CN107884570A - A kind of pregnant woman's pre-eclampsia detection kit - Google Patents
A kind of pregnant woman's pre-eclampsia detection kit Download PDFInfo
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- CN107884570A CN107884570A CN201610865129.8A CN201610865129A CN107884570A CN 107884570 A CN107884570 A CN 107884570A CN 201610865129 A CN201610865129 A CN 201610865129A CN 107884570 A CN107884570 A CN 107884570A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
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Abstract
The present invention relates to a kind of pregnant woman's pre-eclampsia detection kit,Including PIGF and the immunochromatographydetecting detecting test strips of sFlt 1,Or including PIGF immunochromatographydetecting detecting test strips and the immunochromatographydetecting detecting test strips of sFlt 1,Preferably,There is PIGF to detect immune labeled antibody and PIGF detection antibody respectively on the pad and nitrocellulose filter of PIGF immunochromatographydetecting detecting test strips,There is sFlt 1 to detect immune labeled antibody and sFlt 1 respectively on the pad and nitrocellulose filter of the immunochromatographydetecting detecting test strips of sFlt 1 and detect antibody,Or above-mentioned immune labeled antibody and antibody are all coated on the pad and nitrocellulose filter of PIGF and the immunochromatographydetecting detecting test strips of sFlt 1 respectively,The present invention can detect PIGF and sFlt 1 simultaneously by primary sample,Using simplicity,And it can accurately obtain ratio between the two,And then it can predict that the onset risk of eclampsia occurs for pregnant woman exactly,Strong foundation is provided for accurate treatment in next step,Suitable for large-scale promotion application.
Description
Technical field
The present invention relates to disease detection technical field, and in particular to disease detection kit technical field, more particularly to one
Kind pregnant woman's pre-eclampsia detection kit.
Background technology
Eclampsia is one of the main reason for causing maternal death, and the global incidence of disease is about 3~5%, developing country
The incidence of disease close to 10%.Harm of the eclampsia to pregnant woman include twitch, placental abruption, disseminated intravascular coagulation, Delayed graft function,
Pulmonary edema, cerebral hemorrhage, encephaledema, thromboembolism and death；Harm to fetus simultaneously includes that intrauterine growth is bad, intrauterine growth
Limited, premature labor and stillborn foetus.
Placental ischemia theoretical research in recent years thinks, soluble class fms EGFR-TKs -1 during blood circulation
(sFlt-1) and placenta growth factor (PIGF) is two important factors for causing and aggravating eclampsia symptom, according to its biology work(
Can be the expression in placenta in preeclampsia, can be as the important indicator of screening, prediction and diagnosis eclampsia.
Clinical research has confirmed, sFlt-1 and PIGF concentration proportion in blood, can predict pregnant woman exactly
The onset risk of eclampsia, also, the kit for the kit and detection PIGF for detecting sFlt-1 has listed for many years.
Accurate kit is detected to PIGF and sFlt-1 is detected as described above, clinically having existed at present
Accurate kit.But sFlt-1 and PIGF are close associations but relatively independent two indices；It is existing
SFlt-1 detection kits and PIGF detection kits, it is two independent products, to two index separate detections, at sample
Reason, loading etc. can all have differences, and so as to be impacted to ratio between the two, and then influence prediction pregnant woman and eclampsia occurs
The accuracy of onset risk, and further influence accurate treatment in next step.
In order to solve existing above mentioned problem simultaneously, it is desirable to provide a kind of kit, it can be detected by primary sample
PIGF and sFlt-1, using simplicity, and ratio between the two can be accurately obtained, and then pregnant woman can be predicted exactly
The onset risk of eclampsia, strong foundation is provided for accurate treatment in next step.
The content of the invention
The purpose of the present invention be overcome it is above-mentioned in the prior art the shortcomings that, there is provided a kind of pregnant woman's pre-eclampsia detection reagent
Box, pregnant woman's pre-eclampsia detection kit can detect PIGF and sFlt-1 simultaneously by primary sample, using simplicity, and energy
The enough accurate ratio obtained between the two, and then can predict that the onset risk of eclampsia occurs for pregnant woman exactly, it is accurate in next step
Really treatment provides strong foundation, suitable for large-scale promotion application.
