CN103175968A - Detection kit for premature rupture of fetal membranes and preparation method thereof - Google Patents
Detection kit for premature rupture of fetal membranes and preparation method thereof Download PDFInfo
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- CN103175968A CN103175968A CN2011104306192A CN201110430619A CN103175968A CN 103175968 A CN103175968 A CN 103175968A CN 2011104306192 A CN2011104306192 A CN 2011104306192A CN 201110430619 A CN201110430619 A CN 201110430619A CN 103175968 A CN103175968 A CN 103175968A
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Abstract
The invention discloses a detection kit for premature rupture of fetal membranes, which comprises a detection test paper strip or a test paper card for premature rupture of fetal membranes, a buffer and a cotton swab, the test paper strip or the test paper card comprise a detection zone (T) coated with anti-human insulin-like growth factor binding protein-1 monoclonal antibody 1 and a quality control zone (C) coated with anti-mice polyclonal antibody and colloidal gold coated with anti-human insulin-like growth factor binding protein-1 monoclonal antibody 2. The preparation method of the kit comprises the following steps: preparing a treating liquid, preparing the kit, preparing a colloidal gold compound, spraying metal, cutting, assembling, cutting, packaging and the like. The detection kit for premature rupture of fetal membranes has the advantages of good specificity, high sensitivity, rapid and simple detection, and no pain in a usage process, and can be used for self-test in household and test on bed.
Description
Technical field
The present invention relates to the detection kit field, more specifically to a kind of kit for detecting premature rupture of membrane and preparation method.
Background technology
Rupture of membranes just before giving birth is called premature rupture of fetal membranes (premature rupture of fetal membrane, prom), is a kind of common complication of obstetrics, and the domestic report of its incidence of disease is about 10%.The concurrent uterine cavity of prom infects can cause premature labor, it is dead to enclose living youngster, is one of ill and dead major reason of female youngster.
In prior art, adopting goldstandard is a kind of accurate method of Diagnosis of Premature Rupture, namely inject dyestuff (as methylene blue) in pregnant woman's amniotic cavity, can be diagnosed as premature rupture of fetal membranes if there is infected painted liquid to flow out in vagina, but adopt goldstandard to detect clinical very difficult operation, and cause patient's misery, therefore clinical seldom use.Usually adopt clinically inquiry medical history, patient's private prosecution vagina to have amniotic fluid outflow, the spoiled meta-alkalescence of vaginal fluid pH value (normal vaginal fluid pH is 4.5～5.5), microscopically to look into the methods such as occurring amniotic fluid crystal in the vaginal fluid smear of seeing, although these methods are simple to operate, accuracy, susceptibility are all lower.
Highly-pathogenic avian influenza is to use colloidal gold-labeled method, as tracer, is applied to a kind of Novel immune labelling technique of antigen-antibody reaction with collaurum.In chromatography process, the gold label is in prior immobilization in chromatographic material: be that antigen on the NC film or antibody specific immune response occur and is trapped as nitrocellulose filter, further enrichment forms the visible aubergine band of naked eyes, thereby obtain experimental result intuitively, reach the purpose of detection.International monopoly PCT/US2003/025125 discloses a kind of apparatus and method that adopt the double antibodies sandwich colloidal gold immunity chromatography to detect amniotic fluid in vaginal fluid, and two kinds of monoclonal antibodies of its use are for the different epitopes of a1-microglobulin (PAMG-1).Chinese patent application 200620083734.1 discloses a kind of disposable premature rupture of fetal membranes pH test paper diagnosis rod, but its susceptibility is not high, easily causes false negative.International monopoly WO2007030890 discloses a kind of method that the ELISA of utilization method detects amniotic fluid, and this method length consuming time needs operating instrument, and operating personnel are had relatively high expectations.Therefore need a kind of highly sensitive convenient operation again, novel kit for detecting premature rupture of membrane efficiently badly.
