CN107271664A - A kind of amniotic fluid Test paper and preparation method thereof - Google Patents
A kind of amniotic fluid Test paper and preparation method thereof Download PDFInfo
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- CN107271664A CN107271664A CN201610228107.0A CN201610228107A CN107271664A CN 107271664 A CN107271664 A CN 107271664A CN 201610228107 A CN201610228107 A CN 201610228107A CN 107271664 A CN107271664 A CN 107271664A
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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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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Abstract
The invention discloses a kind of amniotic fluid Test paper and preparation method thereof, the test strips are coated with the collaurum mouse anti-Human Insulin's like growth factor Binding Protein 1 monoclonal antibody 2 adsorbed on sheep anti mouse polyclonal antibody and polyester film in the coated mouse anti-Human Insulin like growth factor Binding Protein 1 monoclonal antibody 1 on detection zone (T) nitrocellulose filter and quality control region (C) nitrocellulose filter and constituted, when containing hIGFBP-1 in sample, combined first with gold labeling antibody 2, the antibody 1 being coated in chromatography process is captured, form band, Quality Control band is formed on the coated nitrocellulose filter of sheep anti mouse polyclonal antibody simultaneously.The preparation method of above-mentioned test paper includes the steps such as configuration, the preparation of test strips, the preparation of colloidal gold composite for the treatment of fluid.The amniotic fluid Test paper can detect amniotic fluid of pregnant woman seepage in real time, and its specificity is good, sensitivity is high, detection is quick and easy using process safety, is tested oneself available for family.
Description
Technical field
The present invention relates to in-vitro diagnosis field, more specifically to a kind of amniotic fluid Test paper and preparation method thereof.
Background technology
Amniotic fluid (amniotic fluid) is the liquid in amniotic cavity.Effect is to protect fetus outer from damage, buffering
Carrying out pressure makes embryo from shaking, preventing the adhesion of amnion and idiosome and provide embryo's free growth movable condition, maintains
Liquid environment needed for embryonic development.During childbirth, contribute to cervix dilating, cleaning and lubrication birth canal, in order to which fetus produces
Go out.Capacity, source and its composition of amniotic fluid are changed with the gravidic difference of people.First Trimester amniotic fluid is limpid, and 98%
For moisture, containing inorganic salts, protein, glucose, enzyme, fat and hormone etc..These materials are mainly amnion cell institute
Secretion.The amniotic fluid of latter half of gestation slightly has muddiness because add the secretion of fetus, excreta for example urea, uric acid,
The epithelial cell that sebum matter, hormone and fetus come off.Amniotic fluid examination is to diagnose one of important method of obstetric conditions, and
Shi Faxian leakage of amniotic fluid, could reduce pregnancy period risk.
Because rupture of membranes or early break cause amniotic fluid of pregnant woman seepage to be unconscious, it is impossible to control, and be continuation,
Leakage of amniotic fluid to a certain extent can caused by hapamnion fetal asphyxia, it is very harmful.
In existing monitoring leakage of amniotic fluid technology, the goldstandard of use is a kind of accurate method of monitoring, i.e., in pregnant woman sheep
Through puncturing injection dyestuff (such as methylene blue) in membrane cavity, if the liquid outflow that intravaginal has infected coloring can be diagnosed as amniotic fluid
Seepage, but, and cause the pain of patient using goldstandard detection clinical risk greatly, thus it is clinical it is basic without.And face
Generally there are amniotic fluid outflow, the spoiled meta-alkalescence of vaginal fluid pH value (just using inquiry medical history, patient's private prosecution vagina on bed
Normal vaginal fluid pH is 4.5~5.5), under microscope looks into and sees occur the methods such as amniotic fluid crystal in vaginal fluid smear,
These methods are although simple to operate, but accuracy, sensitiveness are relatively low, are also not use.
Amniotic fluid Test paper application colloidal gold-labeled method, using collaurum as tracer, utilizes antigen-antibody reaction
A kind of novel immune labelling technique.In chromatography process, golden label is in prior immobilization in chromatographic material：As nitric acid is fine
The plain film of dimension is that antigen or antibody on NC films occur specific immune response and is trapped, and further enrichment forms meat
The visible aubergine band of eye, so as to obtain intuitively experimental result, reaches the purpose of detection.