To achieve these goals, pregnant woman's pre-eclampsia detection kit of the invention has following form：
Pregnant woman's pre-eclampsia detection kit, is characterized in, including PIGF and sFlt-1 immunochromatographydetecting detecting test strips,
Or including PIGF immunochromatographydetecting detecting test strips and sFlt-1 immunochromatographydetecting detecting test strips.
It is preferred that there is the immune mark of PIGF detections on the pad of the PIGF and sFlt-1 immunochromatographydetecting detecting test strips
Remember that antibody and sFlt-1 detect immune labeled antibody, the nitrocellulose of the PIGF and sFlt-1 immunochromatographydetecting detecting test strips
PIGF detection antibody and sFlt-1 detection antibody are coated with film.
It is preferred that on the pad of the PIGF immunochromatographydetecting detecting test strips there is PIGF to detect immune labeled antibody,
PIGF detection antibody is coated with the nitrocellulose filter of the PIGF immunochromatographydetecting detecting test strips, layer is immunized in the sFlt-1
Analysing on the pad of test strip, there is sFlt-1 to detect immune labeled antibody, the sFlt-1 immunochromatographydetecting detecting test strips
Nitrocellulose filter on be coated with sFlt-1 detection antibody.
More preferably, it is the immune labeled antibody of anti-human placenta growth factor that the PIGF, which detects immune labeled antibody, described
PIGF detection antibody is anti-human plgf antibody, and it is anti-human soluble class that the sFlt-1, which detects immune labeled antibody,
- 1 immune labeled antibody of fms EGFR-TKs, the sFlt-1 detections antibody are that anti-human soluble class fms EGFR-TKs -1 are anti-
Body.
More preferably, it is PIGF detections fluorescent labeled antibody, PIGF detection collaurums that the PIGF, which detects immune labeled antibody,
Labelled antibody or PIGF detection latex labelled antibodies, it is sFlt-1 detection fluorescence labelings that the sFlt-1, which detects immune labeled antibody,
Antibody, sFlt-1 detection colloidal gold labeled monoclonal antibodies or sFlt-1 detection latex labelled antibodies.
It is preferred that the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips are set side by side
Put.
It is preferred that pregnant woman's pre-eclampsia detection kit also includes bottom plate, the PIGF immunity chromatography detection test papers
Bar and the sFlt-1 immunochromatographydetecting detecting test strips are disposed in parallel on the bottom plate or are fitted in respectively the upper of the bottom plate
On lower surface.
It is preferred that pregnant woman's pre-eclampsia detection kit also includes two bottom plates, the PIGF immunochromatographies detection
Test strips and the sFlt-1 immunochromatographydetecting detecting test strips are set up in parallel and are separately positioned on two bottom plates.
The beneficial effects of the present invention are：
(1) present invention includes PIGF and sFlt-1 immunochromatographydetecting detecting test strips, or is detected including PIGF immunochromatographies
Test strips and sFlt-1 immunochromatographydetecting detecting test strips, so as to primary sample, you can detect, avoid to PIGF and sFlt-1
Sampling causes patient uncomfortable repeatedly, simplifies detection operation.
(2) while detect that PIGF and sFlt-1 two indices are very necessary, the height one such as the sample-adding amount of detection, sample process
Cause, it is higher relative to two indices separate detection, accuracy.
Therefore, the present invention can detect PIGF and sFlt-1 simultaneously by primary sample, using simplicity, and can accurately obtain
Ratio between the two is taken, and then can predict that the onset risk of eclampsia occurs for pregnant woman exactly, is carried for accurate treatment in next step
Strong foundation is supplied, suitable for large-scale promotion application.