The present inventor finds by research, non-phosphorylating IGFBP-1 (IGFBP-1) be gestation after 3 months until one of topmost protein in the childbirth forewaters, when rupture of membranes occured, the IGFBP-1 in amniotic fluid can escape in the uterine neck vagina by the fetal membrane cut.And high 100～1000 times in Volume fraction mother blood of IGFBP-1 in amniotic fluid, uterine neck mucus, seminal fluid and urine are on testing all without impact, detect IGFBP-1 diagnosis prom and pregnant age big or small irrelevant, mature and incomplete pregnancy pregnant woman are had same diagnostic value, thereby completed the present invention.
Summary of the invention
The object of the present invention is to provide a species specificity good, highly sensitive, detect fast and to the kit for detecting premature rupture of membrane of patient's no pain, can be used for that family tests oneself and POCT.
Another object of the present invention is to provide the preparation method of this kit for detecting premature rupture of membrane.
To achieve these goals, the present invention adopts following technical scheme:
A kind of kit for detecting premature rupture of membrane, it comprises premature rupture of fetal membranes Test paper, damping fluid and cotton swab, described Test paper comprises detection zone (T), Quality Control district (C) and collaurum, it is characterized in that described detection zone (T) is coated with anti-human IGFBP-1 monoclonal antibody 1, described Quality Control district (C) is coated with anti-mouse polyclonal antibody, and described collaurum is coated with anti-human IGFBP-1 monoclonal antibody 2.
In a preferred embodiment, described Test paper is test strips.At this moment, kit for detecting premature rupture of membrane comprises premature rupture of fetal membranes test strip, damping fluid and cotton swab.Wherein, described premature rupture of fetal membranes test strip forms by immersing district, collaurum, detection zone, Quality Control district and holding area from lower to upper successively.
In a preferred embodiment, described Test paper is test card.At this moment, kit for detecting premature rupture of membrane also includes dropper, and namely kit for detecting premature rupture of membrane comprises premature rupture of fetal membranes Test paper card, damping fluid, cotton swab and dropper.Wherein, described premature rupture of fetal membranes Test paper card also comprises well, and namely described premature rupture of fetal membranes Test paper card is comprised of well, collaurum, detection zone, Quality Control district and holding area from lower to upper successively.
A kind of preparation method of kit for detecting premature rupture of membrane is characterized in that processing step is as follows:
(1) configuration step for the treatment of fluid: by concentration ratio configuration gold mark pad treating fluid, sample pad treating fluid, some membrane antibody dilution, labelled antibody dilution and colloidal gold composite dilution;
(2) preparation process of kit: the cutting of the cutting of the preparation of the processing by the film material, some film, colloidal gold composite, metal spraying, film material, assembling, large plate and packaging step obtain the complete kit of aluminium foil bag encapsulation.
When some film dosing, get the sheep anti mouse polyclonal antibody, be diluted to the antibody-solutions of 0.5mg/ml with PBS.Get IGFBP-1 monoclonal antibody 1, be diluted to the antibody-solutions of 1.0mg/ml with PBS.
In the preparation process of colloidal gold composite, in described colloidal gold composite, every 1ml collaurum adds 5～20 μ lK
2CO
3, selection can keep the constant minimum pH value of color, generally selects the 1ml collaurum to add 15 μ l1K
2CO
3
In the preparation process of colloidal gold composite, in described colloidal gold composite, every 1ml collaurum is in conjunction with the anti-human IGFBP-1 monoclonal antibody 2 of 5～15 μ g.Selection can keep the protein content of the constant minimum of color, is generally the 1ml collaurum in conjunction with the albumen of 10 μ g, and every 1ml collaurum is added 10% antibody protein more in practical operation.
Advantage of the present invention has: (1) can detect few amniotic fluid to 1ul in vaginal fluid, finds in advance clinical still unconscious small rupture of membranes; (2) sensitivity of this index diagnosis prom is 98.0%, and specificity reaches 100.0%; (3) this kit specificity good, highly sensitive, detect fast and use patient's no pain; (4) simple to operate, can obtain result in 5 minutes, have higher practical value in the clinical diagnosis of premature rupture of fetal membranes, can be used as the routine inspection method and promoted.
