CN103235134B - A kind of immune chromatography test paper detecting premature rupture of fetal membranes and preparation method thereof - Google Patents
A kind of immune chromatography test paper detecting premature rupture of fetal membranes and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of immune chromatography test paper, is specifically related to a kind of immune chromatography test paper detecting premature rupture of fetal membranes.Described immune chromatography test paper is provided with many detection lines, every bar detection line is formed by different antibody bags, and described antibody is selected from goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody, rabbit anti-human alpha-fetoprotein polyclonal antibody and the anti-human Axl polyclonal antibody of rabbit; Nature controlling line is for be formed by sheep anti-mouse igg polyclonal antibody bag; Immune chromatography test paper of the present invention is when detecting premature rupture of fetal membranes, and the sensitivity of its testing result and specificity have obvious raising, can reach more than more than 99% and 96% respectively, thus reduces the incidence of mistaken diagnosis.
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 detecting premature rupture of fetal membranes.
Background technology
Immune chromatography test paper is that the target detection molecule in liquid to be detected or composition etc. are flowed to detection line (T line) and nature controlling line (C line) by a kind of chromatographic theory that utilizes in test strips, when the change of detection line generation color then illustrates that testing result is positive, otherwise be negative, when nature controlling line generation color changes, then illustrate that testing result is effective, otherwise invalid.
Immune chromatography test paper is in detection or diagnose during some disease and have conveniently advantage, and therefore it is by the detection that fully applies to various diseases or symptom or diagnostic procedure, such as early pregnancy test paper.Generally, immune chromatography test paper includes following several structure: sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate, and described sample pad, gold conjugation pad, chromatographic film and adsorptive pads are pasted onto on offset plate successively; Described chromatographic film is disposed with detection line and nature controlling line; Its principle of work is: add in sample pad by drop to be measured, and liquid to be measured utilizes chromatographic theory to flow to gold conjugation pad, and liquid to be measured dissolves the colloidal gold conjugate in gold conjugation pad, and jointly flows to detection line in chromatographic film and nature controlling line successively with it; When containing target detection molecule in liquid to be measured, then the antibody component that target detection molecule, colloidal gold conjugate can comprise in detection line is combined thus forms immune complex, and detection line color changes simultaneously; When liquid to be measured flow to nature controlling line, the antibody component comprised in nature controlling line can be combined with colloidal gold conjugate, thus nature controlling line color changes.
Rupture of membranes (Rupture of Fetal Membrane is called for short ROM) may occur at any time in the pregnancy period, and the rupture of membranes occurred before is just before giving birth called premature rupture of fetal membranes (PROM).After mature, the incidence of (after 37 pregnant weeks) PROM is about 10%, and before mature, the incidence of (before 37 pregnant weeks) PROM is 2-3.5%.Premature rupture of fetal membranes causes that obstetrical patient is antenatal, the principal element of postpartum complication, is also the main cause causing fetus premature labor and neonate must move in intensive care unit.Have in prolong pregnancy week due to medical worker and in utero and between the risk of infection of pregnant women and Fetal Lung growth problem ask balance, thus, difficulty high to the handling cost of Patients with Preterm Premature Rupture of Membranes is large, accurately and timely diagnoses pregnant woman whether premature rupture of fetal membranes to occur most important.
In the method for existing detection premature rupture of fetal membranes except the methods such as bed-side query medical history in the past, mainly with the Soluble ICAM-1 (be called for short sICAM-1) in gravid woman's vaginal fluid for the testing tool of Testing index is newer method.Be wherein that the detection premature rupture of fetal membranes immune chromatography test paper of Testing index is diagnosed in time for obstetrical patient and whether premature rupture of fetal membranes occurs served good effect with sICAM-1; The detection line endoperidium of described immune chromatography test paper has goat-anti people sICAM-1 polyclonal antibody working fluid, and nature controlling line endoperidium has sheep anti-mouse igg polyclonal antibody working fluid, containing mouse-anti people sICAM-1 monoclonal antibody in colloidal gold conjugate.