Brief description of the drawings
Fig. 1 is the schematic front view of the first specific embodiment of the present invention.Wherein 1 is that pad (contains anti-human placenta growth
Factor fluorescent labeled antibody and the anti-human soluble fluorescent labeled antibody of class fms EGFR-TKs -1)；2 be arrow；3 be part not
Transparent protective film；4 be the first detection line (being coated with anti-human plgf antibody)；5 be that (coating is anti-human solvable for the second detection line
The antibody of property class fms EGFR-TKs -1)；6 be nature controlling line；7 be nitrocellulose filter；8 be opaque diaphragm.
Fig. 2 is the schematic front view of the second specific embodiment of the present invention.Wherein 9 be that pad (contains anti-human placenta growth
Factor colloidal gold labeled monoclonal antibody)；10 be pad (containing the anti-human soluble colloidal gold labeled monoclonal antibody of class fms EGFR-TKs -1)；2
It is arrow；3 be the opaque diaphragm in part；4 be the first detection line (being coated with anti-human plgf antibody)；5 be the second inspection
Survey line (the anti-human soluble antibody of class fms EGFR-TKs -1 of coating)；6 be nature controlling line；7 be nitrocellulose filter；8 be opaque
Diaphragm, 11 be bottom plate.
Fig. 3 is the schematic top plan view of the 3rd specific embodiment of the present invention.Wherein 9 be that pad (contains anti-human placenta growth
Factor latex labelled antibody)；10 be pad (containing the anti-human soluble latex labelled antibody of class fms EGFR-TKs -1)；2 be arrow
Head；3 be the opaque diaphragm in part；4 be the first detection line (being coated with anti-human plgf antibody)；5 be the second detection line
(the anti-human soluble antibody of class fms EGFR-TKs -1 of coating)；6 be nature controlling line；7 be nitrocellulose filter；8 be opaque protection
Film, 11 be bottom plate.
Fig. 4 is the schematic front view of the 4th specific embodiment of the present invention.Wherein 9 be that pad (contains anti-human placenta growth
Factor colloidal gold labeled monoclonal antibody)；10 be pad (containing the anti-human soluble colloidal gold labeled monoclonal antibody of class fms EGFR-TKs -1)；2
It is arrow；3 be the opaque diaphragm in part；4 be the first detection line (being coated with anti-human plgf antibody)；5 be the second inspection
Survey line (the anti-human soluble antibody of class fms EGFR-TKs -1 of coating)；6 be nature controlling line；7 be nitrocellulose filter；8 be opaque
Diaphragm, 11 be bottom plate.
Fig. 5 is the schematic front view of the 5th specific embodiment of the present invention.Wherein 9 be that pad (contains anti-human placenta growth
Factor fluorescent labeled antibody)；10 be pad (containing the anti-human soluble fluorescent labeled antibody of class fms EGFR-TKs -1)；2 be arrow
Head；3 be the opaque diaphragm in part；4 be the first detection line (being coated with anti-human plgf antibody)；5 be the second detection line
(the anti-human soluble antibody of class fms EGFR-TKs -1 of coating)；6 be nature controlling line；7 be nitrocellulose filter；8 be opaque protection
Film.
Embodiment
In order to be more clearly understood that the technology contents of the present invention, described in detail especially exemplified by following examples.Wherein Fig. 1 shows
What is shown is the first specific embodiment of the present invention, and the second specific embodiment of the present invention is shown in Fig. 2, and this is shown in Fig. 3
The 4th specific embodiment of the present invention is shown in 3rd specific embodiment of invention, Fig. 4, and the of the present invention is shown in Fig. 5
Five specific embodiments.Wherein identical part uses identical reference.
