CN101750500A - Colloidal gold kit for judging premature delivery risk of pregnant women and preparing method thereof - Google Patents
Colloidal gold kit for judging premature delivery risk of pregnant women and preparing method thereof Download PDFInfo
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- CN101750500A CN101750500A CN200810186280A CN200810186280A CN101750500A CN 101750500 A CN101750500 A CN 101750500A CN 200810186280 A CN200810186280 A CN 200810186280A CN 200810186280 A CN200810186280 A CN 200810186280A CN 101750500 A CN101750500 A CN 101750500A
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Abstract
The present invention discloses a colloidal gold kit for judging the premature delivery risk of pregnant women and a preparing method thereof. The kit comprises a detecting card, a dropper, a sample feeding cup and a disposable syringe. The detecting card is formed by orderly connecting a sample pad, a combined pad marked with colloidal gold, a nitrocellulose membrane and a water absorbing pad. In the present invention, monoclonal antibodies resisting fetal fibronectin and monoclonal antibodies resisting interleukin-6 are respectively fixed in different positions of the nitrocellulose membrane or a nylon membrane; the two kinds of monoclonal antibodies are labeled with gold particles, and the gold combined pad is prepared by mixing the antibodies labeled with gold; the sample pad and the water absorbing pad are added, and the detecting card is combined. Clinic research shows that the kit can accurately judge the premature delivery risk of pregnant women. The detectable rate of premature delivery is obviously improved. The kit has high sensitivity and high specificity. The whole detecting process only takes 3 minutes. The results can be directly read by eyes without special apparatus.
Description
Technical field
The present invention relates to a kind of kit, relate in particular to a kind of colloidal gold kit with fetal fibronectin (fFN) and interleukin 6 (IL-6) associating judging premature delivery risk of pregnant women, the invention still further relates to the preparation method of this kit, belong to the reagent field of judging premature delivery risk of pregnant women.
Background technology
Premature labor is a kind of serious problem that poses a health risk, and the premature suffers from the dangerous higher of neonate's health complications (depauperation).As: feeblemindedness, brain paralysis, therefore lung and alimentary canal problem to such an extent as to blind and sense of hearing death, predict that premature labor is significant for reducing premature labor harm.
Since 1981, women's productive rate morning has increased more than 30%.In in the past 20 years, although science and technology and medical science are constantly progressive, early productive rate approximately increased by 10% in still per 10 years.At present, the baby of the U.S. about 12.5% is the premature, and childbirth ahead of time is U.S. baby morbidity and the first deadly reason.In China, the incidence of preterm birth that counts on is 8-10%.
The baby that the gestational period is less than the birth of 36 weeks is called as the premature, and wherein the gestational period accounts for 70% in 34-36 week, and the gestational period accounts for 13% in 32-33 week, and the gestational period accounts for 10% in 28-31 week, and other had 6% 28 weeks of the less than gestational period.Premature labor is about the total 8-15% of childbirth in China, premature's developmental immaturity, body weight light (generally being lower than 2500 grams).The premature death rate of China has lifelong sequelae up to 15%, 10% premature.Premature labor is a kind of serious problem that poses a health risk, and family, society, the hospital of delivering a child, doctor and premature are had bigger mental burden and material burden.
Current prediction premature labor mainly contain physical index and two kinds of methods of biochemical indicator:
1, physical index and related detecting method:
Uterine neck is estimated
Uterine neck index (funnel length+1/ cervical canal length)
Vagina refers to inspection: subjectivity, and traumatic, easy infection
The uterine neck transvaginal sonography is checked: poor specificity, influence factor is more.
2, biochemical indicator and related detecting method
The content of various hormones in blood and/or body fluid
The adrenotrophic hormone liberin (corticotrophin-releasing hormone, CRH), the measurable premature labor of CRH level in the blood plasma, especially irritability premature labor;
Estriol (estriol, E3): the measurable premature labor of the variation of E3 content in the saliva (be subjected to environmental interference such as diet bigger, poor specificity, error is big);
The inflammatory factor interleukin 6,8 (IL-6,8): the content prediction premature labor of interleukin 6 in body fluid, especially infectious premature labor.
