CN103235134A - Immunochromatography test paper for detecting premature rupture of fetal membranes - Google Patents
Immunochromatography test paper for detecting premature rupture of fetal membranes Download PDFInfo
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- CN103235134A CN103235134A CN201310166955XA CN201310166955A CN103235134A CN 103235134 A CN103235134 A CN 103235134A CN 201310166955X A CN201310166955X A CN 201310166955XA CN 201310166955 A CN201310166955 A CN 201310166955A CN 103235134 A CN103235134 A CN 103235134A
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Abstract
The invention discloses immunochromatography test paper, and in particular relates to immunochromatography test paper for detecting premature rupture of fetal membranes. The immunochromatography test paper is provided with a plurality of detection lines, each detection line is formed by coating different antibodies, and the antibodies are selected from a soluble goat anti-human intercellular adhesion molecule-1 polyclonal antibody, a rabbit anti-human insulin-like growth factor binding protein-1 antibody, a rabbit anti-human placenta alpha-microglobulin-1 antibody, a rabbit anti-human alpha fetoprotein polyclonal antibody and a rabbit anti-human Axl polyclonal antibody; a quality control line is formed by coating a goat anti-mouse IgG polyclonal antibody. When the immunochromatography test paper is used for detecting the premature rupture of fetal membranes, the sensitivity and specificity of the detection result are obviously improved respectively to more than 99% and 96%, so that the misdiagnosis occurrence rate can be reduced.
Description
Technical field
The present invention relates to a kind of immune chromatography test paper, be specifically related to a kind of immune chromatography test paper that detects premature rupture of fetal membranes.
Background technology
Immune chromatography test paper is a kind ofly to utilize chromatographic theory that the target detection molecule in the liquid to be detected or one-tenth are graded to flow to detection line (T line) and nature controlling line (C line) in test strips, when detection line generation change color illustrates that then testing result is positive, otherwise it is negative, when nature controlling line generation change color, illustrate that then testing result is effective, otherwise invalid.
Immune chromatography test paper has advantage very easily when detecting or diagnosing some disease, so it has fully been applied in the detection or diagnostic procedure of multiple disease or symptom, for example the early pregnancy test paper.Generally speaking, immune chromatography test paper includes following several structure: sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate, and described sample pad, collaurum pad, chromatographic film and adsorptive pads stick on the offset plate successively; Be disposed with detection line and nature controlling line on the described chromatographic film; Its principle of work is: drop to be measured is added on the sample pad, and liquid to be measured utilizes chromatographic theory to flow to the collaurum pad, the collaurum bond in the liquid to be measured dissolving collaurum pad, and flow to detection line and nature controlling line on the chromatographic film jointly successively with it; Contain the target detection molecule in liquid to be measured, thereby then target detection molecule, the collaurum bond antibody component that can comprise in detection line is combined and is formed immune complex, the detection line color changes simultaneously; When liquid to be measured flow to nature controlling line, the antibody component that comprises in the nature controlling line can be combined with the collaurum bond, thereby the nature controlling line color changes.
Rupture of membranes (Rupture of Fetal Membrane is called for short ROM) may take place at any time in the pregnancy period, and the rupture of membranes that takes place before just before giving birth is called premature rupture of fetal membranes (PROM).The incidence of mature back (after 37 pregnant weeks) PROM is about 10%, and the incidence of mature preceding (before 37 pregnant weeks) PROM is 2-3.5%.Premature rupture of fetal membranes is to cause that the obstetrical patient is antenatal, the principal element of postpartum complication, also is to cause the fetus premature labor and main cause that the neonate must move in the intensive care unit.Because the medical worker has to ask balance prolonging between pregnant week and the risk that in utero reaches pregnant woman's infection and the fetus lung development problem, thereby big to premature rupture of fetal membranes patient's handling cost height, difficulty, it is most important accurately and timely to diagnose the pregnant woman whether premature rupture of fetal membranes to take place.