Pregnant woman's pre-eclampsia detection kit of the present invention includes PIGF and sFlt-1 immunochromatographydetecting detecting test strips, or
Including PIGF immunochromatographydetecting detecting test strips and sFlt-1 immunochromatographydetecting detecting test strips.It is shown in Figure 1, the present invention's
In first specific embodiment, pregnant woman's pre-eclampsia detection kit includes PIGF and sFlt-1 immunity chromatography detection test papers
Bar, that is, a test strip is only included, PIGF and sFlt-1 can be detected simultaneously.Refer to shown in Fig. 2-Fig. 5, in the present invention
Second the-the five specific embodiment of specific embodiment in, pregnant woman's pre-eclampsia detection kit includes PIGF immunochromatographies
Test strip and sFlt-1 immunochromatographydetecting detecting test strips, i.e., including two test strips, detect simultaneously respectively PIGF and
sFlt-1。
It is well known that immunochromatographydetecting detecting test strip is general from top to bottom successively by sample pad, pad, nitrocellulose
Film and adsorptive pads composition；Immune labeled antibody is coated with the pad, antibody is coated with nitrocellulose filter.Detection
When, immune labeled antibody and antibody will be combined so as to form interlayer structure with tested substance respectively.
PIGF the and sFlt-1 immunochromatographydetecting detecting test strips can be suitably composed with any, refer to Fig. 1 institutes
Show in the first specific embodiment of the present invention, have on the pad of the PIGF and sFlt-1 immunochromatographydetecting detecting test strips
There is PIGF to detect immune labeled antibody and sFlt-1 and detect immune labeled antibody, PIGF the and sFlt-1 immunochromatographies detection examination
PIGF detection antibody and sFlt-1 detection antibody are coated with the nitrocellulose filter of paper slip.
The PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips can have any
It is suitably composed, refers to shown in Fig. 2-Fig. 5, it is described in second the-the five specific embodiment of specific embodiment of the present invention
There is PIGF to detect immune labeled antibody, the PIGF immunochromatographies detection on the pad of PIGF immunochromatographydetecting detecting test strips
PIGF detection antibody, the pad of the sFlt-1 immunochromatographydetecting detecting test strips are coated with the nitrocellulose filter of test strips
It is upper that there is sFlt-1 to detect immune labeled antibody, it is coated with the nitrocellulose filter of the sFlt-1 immunochromatographydetecting detecting test strips
There is sFlt-1 to detect antibody.
The PIGF detects immune labeled antibody, the PIGF detects antibody, the sFlt-1 detects immune labeled antibody,
The sFlt-1 detections antibody can be any suitable antibody, refer to shown in Fig. 1-Fig. 5, and five in the present invention are specific real
Apply in example, it is the immune labeled antibody of anti-human placenta growth factor that the PIGF, which detects immune labeled antibody, and the PIGF detections are anti-
Body is anti-human plgf antibody, and it is anti-human soluble class fms tyrosine-kinases that the sFlt-1, which detects immune labeled antibody,
The immune labeled antibody of enzyme -1, the sFlt-1 detections antibody is the anti-human soluble antibody of class fms EGFR-TKs -1.Obviously, its
In the immune labeled antibody of anti-human placenta growth factor and anti-human plgf antibody combination Human plactnta growth factor not
Same position；Anti-human soluble -1 immune labeled antibody of class fms EGFR-TKs therein and anti-human soluble class fms tyrosine-kinases
The different parts of the antibody binding human soluble class fms of enzyme -1 EGFR-TKs -1.
Immune labeled antibody is using conventional immune labeled means mark.More preferably, the PIGF detections are immune labeled anti-
Body is that PIGF detections fluorescent labeled antibody, PIGF detection colloidal gold labeled monoclonal antibodies or PIGF detect latex labelled antibody, described
SFlt-1 detect immune labeled antibody be sFlt-1 detections fluorescent labeled antibody, sFlt-1 detection colloidal gold labeled monoclonal antibodies or
SFlt-1 detects latex labelled antibody.Refer to shown in Fig. 1 and Fig. 5, it is specific in the first specific embodiment of the present invention and the 5th
In embodiment, it is anti-human placenta growth factor fluorescent labeled antibody that the PIGF, which detects immune labeled antibody, the sFlt-1 inspections
It is the anti-human soluble fluorescent labeled antibody of class fms EGFR-TKs -1 to survey immune labeled antibody.Refer to shown in Fig. 2 and Fig. 4,
In the second specific embodiment and the 4th specific embodiment of the present invention, it is anti-human placenta life that the PIGF, which detects immune labeled antibody,
Long factor colloidal gold labeled monoclonal antibody, it is anti-human soluble class fms EGFR-TKs -1 that the sFlt-1, which detects immune labeled antibody,
Colloidal gold labeled monoclonal antibody.Shown in Figure 3, in the 3rd specific embodiment of the present invention, the PIGF detections are immune labeled
Antibody is anti-human placenta growth factor latex labelled antibody, and it is anti-human soluble class that the sFlt-1, which detects immune labeled antibody,
The latex labelled antibody of fms EGFR-TKs -1.
The PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips can use any
Suitable form is placed.It is shown in Figure 2, in the second specific embodiment of the present invention, pregnant woman's pre-eclampsia detection
Kit also includes bottom plate, and the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips are simultaneously
Row are arranged on the bottom plate.It is shown in Figure 3, in the 3rd specific embodiment of the present invention, pregnant woman's pre-eclampsia
Detection kit also includes bottom plate, the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips
It is fitted in respectively in the upper and lower surface of the bottom plate.It is shown in Figure 4, it is described in the 4th specific embodiment of the present invention
Pregnant woman's pre-eclampsia detection kit also includes two bottom plates, and the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 exempt from
Epidemic disease chromatography detecting test paper strip is set up in parallel and is separately positioned on two bottom plates.It is shown in Figure 5, the of the present invention
In five specific embodiments, pregnant woman's pre-eclampsia detection kit does not include bottom plate, the PIGF immunity chromatography detection test papers
Bar and the sFlt-1 immunochromatographydetecting detecting test strips are set up in parallel.
Pregnant woman's pre-eclampsia detection kit of the present invention can also include the other common of immunochromatographytest test kit
Reagent, such as sample sustained-release liquid etc..
The preparation process of the test strip of pregnant woman's pre-eclampsia detection kit of the present invention is summarized as follows, it should be appreciated that
These embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.Unreceipted actual conditions in the following example
Experimental method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:
Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to proposed by manufacturer
Condition.
The fluorescence labeling of embodiment 1
Cleaning：Take fluorescent microsphere (Bangs Lab, article No.：11233) into centrifuge tube, 0.1M MES (morpholine second sulphurs are added
Acid, pH 5.0) buffer solution mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, supernatant is abandoned, with 0.1M MES (pH 5.0)
Buffer solution is resuspended stand-by.Activate and clean：By activator EDC (1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid
Salt), NHS (1- hydroxypyrrole quinoline -2,5- diketone) and fluorescent microsphere be according to mass ratio 2:1:2 amount is activated, concrete operations
It is as follows：
Weigh EDC, NHS add 0.1M MES (pH 5.0) buffer solution in dissolve, take rapidly in right amount to cleaning finish it is glimmering
In light microballoon, sealed with sealed membrane and be placed on room temperature on 200rpm shaking tables and shake up 30min, 13000rpm, 30min, 4 DEG C after taking-up
Supernatant is centrifuged off, ultrasound mixing is resuspended with equivalent 0.1M MES (pH 6.5) buffer solution and cleans, is repeated more than once operation
Clean twice.It is stand-by that supernatant is discarded after the completion of centrifugation.
Mark：Latex after activation is resuspended into 0.1M MES (pH 6.5) buffer solution, be rapidly added antibody (Sigma,
The anti-human placenta growth factor monoclonal antibody of mouse, clone number：37203.111,0.5mg/ bottles), mix, placement room temperature, on 200rpm shaking tables
Shake up 4 hours.
