CN103175959B - A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper - Google Patents
A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper Download PDFInfo
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Abstract
The present invention discloses a kind of preparation method of immune chromatography test paper, is specifically related to a kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper.Gold conjugation pad in described preparation method is for prepare gained by the following method: add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min in collaurum after, add bovine serum albumin(BSA) and close centrifuging after 20-50min, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 10-30mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour; Preparation method of the present invention prepares the sensitivity of the detection premature rupture of fetal membranes immune chromatography test paper of gained and specificity has obvious raising, can reach more than more than 97% and 93% respectively, thus reduces the incidence of mistaken diagnosis.
Description
Technical field
The present invention relates to a kind of preparation method of immune chromatography test paper, be specifically related to a kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper.
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 formation: 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 intercellular adhesion molecule-1 (be called for short ICAM-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 ICAM-1; The detection line endoperidium of described immune chromatography test paper has goat-anti H-ICAM-1 polyclonal antibody working fluid, and nature controlling line endoperidium has sheep anti-mouse igg working fluid, containing mouse-anti H-ICAM-1 monoclonal antibody in colloidal gold conjugate.
Be although that the detection premature rupture of fetal membranes immune chromatography test paper of Testing index is provided convenience for obstetrical patient with sICAM-1 existing, but its sensitivity, specificity and accuracy are all 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 preparation method detecting premature rupture of fetal membranes immune chromatography test paper, this preparation method prepares the sensitivity of the detection premature rupture of fetal membranes immune chromatography test paper of gained and specificity has obvious raising, more than more than 97% and 93% 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 preparation method detecting premature rupture of fetal membranes immune chromatography test paper, and described preparation method includes following steps:
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close centrifuging after 20-50min, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 10-30mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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.
Preferably, the Sodium azide containing 20mg/100mL in phosphate buffer in described step B.
Preferably, the ratio that the amount of the mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody added in described step B accounts for collaurum is 150-180 μ g/mL.
Preferably, the ratio that the amount of the bovine serum albumin(BSA) added in described step B accounts for collaurum is 150-200mg/100mL.
Preferably, in described step A, detection line working fluid concentration is 1.5-2.5mg/mL, and nature controlling line working fluid concentration is 0.5-1.5mg/mL; Through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film.
Preferably, in described step A, detection line working fluid concentration is 2.5mg/mL, and nature controlling line working fluid concentration is 1.5mg/mL.
Preferably, in described step A through bag by after nitrocellulose filter at 38 DEG C dry 10 hours.
Preferably, in described step C, sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour.
Preferably, the BSA of Triton-X100 and 0.5-2g/100mL also containing 0.5-3.0mL/100mL in phosphate buffer in described step C.
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 preparation method detecting premature rupture of fetal membranes immune chromatography test paper, and described preparation method includes following steps:
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close centrifuging after 20-50min, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 10-30mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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.
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 extracellular matrix, 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 ICAM-1 and the impact of neutrophil leucocyte on the monocyte in fetal membrane and amniotic fluid, ICAM-1 content in its vaginal fluid obviously raises, and comprises the content of membranous type ICAM-1 and sICAM-1.Now existing by ICAM-1 be target molecule to detect the testing tool of premature rupture of fetal membranes, but in actual application, its sensitivity and specificity are all lower, easily produce mistaken diagnosis.
The present inventor, through studying discovery fully, is mainly the change of sICAM-1 content in the reason occurring to cause ICAM-1 content to raise in the vaginal fluid of the gravid woman of premature rupture of fetal membranes, instead of the membranous type ICAM-1 that content is less; The present inventor found through experiments, 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, except the membranous type ICAM-1 on fetal membrane superficial cell can flow to except vagina with amniotic fluid, main or based on amniotic fluid, in amniotic fluid content more sICAM-1 then appear in vaginal fluid.Therefore, the sICAM-1 in gravid woman vaginal fluid is adopted as detection target molecule relative to being detect target molecule can have better sensitivity and specificity to detect premature rupture of fetal membranes with ICAM-1.
In view of the abundant research of above-mentioned experimental result and the present inventor, adopt goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody as detection line working fluid in the preparation method of the detection premature rupture of fetal membranes immune chromatography test paper described in technical solution of the present invention, sheep anti-mouse igg polyclonal antibody is as nature controlling line working fluid.