Though more than the method for prediction premature labor respectively has characteristics, but still lack a kind of reliably, simple and convenient, economical quick, sensitivity and specificity all good clinical detection method assist the ob-gyn to predict premature labor.
Fetus fiber adhesion albumen (fFN) is a kind of connection foetal sac and endometrial xanthan gum.Between 22 to 35 weeks, because the fusion of chorion and decidua, the tire glue protein content in its body fluid is extremely low at normal pregnancy for the pregnant woman.During childbirth, the tire glue protein can separate with fetus with parent and is dissolved in amniotic fluid and dissolved by uterine neck and vaginal fluid.The existence of fetal fibronectin is the final sensitive mark of fetus childbirth in pregnant woman's uterine neck vaginal fluid.Matthewa G et al etc. find under study for action, IL-6 in the uterine neck vaginal fluid predicts that high-risk women's premature labor is independently, and be to be equal to cervical dilatation and the status of FFN in the prediction premature labor at least, and the susceptibility 77% of prediction premature labor during report IL-6＞35pg/ml, specificity 60%, positive predictive value 12%, negative predictive value 97%.
A large amount of professional achievement in research and experimental results show that tire glue protein content is with the closely related property of premature labor in pregnant woman's body fluid.During pregnant 22-35 week, the existence of tire glue protein shows that " colloid " breaks away from advance than expection in pregnant woman's uterine neck vaginal fluid, it is the high-risk signal of premature labor, research is at present thought: early the generation mechanism that the fFN level increases in the antenatal cervical secretions mainly contains two kinds: a kind of is early antenatal protein breakdown and mechanical destruction fine hair-demoulding extracellular matrix surface of contact, and complete release of fFN or degraded are entered in the uterine neck vaginal fluid; Another kind is with the closely-related inflammatory process of premature labor proteolytic enzyme to be discharged, and destroys the villus cell epimatrix, and fFN is discharged to be increased.Therefore, the appearance of fFN is the comparatively ideal index of prediction premature labor in the uterine neck vaginal fluid.
FFN in the cervical secretions and IL-6 level and premature labor are closely related, and FFN and IL-6 have correlativity.FFN and IL-6 all are comparatively responsive indexs of prediction premature labor and childbirth in the week, and two indexs of joint-detection will improve the predictive ability of premature labor and childbirth in the week, can be used as a kind of new prediction premature labor and the method for childbirth in the week.FFN and IL-6 and be sampled to sky, delivery interval number average and be negative correlation, increase along with their levels, obviously shorten the delivery interval, the possibility that premature labor takes place is bigger, and meaning and the value of the badness come-off fFN of FFN and the IL-6 positive and gestation in the premature labor prediction receives publicity day by day, its predictive value in premature labor, especially negative predictive value is comparatively sure.Owing to premature labor is not that list-reason causes, therefore, fFN detects with the IL-6 joint-detection significant for improving the premature labor predicted value.
Immunochromatographic method (Immunochromatography) is a kind of quick diagnosis technology based on immune colloidal gold technique that the nineties is risen in the U.S., the product of Ying Yonging mainly adopts the design of double-antibody sandwich clinically, the outstanding advantage that this technology is compared with any one detection technique in the past, (all testing process only needs 3-20 minute) fast, accurately, easy, cheap, the manual operation error is little, outside the characteristics such as good stability, because this chromatography is for to carry out cross flow by nitrocellulose filter, allow a plurality of reagent strip in parallel to carry out the many index joint-detection, thereby reach the purpose of joint inspection.
Summary of the invention
The object of the invention is to use immunochromatographic method and develops a kind of reliable clinical detection reagent, by detecting fFN and two kinds of premature labor marks of IL-6, thereby improve pregnant woman's premature labor predicted value greatly, be fit to pregnant women oneself detection and clinical hospital and detect the risk whether premature labor is arranged.