Closing：The good microballoon of above-mentioned mark is taken to be centrifuged off supernatant in 13000rpm, 30min, 4 DEG C, immediately into microballoon
Equivalent confining liquid (the 0.01M PB solution (pH7.5) containing 2%BSA) is added, ultrasound places room temperature, 200rpm shaking tables again after being resuspended
On shake up 1 hour.13000rpm, 20min, 4 DEG C of centrifugations, take supernatant to be checked after the completion of reaction.
| Detection content | Quality standard | Detection method |
| Package amount | The μ gPIGF antibody of package amount >=40/mg microballoons | BCA determination of protein concentration methods |
＆ is cleaned to be resuspended：Re-suspension liquid (PB (pH7.5) of the 0.01M containing 0.5%BSA) is added, ultrasound piping and druming is resuspended, with height
Fast refrigerated centrifuge 13000rpm, 20min, 4 DEG C of centrifugations, discard supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.
The microspheres solution being resuspended carries out mark, standby.
Antibody (Sigma, the anti-human soluble monoclonal antibody of class fms EGFR-TKs -1 of mouse, clone number for sFlt-1 diagnosis：
Clone FLT-11,0.2ml/ bottle) fluorescence labeling method is same as above, the μ g sFlt-1 antibody of package amount >=40/mg microballoons.
The specking of the pad of embodiment 2
Specking method for antibody (anti-human placenta growth factor) pad of PIGF diagnosis is as follows：Fluorescent labeled antibody
Solution dilutes：The fluorescent labeled antibody conjugate of above-mentioned preparation is diluted 4 times with fluorescent labeled antibody re-suspension liquid；Set point film
Instrument, the power supply of point film instrument is opened, set specking program, specking amount is 8ul/cm；No. 1 pipeline is specking passage；Point film instrument is initial
Change：No. 1 pipeline is placed in into fluorescent labeled antibody to be resuspended in solution, selects initialization program, initializes 6 circulations；Specking：Will knot
Close pad to lie in point film instrument by fixed position, press on control panel " GO " key and start specking, removed after having put, check that specking is good
Pad, the fluorescent labeled antibody band of specking uniformly, continuous and the whole pad of insertion straight line be qualified specking product, two
It is unqualified specking product to occur breakpoint in bar straight line；A piece of pad is often put, it is specking to press " GO " key on a control panel
Once (a piece of)；Specking terminates, and the pad of specking is placed in room temperature and spontaneously dried 1 hour, specking trace should be can't see on film
Mark.
Antibody (anti-human soluble class fms EGFR-TKs -1) pad specking method for sFlt-1 diagnosis is same as above.
The preparation of the nitrocellulose filter of embodiment 3
Antibody (anti-human placenta growth factor) nitrocellulose membrane preparation method for PIGF diagnosis is as follows：Take goat-anti
People PIGF polyclonal antibodies (Sigma, goat-anti Human plactnta growth factor is more anti-, 0.1ml/ bottles) 500ug, is added to the centrifugation of 5ml scales
Guan Zhong, antibody diluent to 1ml, Container Tag T flag.Take sheep anti-mouse igg antibody (the grand base in Hangzhou biology, goat anti-mouse igg,
10mg/ bottles) 25ul, it is added in 5ml graduated centrifuge tubes, antibody diluent to 1ml, Container Tag C marks.Point film instrument is set, is opened
The power supply of point film instrument is opened, sets specking program, specking amount is 1ul/cm；No. 1 pipeline is detection band specking passage, and No. 2 pipelines are
Control band specking passage；Point film instrument initializes：No. 1 pipeline is placed in detection band solution, No. 2 pipelines are placed in control band solution
In, initialization program is selected, initializes 6 circulations；Specking：Nitrocellulose filter is lain in point film instrument by fixed position,
Press on control panel " GO " key and start specking, removed after having put, check the good nitrocellulose filter of specking, detection band and control band
It is qualified specking product for two uniform, continuous and the whole nitrocellulose filter of insertion straight lines, breakpoint occur in two straight lines is
Unqualified specking product；A piece of nitrocellulose filter is often put, presses " GO " key on a control panel as specking once (a piece of)；Spray
Point terminates, and the nitrocellulose filter of specking is placed in room temperature and spontaneously dried 1 hour, specking vestige should be can't see on film.