Description of drawings
Fig. 1 is premature rupture of fetal membranes test strip structural representation.
Fig. 2 is premature rupture of fetal membranes test strip structural representation.
Embodiment
Below in conjunction with Fig. 1, the present invention is done further expansion explanation, but it is pointed out that premature rupture of fetal membranes test strip of the present invention is not limited to this specific shape or structure.Obviously be understandable that for those skilled in the art, even the following description content does not make any adjustments or revises, also can be directly applied for the test strips in these unspecified other types or structure.
Fig. 1 is premature rupture of fetal membranes test strip structural representation, comprising: wherein, immerse district 1, collaurum 2, detection zone 3, Quality Control district 4, holding area 5.Be coated with anti-human IGFBP-1 monoclonal antibody 1 at detection zone (T) 3, Quality Control district (C) 4 is coated with anti-mouse polyclonal antibody, and collaurum 2 is coated with anti-human IGFBP-1 monoclonal antibody 2.
At this moment, kit for detecting premature rupture of membrane comprises premature rupture of fetal membranes test strip, damping fluid and cotton swab.In use, should first check all article in box.Before not carrying out the test preparation, do not tear in advance aluminium foil bag.Using method is as follows: 1) aluminium foil bag in kit and damping fluid are taken out at room temperature balance.2) open aluminium foil bag, take out test strip, indicate test sample and patient information.Take out test strips from aluminium foil bag after, should use as early as possible in 1 hour.3) open damping fluid, place vertically at desktop, the vagina cotton swab that is collected sample is put into the damping fluid bottle, the rotation dilution is 1 minute in damping fluid.4) test strips is inserted in the damping fluid of dilution after sample vertically downward along the direction of arrow.5) take out test strips when colour band appears in test strips, test strips is placed desktop, reading result in 15 minutes.
Can be as follows by the colour band situation judged result of detection zone and Quality Control district's appearance: the 1) positive: a colour band respectively occurs at detection zone (T) and control zone (C), testing result be positive.2) feminine gender: only a colour band appears in (C) in the control zone, and testing result is negative.3) invalid: control zone (C) colourless band occurs, and shows invalidate the test, should re-start detection.The limit of identification of the detected human insulin-like growth factor binding protein of this kit energy white-1 is 20ng/ml.
Fig. 2 is premature rupture of fetal membranes Test paper card structure schematic diagram, comprising: wherein, and collaurum 2, detection zone 3, Quality Control district 4, holding area 5, well 6.The Test paper of this kind pattern only independently is provided with well, and this just needs in kit with dropper, testing sample solution to be dropped to well, other steps and determination methods is the same as a result, then this does not do and gives unnecessary details.
The preparation method of kit for detecting premature rupture of membrane comprises the configuration for the treatment of fluid and two large steps of preparation of kit, and the concrete steps detail is as follows:
1) preparation for the treatment of fluid
The preparation of A gold mark pad treating fluid: Tris-Hcl (10mM, PH=7.4)+0.5%PVP+0.5%TW-20
B sample pad treating fluid preparation: Tris-Hcl (0.1MPH=9.0)+1%PVP+1.0%S17+0.8%Casein+0.3%TritonX-100
C point membrane antibody diluent preparing: PBS{PB (10mM, PH=7.4), 0.85%NaCl}
The preparation of D labelled antibody dilution: PB (10mM, PH=7.4)
The preparation of E colloidal gold composite dilution: PB (10mM, PH=7.4)+20% sucrose+1%BSA
2) preparation of kit
The processing of A film material:
The processing of a sample pad: with the sample pad (SB08) of receiving, soak with the treating fluid that has prepared, fully soak and be placed on drying room and dry by the fire to drop to below 30% to humidity and just can collect the film material, the film material of collecting will be positioned in aluminium foil bag (will place some drying agent in aluminium foil bag), seals at last stand-by.