Concrete, the present invention seeks to be achieved through the following technical solutions:
A kind of collaurum check reagent box of judging premature delivery risk of pregnant women comprises test card, inhales dropper, sample adding cup and disposable syringe; Be fixed in after described test card is linked together by the edge separately of pad, nitrocellulose filter and the adsorptive pads of sample pad, colloid gold label from bottom to top successively on the backing and form; Wherein, contain on the described nitrocellulose filter by anti-fetus fiber adhesion protein monoclonal antibody and anti-interleukin-6 (IL-6) monoclonal antibody and wrap 2 detection lines and 1 control line that is formed by the sheep anti-mouse igg bag that is formed respectively; Wherein, the sheep anti-mouse igg control line is near thieving paper one end, and 2 detection lines are near collaurum pad one end.
The pad of described colloid gold label prepares in accordance with the following methods: after will resisting fetus fiber adhesion protein monoclonal antibody and anti-interleukin-6 monoclonal antibody to use aurosol grain mark respectively, with its mixing, bag is by on glass fibre, promptly;
Wherein, described nitrocellulose filter can be by replacements such as nylon membranes.
Described backing can be the holder of various hard, all can be used for the present invention as long as have the function that certain rigidity has appendix or support, for example can be plastic plate (PVC), cardboard etc., is preferably PVC.
The using method of kit of the present invention and judgment criteria:
(1) detected object: be used for detecting vagina secretion fFN and IL-6;
(2) collection of specimens: the routine disinfection vulva, the specula vaginal dilation exposes uterine neck, and aseptic cotton carrier is placed 10s in the cervical canal, takes out to be placed among the PBS liquid 1mL, is placed on one 70 ℃ of refrigerators and preserves stand-by;
(3) detect
With inhaling dropper sample (vaginal fluid) is collected in the sample adding cup, in the following immersion of test paper scale mark (mark) sample.Waited about 1 minute, and after sample is absorbed fully, took out the evaluation sheet and identify;
(4) measuring principle
Detection kit of the present invention adopts fFN and the IL-6 in 2 sandwich solid-phase immunity chromatography qualitative detection vaginal fluids.Detection zone at kit is coated with anti-fFN and IL-6 monoclonal antibody respectively, and the Quality Control district is coated with sheep anti-mouse igg antibody.The anti-fFN of colloid gold label or the IL-6 monoclonal antibody of bag quilt are combined into bond in advance on fFN during detection in the vaginal fluid or IL-6 and the kit, this bond is along with the capillarity of film is upwards moved the arrival detection zone, react with anti-fFN or IL-6 antibody on the film, one or two red stripes occur.No matter whether have fFN or IL-6 in the sample, when liquid level continued to migrate to fixedly sheep anti-mouse igg district band, a red stripes should be able to appear in the Quality Control district.If if two red stripes appear in detection zone (T) simultaneously, a red stripes appears in the Quality Control district, and it is very high to show that then the risk of premature labor appears in the measured; If a red stripes only appears in detection zone (T), a red stripes appears in the Quality Control district, shows that then the measured has certain premature delivery risk; If have only Quality Control district (C) red stripes to occur, and red stripes does not appear in detection zone (T), it is extremely low to show that then the possibility of premature labor appears in the measured; Whether if red stripes does not appear in Quality Control district (C), it is normal etc. to need then to judge again whether sample size enough maybe needs to analyze the chromatography process
The present invention is an immunochromatography collaurum Fast Detection Technique, fetal fibronectin and IL-6 in the qualitative or half-quantitative detection vaginal fluid, with the anti-fFN of purifying and the automatic micro-specking equipment of monoclonal antibody utilization of IL-6, be separately fixed at and pass through on the pre-service nitrocellulose filter, again will be separately fixed on the glass fibre membrane with the anti-fFN and the IL-6 monoclonal gold labeling antibody of colloid gold label, be prepared into golden bond pad, be aided with appropriate sample preparation pad (polyester film) again, be combined into test card.Adopt the double antibody sandwich method principle, but fFN and IL-6 in the pregnant early stage woman vagina secretion of fast detecting.