International monopoly PCT/US2003/025125 discloses a kind of using the inspection of double antibodies sandwich colloidal gold immunity chromatography
The apparatus and method for surveying amniotic fluid in vaginal fluid, its two kinds of monoclonal antibody used are to be directed to a1- microglobulins
(PAMG-1) different epitopes have.Chinese patent application 200620083734.1 discloses a kind of disposable amniotic fluid
Seepage pH test paper diagnoses rod, but its susceptibility is not high, easily causes false negative.International monopoly WO2007030890 is disclosed
A kind of method that utilization ELISA method detects amniotic fluid, time-consuming for the method, it is necessary to operating instrument, will to operating personnel
Ask higher.Therefore need a kind of sensitivity height badly and be easy to operation, efficiently novel sheep water monitoring test paper.
The present invention is optimized to a pair of IGFBP-1 (IGFBP-1) antibody by research,
With optimal coating and labelled antibody concentration, amniotic fluid Test paper is prepared, the test paper specificity is good, sensitivity is high, inspection
Survey is quick and easy using process safety, and Ovulation prediction, seminal fluid and urine, without influence, are answered test paper better than current clinic
The methods such as PH test paper.
In the very long pregnancy period, pregnant woman can cause rupture of membranes because of various accidents, produce leakage of amniotic fluid, and go to hospital then
Endless wait is faced, even a small minor inspection, this carrys out endless risk to suspender for pregnant woman again.Test paper of the present invention is adapted to length
Phase monitoring is used, and the family easy to operate that can carry out tests oneself.
The content of the invention
It is an object of the invention to provide a species specificity is good, sensitivity is high, detection is quick and to pregnant woman user
Just amniotic fluid Test paper：Vaginal fluid is taken to carry out vitro detection when using, it is safe and reliable, tested oneself available for family
And bedside is examined in time.
Another object of the present invention is to provide the preparation method of this amniotic fluid Test paper.
To achieve these goals, the present invention is adopted the following technical scheme that：
A kind of amniotic fluid Test paper and preparation method thereof, it is characterised in that：Test strips are by detection zone (T) nitric acid
The coated monoclonal antibody 1 of mouse anti-Human Insulin like growth factor associated proteins -1 and quality control region (C) nitre on cellulose membrane
Collaurum-mouse anti-Human Insulin sample the growth adsorbed on sheep anti mouse polyclonal antibody and polyester film is coated with acid cellulose film
The monoclonal antibody of factor bindin -1 2 is constituted,
In a preferred embodiment, the Test paper is test strips, from lower to upper successively by immersing
Area, collaurum chelant area, detection zone, quality control region and holding area composition.
A kind of preparation method of amniotic fluid Test paper, it is characterised in that processing step is as follows：
(1) configuration step for the treatment of fluid：By concentration ratio configuration gold standard pad treatment fluid, sample pad treatment fluid,
Point membrane antibody dilution, labelled antibody dilution and colloidal gold composite dilution；
(2) preparation process of test paper：By the processing of membrane material, point film, the preparation of colloidal gold composite,
Metal spraying, the cutting of membrane material, assembling, the cutting of big plate and packaging step obtain aluminium foil bag and encapsulate complete test paper.
When a film matches somebody with somebody liquid, sheep anti mouse polyclonal antibody is taken, 0.5mg/ml antibody-solutions are diluted to PBS.
IGFBP-1 monoclonal antibody 1 is taken, 1.0mg/ml antibody-solutions are diluted to PBS.
In the preparation process of colloidal gold composite, added in the colloidal gold composite per 1ml collaurums
5~20 μ lK2CO3, select that the constant minimum pH value of color can be kept, be typically chosen the addition of 1ml collaurums
15μlK2CO3
In the preparation process of colloidal gold composite, combined in the colloidal gold composite per 1ml collaurums
5~15 μ g monoclonal antibody 2 of mouse anti-Human Insulin's like growth factor associated proteins -1.Selection can keep color not
The minimum protein content of change, generally 1ml collaurums combine 10 μ g albumen, per 1ml collaurums in practical operation
Add 10% antibody protein.