Except methods such as bed-side query medical history in the past, mainly be called for short sICAM-1 with the soluble intercellular adhesion molecule in gravid woman's vaginal fluid-1(in the existing method that detects premature rupture of fetal membranes) serve as that the testing tool of detection index is newer method.Wherein be whether the detection premature rupture of fetal membranes immune chromatography test paper that detects index for the timely diagnosis of obstetrical patient premature rupture of fetal membranes takes place and played effect preferably with sICAM-1; The detection line endoperidium of described immune chromatography test paper has goat-anti people sICAM-1 polyclonal antibody working fluid, and the nature controlling line endoperidium has sheep anti-mouse igg polyclonal antibody working fluid, contains mouse-anti people sICAM-1 monoclonal antibody in the collaurum bond.
In the existing clinical diagnostic procedure to premature rupture of fetal membranes, the obstetrical patient that part fetal membrane cut is big, vagina amniotic fluid flow is big can access diagnosis timely, and for the obstetrical patient little for part fetal membrane cut, that break location is high, vagina amniotic fluid flow is little, still there is certain difficulty in clinical diagnosis at present, thereby delays the time that the obstetrical patient in time goes to a doctor easily; And existing only be to detect for the obstetrical patient that immune chromatography test paper is little for aforesaid fetal membrane cut, break location is high, vagina amniotic fluid flow is little of detection premature rupture of fetal membranes of index with sICAM-1, the sensitivity of its testing result and specificity are often limited, not ideal enough, thereby cause mistaken diagnosis easily, concerning the obstetrical patient, can bring inconvenience equally.
Summary of the invention
In view of this, the invention provides a kind of immune chromatography test paper that detects premature rupture of fetal membranes, this immune chromatography test paper is when detecting premature rupture of fetal membranes, and the sensitivity of its testing result and specificity have more significantly raising, can reach respectively more than 99% and more than 96%, thereby reduce the incidence of mistaken diagnosis.
For solving above technical matters, technical scheme of the present invention is to adopt a kind of immune chromatography test paper that detects premature rupture of fetal membranes, and this immune chromatography test paper includes sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with detection zone and Quality Control district on the described chromatographic film, described detection zone is near collaurum pad one end, and described Quality Control district is near adsorptive pads one end; Be provided with detection line in the described detection zone, it is main detection line and secondary detection line that described detection line sets gradually, and described main detection line is for to be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line one or both different antibody of serving as reasons wrap respectively and are formed one or two secondary detection lines; The antibody of bag quilt is for being selected from white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody, the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody in the described secondary detection line; Described Quality Control is provided with nature controlling line in the district, and described nature controlling line is for to be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1-2.5mg/mL, white-1 antibody 0.2-2mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-2.5mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 0.5-2.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 0.5-2mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Preferably, the antibody of bag quilt is selected from white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody for one or both in the described secondary detection line.
Preferably, the antibody of bag quilt is respectively white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody in the described secondary detection line.
Preferably, the working fluid concentration of each antibody is white-1 antibody 1-2mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL in described detection line and the nature controlling line.
Preferably, the working fluid concentration of each antibody is white-1 antibody 1.5mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL in described detection line and the nature controlling line.
The present invention compared with prior art, it is described in detail as follows:
The technical solution used in the present invention is: detect the immune chromatography test paper of premature rupture of fetal membranes, this immune chromatography test paper includes sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with detection zone and Quality Control district on the described chromatographic film, described detection zone is near collaurum pad one end, and described Quality Control district is near adsorptive pads one end; Be provided with detection line in the described detection zone, it is main detection line and secondary detection line that described detection line sets gradually, and described main detection line is for to be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line one or both different antibody of serving as reasons wrap respectively and are formed one or two secondary detection lines; The antibody of bag quilt is for being selected from white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody, the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody in the described secondary detection line; Described Quality Control is provided with nature controlling line in the district, and described nature controlling line is for to be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1-2.5mg/mL, white-1 antibody 0.2-2mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-2.5mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 0.5-2.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 0.5-2mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
At first, ICAIU (intercellular adhesive molecule1, be called for short ICAM-1) be interactional molecule general designation between a kind of participation cell and the cell and between cell and the epimatrix, include the soluble intercellular adhesion molecule (soluble intercellular adhesive molecule1 is called for short sICAM-1) that is present in the body fluid and the membranous type ICAIU (being called for short membranous type ICAM-1) that is present in cell surface.Wherein, sICAM-1 has not only kept the biological characteristics of membranous type ICAM-1, and it still is the soluble form of membranous type ICAM-1 in body fluid.The inventor is through discovering fully, the gravid woman that premature rupture of fetal membranes takes place is subjected to the expression of sICAM-1 on the monocyte in the amniotic fluid and the influence of neutrophil leucocyte, sICAM-1 content in its vaginal fluid obviously raises, now existing is the testing tool that target molecule detects premature rupture of fetal membranes with sICAM-1, as immune chromatography test paper, but in actual application, it is single that it detects target molecule, when if premature rupture of fetal membranes takes place, it is little the fetal membrane cut to occur, the cut height, during the little situation of vagina amniotic fluid flow, then easily cause sensitivity and specificity all lower, be easy to generate mistaken diagnosis.
