;
(4) the Streptavidin Streptavidin-horseradish peroxidase:R & D company of horseradish peroxidase-labeled, DY998;
(5) TMB:Sigma company, TO440-1L;
(6) PBS: autogamy, pH7.4,137mM NaCl, 2.7mM KCl, 8.1mM Na
2hPO
4, 1.5mMKH
2pO
4, pH7.2-7.4,0.2 μm of filtered;
(7) Tween-20: the raw work in Shanghai;
(8) bag is buffered liquid: autogamy, pH9.6,50mM carbonate buffer solution, Na
2cO
31.59g, NaHCO
32.93g, adding distil water is to 1L;
(9) cleansing solution: autogamy, PBS-T (PBS, containing 0.02%Tween-20)
(10) stop buffer: autogamy, 2MH
2sO
4;
(11) skimmed milk power: YiLi corporation;
(12) bovine serum albumin(BSA) (BSA): Roche Holding Ag;
(13) hyclone (FBS): GIBCO company;
(14) removable 96 hole elisa plates: COSTAR company.
3, experimental technique
(1) after institute's test sample originally being put at room temperature recovery, then detect;
(2) bag is by Capture Antibody, 4 μ g/ml, 100 μ l/well, and (about 16-20h) is spent the night in 4 DEG C of placements;
(3) 3%BSA solution, 3%FBS solution or be 5% skim milk powder solution close, 100 μ l/well, hatch 2h for 37 DEG C;
(4) PBS-T washes plate three times; Add 8 variable concentrations PLGF standard items (concentration is respectively 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml) and testing sample respectively, 100 μ l/well, two multiple holes are all set, two blank well are set simultaneously, hatch 2h for 37 DEG C;
(5) PBS-T washes plate three times; Add Detection Antibody respectively, 1 μ g/ml, 100 μ l/well, hatch 2h for 37 DEG C;
(6) PBS-T washes plate three times; Add streptavidin-horseradish peroxidase, 1:200,100 μ l/well, room temperature, 1h;
(7) PBS-T washes plate three times; Add TMB, 100 μ l/well, lucifuge hatches 10min in 37 DEG C;
(8) directly stop buffer is added, 50 μ l/well; OD value is detected at 450nm place, drawing standard curve, calculates the PLGF level of measuring samples.
The correlativity of embodiment 3PLGF level and fetus No. 7 chromosome abnormalities
Detect blood and the urine specimen of embodiment 1 collection respectively according to the method for embodiment 2, statistics is as shown in table 1 ~ 2:
The correlativity of table 1 fetus No. 7 chromosome abnormalities and blood-serum P LGF level
As shown in table 1, during normal fetal, pregnancy serum PLGF is 228.07pg/ml, and during fetus No. 7 chromosome abnormalities, pregnancy serum PLGF is low to moderate 15.6pg/ml, and the former is than the latter's height 212.47pg/ml, is 14.62 times of the latter.
The correlativity of table 2 fetus No. 7 chromosome abnormalities and urine PLGF level
As shown in table 2, during normal fetal, pregnant woman urine PLGF is 164.66pg/ml, and during fetus No. 7 chromosome abnormalities, pregnant woman urine PLGF is 105.23pg/ml, and the former is than the latter's height 59.43pg/ml, is 1.564 times of the latter.
Meanwhile, pregnant woman's absent cardiovascular of fetus No. 7 chromosome abnormalities, liver, the complication such as kidney and hemopoietic system, without other primary diseases and mental patient, also without medical history, allergies, pregnant mode is natural conception, and body mass index is normal.
By above-mentioned experimental result, pregnancy serum and urine PLGF level and fetus No. 7 chromosome abnormality significant correlations can be found out.Although the case of fetus No. 7 chromosome abnormalities is rarer, when pregnancy serum PLGF level is lower than 15.6pg/ml, or pregnant woman urine PLGF level lower than 105.23pg/ml time, can sensitive prompting fetus to suffer from the possibility of No. 7 chromosome abnormalities large.Therefore, can by detecting pregnancy serum or urine PLGF level, whether examination fetus has No. 7 chromosomal disorders.
Embodiment 4 kit for screening of the present invention
1, the component of kit for screening of the present invention and content (50 person-portion):
2, the using method of kit of the present invention
Blood sample: adopt the test tube containing EDTA to gather pregnant woman venous blood (on an empty stomach) 4ml, by its supernatant after 4 DEG C of low-temperature centrifugations (4000rpm, 10min), it is to be measured that numbering is stored in-80 DEG C of refrigerator freezings.