The processing of b sample pad: the gold mark pad (KB50) that will receive, soak with the treating fluid that has prepared, fully soak and be placed on drying room and dry by the fire to drop to below 30% to humidity and just can collect the film material, the film material of collecting will be positioned in aluminium foil bag (will place some drying agent in aluminium foil bag), seals at last stand-by.
B point film:
A dosing: get the sheep anti mouse polyclonal antibody, be diluted to the antibody-solutions of 0.5mg/ml with PBS.Get IGFBP-1 monoclonal antibody 1, be diluted to the antibody-solutions of 1.0mg/ml with PBS.
B pad pasting: get NC film and PVC offset plate, the NC film is affixed on the appointed area of PVC offset plate.
C point film: opening point film gold spraying instrument, set corresponding parameter, some film concentration is 1 μ l/cm, the starting point film.The offset plate of the good film of point is placed in 37 ℃ of baking ovens, dries by the fire 12 hours, opens and can collect offset plate after dehumidifier makes the residing environment temperature of baking oven reduce to 30%, and the offset plate of collecting is placed in aluminium foil bag (aluminium foil bag will be placed some drying agent), seals at last standby.
The preparation of C colloidal gold composite
The mensuration of the best pH value of a
By upper table preparation colloidal gold composite, selection can keep the constant minimum pH value of color, generally selects 1ml collaurum+15 μ lK
2CO
3
The minimum protein combination amount of b
By upper table preparation colloidal gold composite, selection can keep the protein content of the constant minimum of color, is generally the 1ml collaurum in conjunction with the albumen of 10u g, and every 1ml collaurum is added 10% antibody protein more in practical operation.Usually, collaurum of every replacing or antibody will be looked for best pH value and minimum protein combination amount again.
The preparation of c colloidal gold composite: get a certain amount of collaurum, add the 0.1MK of respective amount
2CO
3Mixing on magnetic stirring apparatus, the antibody-solutions that adds respective amount, magnetic agitation 30min, adding PEG20000 to make final concentration is 0.1% again, then magnetic agitation 30min, 4 ℃ of centrifugal 40min of 8000r, abandon supernatant, precipitation is marked dilution redissolution (the last simmer down to 1ml of every 20ml collaurum) with gold.4 ℃ store for future use.
The D metal spraying
A debugging: open gold spraying instrument, take out handled well gold mark pad, carry out metal spraying with the colloidal gold composite for preparing, first debug different metal spraying concentration (as 2 μ l/cm, 3 μ l/cm, 4 μ l/cm, 5 μ l/cm) and be placed in 37 ℃ of baking ovens, dried by the fire two hours, select suitable starting material and be assembled into plate, determine that finally which kind of metal spraying concentration is most suitable.
The b metal spraying: the result according to debugging is carried out metal spraying with some film gold spraying instrument, in the good rearmounted 37 ℃ of baking ovens of spray, dried by the fire two hours, open and to collect gold mark pad after dehumidifier makes the residing environment temperature clothes of baking oven reduce to 30%, the gold mark pad of collecting is placed in aluminium foil bag (aluminium foil bag will be placed some drying agent), seals at last standby.
The cutting of E film material: get thieving paper CH27, the sample pad of having processed and the gold mark pad of metal spraying, open dehumidifier, after making the temperature of environment reduce to below 30%, cut according to certain size, gold mark pad after well cutting, sample pad, thieving paper are placed in different aluminium foil bag (aluminium foil bag will be placed some drying agent), seal at last standby.
F assembling: get PE film bar and MAX line, open dehumidifier, take out gold mark pad, sample pad, the thieving paper of well cutting after making the humidity of environment reduce to below 30% and put the offset plate of film, take the joint strip of gold mark pad position off, stick gold mark pad, make gold mark pad small part press the NC film, stick again sample pad, make sample pad partly press gold mark pad, stick thieving paper, the thieving paper small part is being pressed the NC film, sticks at last PE film bar and MAX line.The offset plate that assembles is placed in aluminium foil bag (aluminium foil bag will be placed some drying agent), seals at last standby.