Sensitivity of the present invention is the number percent that the sample that draws positive detection among the ill crowd accounts for patient's sum, and specificity is to draw the number percent that the negative sample that detects accounts for healthy people's sum in the healthy population.It is more to influence immune chromatography test paper testing result sensitivity of the present invention and specific factor, include the difference of immune chromatography test paper itself, as the selection of detection line working fluid, the concentration of detection line working fluid and the concentration of nature controlling line working fluid etc., in addition, the manufacture craft of test paper itself can have certain influence to sensitivity and the specificity of testing result too.
The inventor is through discovering fully, and sICAM-1 appears at one of main protein in the amniotic fluid third trimester of pregnancy, and content is generally about 14ng/mL; And when premature rupture of fetal membranes took place, the more sICAM-1 of content then can appear in the vaginal fluid in the amniotic fluid.In addition, except above-mentioned sICAM-1 content in the woman vagina fluid samples that premature rupture of fetal membranes takes place was more, the antigen of other content showed increased also had insulin-like growth factor binding protein-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl.Therefore, the present invention mainly adopts with the multiple antigenic component in gravid woman's vaginal fluid as the common target molecule that detects.
The present invention finds through the protein chip shaker test, the premature rupture of fetal membranes puerpera who makes a definite diagnosis compares (two groups of puerperas' age and coupling in age in gestation week with the non-premature rupture of fetal membranes puerpera who makes a definite diagnosis, the foundation-free disease, no pregnancy complication), the concentration of sICAM-1, insulin-like growth factor binding protein-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl has notable difference in its vaginal fluid sample; Wherein, in the premature rupture of fetal membranes puerpera vaginal fluid sample of making a definite diagnosis the concentration of sICAM-1 more than 90% all more than 2ng/mL, and in the non-premature rupture of fetal membranes puerpera vaginal fluid sample of making a definite diagnosis the concentration of sICAM-1 more than 90% all below 1ng/mL; The mean concentration of the mean concentration of sICAM-1 in the premature rupture of fetal membranes puerpera who makes a definite diagnosis and the vaginal fluid sample of normal healthy controls group, the mean concentration of insulin-like growth factor binding protein-1 and Axl can see table (seeing embodiment 1 for details); But placenta α-microglobulin-1 is at data list of references: the Guo Guangling such as application that detect gravid woman's premature rupture of fetal membranes, Liu Yongzhen, Deng. detecting the value [J] of placenta α microglobulin-1 diagnosis premature rupture of fetal membranes in the vaginal secretion. Chinese doctor studies magazine, and 2010,33(15); The mean concentration of alpha-fetoprotein can be referring to document: Yi Yuanyuan, its wooden lattice, etc. detect the clinical value [J] of vaginal secretion HCG and AFP level diagnosis premature rupture of fetal membranes. Inner Mongol medical journal, 2008,40(7): repeat no more among 794-797. the present invention.
In view of above-mentioned experimental result, in order to improve immune chromatography test paper of the present invention to sensitivity and the specificity of the testing result of premature rupture of fetal membranes, the inventor is through research fully, employing becomes the mode of multiple detection line to detect premature rupture of fetal membranes jointly with the Antibody Preparation of multiple detection target molecule, thereby more whether the qualitative analysis gravid woman premature rupture of fetal membranes takes place; The present invention detects the immune chromatography test paper of premature rupture of fetal membranes can effectively avoid some disturbing factor, thereby the diagnosis of premature rupture of fetal membranes is had analysis more accurately, thereby improves sensitivity and the specificity of testing result.