Antibody nitrocellulose membrane preparation method for sFlt-1 diagnosis is same as above, and marks the solution of T flag to use rabbit-anti
The polyclonal antibody of human soluble class fms EGFR-TKs -1 (Sigma, rabbit-anti human soluble class fms EGFR-TK more than -1 is anti-,
0.1mg/ bottles) prepare, the solution of mark C marks is using sheep anti-mouse igg antibody (the grand base biology in Hangzhou, goat anti-mouse igg, 10mg/
Bottle) prepare.
The pad pasting of embodiment 4, cut film
The protection sheet of wider portion on bottom plate is removed, along the lower edge of protection sheet above, the cellulose nitrate of line will be pulled
Plain film, by C lines up in a manner of be attached on bottom plate plate；Pad is attached to below T lines, contacted a little with NC films；By sample
Pad is attached to below pad, is contacted a little with pad；Then top protection sheet is removed, blotting paper is attached to the top of NC films,
Contacted a little with NC films；Protection sheet and index strip paper are attached to one by one outside the test strips assembled, are assembled into kilocalorie.
Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 4mm；Kilocalorie certified products are kept flat into cutting
In machine platform track, " GO " key face-up, is pressed on guidance panel, starts to cut；A piece of kilocalorie certified products are often put, by operating surface
On plate " GO " key once, until cut all kilocalorie certified products；After the completion of cutting, it will be used to detect PIGF and sFlt-1 respectively
Test strips stick in side by side on bottom plate, form PIGF and the sFlt-1 test strip of block form.
The kit of embodiment 5 assembles
Above-mentioned test strips are fitted into cartridge, form detection card.
Take aluminium foil bag and drier；Open heat sealing machine, preheating；Detection card to be packed, 1 bag of drier are loaded into aluminium foil bag
In；According to aluminium foil bag of the length cut-out of regulation equipped with detection card and drier；Aluminium foil bag is sealed with heat sealing machine；It is labelled.
The clinical sample of embodiment 6 is verified
By above-mentioned detection card, verified with clinical sample.
Sample to be tested is added drop-wise in the sample aperture (left position for being located at test strips in Fig. 1-Fig. 5) of detection card, then
It is placed in fluorescence detector and carries out reading.
Testing result and Follow-up results are as follows (to normal labor after Autonomous test)：
If band i.e. nature controlling line 6 occur in C areas during detection, there is two bands (the first detection line 4 and the second detection in T areas
Line 5) represent sample for the positive；There is a band or do not occur band then representing negative.
If C areas do not occur band during detection, then it represents that test strips fail.
Testing result is as follows：
| Follow-up results：Eclampsia | Follow-up results：Normally | It is total | |
| Detection is positive | 20 | 11 | 31 |
| Detection is negative | 7 | 121 | 128 |
| It is total | 27 | 132 | 159 |
The detection sensitivity of the test strips is：20/ (20+7) × 100%=74.07%；
The detection specificity of the test strips is：121/ (121+11)=91.67%.
Above-described embodiment result shows that the accuracy rate of this detection card is very high, meets clinical practice.
Therefore, the present invention can not only be detected by primary sample to PIGF and sFlt-1, be avoided repeatedly
Sampling detection causes the interference that result is inaccurate and environment is to testing result, and by detecting PIGF and sFlt-1 two simultaneously
Individual index, the accuracy of pre-eclampsia diagnosis is improved, and can be carried out according to the testing result of PIGF and sFlt-1 two indices
The stronger therapeutic scheme of specific aim.
Therefore, the present invention can detect PIGF and sFlt-1 simultaneously by primary sample, using simplicity, and can be accurate
Ratio between the two is obtained, and then can predict that the onset risk of eclampsia occurs for pregnant woman exactly, for accurate treatment in next step
Strong foundation is provided, suitable for large-scale promotion application.
To sum up, pregnant woman's pre-eclampsia detection kit of the invention can by primary sample simultaneously detect PIGF and
SFlt-1, using simplicity, and ratio between the two can be accurately obtained, and then can predict that eclampsia occurs for pregnant woman exactly
Onset risk, strong foundation is provided for accurate treatment in next step, suitable for large-scale promotion application.