The cutting of the large plate of G: open dehumidifier, make the humidity of environment reduce to the large plate that below 30%, rear taking-up has assembled, then open cutting machine, according to certain size cutting, the test strips of well cutting is placed in aluminium foil bag (aluminium foil bag will be placed some drying agent), seals at last standby.
Seal mouth in H: test strips and drying agent are put into aluminium foil bag, and an aluminium foil bag is put a test strips and drying agent.Adjust the capper character code by the date of manufacture, open capper, be preheated to 180 ℃, seal.Close capper after sealing, paste to be verified and the card that flows, indicate the name of an article, lot number, specification, date of manufacture, operator.Enter through-station or outsourcing, fill in record.
Without any restriction, as long as can realize technical scheme of the present invention, the test paper width should be not less than 2.5mm usually, should be not less than 10mm/min to guarantee the liquid speed of dividing a word with a hyphen at the end of a line for the width that cuts test paper in the present invention.The packing of final kit for detecting premature rupture of membrane is without any restriction, the quantity of kit inner packing can be random, for example can be 1 person-portion/box, 2 person-portions/box, 3 person-portions/box, 5 person-portions/box, 10 person-portions/box, 20 person-portions/box, 50 person-portions/box, 100 person-portions/box etc.
Embodiment
Kit for detecting premature rupture of membrane of the present invention (IGFBP-1 detection kit) (colloidal gold immunity chromatography) has been carried out the demonstration test of 200 routine clinical samples by adopting aforementioned using method, the diagnosing premature rupture of fetal membrane standard that the control group employing is traditional namely adopts pH detection paper vaginal fluid pH＞7 or direct microscopy vaginal fluid to see fernlike crystal and the visible a small amount of liquid of vaginoscopy is criterion from the outflow of uterine neck mouth.Result shows test products and control group there was no significant difference, and test products reaches the golden standard result.
Although above the specific embodiment of the present invention has been given to describe in detail and explanation; but what should indicate is; we can carry out various equivalences to above-mentioned embodiment according to conception of the present invention and change and revise; when its function that produces does not exceed spiritual that instructions and accompanying drawing contain yet, all should be within protection scope of the present invention.
Claims (11)
1. kit for detecting premature rupture of membrane, comprise premature rupture of fetal membranes Test paper, damping fluid and cotton swab, described Test paper comprises detection zone (T), Quality Control district (C) and collaurum, it is characterized in that described detection zone (T) is coated with anti-human IGFBP-1 monoclonal antibody 1, described Quality Control district (C) is coated with anti-mouse polyclonal antibody, and described collaurum is coated with anti-human IGFBP-1 monoclonal antibody 2.
2. kit for detecting premature rupture of membrane as claimed in claim 1, is characterized in that described Test paper is test strips.
3. kit for detecting premature rupture of membrane as claimed in claim 2, is characterized in that described test strips forms by immersing district, collaurum, detection zone, Quality Control district and holding area from lower to upper successively.
4. kit for detecting premature rupture of membrane as claimed in claim 1, is characterized in that described Test paper is test card.
5. kit for detecting premature rupture of membrane as claimed in claim 4, is characterized in that described kit also comprises dropper.
6. kit for detecting premature rupture of membrane as described in claim 4 or 5, is characterized in that described test card also comprises well, is comprised of well, collaurum, detection zone, Quality Control district and holding area successively from lower to upper.
7. the preparation method of claim 1-6 any one kit for detecting premature rupture of membrane is characterized in that processing step is as follows:
(1) configuration step for the treatment of fluid: by concentration ratio configuration gold mark pad treating fluid, sample pad treating fluid, some membrane antibody dilution, labelled antibody dilution and colloidal gold composite dilution;
(2) preparation process of kit: the cutting of the cutting of the preparation of the processing by the film material, some film, colloidal gold composite, metal spraying, film material, assembling, large plate and packaging step obtain the complete kit of aluminium foil bag encapsulation.
8. the preparation method of kit for detecting premature rupture of membrane as claimed in claim 7, the concentration that it is characterized in that described sheep anti mouse Anti-TNF-α liquid solution is 0.5mg/ml.