The variable concentrations of antibody has different effects for the colour developing degree that detects the antigenic component in the sample in the detection line; Antibody concentration in detection line is low excessively, and then it is less with the amount that antigen plays association reaction, thereby it is not obvious to cause developing the color, and causes should detecting among the ill crowd negative findings that detects of positive findings, and sensitivity is on the low side; Antibody concentration in detection line is too high, and then it is more with the amount that antigen plays association reaction, thereby causes colour developing too obvious, causes should detecting in the healthy population positive findings that detects of negative findings, and specificity is on the low side.
The variable concentrations of antibody has different effects for the judgement of the testing result validity of test paper in the nature controlling line; When detecting liquid through detection line, antibody in antigen wherein and the collaurum pad (among the present invention for mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody) can the antibody in detection line be combined formation and be had the bond of certain color, part does not have the interior antibody of collaurum pad of combination then can continue to move to nature controlling line, thereby be combined into another kind of bond with the antibody component in the nature controlling line, the nature controlling line change color, thus the explanation testing result is effective.When the excessive concentration of detection line working fluid, the antibody content of collaurum pad that then can cause moving to nature controlling line is less, thereby causes the nature controlling line change color obvious inadequately, causes testing result to lose efficacy; On the low side when the concentration of detection line working fluid, the antibody content of collaurum pad that then can cause moving to nature controlling line is more, thereby causes the waste on the nature controlling line working fluid cost.
In view of the foregoing, iff independent adjustment detection line working fluid concentration and nature controlling line working fluid concentration, what then obtain is both optium concentrations when using separately, but because detection line and nature controlling line are for using simultaneously in the immune chromatography test paper, therefore the preferred concentration after the combination between the two need be passed through a large amount of experiments and screened and verify.Simultaneously, because the detection line of immune chromatography test paper of the present invention is multiple detection line, the antibody component difference of the quilt that wraps in the different detection lines, therefore, the working fluid concentration of each bar detection line is all influential to sensitivity and the specificity of testing result in the immune chromatography test paper of the present invention; In view of the foregoing, the present invention need study the preferable combined concentration of multiple detection line working fluid, and this research is comparatively loaded down with trivial details.
In addition, become detection line itself just to have multiple array mode various detection Antibody Preparation, the present invention screens on the target molecule of the multiple gravid woman's of detection premature rupture of fetal membranes, draw various antibody test composition of the present invention, include goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody, the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody; Simultaneously, the inventor studies through sufficient in the various combinations of above-mentioned various antibody test compositions, find that mode of the present invention is prepared into main detection line with goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, and the mode that all the other various antibody are prepared into one or two different secondary detection line is respectively applied to immune chromatography test paper can obtain better testing result, its sensitivity and specificity all are greatly improved, and can reach more than 99% and 96% respectively.
Immune chromatography test paper of the present invention is in the process of using, antigenic component in the sample to be checked at first with the collaurum pad in the collaurum bond of mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody carry out combination, the gained bond moves to main detection line and secondary detection line successively under the effect of chromatographic film then, if contain antigenic component in the sample to be checked or antigenic component content is enough, then each detection line all should be transformed into purplish red colo(u)r streak; But in the actual mechanical process, because the influence of some unkownable factors, thereby cause that some antigenic component contains quantity not sufficient in the sample to be checked; In view of the foregoing, the working fluid concentration of employed antibody is through a large amount of experimental studies in the immune chromatography test paper of the present invention, and the present invention adopts the working fluid concentration of each antibody to be set to goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1-2.5mg/mL, white-1 antibody 0.2-2mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-2.5mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 0.5-2.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 0.5-2mg/mL, during sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL.
Confirm that through experimental study gained immune chromatography test paper of the present invention testing result is in use judged as follows: when nature controlling line changes purplish red colo(u)r streak on the immune chromatography test paper of the present invention, illustrate that the result is effective, otherwise invalid; When only having a detection line to change purplish red colo(u)r streak into or not having detection line and change purplish red colo(u)r streak into, the result is judged to be feminine gender, and premature rupture of fetal membranes does not namely take place; When two above detection lines changed purplish red colo(u)r streak into, the result was judged to be the positive; Immune chromatography test paper of the present invention adopts the common mode that detects of multiple detection line can avoid the influence of some factor, thereby better judge whether premature rupture of fetal membranes takes place, and the sensitivity of its testing result and specificity can reach more than 99% and 98% respectively.