Advantages of the present invention has：(1) as little as 1ul amniotic fluid can be detected in vaginal fluid, finds to face in advance
The still unconscious small rupture of membranes of bed；(2) sensitivity of index diagnosis leakage of amniotic fluid is 98.0%, specific up to 100.0%；
(3) the test paper specificity is good, sensitivity is high, detection is quick and uses to patient's no pain；(4) it is simple to operate, 5
Minute can obtain result, have higher practical value in the clinical diagnosis of leakage of amniotic fluid, can be as routine inspection side
Method is promoted.
Brief description of the drawings
Fig. 1 is leakage of amniotic fluid test strip structural representation.
Embodiment
Explanation is further spread out to the present invention with reference to Fig. 1, it is to be noted that the sheep of the present invention
Water leak detection test strips are not limited to this specific shape or structure.Meeting art personnel for this area obviously can be with
Understand, even if the following description content does not make any adjustments or corrected, can also be directly applied for not referring to herein
Bright other types or the test strips of structure.
Fig. 1 is leakage of amniotic fluid test strip structural representation, including：Wherein, immersion area 1, collaurum
Chelant area 2, detection zone 3, quality control region 4, holding area 5.Mouse anti-Human Insulin is coated with detection zone (T) 3
The monoclonal antibody 1 of like growth factor associated proteins -1, quality control region (C) 4 is coated with sheep anti mouse polyclonal antibody,
Collaurum chelant area 2 is coated with the monoclonal antibody 2 of mouse anti-Human Insulin's like growth factor associated proteins -1.
Test paper application method is as follows：1) aluminium foil bag in test paper and buffer solution are taken out and balanced at room temperature.2)
Aluminium foil bag is opened, test strip is taken out, indicates test sample and patient information.Test strips are taken out from aluminium foil bag
Afterwards, should as early as possible it be used in 1 hour.3) buffer solution is opened, places vertically in desktop, the vagina of sample will be collected
Cotton swab is put into buffer solution bottle, revolving dilution 1 minute in buffer solution.4) test strips are hung down in the direction of the arrow
In the straight buffer solution being downwardly into after dilution sample.5) test strips, test strips are taken out when colour band occur in test strips
Desktop is placed, result is read in 15 minutes.
The colour band situation judged result that can occur by detection zone and quality control region is as follows：1) it is positive：In detection
Respectively there is a colour band in area (T) and control zone (C), and testing result is the positive.2) it is negative：Only in control zone
(C) there is a colour band, testing result is feminine gender.3) it is invalid：The colourless band in control zone (C) occurs, and shows
Experiment is invalid, should re-start detection.The human insulin-like growth factor binding protein white -1 that the test paper can be detected
Limit of identification be 20ng/ml.
Family need to read over specification when testing oneself, and operating procedure is carried out to specifications in detail, is such as occurred different
Reason condition, goes to hospital to do and further detects and make a definite diagnosis in time.
The preparation method of leakage of amniotic fluid Test paper, including the configuration for the treatment of fluid and the two big step of the preparation of test paper
Suddenly, specific steps are detailed as follows：
1) preparation for the treatment of fluid
The preparation of A gold standard pad treatment fluids：Tris-Hcl (10mM, PH=7.4)+0.5%PVP+0.5%TW-20
B sample pads treatment fluid is prepared：Tris-Hcl (0.1M, PH=9.0)
+ 1%PVP+1.0%S17+0.8%Casein+0.3% TritonX-100
C point membrane antibody diluent preparings：PBS { PB (10mM, PH=7.4), 0.85%NaCl }
The preparation of D labelled antibody dilutions：PB (10mM, PH=7.4)
The preparation of E colloidal gold composite dilutions：Sucrose+the 1%BSA of PB (10mM, PH=7.4)+20%
2) preparation of test paper
The processing of A membrane materials：
The processing of a sample pads：The miillpore filter got ready is affixed in sample pad (SB08), with formulated
Good treatment fluid immersion, membrane material can just be collected by fully soaking rearmounted hand drying room and being dried to humidity and drop to less than 30%, be received
The membrane material taken will be positioned in aluminium foil bag and (some drier be placed in aluminium foil bag), finally seal stand-by.