Existing clinical in the diagnostic procedure of premature rupture of fetal membranes, the obstetrical patient that part fetal membrane cut is large, vagina amniotic fluid flow is large can be diagnosed timely, and for the obstetrical patient little for part fetal membrane cut, break location is high, vagina amniotic fluid flow is little, still there is certain difficulty in current clinical diagnosis, thus easily delays the time that obstetrical patient goes to a doctor in time; And existing with sICAM-1 be only the detection premature rupture of fetal membranes of Testing index 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 for, the sensitivity of its testing result and specificity are often limited, not ideal enough, thus easily cause mistaken diagnosis, can bring inconvenience equally concerning obstetrical patient.
Summary of the invention
In view of this, the invention provides a kind of immune chromatography test paper detecting 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 obvious raising, more than more than 99% and 96% can be reached respectively, thus reduce the incidence of mistaken diagnosis.
For solving above technical matters, technical scheme of the present invention adopts a kind of immune chromatography test paper detecting premature rupture of fetal membranes, and this immune chromatography test paper includes sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with detection zone and quality control region, described detection zone is near gold conjugation pad one end, and described quality control region is near adsorptive pads one end; Be provided with detection line in 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 be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line is wrapped respectively by one or both different antibody to be formed one or two secondary detection lines; The antibody of quilt is wrapped for being selected from rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody, rabbit anti-human alpha-fetoprotein polyclonal antibody and the anti-human Axl polyclonal antibody of rabbit in described secondary detection line; Be provided with nature controlling line in described quality control region, described nature controlling line is for 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, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 0.2-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-2.5mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 0.5-2.5mg/mL, rabbit anti-human Axl polyclonal antibody 0.5-2mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Preferably, the antibody wrapping quilt in described secondary detection line is selected from rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody for one or both.
Preferably, the antibody wrapping quilt in described secondary detection line is respectively rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody.
Preferably, in described detection line and nature controlling line, the working fluid concentration of each antibody is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL.
Preferably, in described detection line and nature controlling line, the working fluid concentration of each antibody is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1.5mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL.
Compared with prior art, it is described in detail as follows in the present invention:
The technical solution used in the present invention is: the immune chromatography test paper detecting premature rupture of fetal membranes, and this immune chromatography test paper includes sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with detection zone and quality control region, described detection zone is near gold conjugation pad one end, and described quality control region is near adsorptive pads one end; Be provided with detection line in 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 be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line is wrapped respectively by one or both different antibody to be formed one or two secondary detection lines; The antibody of quilt is wrapped for being selected from rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody, rabbit anti-human alpha-fetoprotein polyclonal antibody and the anti-human Axl polyclonal antibody of rabbit in described secondary detection line; Be provided with nature controlling line in described quality control region, described nature controlling line is for 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, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 0.2-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-2.5mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 0.5-2.5mg/mL, rabbit anti-human Axl polyclonal antibody 0.5-2mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
First, ICAIU (intercellular adhesive molecule1, be called for short ICAM-1) be a kind of to participate between cell and cell and between cell and epimatrix, interactional molecule is referred to as, include the soluble intercellular adhesion molecule (soluble intercellular adhesive molecule1 is called for short sICAM-1) be present in body fluid and the membranous type ICAIU (being called for short membranous type ICAM-1) being present in cell surface.Wherein, sICAM-1 not only remains the biological characteristics of membranous type ICAM-1, itself or the soluble form of membranous type ICAM-1 in body fluid.The present inventor is through studying discovery fully, there is the gravid woman of premature rupture of fetal membranes by the expression of sICAM-1 and the impact of neutrophil leucocyte on the monocyte in amniotic fluid, sICAM-1 content in its vaginal fluid obviously raises, now existing is that target molecule is to detect the testing tool of premature rupture of fetal membranes by sICAM-1, as immune chromatography test paper, but in actual application, it is single that it detects target molecule, if when there is premature rupture of fetal membranes, occur that fetal membrane cut is little, cut is high, during the little situation of vagina amniotic fluid flow, then easily cause sensitivity and specificity all lower, easy generation mistaken diagnosis.
Sensitivity of the present invention is show in patient groups that the sample of positive detection accounts for the number percent of patient's sum, and specificity is show in healthy population that the negative sample detected accounts for the number percent of Healthy People sum.Affect immune chromatography test paper testing result sensitivity of the present invention and specific many factors, include the difference of immune chromatography test paper itself, as the concentration etc. of the selection of detection line working fluid, the concentration of detection line working fluid and nature controlling line working fluid, in addition, the manufacture craft of test paper itself can have a certain impact to the sensitivity of testing result and specificity too.