Further, immune chromatography test paper of the present invention also can adopt the mode that is more preferably, and namely the preferred antibody of bag quilt in the described secondary detection line that adopts is selected from white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody for one or both.
Further, immune chromatography test paper of the present invention also can adopt the mode that is more preferably, and namely the preferred antibody of bag quilt in the described secondary detection line that adopts is respectively white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody.
Further, immune chromatography test paper of the present invention also can adopt the mode that is more preferably, and namely the working fluid concentration of each antibody is white-1 antibody 1-2mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL in the preferred described detection line of employing and the nature controlling line.
Further, immune chromatography test paper of the present invention also can adopt the mode that is more preferably, and namely the working fluid concentration of each antibody is white-1 antibody 1.5mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL in the preferred described detection line of employing and the nature controlling line.
Description of drawings
Fig. 1 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of premature rupture of fetal membranes group vaginal fluid sample;
Fig. 2 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of normal healthy controls group vaginal fluid sample;
Fig. 3 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of healthy gravid woman's amniotic fluid sample;
Fig. 4 is the testing result figure of the enzyme linked immunosorbent assay of sICAM-1;
Fig. 5 is the testing result figure of the enzyme linked immunosorbent assay of insulin-like growth factor binding protein-1;
Fig. 6 is the testing result figure of the enzyme linked immunosorbent assay of Axl.
Embodiment
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is described in further detail below in conjunction with specific embodiment.
Reaching the puerpera who is in hospital with West China No.2 Hospital, Sichuan University's outpatient service is experimental subjects, according to existing detection method experimental subjects is divided into premature rupture of fetal membranes group, normal healthy controls group and amniotic fluid sample group.
Premature rupture of fetal membranes group: 110 routine premature rupture of fetal membranes gravid woman (the wherein mature preceding premature rupture of fetal membranes of 80 routine mature prematures rupture of fetal membranes and 30 examples);
Normal healthy controls group: 110 routine prepartal intact fetal membranes gravid woman;
Amniotic fluid sample group: 30 customary caesarean delivered intact fetal membranes gravid woman.
The clinical screening criteria of experimental subjects:
Premature rupture of fetal membranes group-(i) just before giving birth before observe amniotic fluid to spill;
The (ii) vaginal fluid sample pH detection paper positive;
(iii) the lobate crystallization of pteridophyte detects positive.
Normal healthy controls group-(i) is not observed amniotic fluid before just before giving birth and is spilt;
(ii) vaginal fluid sample pH detection paper feminine gender;
(iii) the lobate crystallization of pteridophyte detects negative.
Experimental subjects age distribution: 80 routine mature 28 years old premature rupture of fetal membranes mean age (the range of age 19-35 year), 31 years old premature rupture of fetal membranes mean age before 30 examples are mature (the range of age 19-45 year), 30 years old normal healthy controls group mean age (the range of age 18-43 year).
The sample collection method: the gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), peep out vagina after, stretch into the posterior fornix place with disposable sterilized cotton swab, rotation 5 circles are taken a sample.Cotton swab head is inserted in the 1mL sample dilution (phosphatebuffer buffer system), rotate 5 times after, be close to tube wall extruding rotation 2 times, obtain sample to be tested.
A, experimental technique: cell factor/chemotactic factor (CF) antibody chip qualitative detection
Experiment condition:
1, the film (RayBiotech, the U.S.) that will be marked with 174 cytokine antibodies is in advance put into the detection box, adds the 2ml confining liquid then, room temperature sealing 6 hours;
2, discard confining liquid, add 1 part in 1.2ml sample to be checked, 4 ℃ of night incubation;
3, discard sample, clean 5 times with the 2ml lavation buffer solution;
4, the detection antibody that adds the coupling of 1ml biotin, incubated at room 2 hours;
5, discard antibody, clean 5 times with the 2ml lavation buffer solution;
6, the streptomysin Avidin that adds 2ml horseradish peroxidase (HRP) coupling, incubated at room 2 hours;
7, discard liquid, clean 5 times with the 2ml lavation buffer solution;
8, add chemical illuminating reagent 500 μ l, lucifuge effect 2 minutes, observations at film.