The present invention has adopted two kinds of markers in detecting premature labors, has overcome the shortcoming of the susceptibility difference of former separate detection method, and the present invention can judged result after 3 minutes, does not need specific apparatus, the direct sentence read result of naked eyes.Accurately, quick, simple to operate, can be single part use, be applicable to that the self-service detection of family etc. needs the purposes of fast detecting.
Description of drawings
Test card synoptic diagram in Fig. 1 kit of the present invention; Be fixed on the backing after linking together by pad, nitrocellulose filter and the adsorptive pads of sample pad, colloid gold label successively from left to right.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
The preparation of embodiment 1 colloidal gold diagnosis kit of the present invention
1, the monoclonal antibody of anti-fFN of preparation and IL-6
Use the fFN (please provide) and the IL-6 memebrane protein immunity mouse inbred lines of purifying respectively, when mice serum produces corresponding antibody, can be with mouse boosting cell and SP2/0 Fusion of Cells.Utilize HAT to select hybridoma, and McAb is detected with enzyme linked immunosorbent assay (ELISA).Using limiting dilution assay for the hybridoma that detects antibody positive clones, preserve the positive cell strain simultaneously, utilize the BALB/c mouse inbred lines to prepare a large amount of monoclonal antibodies, the evaluation of the specific evaluation of antagonist simultaneously, identification epitope, the evaluation and the paired experiment of affinity filter out the best a pair of cell line of effect.The cell line that screens is implanted in the mouse peritoneal again, spent 5-6 days and take out ascites, utilize ammonium sulfate precipitation and affinitive layer purification to obtain monoclonal antibody.
2, the preparation of colloidal gold strip
2.1 the preparation of collaurum:
(1) tetra chlorauric acid of getting 3ml 1% with 5 milliliters of micropipette rifles is in the round-bottomed flask of 500ml, and the ultrapure water of measuring 297ml also adds in the flask, is mixed with 0.01% tetra chlorauric acid reactant liquor, fully stirring and evenly mixing;
(2) serpentine condenser in the connection is opened condensate water, and is placed on the magnetic force heating stirrer heated and boiled;
(3) put into stirrer, vigorous stirring, disposable then, add the 3ml sodium citrate solution fast and accurately;
(4) flavous aqueous solution of chloraurate grizzle at first becomes aubergine after about 2 minutes, continues to boil 5 minutes;
(5) close the magnetic force heating stirrer, treat collaurum cooling after, be sub-packed in the glass reagent bottle of 500ml, the outside is covered with aluminium foil, and is labelled in accordance with regulations;
The collaurum of preparation should present bright aubergine, does not have polymkeric substance and macroscopic precipitation; Get in right amount and measure in 530nm wavelength place, ultraviolet absorption value is between 1.1-1.3.
2.2 preparation immunity gold
(1) go out the total amount of needed protein to be marked according to calculation of total with the collaurum of intending mark, the every ml colloid gold label of this technology 12ug/ antibody, the antibody amount of mark is 3.6mg;
(2) pH value of regulating collaurum with sal tartari or the 0.1M hydrochloric acid of 0.1M is 7.8;
(3) under electromagnetic agitation, antibody protein solution is added in the colloidal gold solution, should dropwise add when adding protein, the about 5min of the protein of 1mg adds;
(4) antibody and colloidal gold reaction be after 5 minutes, and adding 5% bovine serum albumin(BSA) (BSA) under magnetic agitation, to make its final concentration be 1%;
After (5) 10 minutes, add 3% PEG20000 to final concentration be 0.3%;
(6) continue to react 1 hour or spent the night;
(7) mark is good colloidal gold solution is in 2000r/min, 4 ℃ of centrifugal 10min, and the sucking-off supernatant discards precipitation, to remove big polymkeric substance;
(8) rotating speed of adjusting hydro-extractor is 10000g/min, 4 ℃ centrifugal 30 minutes, abandon supernatant, with 0.01mol./1 PBS pH8.2 (the include 1%BSA) dissolving of precipitation with original volume, repeated centrifugation 3 times;
(9) last careful supernatant discarded, precipitation is dissolved among the 1/50PBS (including 1%BSA) of original volume, promptly gets immune gold;
2.3 detection line and control line bag are by (will detect antibody is fixed on the nitrocellulose membrane)
Detection line line: will resist fFN and IL-6 memebrane protein monoclonal antibody to pack into (albumen trace spray film system) in the BioDot Membrane jetter respectively, (the film specification is 25mm * 310mm) drawing by the amount of 1 μ l/cm on the nitrocellulose filter;
Control line line: the sheep anti-mouse antibody coating buffer is packed into (albumen trace spray film system) in the BioDot Membrane jetter, and (the film specification is 25mm * 310mm) drawing by the amount of 1 μ l/cm on the nitrocellulose filter;
The bag quilt: 37 ℃ of bags were by 2 hours;
Sealing: 37 ℃ were sealed 30 minutes;
Dry: the nitrocellulose filter that will wrap behind the quilt was put into the vacuum dryer inner drying 20 hours, and airtight preservation is stand-by.