The present inventor is through studying discovery fully, and sICAM-1 is one of main protein appeared at third trimester of pregnancy in amniotic fluid, and content is generally about 14ng/mL; And when there is premature rupture of fetal membranes, the sICAM-1 that in amniotic fluid, content is more then appears in vaginal fluid.In addition, except above-mentioned sICAM-1 content in the woman vagina fluid samples that premature rupture of fetal membranes occurs is more, the antigen of other content showed increased also has IGFBP-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl.Therefore, the present invention mainly adopts using the plurality of antigens composition in gravid woman's vaginal fluid as jointly detecting target molecule.
The present invention finds through protein chip shaker test, (mated with age in gestation week by the age of two groups of puerperas compared with the non-premature rupture of fetal membranes puerpera made a definite diagnosis for the premature rupture of fetal membranes puerpera made a definite diagnosis, foundation-free disease, without pregnancy complication), in its vaginal fluid sample, the concentration of sICAM-1, IGFBP-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl has notable difference; Wherein, in the premature rupture of fetal membranes puerpera vaginal fluid sample made a definite diagnosis, the concentration more than 90% of sICAM-1 is all at more than 2ng/mL, and in the non-premature rupture of fetal membranes puerpera vaginal fluid sample made a definite diagnosis the concentration more than 90% of sICAM-1 all at below 1ng/mL; The mean concentration of the mean concentration of sICAM-1, the mean concentration of IGFBP-1 and Axl in the premature rupture of fetal membranes puerpera made a definite diagnosis and the vaginal fluid sample of normal healthy controls group can see table (detailed in Example 1); The data such as application that placenta α-microglobulin-1 is detecting gravid woman premature rupture of fetal membranes can list of references: Guo Guangling, Liu Yongzhen, Deng. detecting the value [J] of placenta α microglobulin-1 Diagnosis of Premature Rupture in vaginal secretion. Chinese doctor studies magazine, and 2010,33(15); The mean concentration of alpha-fetoprotein can see document: Yi Yuanyuan, its wooden lattice, etc. detect the clinical value [J] of the horizontal Diagnosis of Premature Rupture of vaginal secretion HCG and AFP. Inner mongolia medical journal, 2008,40(7): repeat no more in 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 the sensitivity of the testing result of premature rupture of fetal membranes and specificity, the present inventor is through research fully, the mode that employing is prepared into Multiple detection line with the antibody of multiple detection target molecule detects premature rupture of fetal membranes jointly, thus more whether qualitative analysis gravid woman premature rupture of fetal membranes occurs; The immune chromatography test paper that the present invention detects premature rupture of fetal membranes can effectively avoid some disturbing factor, thus has the diagnosis of premature rupture of fetal membranes and analyze more accurately, thus improves sensitivity and the specificity of testing result.
In detection line, the variable concentrations of antibody has different effects for the colour developing degree of the antigenic component detected in sample; When the antibody concentration in detection line is too low, then to play the amount of association reaction less for itself and antigen, thus cause colour developing not obvious, and what cause should detecting in patient groups positive findings detects negative findings, and sensitivity is on the low side; When the antibody concentration in detection line is too high, then to play the amount of association reaction more for itself and antigen, thus cause colour developing too obvious, and what cause should detecting in healthy population negative findings detects positive findings, and specificity is on the low side.
In nature controlling line, the variable concentrations of antibody has different effects for the judgement of the testing result validity of test paper; When detection liquid is through detection line, antibody (being mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in the present invention) in antigen wherein and gold conjugation pad can be combined the bond of formation with certain color by the antibody in detection line, antibody in the gold conjugation pad that part does not combine then can continue to move to nature controlling line, thus be combined into another kind of bond with the antibody component in nature controlling line, nature controlling line color changes, thus illustrates that testing result is effective.When the excessive concentration of detection line working fluid, then the antibody content of the gold conjugation pad moving to nature controlling line can be caused less, thus it is obvious not to cause nature controlling line color to change, and causes testing result to lose efficacy; When the concentration of detection line working fluid is on the low side, then the antibody content of the gold conjugation pad moving to nature controlling line can be caused more, thus cause the waste on nature controlling line working fluid cost.