Each sample to be tested of collecting all adopts above-mentioned experiment condition to detect from premature rupture of fetal membranes group, normal healthy controls group and amniotic fluid sample group, gained result such as Fig. 1, Fig. 2 and shown in Figure 3.
B, experimental technique: enzyme linked immunosorbent assay quantitatively detects
Experiment condition:
Experiment material: sICAM-1ELISA detection kit (R﹠amp; D company)
1. the preparation of reagent
Lavation buffer solution (Wash Buffer)
Substrate solution (Substrate Solution): before the use, the developer A in the kit and developer B equivalent, lucifuge were mixed 15 minutes, each hole needs the potpourri 200 μ l of two kinds of developers.
SICAM-1 standard items: the sICAM-1 standard items are diluted with the 1ml deionized water.Standard items original liquid concentration after the dilution is 250ng/ml.For guaranteeing the abundant mixing of standard items, before further dilution, standard items are put in rock mixing on the shaking table gently more than at least 15 minutes.
Standard items with 250ng/ml are configured to the sICAM-1 examination criteria product that concentration is 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, 1.56ng/ml respectively; RD5-7 calibration thinning agent is as blank (0ng/ml)
2. trace routine
In each micropore, add 100 μ l sICAM-1Conjugate; The addition of standard items, tester, sample is 100 μ l, and joins in the corresponding micropore.Micropore is built with the adhesive tape that kit provides.Place on the horizontal shaking table 500 ± 50 rev/mins to cultivate at ambient temperature 1.5 hours microwell plate.Wash plate; Every hole adds substrate solution (Substrate Solution) 200 μ l, under the room temperature condition, lucifuge, keeps flat and cultivates 30 minutes; Every hole adds 50 μ l stop buffers (Stop Solution).The color of solution will become yellow by blueness in the hole.If solution colour is that green or change color are inconsistent in the hole, pat flat board gently, to guarantee the abundant mixing of solution; Survey the 450nm light absorption value in 30 minutes.
3. the production standard curve calculates sample content.
4. will calculate gained each sample to be tested content statistics and be depicted as chart, the gained chart is seen accompanying drawing 4.
The quantitative detection of the enzyme linked immunosorbent assay of insulin-like growth factor binding protein-1 and Axl can quantitatively detect with reference to the enzyme linked immunosorbent assay of above-mentioned sICAM-1 to be carried out, and the detection kit that adopts is all available from same company, and the gained experimental result is as follows.
Experimental result: among Fig. 4-Fig. 6, AF of control is the amniotic fluid sample, and CVF of PROM is the vaginal fluid of premature rupture of fetal membranes group, and CVF of control is the vaginal fluid of normal healthy controls group.
As can be seen from Figure 4, the concentration of sICAM-1 in healthy gravid woman's amniotic fluid sample is the highest, on average can reach 80ng/mL; The concentration of sICAM-1 in premature rupture of fetal membranes group vaginal fluid sample can reach more than the 2ng/mL more than 90%; The concentration of sICAM-1 in normal healthy controls group vaginal fluid sample more than 90% below 1ng/mL.
As can be seen from Figure 5, the concentration of insulin-like growth factor binding protein-1 in healthy gravid woman's amniotic fluid sample is the highest, on average can reach 270ng/mL; The concentration of insulin-like growth factor binding protein-1 in premature rupture of fetal membranes group vaginal fluid sample can reach more than the 15ng/mL more than 90%; The concentration of insulin-like growth factor binding protein-1 in normal healthy controls group vaginal fluid sample more than 90% below 5ng/mL.
As can be seen from Figure 6, the concentration of Axl in healthy gravid woman's amniotic fluid sample is the highest, on average can reach 80ng/mL; The concentration of Axl in premature rupture of fetal membranes group vaginal fluid sample can reach more than the 1ng/mL more than 90%; The concentration of Axl in normal healthy controls group vaginal fluid sample more than 90% below 0.5ng/mL.
Experimental result from embodiment 1 and aforementioned documents data as can be known, the antigen that content takes place to take place in premature rupture of fetal membranes gravid woman's the vaginal fluid includes soluble intercellular adhesion molecule sICAM-1, insulin-like growth factor binding protein-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl; Described antigen mainly comes from the amniotic fluid of rupture of membranes.