9. the preparation method of kit for detecting premature rupture of membrane as claimed in claim 7, the concentration that it is characterized in that described IGFBP-1 monoclonal antibody 1 solution is 0.5mg/ml.
10. the preparation method of kit for detecting premature rupture of membrane as claimed in claim 7, is characterized in that in described colloidal gold composite, every 1ml collaurum adds 5～20 μ lK
2CO
3
11. the preparation method of kit for detecting premature rupture of membrane as claimed in claim 7 is characterized in that in described colloidal gold composite, every 1ml collaurum is in conjunction with the anti-human IGFBP-1 monoclonal antibody 2 of 5～15 μ g.
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| CN2011104306192A CN103175968A (en) | 2011-12-20 | 2011-12-20 | Detection kit for premature rupture of fetal membranes and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011104306192A CN103175968A (en) | 2011-12-20 | 2011-12-20 | Detection kit for premature rupture of fetal membranes and preparation method thereof |
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| CN2011104306192A Pending CN103175968A (en) | 2011-12-20 | 2011-12-20 | Detection kit for premature rupture of fetal membranes and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103645321A (en) * | 2013-12-03 | 2014-03-19 | 张超 | Test paper for screening procalcitonin and preparation method of test paper |
| CN104198731A (en) * | 2014-08-28 | 2014-12-10 | 宁波瑞源生物科技有限公司 | C-reactive protein (CRP) semi-quantitative detection reagent and test paper using reagent |
| CN106932573A (en) * | 2017-04-28 | 2017-07-07 | 天津医科大学总医院 | Detect immunity colloidal gold test paper strip of thyroglobulin and preparation method thereof |
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| US5554504A (en) * | 1990-12-31 | 1996-09-10 | Oy Medix Biochemica Ab | Diagnostic method for detecting the rupture of fetal membranes |
| CA2533915A1 (en) * | 2002-08-13 | 2004-02-19 | N-Dia, Inc. | Devices and methods for detecting amniotic fluid in vaginal secretions |
| CN201697917U (en) * | 2010-08-16 | 2011-01-05 | 李民友 | Detection test paper for insulin-like growth factor binding protein-1 |
| CN201876456U (en) * | 2010-10-15 | 2011-06-22 | 无锡市凯奥善生物医药科技有限公司 | Joint inspection kit for premature birth of pregnant woman and premature rupture of membrane |
| CA2798144A1 (en) * | 2010-06-03 | 2011-12-08 | Biosynex | Method of detecting rupture of membranes |
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2011
- 2011-12-20 CN CN2011104306192A patent/CN103175968A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5554504A (en) * | 1990-12-31 | 1996-09-10 | Oy Medix Biochemica Ab | Diagnostic method for detecting the rupture of fetal membranes |
| CA2533915A1 (en) * | 2002-08-13 | 2004-02-19 | N-Dia, Inc. | Devices and methods for detecting amniotic fluid in vaginal secretions |
| CA2798144A1 (en) * | 2010-06-03 | 2011-12-08 | Biosynex | Method of detecting rupture of membranes |
| CN201697917U (en) * | 2010-08-16 | 2011-01-05 | 李民友 | Detection test paper for insulin-like growth factor binding protein-1 |
| CN201876456U (en) * | 2010-10-15 | 2011-06-22 | 无锡市凯奥善生物医药科技有限公司 | Joint inspection kit for premature birth of pregnant woman and premature rupture of membrane |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103645321A (en) * | 2013-12-03 | 2014-03-19 | 张超 | Test paper for screening procalcitonin and preparation method of test paper |
| CN104198731A (en) * | 2014-08-28 | 2014-12-10 | 宁波瑞源生物科技有限公司 | C-reactive protein (CRP) semi-quantitative detection reagent and test paper using reagent |
| CN106932573A (en) * | 2017-04-28 | 2017-07-07 | 天津医科大学总医院 | Detect immunity colloidal gold test paper strip of thyroglobulin and preparation method thereof |
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Application publication date: 20130626 |