In view of the foregoing, iff adjusting detection line working fluid concentration and nature controlling line working fluid concentration separately, what then obtain is both optium concentrations when being used alone, but because detection line in immune chromatography test paper and nature controlling line are for use simultaneously, the preferred concentration therefore after combination between the two needs to carry out screening and verifying through a large amount of experiments.Simultaneously, detection line due to immune chromatography test paper of the present invention is Multiple detection line, in different detection line, the antibody component of institute's bag quilt is different, and therefore, in immune chromatography test paper of the present invention the working fluid concentration of each detection line all has impact to the sensitivity of testing result and specificity; In view of the foregoing, the present invention needs the better combined concentration studying multiple detection line working fluid, and this research is comparatively loaded down with trivial details.
In addition, various detection antibody is prepared into detection line inherently there is multiple array mode, 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, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody, rabbit anti-human alpha-fetoprotein polyclonal antibody and the anti-human Axl polyclonal antibody of rabbit; Simultaneously, the present inventor studies through sufficient in the various combinations of above-mentioned various antibody test composition, find that goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody preparation is become main detection line by mode of the present invention, and the mode all the other various antibody being prepared into respectively one or two different secondary detection line applies to immune chromatography test paper can obtain better testing result, its sensitivity and specificity are all greatly improved, and can reach more than 99% and 96% respectively.
Immune chromatography test paper of the present invention is in the process used, first antigenic component in sample to be checked combines with the colloidal gold conjugate of the mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody in gold conjugation pad, then gained bond moves to main detection line and secondary detection line successively under the effect of chromatographic film, if enough containing antigenic component or antigenic component content in sample to be checked, then each detection line all should be transformed into purplish red colo(u)r streak, but in actual mechanical process, because some unkownable factors affects, thus cause some antigenic component in sample to be checked to contain quantity not sufficient, in view of the foregoing, the working fluid concentration of the antibody used in immune chromatography test paper of the present invention is through a large amount of experimental studies, the present invention adopts and the working fluid concentration of each antibody is set to goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1-2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 0.2-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-2.5mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 0.5-2.5mg/mL, rabbit anti-human Axl polyclonal antibody 0.5-2mg/mL, during sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL.
Confirm through experimental study, gained immune chromatography test paper of the present invention testing result in use judges as follows: when on immune chromatography test paper of the present invention, nature controlling line changes purplish red colo(u)r streak into, illustrates that result is effective, otherwise invalid; When only there being a detection line change purplish red colo(u)r streak into or change purplish red colo(u)r streak into without detection line, result is judged to be feminine gender, namely premature rupture of fetal membranes does not occur; When there being more than two detection lines to change purplish red colo(u)r streak into, result is judged to be the positive; The mode that immune chromatography test paper of the present invention adopts multiple detection line jointly to detect can avoid the impact of some factor, thus better judges whether premature rupture of fetal membranes occurs, 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 be more preferably, and namely preferably adopts the antibody wrapping quilt in described secondary detection line to be selected from rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody for one or both.
Further, immune chromatography test paper of the present invention also can adopt the mode be more preferably, and namely preferably adopts the antibody wrapping quilt in described secondary detection line to be respectively rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody.
Further, immune chromatography test paper of the present invention also can adopt the mode be more preferably, and the working fluid concentration namely preferably adopting each antibody in described detection line and nature controlling line is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL.
Further, immune chromatography test paper of the present invention also can adopt the mode be more preferably, and the working fluid concentration namely preferably adopting each antibody in described detection line and nature controlling line is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1.5mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL.
Accompanying drawing explanation
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 IGFBP-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, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
With West China No.2 Hospital, Sichuan University's outpatient service and the puerpera that is in hospital for 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 (wherein 80 routine mature prematures rupture of fetal membranes and the mature front premature rupture of fetal membranes of 30 example);
Normal healthy controls group: the complete gravid woman of 110 routine prepartal fetal membrane;
Amniotic fluid sample group: the complete gravid woman of 30 customary caesarean delivered fetal membrane.
Experimental subjects screening standard:
Premature rupture of fetal membranes group-(i) just before giving birth before observe amniotic fluid to spill;
(ii) vaginal fluid sample pH detection paper is positive;
(iii) the lobate crystallization of pteridophyte detects positive.
Normal healthy controls group-(i) just before giving birth before do not observe amniotic fluid and spill;
(ii) vaginal fluid sample pH detection paper is negative;
(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), 30 example mature front 31 years old premature rupture of fetal membranes mean age (the range of age 19-45 year), 30 years old normal healthy controls group mean age (the range of age 18-43 year).