Using method:
(1), after institute's test sample originally being put at room temperature recovery, detect;
(2) bag is by Capture Antibody, 4 μ g/ml, 100 μ l/well, and (about 16-20h) is spent the night in 4 DEG C of placements;
(3) 3%BSA closes, and 100 μ l/well, hatch 2h for 37 DEG C;
(4) PBS-T washes plate three times; Add PLGF standard items (concentration is respectively 2000pg/ml, 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.25pg/ml, 15.625pg/ml) and the testing sample of 8 variable concentrations respectively, 100 μ l/well, two multiple holes are all set, two blank well are set simultaneously, hatch 2h for 37 DEG C;
(5) PBS-T washes plate three times; Add Detection Antibody respectively, 1 μ g/ml, 100 μ l/well, hatch 2h for 37 DEG C;
(6) PBS-T washes plate three times; Add streptavidin-horseradish peroxidase, 1:200,100 μ l/well, room temperature, 1h;
(7) PBS-T washes plate three times; Add TMB, 100 μ l/well, lucifuge hatches 10min in 37 DEG C;
(8) directly stop buffer is added, 50 μ l/well; OD value is detected at 450nm place, drawing standard curve, calculates the PLGF level of measuring samples.
To sum up, use kit for screening of the present invention to detect pregnant woman and carry out blood or urine, can No. 7 chromosomes of examination fetus whether abnormal, for the timely treatment of abnormal crowd provides foundation, potential applicability in clinical practice is good.
Claims (5)
1. detect the purposes of reagent in preparation fetus No. 7 chromosome abnormality screening agents of pregnancy serum or urine PLGF level.
2. purposes according to claim 1, is characterized in that: the reagent of described detection pregnancy serum or urine PLGF level is ELISA detection reagent.
3. purposes according to claim 2, is characterized in that: described ELISA detection reagent is ELISA direct method, ELISA indirect method, ELISA double antibody sandwich method or ELISA competition law detection reagent.
4. purposes according to claim 3, is characterized in that: described ELISA double antibody sandwich method detection reagent comprises anti-human PLGF antibody, people PLGF standard items, ELISA detection enzyme and ELISA detection substrate.
5. purposes according to claim 4, is characterized in that: described anti-human PLGF antibody comprises anti-human PLGF monoclonal antibody and biotin labeled anti-human PLGF polyclonal antibody; Described ELISA detection enzyme is horseradish peroxidase-labeled Streptavidin; Described ELISA detection substrate is TMB.
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| CN103207274B true CN103207274B (en) | 2015-02-25 |
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| CN112578123B (en) * | 2019-09-27 | 2022-10-25 | 成都中医药大学 | Application of reagent for detecting content of calprotectin in preparation of uterine lesion screening kit |
| WO2021057987A1 (en) * | 2019-09-27 | 2021-04-01 | 成都中医药大学 | Application of reagent for measuring content of faecal calprotectin in preparation of teratozoospermia screening kit |
| WO2021057986A1 (en) * | 2019-09-27 | 2021-04-01 | 成都中医药大学 | Use of reagent for detecting content of faecal calprotectin in preparation of kit for screening ovarian lesions |
| CN113804899A (en) * | 2021-08-26 | 2021-12-17 | 宁波奥丞生物科技有限公司 | Immunochromatography reagent strip for detecting urine placenta growth factor of pregnant woman and preparation method thereof |
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| ES2371084T3 (en) * | 2004-12-21 | 2011-12-27 | Yale University | DIAGNOSIS OF PREECLAMPSY. |
| WO2007051069A2 (en) * | 2005-10-27 | 2007-05-03 | Yale University | Urinary proteomic biomarker patterns in preeclampsia |
| JP2009538915A (en) * | 2006-05-31 | 2009-11-12 | ベス イスラエル ディーコネス メディカル センター | How to diagnose and treat pregnancy complications |
| US8647832B2 (en) * | 2008-01-25 | 2014-02-11 | Perkinelmer Health Sciences, Inc. | Methods for determining the risk of prenatal complications |
| US8741287B2 (en) * | 2008-06-18 | 2014-06-03 | Abbott Laboratories | PlGF-1 assay and kits and components thereof |
| CA2746120A1 (en) * | 2008-12-23 | 2010-07-01 | Genentech, Inc. | Methods and compositions for diagnostic use in cancer patients |
| CN102219854A (en) * | 2010-04-15 | 2011-10-19 | 苏州思坦维生物技术有限责任公司 | Monoclonal antibody of anti-human PIGF (placental growth factor) protein as well as preparation method and application thereof |
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