Below be with aforementioned each antigen in gravid woman's vaginal fluid as the common target molecule that detects, and prepare the reference examples of the immune chromatography test paper of gained according to the existing conventional method for preparing immune chromatography test paper, each reference examples is as follows:
Reference examples 1
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with the detection line that is formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 2
Reference examples 2 immune chromatography test papers: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit and the anti-human a-fetoprotein polyclonal antibody of rabbit bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: insulin-like growth factor binding protein-1 antibody 3mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 3mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 3
Reference examples 3 immune chromatography test papers: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: white-1 antibody 1mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 3mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 4
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by the anti-people's placenta of rabbit α-microglobulin-1 antibody and the anti-people Axl of rabbit polyclonal antibody bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: the anti-people's placenta of rabbit α-microglobulin-1 antibody 2.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 5
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: the anti-human a-fetoprotein 2mg/mL of rabbit, the anti-people Axl of rabbit polyclonal antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 6
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and the anti-human a-fetoprotein polyclonal antibody of rabbit bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 3mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 0.5mg/mL, sheep anti-mouse igg polyclonal antibody 3mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 7
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1mg/mL, the anti-people's placenta of rabbit α-microglobulin-1 antibody 3mg/mL, sheep anti-mouse igg polyclonal antibody 1mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 8
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and the anti-human a-fetoprotein polyclonal antibody of rabbit bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 9
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that are formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and the anti-people Axl of rabbit polyclonal antibody bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 10
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively three detection lines that are formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody bag and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 2.5mg/mL, the anti-people's polyclonal antibody of rabbit Axl2mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 11
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and white-1 antibody sandwich of the anti-human insulin-like growth factor binding protein of rabbit and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit white-1 antibody 0.2mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 12
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-people's placenta of rabbit α-microglobulin-1 antibody 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 13
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5mg/mL, white-1 antibody 1mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 14
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, white-1 antibody 2mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 1.5mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Reference examples 15
Reference examples 1 immune chromatography test paper: include sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Be disposed with respectively two detection lines that formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit and the anti-people's placenta of rabbit α-microglobulin-1 antibody sandwich and the nature controlling line that is formed by sheep anti-mouse igg polyclonal antibody bag on the described chromatographic film;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, white-1 antibody 1.5mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
Material source among the above-mentioned reference examples 1-15 is as follows:
Goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody: available from R﹠amp; D company;
Sheep anti-mouse igg polyclonal antibody: available from the female bioengineering of last Shanghai's style company limited;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody: available from R﹠amp; D company;
White-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit: available from R﹠amp; D company;
The anti-people's placenta of rabbit α-microglobulin-1 antibody: available from R﹠amp; D company;
The anti-human a-fetoprotein polyclonal antibody of rabbit: available from Zhengzhou hundred basic biotech firms;
The anti-people Axl of rabbit polyclonal antibody: available from Beijing An Biqi biotech firm;
Sample pad (commercially available): available from Ahlstrom company;
Adsorptive pads (commercially available): available from the outstanding biological Science and Technology Ltd. in Shanghai;
Collaurum (commercially available): available from the prompt peaceful bio tech ltd in Shanghai;
Bovine serum albumin(BSA) (BSA): Roche company;
The reference examples 1-15 immune chromatography test paper that is prepared from is respectively applied to detect the clinical sample of same gravid woman's vaginal fluid, include premature rupture of fetal membranes group and normal healthy controls group, wherein the premature rupture of fetal membranes group is 500 examples, the normal healthy controls group is 500 examples, and subjects all adopts the method for embodiment 1 to carry out clinical diagnosis and screening; Wherein be the little or high less obstetrical patient of vagina amniotic fluid flow who causes of cut of fetal membrane cut in the premature rupture of fetal membranes group more than 70%.
Sample collection: the gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), peep out vagina after, stretch into the posterior fornix place with disposable sterilized cotton swab, rotation 5 circles are taken a sample.Cotton swab head is inserted in the 1mL sample dilution (phosphatebuffer buffer system), rotate 5 times after, be close to tube wall extruding rotation 2 times, obtain sample.