Immune chromatography test paper is show in patient groups that the sample of positive detection accounts for the number percent of patient's sum for sensitivity when detecting disease, and specificity is show in healthy population that the negative sample detected accounts for the number percent of Healthy People sum.Affect sensitivity and specific many factors, include the difference of the manufacturing process of test paper itself and individual and operate whether specification etc., but basic or based on the manufacturing process of test paper itself.In the manufacturing process of immune chromatography test paper, include several committed step, be mainly the preparation of chromatographic film, collaurum, gold conjugation pad and sample pad.
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 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.
In view of above-mentioned experimental result, correctly can detect to allow immune chromatography test paper whether gravid woman premature rupture of fetal membranes occurs, the preparation method of immune chromatography test paper just needs through certain adjustment, and the present invention is then mainly to adjust the preparation method of colloidal gold conjugate in wherein gold conjugation pad to improve sensitivity and the specificity of immune chromatography test paper.
Colloidal gold conjugate of the present invention adopts above-mentioned preparation method, while holding time of gained colloidal gold conjugate is ideal, the chromatographic effect of its gold conjugation pad prepared is also ideal, thus the sensitivity of gained immune chromatography test paper and specific performance access and comparatively significantly improve.
Colloidal gold conjugate of the present invention is the bond of mouse-anti human soluble ICAIU monoclonal antibody and collaurum.Usually can add sodium chloride solution in the preparation method of existing colloidal gold conjugate to extend its pot-life, Sodium azide can be added simultaneously better play the object extending the pot-life in phosphate buffer.Although sodium chloride and Sodium azide add the pot-life that can extend colloidal gold conjugate, both add the chromatography ability that membership reduces gained gold conjugation pad to a certain extent, thus reduce sensitivity and the specificity of immune chromatography test paper.
Sodium chloride solution is not added in the preparation method of colloidal gold conjugate of the present invention, and the concentration of Sodium azide is adjusted to 10-30mg/100mL, the chromatography capacity of this preparation condition to gained gold conjugation pad is less, sensitivity and the specificity of gained immune chromatography test paper are effectively improved, meanwhile, the pot-life of gained colloidal gold conjugate can not be affected.
Further, the present invention preferably adopts the Sodium azide containing 20mg/100mL in described phosphate buffer.The present invention, on the basis of above-mentioned preparation condition, can adjust the concentration of Sodium azide in phosphate buffer further, is specifically preferably 20mg/mL.
Further, the present invention preferably adopt described in the amount of mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody that the adds ratio that accounts for collaurum be 150-180 μ g/100mL.In the preparation process of colloidal gold conjugate of the present invention can further preferably mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody the amount ratio that accounts for collaurum be 150-180mg/100mL, under described preferred implementation, the chromatography ability of gained gold conjugation pad is better, and sensitivity and the specificity of gained immune chromatography test paper improve.Further, the present invention preferably adopt described in the amount of bovine serum albumin(BSA) that the adds ratio that accounts for collaurum be 150-200mg/100mL.
Further, the present invention preferably adopts sample pad in described step C to be adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour.Inventive samples pad can adopt existing known preparation method to be prepared gained, also can adopt and buy commercially available product; The present invention preferably adopts described sample pad to prepare gained for adopting above-mentioned optimal way, and this method gained sample pad is for the sensitivity of gained test paper and the specificity effect of improving.
Further, in described step A, detection line working fluid concentration is 1.5-2.5mg/mL, and nature controlling line working fluid concentration is 0.5-1.5mg/mL; Through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film.
The variable concentrations of antibody in detection line (being goat-anti human soluble ICAIU polyclonal antibody in the present invention) has different effects for the colour developing degree of the antigenic component detected in sample (being the soluble intercellular adhesion molecule in gravid woman's vaginal fluid in the present invention); 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.
The variable concentrations of antibody in nature controlling line (being sheep anti-mouse igg polyclonal antibody in the present invention) 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.The present inventor is through studying discovery fully, detect on the basis of premature rupture of fetal membranes immune chromatography test paper method in existing preparation, when detection line working fluid concentration and nature controlling line working fluid concentration are adjusted to 1.5-2.5mg/mL and 0.5-1.5mg/mL respectively, gained test paper can have better sensitivity and specificity under identical using method.