2.4 pad preparation
(1) opening point film instrument, preheating 30 minutes is done 10 circulations with distilled water;
(2) adjusting the golden OD value of immunity is 30, the immunity gold is added in the plastic containers on the machine left side, and setting program, adjusting 2# shower nozzle discharge rate is 2.5UL/CM, and immune gold evenly is sprayed on the glass fiber conjugate pad, sprays the central authorities of the position of line at pad;
(3) sprayed pad after, placed 37 ℃ of baking ovens dry 30 minutes; Dry: glass fibre behind the mark was put into the vacuum dryer inner drying 20 hours, and it is stand-by to take out airtight preservation.
2.5 the preparation of test card
(1) tears adhesive tape above being overlying on off in the central authorities of plastic bottom board, stick bag by the nitrocellulose membrane of good antibody;
(2) tearing wide below the plastic plate central authorities off is to stick the pad of spraying collaurum, the overlapping 2mm of pad front end and nitrocellulose membrane by the adhesive tape of 5mm;
(3) tear the bottom wide adhesive tape of 20mm that be of plastic plate off, stick and pass through pretreated sample pad, the overlapping about 5mm of the front end of sample pad and pad;
(4) the top that tears plastic bottom board off is about the adhesive tape of 25mm, sticks thieving paper, the overlapping about 2mm of thieving paper and nitrocellulose membrane;
(5) assembled after, with all compactings of each accessory, check and pasting effect the kilocalorie that assembles to be cut into the wide test strips of 3mm with cutting cutter;
(6) test strips that cuts is contained in the test card, promptly.
2.6 the assembling of kit
Be assembled in the box inhaling dropper, sample pipetting volume cup, disposable syringe, the test card for preparing and operation instructions, promptly get kit of the present invention;
The test of the clinical judging premature delivery risk of pregnant women probability of test example 1 kit of the present invention
Select September 1 year March in 2007 in the People's Hospital, Heilongjiang Province antenatal clinic and hospitalization pregnant 28～34 all primipara's 141 examples, be divided into 3 groups.I organizes 41 examples, inclusion criteria: 1. irregular uterine contraction 〉=4 time/h is arranged; 2. dilatation of cervix＜2cm; 3. intact fetal membranes.II organizes 40 examples, inclusion criteria: 1. regular uterine contractile; 2. cervical dilatation 〉=2cm or have the proliferative cervical of carrying out to hold and cervical dilatation; 3. courageous and upright secretion of vagina or ruptured fetal membranes are arranged.2 groups are single pregnancy.III organizes (control group) 60 examples, is single tire normal pregnancies in identical pregnant week.All pregnant woman do not have obstetrics and inside and outside section complication.3 groups age, pregnant time and the pregnant all there was no significant differences of taking a sample.