Sample collection method: gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), after peeping out vagina, stretches into posterior fornix place with disposable sterilized cotton swab, rotates 5 circle samplings.Cotton swab head is inserted in 1mL Sample dilution (phosphatebuffer buffer system), after rotating 5 times, 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.) being marked with 174 cytokine antibodies is in advance put into detection box, then add 2ml confining liquid, room temperature closes 6 hours;
2, discard confining liquid, add 1 part, 1.2ml sample to be checked, 4 DEG C of night incubation;
3, discard sample, clean 5 times with 2ml lavation buffer solution;
4, the detection antibody of 1ml biotin coupling is added, incubated at room 2 hours;
5, discard antibody, clean 5 times with 2ml lavation buffer solution;
6, the streptomysin Avidin of 2ml horseradish peroxidase (HRP) coupling is added, incubated at room 2 hours;
7, discard liquid, clean 5 times with 2ml lavation buffer solution;
8, on film, chemical illuminating reagent 500 μ l is added, lucifuge effect 2 minutes, observations.
From premature rupture of fetal membranes group, normal healthy controls group and amniotic fluid sample group, each sample to be tested collected all adopts above-mentioned experiment condition to detect, acquired results as shown in Figure 1, Figure 2 and Figure 3.
Experimental result: the mark 1 in Fig. 1, Fig. 2 and Fig. 3 is the expression signal of IGFBP-1, mark 2 is the expression signal of sICAM-1, and mark 3 is the expression signal of Axl.As can be seen from Fig. 1, Fig. 2 and Fig. 3, in the vaginal fluid of premature rupture of fetal membranes group and amniotic fluid sample group, the expression of IGFBP-1, sICAM-1, Axl is stronger, and the expression of above-mentioned three is more weak in healthy gravid woman's vaginal fluid sample, in above-mentioned three, the strongest with the expression of sICAM-1 again.
B, experimental technique: enzyme linked immunosorbent assay quantitatively detects
Experiment condition:
Experiment material: sICAM-1ELISA detection kit (R & D company)
1. the preparation of reagent
Lavation buffer solution (Wash Buffer)
Substrate solution (Substrate Solution): before use, mixes 15 minutes by the developer A in kit and developer B equivalent, lucifuge, and each hole needs the potpourri 200 μ l of two kinds of developers.
SICAM-1 standard items: sICAM-1 standard items are diluted with 1ml deionized water.Standard items original liquid concentration after dilution is 250ng/ml.For guaranteeing that standard items fully mix, before diluting further, standard items being put on shaking table and rocking mixing more than at least 15 minutes gently.
Respectively the standard items of 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; RD5-7 calibrates thinning agent as blank (0ng/ml)
2. trace routine
100 μ l sICAM-1Conjugate are added in each micropore; The addition of standard items, tester, sample is 100 μ l, and joins in corresponding micropore.The adhesive tape that micropore kit provides is built.Microwell plate to be placed at ambient temperature on horizontal shaker 500 ± 50 revs/min to cultivate 1.5 hours.Wash plate; Every hole adds substrate solution (Substrate Solution) 200 μ l, under room temperature condition, lucifuge, keeps flat cultivation 30 minutes; Every hole adds 50 μ l stop buffers (Stop Solution).In hole, the color of solution will become yellow from blueness.If solution colour is that green or color change are inconsistent in hole, pat flat board gently, to guarantee that solution fully mixes; 450nm light absorption value is surveyed in 30 minutes.
3. production standard curve, calculates sample content.
4. added up and be depicted as chart by the content of each sample to be tested calculating gained, accompanying drawing 4 is shown in by gained chart.
The enzyme linked immunosorbent assay of IGFBP-1 and Axl quantitatively detects the enzyme linked immunosorbent assay that can refer to above-mentioned sICAM-1 and quantitatively detects and carry out, and the detection kit adopted is all purchased from same company, and experimental results is as follows.
Experimental result: in Fig. 4-Fig. 6, AF of control is 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 more than 90% of sICAM-1 in premature rupture of fetal membranes group vaginal fluid sample can reach more than 2ng/mL; The concentration more than 90% of sICAM-1 in normal healthy controls group vaginal fluid sample is at below 1ng/mL.