In addition, the present invention detects gained detection line and nature controlling line bag in the preparation method of premature rupture of fetal membranes immune chromatography test paper is needed within dry 8-12 hour at 36-38 DEG C, obtain chromatographic film by the nitrocellulose filter crossed.Wrap by the nitrocellulose filter crossed after the drying of uniform temperature, its sensitivity for test paper and specificity also can play certain raising effect.
Further, the present invention preferably adopts detection line working fluid concentration in described step A to be 2.5mg/mL, and nature controlling line working fluid concentration is 1.5mg/mL.In preparation method of the present invention, detection line working fluid concentration is 1.5-2.5mg/mL, and nature controlling line working fluid concentration is 0.5-1.5mg/mL; The present invention passes through screening and test in above-mentioned concentration ranges, detection line working fluid concentration is preferably adopted to be 2.5mg/mL in preparation method of the present invention, nature controlling line working fluid concentration is 1.5mg/mL, the present invention preferably adopts the combination of above-mentioned two kinds of concentration, and sensitivity and specificity that obtained immune chromatography test paper is detecting gravid woman's premature rupture of fetal membranes are all significantly improved.
Further, the present invention preferably adopt in described step A through bag by after nitrocellulose filter at 38 DEG C dry 10 hours.In preparation method of the present invention through bag by after nitrocellulose filter be dry 8-12 hour at 36-38 DEG C, the present invention preferably to adopt at 38 DEG C drying 10 hours.
Further, the present invention preferably adopts sample pad in described step C to be adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour.Inventive samples pad can adopt existing known preparation method to be prepared gained, also can adopt and buy commercially available product; The present invention preferably adopts described sample pad to prepare gained for adopting above-mentioned optimal way, and this method gained sample pad is for the sensitivity of gained test paper and the specificity effect of improving.
Further, the present invention preferably adopts the BSA of Triton-X100 and 0.5-2g/100mL also containing 0.5-3.0mL/100mL in phosphate buffer in described step C.Triton-X100 of the present invention is Triton X-100, and it is a kind of surfactant; BSA is bovine serum albumin(BSA); Adopt in the present invention and be used for soaking glass fibre by above-mentioned two kinds of material co-formulation in phosphate buffer, its sample pad preparing gained is better for the buffer action effect of testing sample, thus is significantly improved to the sensitivity of test paper and specificity.
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.
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
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:
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.
A, experimental technique: cell factor/chemotactic factor (CF) antibody chip qualitative detection
Experiment condition:
1) 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.
Experimental result: in Fig. 4, 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.
From the experimental result of embodiment 1, in the vaginal fluid of generation premature rupture of fetal membranes gravid woman, that one of main generation content is soluble intercellular adhesion molecule sICAM-1, and sICAM-1 mainly comes from the amniotic fluid of rupture of membranes.
Be below with sICAM-1 in gravid woman's vaginal fluid for detecting target molecule, and prepare the reference examples of the immune chromatography test paper of gained according to different preparation methods, the preparation method of each reference examples is as follows:
Reference examples 1
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 10g/100mL, centrifuging, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 90mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads offset plate is prepared into reference examples 1-detects premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 2
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 5g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 80mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads offset plate is prepared into reference examples 2-detects premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 3
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 5g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 5mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads offset plate is prepared into reference examples 3-detects premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 4
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 1g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 10mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads offset plate is prepared into reference examples 4-detects premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are buy commercially available product.
Reference examples 5
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 5g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
C, the chromatographic film of A and step B gained and gold conjugation pad be pasted onto successively according to the order of sample pad, gold conjugation pad, chromatographic film, adsorptive pads offset plate is prepared into reference examples 5-detects premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 6
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 5g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 30mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 7
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close 20-50min, then add the sodium chloride of 1g/100mL, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 25mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 8
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 150mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available product.
Reference examples 9
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 180 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 150mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 10
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 11
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 2mg/mL, and nature controlling line working fluid concentration is 2mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 12
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 3mg/mL, and nature controlling line working fluid concentration is 3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 13
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1.5mg/mL, nature controlling line working fluid concentration is 0.5mg/mL, described detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 14
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1.5mg/mL, nature controlling line working fluid concentration is 1.5mg/mL, described detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 15
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 2.5mg/mL, nature controlling line working fluid concentration is 0.5mg/mL, described detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 16
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 2.5mg/mL, nature controlling line working fluid concentration is 1.5mg/mL, described detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, through bag by after nitrocellulose filter at 38 DEG C dry 10 hours chromatographic film;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad, adsorptive pads are buys commercially available prod.