Sampling test object age distribution: 500 routine 27 years old premature rupture of fetal membranes mean age (the range of age 19-36 year), 500 routine 30 years old normal healthy controls group mean age (the range of age 18-44 year).
Utilize reference examples 1-15 respectively 1000 collected routine samples to be detected, the feminine gender of gained testing result and positive routine number statistics see Table one:
Table one
In the last table one, reference examples 1 is existing immune chromatography test paper, and reference examples 2-7 is the immune chromatography test paper of the common multiple antibody detection line of employing, and reference examples 8-15 is the immune chromatography test paper of embodiment of the present invention; Wherein, the difference of reference examples 8-15 and reference examples 2-7 is the selection of antibody test composition, the difference of sequential scheduling successively of each antibody in the working fluid concentration of antibody and the different detection line in the detection line; Reference examples 11-12 carries out preferred embodiment for going on foot at immune chromatography test paper of the present invention enterprising, and reference examples 13-15 is further more preferred implementation.Testing result from table one as can be seen, the immune chromatography test paper of embodiment of the present invention is for the immune chromatography test paper of existing immune chromatography test paper and the common multiple antibody detection line of employing, have better sensitivity and specificity, can reach respectively more than 99% and 96%; In addition, from the immune chromatography test paper of reference examples 11-15 embodiment as can be seen, it has effect more significantly sensitivity and specificity with respect to reference examples 8-10, and especially specific lifting is comparatively remarkable; More further, the immune chromatography test paper of reference examples 15 embodiments has better effect in specificity.
Only be preferred implementation of the present invention below, should be pointed out that above-mentioned preferred implementation should not be considered as limitation of the present invention, protection scope of the present invention should be as the criterion with claim institute restricted portion.For those skilled in the art, without departing from the spirit and scope of the present invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (6)
1. immune chromatography test paper that detects premature rupture of fetal membranes, this immune chromatography test paper includes sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, collaurum pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; It is characterized in that: be disposed with detection zone and Quality Control district on the described chromatographic film, described detection zone is near collaurum pad one end, and described Quality Control district is near adsorptive pads one end; Be provided with detection line in the described detection zone, it is main detection line and secondary detection line that described detection line sets gradually, and described main detection line is for to be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line one or both different antibody of serving as reasons wrap respectively and are formed one or two secondary detection lines; The antibody of bag quilt is for being selected from white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody, the anti-human a-fetoprotein polyclonal antibody of rabbit and the anti-people Axl of rabbit polyclonal antibody in the described secondary detection line; Described Quality Control is provided with nature controlling line in the district, and described nature controlling line is for to be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1-2.5mg/mL, white-1 antibody 0.2-2mg/mL of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-2.5mg/mL, the anti-human a-fetoprotein polyclonal antibody of rabbit 0.5-2.5mg/mL, the anti-people Axl of rabbit polyclonal antibody 0.5-2mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Be coated with mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the described collaurum pad.
2. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 1 is characterized in that: the antibody of bag quilt is selected from the anti-human insulin-like growth factor binding protein of rabbit-1 antibody, the anti-people's placenta of rabbit α-microglobulin-1 antibody in vain for one or both in the described secondary detection line.
3. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 1 is characterized in that: the antibody of bag quilt is respectively white-1 antibody of the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody in the described secondary detection line.
4. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 3 is characterized in that: the working fluid concentration of each antibody is white-1 antibody 1-2mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL in described detection line and the nature controlling line.
5. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 4 is characterized in that: the working fluid concentration of each antibody is white-1 antibody 1.5mg/mL of goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, the anti-human insulin-like growth factor binding protein of rabbit, the anti-people's placenta of rabbit α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL in described detection line and the nature controlling line.
6. preparation method who prepares the immune chromatography test paper of the described detection premature rupture of fetal membranes of claim 1 is characterized in that including following each step:
A, each antibody detection line working fluid and nature controlling line working fluid are wrapped quilt at nitrocellulose filter, the gained nitrocellulose filter 36-38 ℃ down dry 8-12 hour chromatographic film;
B, mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is become the collaurum pad with Preparation of Colloidal Gold;
C, the chromatographic film of A and B step gained and collaurum pad sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on the offset plate.
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