The processing of b gold standard pads：By the gold standard pad got ready (KB50), soaked with the treatment fluid prepared,
Membrane material can just be collected by being fully placed in drying room after immersion and being dried to humidity and drop to less than 30%, and the membrane material collected will be positioned over
(some drier are placed in aluminium foil bag) in aluminium foil bag, finally sealed stand-by.
B point films：
A matches somebody with somebody liquid：Sheep anti mouse polyclonal antibody is taken, 0.5mg/ml antibody-solutions are diluted to PBS.Take mouse
The monoclonal antibody 1 of anti-Human Insulin's like growth factor associated proteins -1,1.0mg/ml antibody is diluted to PBS
Solution.
B pad pastings：NC films and PVC offset plates are taken, NC films are affixed on to the designated area of PVC offset plates.
C point films：Opening point film gold spraying instrument, sets corresponding parameter, and point film concentration is 1 μ l/cm, is started
Point film.The offset plate of the good film of point is placed in 37 DEG C of baking ovens, dries 12 hours, the ring residing for baking oven is made by dehumidifier
Border humidity can collect offset plate after being reduced to 30%, and the offset plate collected is placed in aluminium foil bag (will place in aluminium foil bag
Drier), finally seal standby.
The preparation of C colloidal gold composites
The measure of the optimal pH values of a
Colloidal gold composite is prepared by upper table, selection can keep the constant minimum pH value of color, be typically chosen
The μ lK of 1ml collaurums+152CO3Prepare.
B minimum protein binding amounts
Colloidal gold composite is prepared by upper table, selection can keep the constant minimum protein content of color, generally 1ml
Collaurum combines 10 μ g albumen, in practical operation per 1ml collaurums more add 10% antibody protein.Each
During preparation, a collaurum or antibody are often changed, optimal pH value and minimum protein binding amount will be looked for again.
The preparation of c colloidal gold composites：A certain amount of collaurum is taken, the 0.1MK of respective amount is added2CO3, in magnetic
Mixed on power agitator, the antibody-solutions of addition respective amount, magnetic agitation 30min, adding PEG20000 makes end dense
Spend for 0.1%, then magnetic agitation 30min, 4 DEG C of 8000r centrifuge 40min, abandon supernatant, precipitation gold mark dilution redissolves
(per 20ml collaurums, finally concentration is 1ml).4 DEG C store for future use.
D metal sprayings
A is debugged：Gold spraying instrument is opened, processed good gold standard pad is taken out, is entered with the colloidal gold composite prepared
Row metal spraying, first debugs different metal spraying concentration (such as 2 μ l/cm, 3 μ l/cm, 4 μ l/cm, 5 μ l/cm) and is placed in 37 DEG C of bakings
In case, two hours are dried.
B metal sprayings：Result according to debugging is carried out with point film gold spraying instrument in metal spraying, the good rearmounted 3 DEG C of baking ovens of spray, dries two
Individual hour, opening dehumidifier makes the ambient humidity residing for baking oven to collect gold standard pad after being reduced to 30%, the gold standard pad collected
It is placed in aluminium foil bag and (some drier is placed in aluminium foil bag), finally seals standby.
The cutting of E membrane materials：Blotting paper CH27, processed sample pad and the gold standard pad of metal spraying are taken, unlatching is removed
Wet machine, makes after the humidity of environment is reduced to less than 30%, to be cut according to certain size, gold standard pad after well cutting,
Sample pad, blotting paper are placed in different aluminium foil bags and (some drier are placed in aluminium foil bag), finally seal standby.
F is assembled：Take PE films bar and MAX lines, open dehumidifier, make after the humidity of environment is reduced to less than 30%
Take out the gold standard pad of well cutting, sample pad, blotting paper and put film rhythm offset plate, take the joint strip of gold standard pad position off, paste
Upper gold standard pad, makes gold standard pad small part press against NC films, then sticks sample pad, sample pad part is press against gold standard pad,
Blotting paper is sticked, blotting paper small part press against NC films, finally stick PE films bar and MAX lines.The offset plate assembled
It is placed in aluminium foil bag and (some drier is placed in aluminium foil bag), finally seals standby.
The cutting of the big plates of G：Dehumidifier is opened, the humidity of environment is reduced to and assembled big is taken out after less than 30%
Plate, is then turned on cutting machine, is cut according to certain size, the test strips of well cutting are placed in aluminium foil bag (in aluminium foil bag
Place some drier), finally seal standby.