In this description, the present invention is described with reference to its specific embodiment.But it is clear that it can still make
Various modifications and alterations are without departing from the spirit and scope of the present invention.Therefore, specification and drawings are considered as illustrative
It is and nonrestrictive.
Claims (8)
1. a kind of pregnant woman's pre-eclampsia detection kit, it is characterised in that including PIGF and sFlt-1 immunity chromatography detection test papers
Bar, or including PIGF immunochromatographydetecting detecting test strips and sFlt-1 immunochromatographydetecting detecting test strips.
2. pregnant woman's pre-eclampsia detection kit according to claim 1, it is characterised in that the PIGF and sFlt-1 exempt from
There is PIGF to detect immune labeled antibody and sFlt-1 on the pad of epidemic disease chromatography detecting test paper strip and detect immune labeled antibody, institute
State and PIGF detection antibody and sFlt-1 inspections are coated with the nitrocellulose filter of PIGF and sFlt-1 immunochromatographydetecting detecting test strips
Survey antibody.
3. pregnant woman's pre-eclampsia detection kit according to claim 1, it is characterised in that the PIGF immunochromatographies inspection
Testing on the pad of paper slip, there is PIGF to detect immune labeled antibody, and the nitric acid of the PIGF immunochromatographydetecting detecting test strips is fine
Tie up and PIGF detection antibody is coated with plain film, there is sFlt-1 inspections on the pad of the sFlt-1 immunochromatographydetecting detecting test strips
Immune labeled antibody is surveyed, it is anti-that sFlt-1 detections are coated with the nitrocellulose filter of the sFlt-1 immunochromatographydetecting detecting test strips
Body.
4. pregnant woman's pre-eclampsia detection kit according to Claims 2 or 3, it is characterised in that the PIGF detections are exempted from
Epidemic disease labelled antibody is the immune labeled antibody of anti-human placenta growth factor, and the PIGF detections antibody is that anti-human placenta growth factor resists
Body, it is anti-human soluble -1 immune labeled antibody of class fms EGFR-TKs that the sFlt-1, which detects immune labeled antibody, described
SFlt-1 detection antibody is the anti-human soluble antibody of class fms EGFR-TKs -1.
5. pregnant woman's pre-eclampsia detection kit according to Claims 2 or 3, it is characterised in that the PIGF detections are exempted from
Epidemic disease labelled antibody is that PIGF detections fluorescent labeled antibody, PIGF detection colloidal gold labeled monoclonal antibodies or PIGF detection latex marks are anti-
Body, it is that sFlt-1 detects fluorescent labeled antibody, sFlt-1 detection colloid gold labels resist that the sFlt-1, which detects immune labeled antibody,
Body or sFlt-1 detection latex labelled antibodies.
6. pregnant woman's pre-eclampsia detection kit according to claim 1, it is characterised in that the PIGF immunochromatographies inspection
Test paper slip and the sFlt-1 immunochromatographydetecting detecting test strips are set up in parallel.
7. pregnant woman's pre-eclampsia detection kit according to claim 1, it is characterised in that pregnant woman's pre-eclampsia inspection
Test agent box also includes bottom plate, and the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunochromatographydetecting detecting test strips are simultaneously
Row are arranged on the bottom plate or are fitted in respectively in the upper and lower surface of the bottom plate.
8. pregnant woman's pre-eclampsia detection kit according to claim 1, it is characterised in that pregnant woman's pre-eclampsia inspection
Test agent box also includes two bottom plates, the PIGF immunochromatographydetecting detecting test strips and the sFlt-1 immunity chromatography detection test papers
Bar is set up in parallel and is separately positioned on two bottom plates.
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| CN111007265A (en) * | 2020-01-16 | 2020-04-14 | 郑州大学 | Simoa kit of preeclampsia risk prediction marker PIGF and application thereof |
| 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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