As can be seen from Figure 5, the concentration of IGFBP-1 in healthy gravid woman's amniotic fluid sample is the highest, on average can reach 270ng/mL; The concentration more than 90% of IGFBP-1 in premature rupture of fetal membranes group vaginal fluid sample can reach more than 15ng/mL; The concentration more than 90% of IGFBP-1 in normal healthy controls group vaginal fluid sample is at 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 more than 90% of Axl in premature rupture of fetal membranes group vaginal fluid sample can reach more than 1ng/mL; The concentration more than 90% of Axl in normal healthy controls group vaginal fluid sample is at below 0.5ng/mL.
Known from the experimental result embodiment 1 and aforementioned documents data, the antigen that content occurs to occur in the vaginal fluid of premature rupture of fetal membranes gravid woman includes soluble intercellular adhesion molecule sICAM-1, IGFBP-1, placenta α-microglobulin-1, alpha-fetoprotein and Axl; Described antigen mainly comes from the amniotic fluid of rupture of membranes.
Be below that aforementioned each antigen, as jointly detecting target molecule, and prepares the reference examples of the immune chromatography test paper of gained according to the method preparing immune chromatography test paper of existing routine in gravid woman's vaginal fluid, each reference examples is as follows:
Reference examples 1
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with the detection line be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag and the nature controlling line 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 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 2
Reference examples 2 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by rabbit anti-Human Insulin like growth factor associated proteins-1 antibody and rabbit anti-human alpha-fetoprotein polyclonal antibody bag respectively and the nature controlling line be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: IGFBP-1 antibody 3mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 3mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 3
Reference examples 3 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by rabbit anti-Human Insulin like growth factor associated proteins-1 antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 3mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 4
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by rabbit anti-human placenta α-microglobulin-1 antibody and rabbit anti-human Axl polyclonal antibody bag respectively and the nature controlling line be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: rabbit anti-human placenta α-microglobulin-1 antibody 2.5mg/mL, rabbit anti-human Axl polyclonal antibody 1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 5
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by rabbit anti-human alpha-fetoprotein polyclonal antibody and rabbit anti-human Axl polyclonal antibody bag respectively and the nature controlling line be formed by sheep anti-mouse igg polyclonal antibody bag;
The working fluid concentration of described each antibody is: rabbit anti-human alpha-fetoprotein 2mg/mL, rabbit anti-human Axl polyclonal antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 6
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-human alpha-fetoprotein polyclonal antibody bag respectively and the nature controlling line 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 3mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 0.5mg/mL, sheep anti-mouse igg polyclonal antibody 3mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 7
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line 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 1mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 3mg/mL, sheep anti-mouse igg polyclonal antibody 1mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 8
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-human alpha-fetoprotein polyclonal antibody bag respectively and the nature controlling line 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 2.5mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 9
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-human Axl polyclonal antibody bag respectively and the nature controlling line 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 1.5mg/mL, rabbit anti-human Axl polyclonal antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 10
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with three detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, rabbit anti-human alpha-fetoprotein polyclonal antibody and rabbit anti-human Axl polyclonal antibody bag respectively and the nature controlling line 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 1mg/mL, rabbit anti-human alpha-fetoprotein polyclonal antibody 2.5mg/mL, rabbit anti-human polyclonal antibody Axl2mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 11
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-Human Insulin like growth factor associated proteins-1 antibody bag respectively and the nature controlling line 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 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 0.2mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 12
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line 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 2.5mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 2.5mg/mL, sheep anti-mouse igg polyclonal antibody 2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 13
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line 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 1.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 14
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line 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 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 1.5mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Reference examples 15
Reference examples 1 immune chromatography test paper: include sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; Described chromatographic film is disposed with two detection lines be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody and rabbit anti-human placenta α-microglobulin-1 antibody bag respectively and the nature controlling line 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 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1.5mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
Material source in above-mentioned reference examples 1-15 is as follows:
Goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody: purchased from R & D company;
Sheep anti-mouse igg polyclonal antibody: purchased from the female bioengineering company limited of the upper Shanghai's style;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody: purchased from R & D company;
Rabbit anti-Human Insulin like growth factor associated proteins-1 antibody: purchased from R & D company;
Rabbit anti-human placenta α-microglobulin-1 antibody: purchased from R & D company;
The anti-human alpha-fetoprotein polyclonal antibody of rabbit: purchased from Zhengzhou hundred base biotech firm;
The anti-human Axl polyclonal antibody of rabbit: purchased from Beijing An Biqi biotech firm;
Sample pad (commercially available): purchased from Ahlstrom company;
Adsorptive pads (commercially available): purchased from an outstanding bio tech ltd, Shanghai;
Collaurum (commercially available): purchased from Jie Ning bio tech ltd, Shanghai;
Bovine serum albumin(BSA) (BSA): Roche company;
Embodiment 2
The reference examples 1-15 immune chromatography test paper be prepared from is respectively used to the clinical sample detecting same gravid woman's vaginal fluid, include premature rupture of fetal membranes group and normal healthy controls group, wherein premature rupture of fetal membranes group is 500 examples, normal healthy controls group is 500 examples, and subjects all adopts the method for embodiment 1 to carry out clinical diagnosis and screening; More than 70% be wherein the obstetrical patient that fetal membrane cut is little or the high vagina amniotic fluid flow caused of cut is less in premature rupture of fetal membranes group.