Encapsulating mouth in H：Test strips and drier are put into aluminium foil bag, an aluminium foil bag puts a test strips and dry
Drying prescription.
In above-mentioned preparation process, each step, which all must be turned on dehumidifier, to be made after residing ambient humidity is reduced to 30%
It can carry out, if the effect of chromatography will be influenceed because test paper humidity is excessive, cause test paper to fail,
200 clinical samples have been carried out to the leakage of amniotic fluid Test paper of the present invention by using foregoing application method
Checking test, control group uses traditional leakage of amniotic fluid diagnostic criteria, i.e., using pH test paper detection vaginal fluid pH ＞
7 or direct microscopy vaginal fluids see fernlike crystal and the visible a small amount of liquid of vaginoscopy
Criterion.As a result showing test products, there was no significant difference with control group, and test products reach golden standard knot
Really.
The checking test result of 200 clinical samples see the table below：
Although the embodiment to the present invention gives detailed description and illustrated above, it should be noted that
It is that we can carry out various equivalent changes and modification according to the conception of the present invention to above-mentioned embodiment, produced by it
, all should be within protection scope of the present invention during the spirit that function is still covered without departing from specification and accompanying drawing.
Claims (9)
1. a kind of amniotic fluid Test paper and preparation method thereof, it is characterised in that：Test strips are coated with the collaurum-monoclonal antibody 2 of mouse anti-Human Insulin's like growth factor associated proteins -1 adsorbed on sheep anti mouse polyclonal antibody and polyester film in the coated monoclonal antibody 1 of mouse anti-Human Insulin like growth factor associated proteins -1 on detection zone (T) nitrocellulose filter and quality control region (C) nitrocellulose filter and constituted.
2. amniotic fluid Test paper according to claim 1, it is characterised in that：The quality control region is marked with sheep anti-mouse igg, 0.1~0.5mg/ml of label concentration.
3. amniotic fluid Test paper according to claim 1, it is characterised in that：The sample application zone is pasted with miillpore filter above sample pad, and the impurity in vaginal fluid is removed using miillpore filter, improves chromatography.
4. amniotic fluid Test paper as claimed in claim 1, it is characterised in that the test strips are constituted by immersing area, collaurum chelant area, detection zone, quality control region and holding area successively from lower to upper.
5. the preparation method of any one of claim 1-4 amniotic fluid Test papers, it is characterised in that processing step is as follows：
(1) configuration step for the treatment of fluid：By concentration ratio configuration gold standard pad treatment fluid, sample pad treatment fluid, coating point membrane antibody dilution, labelled antibody dilution and colloidal gold composite dilution；
(2) preparation process of test strips：Aluminium foil bag is obtained by the processing of membrane material, point film, the preparation of colloidal gold composite, metal spraying, the cutting of membrane material, assembling, the cutting of big plate and packaging step and encapsulates complete test paper.
6. the preparation method of amniotic fluid Test paper as claimed in claim 5, it is characterised in that the concentration of the sheep anti mouse Anti-TNF-α liquid solution is 0.1~0.5mg/ml.
7. the preparation method of amniotic fluid Test paper as claimed in claim 5, it is characterised in that the concentration of the solution of -1 monoclonal antibody of the mouse anti-Human Insulin like growth factor associated proteins 1 is 0.1~0.5mg/ml.
8. the preparation method of amniotic fluid Test paper as claimed in claim 5, it is characterised in that add 5~20 μ gK per 1ml collaurums in the colloidal gold composite2CO3。
9. the preparation method of amniotic fluid Test paper as claimed in claim 5, it is characterised in that combine 5~15 μ g monoclonal antibody 2 of mouse anti-Human Insulin's like growth factor associated proteins -1 in the colloidal gold composite per 1ml collaurums.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
-
2016
- 2016-04-09 CN CN201610228107.0A patent/CN107271664A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111551744A (en) * | 2020-05-15 | 2020-08-18 | 安徽中起生物科技有限公司 | Newcastle disease virus N protein IgY antibody colloidal carbon detection test paper and application thereof |
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Application publication date: 20171020 |