The kit of using the present invention's preparation detects, and statistics sees Table 1:
The test findings (%) of table 1 kit clinical detection of the present invention premature labor in pregnant women
Clinical research shows, joint-detection fFN of the present invention and IL-6 collaurum joint inspection diagnostic kit can be passed judgment on out the existing premature delivery risk of pregnant woman accurately, can significantly improve the recall rate of premature labor, highly sensitive, high specificity, and testing process very quick (all testing processes only need 3 minutes).
Claims (6)
1. the collaurum check reagent box of a judging premature delivery risk of pregnant women comprises test card, inhales dropper, sample adding cup and disposable syringe; Wherein, be fixed in after described test card is linked together by the edge separately of pad, nitrocellulose filter and the adsorptive pads of sample pad, colloid gold label from bottom to top successively on the backing and form; Wherein, contain on the described nitrocellulose filter by anti-monoclonal antibody and anti-interleukin-6 monoclonal antibody and wrap 2 detection lines and 1 control line that is formed by the sheep anti-mouse igg bag that is formed respectively.
2. according to the described collaurum check reagent of claim 1 box, it is characterized in that: the pad of described colloid gold label prepares in accordance with the following methods: after will resisting fetus fiber adhesion protein monoclonal antibody and anti-interleukin-6 monoclonal antibody to use aurosol grain mark respectively, with its mixing, bag is by on glass fibre, promptly.
3. according to claim 1 or 2 described collaurum check reagent boxes, it is characterized in that: the sheep anti-mouse igg control line is near thieving paper one end on the described nitrocellulose filter, and 2 detection lines are respectively near collaurum pad one end.
4. according to claim 1 or 2 described collaurum check reagent boxes, it is characterized in that: described nitrocellulose filter is replaced by nylon membrane.
5. according to claim 1 or 2 described collaurum check reagent boxes, it is characterized in that: described backing is the holder of various hard.
6. according to the described collaurum check reagent of claim 5 box, it is characterized in that: described backing is a plastic plate.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810186280A CN101750500A (en) | 2008-12-22 | 2008-12-22 | Colloidal gold kit for judging premature delivery risk of pregnant women and preparing method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810186280A CN101750500A (en) | 2008-12-22 | 2008-12-22 | Colloidal gold kit for judging premature delivery risk of pregnant women and preparing method thereof |
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| CN101750500A true CN101750500A (en) | 2010-06-23 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200810186280A Pending CN101750500A (en) | 2008-12-22 | 2008-12-22 | Colloidal gold kit for judging premature delivery risk of pregnant women and preparing method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445534A (en) * | 2010-10-15 | 2012-05-09 | 无锡市凯奥善生物医药科技有限公司 | Combined assay kit for premature birth of pregnant woman and fetal membrane premature rupture |
| CN103185799A (en) * | 2011-12-27 | 2013-07-03 | 深圳市爱速尔生物技术有限公司 | Uterine neck-vagina rapid detector for early warning of premature delivery |
| CN105445466A (en) * | 2016-01-19 | 2016-03-30 | 苏州市博纳泰科生物技术有限公司 | Detection method for interleukin 6 and reagent kit of detection method |
| CN108780095A (en) * | 2016-03-31 | 2018-11-09 | 积水医疗株式会社 | Utilize the cancer embryo fibronectin detection method of simple immunoassays |
-
2008
- 2008-12-22 CN CN200810186280A patent/CN101750500A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102445534A (en) * | 2010-10-15 | 2012-05-09 | 无锡市凯奥善生物医药科技有限公司 | Combined assay kit for premature birth of pregnant woman and fetal membrane premature rupture |
| CN103185799A (en) * | 2011-12-27 | 2013-07-03 | 深圳市爱速尔生物技术有限公司 | Uterine neck-vagina rapid detector for early warning of premature delivery |
| CN105445466A (en) * | 2016-01-19 | 2016-03-30 | 苏州市博纳泰科生物技术有限公司 | Detection method for interleukin 6 and reagent kit of detection method |
| CN108780095A (en) * | 2016-03-31 | 2018-11-09 | 积水医疗株式会社 | Utilize the cancer embryo fibronectin detection method of simple immunoassays |
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Open date: 20100623 |