Sample collection: gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), after peeping out vagina, stretches into posterior fornix place with disposable sterilized cotton swab, rotates 5 circle samplings.Cotton swab head is inserted in 1mL Sample dilution (phosphatebuffer buffer system), after rotating 5 times, be close to tube wall extruding rotation 2 times, obtain sample.
Sampling test subject age distributes: 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 to detect collected 1000 routine samples respectively, the feminine gender of gained testing result and positive number of cases statistics are in table one:
Table one
In upper table one, reference examples 1 is existing immune chromatography test paper, and reference examples 2-7 is the immune chromatography test paper of common employing Multiple Antibodies detection line, 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 such as order successively of each antibody in the working fluid concentration of antibody and different detection line in detection line; Reference examples 11-12 is for carry out preferred embodiment in the enterprising step of immune chromatography test paper of the present invention, and reference examples 13-15 is more further preferred implementation.Testing result as can be seen from table one, 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 common employing Multiple Antibodies detection line, there is better sensitivity and specificity, more than 99% and 96% can be reached respectively; In addition, as can be seen from the immune chromatography test paper of reference examples 11-15 embodiment, it has effect more significantly sensitivity and specificity relative to reference examples 8-10, and especially specific lifting is comparatively remarkable; More further, the immune chromatography test paper of reference examples 15 embodiment has better effect in specificity.
Below be only the preferred embodiment of the present invention, it 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 limited range.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 (4)
1. detect an immune chromatography test paper for premature rupture of fetal membranes, this immune chromatography test paper includes sample pad, gold conjugation pad, chromatographic film, adsorptive pads, offset plate; Described sample pad, gold conjugation pad, chromatographic film, adsorptive pads are connected with offset plate successively in order; It is characterized in that: described chromatographic film is disposed with detection zone and quality control region, described detection zone is near gold conjugation pad one end, and described quality control region is near adsorptive pads one end; Be provided with detection line in 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 be formed by goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody bag; Described secondary detection line is that two kinds of different antibody wrap respectively and are formed two secondary detection lines; The antibody wrapping quilt in described secondary detection line is respectively rabbit anti-Human Insulin like growth factor associated proteins-1 antibody, rabbit anti-human placenta α-microglobulin-1 antibody; Be provided with nature controlling line in described quality control region, described nature controlling line is for 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, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 0.2-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-2.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-2mg/mL;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody is coated with in described gold conjugation pad.
2. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 1, is characterized in that: in described detection line and nature controlling line, the working fluid concentration of each antibody is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 1.5-2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1-2mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 0.5-1.5mg/mL, sheep anti-mouse igg polyclonal antibody 0.5-1.5mg/mL.
3. the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 2, is characterized in that: in described detection line and nature controlling line, the working fluid concentration of each antibody is goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody 2.5mg/mL, rabbit anti-Human Insulin like growth factor associated proteins-1 antibody 1.5mg/mL, rabbit anti-human placenta α-microglobulin-1 antibody 1mg/mL, sheep anti-mouse igg polyclonal antibody 1.5mg/mL.
4. prepare a preparation method for the immune chromatography test paper of detection premature rupture of fetal membranes according to claim 1, it is characterized in that including following steps:
A, each antibody detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, gained nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film;
B, mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody and collaurum are prepared into gold conjugation pad;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto on offset plate successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper.
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