Reference examples 17
A, goat-anti human soluble intercellular adhesion molecule-1 are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2 hours, within 8 hours, obtains sample pad in 36 DEG C of dryings; Described adsorptive pads is for buying commercially available prod.
Reference examples 18
A, goat-anti human soluble intercellular adhesion molecule-1 are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 6 hours, within 12 hours, obtains sample pad in 38 DEG C of dryings; Described adsorptive pads is for buying commercially available prod.
Reference examples 19
A, goat-anti human soluble intercellular adhesion molecule-1 are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour; The BSA of Triton-X100 and 0.5g/100mL containing 0.5mL/100mL in described phosphate buffer; Described adsorptive pads is for buying commercially available prod.
Reference examples 20
A, goat-anti human soluble intercellular adhesion molecule-1 are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 1-3mg/mL, and nature controlling line working fluid concentration is 1-3mg/mL, described detection line working fluid and nature controlling line working fluid is carried out being packaged as chromatographic film on nitrocellulose filter;
B, after in collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min of 150 μ g in proportion, the bovine serum albumin(BSA) that every 100mL collaurum adds 200mg in proportion closes centrifuging after 20-50min, and the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour;
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; Described sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour; The BSA of Triton-X100 and 2g/100mL containing 3.0mL/100mL in described phosphate buffer; Described adsorptive pads is for buying commercially available prod.
Material source in above-mentioned reference examples 1-20 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;
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 1-20 immune chromatography test paper be prepared from by distinct methods 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 240 examples, normal healthy controls group is 260 examples, and subjects all adopts the method for embodiment 1 to carry out clinical diagnosis and screening.
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:
Utilize reference examples 1-20 to detect collected 500 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, the immune chromatography test paper of reference examples 1-3 prepared by existing preparation method; Reference examples 4-7 is the chromatographic test paper only adjusting colloidal gold conjugate preparation method in gold conjugation pad relative to reference examples 1-3; Reference examples 8-10 is main preparation methods of the present invention; Reference examples 11-20 is the preparation method that the present invention improves further, and wherein reference examples 11-16 mainly further improves the preparation method of colloidal gold conjugate, and reference examples 17-20 mainly further improves the preparation method of sample pad.
Can draw from the difference of upper table one data and reference examples, preparation method of the present invention can significantly improve sensitivity and the specificity of gained immune chromatography test paper; Same, sensitivity and the specificity of the immune chromatography test paper of preparation method's gained that the present invention improves further also have obvious raising.
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 (2)
1. detect a preparation method for premature rupture of fetal membranes immune chromatography test paper, it is characterized in that: described preparation method includes following steps:
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and sheep anti-mouse igg polyclonal antibody is as the working fluid of nature controlling line; Detection line working fluid concentration is 2.5mg/mL, nature controlling line working fluid concentration is 1.5mg/mL, described detection line working fluid and nature controlling line working fluid are carried out bag quilt on nitrocellulose filter, through bag by after nitrocellulose filter at 36-38 DEG C dry 8-12 hour chromatographic film;
B, in collaurum, add mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min after, add bovine serum albumin(BSA) and close centrifuging after 20-50min, the centrifugal product of gained is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, the Sodium azide containing 20mg/100mL in described phosphate buffer; The phosphate buffer adding colloidal gold conjugate is coated on glass fibre, obtained gold conjugation pad in 36-38 DEG C of dry 8-12 hour; The ratio that the amount of the described mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody added accounts for collaurum is 150-180 μ g/100mL; The ratio that the amount of the described bovine serum albumin(BSA) added accounts for collaurum is 150-200mg/100mL;
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; Described sample pad is adopt following methods to be prepared from: soaked by glass fibre in the phosphate buffer of pH7.2-7.4 after 2-6 hour, obtained sample pad in 36-38 DEG C of dry 8-12 hour; The BSA of Triton-X100 and 0.5-2g/100mL also containing 0.5-3.0mL/100mL in phosphate buffer in this step.
2. preparation method according to claim 1, is characterized in that: in described step A through bag by after nitrocellulose filter at 38 DEG C dry 10 hours.
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