CN103134935A - Making method of immunochromatographic test paper for detecting premature rupture of fetal membranes - Google Patents
Making method of immunochromatographic test paper for detecting premature rupture of fetal membranes Download PDFInfo
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- CN103134935A CN103134935A CN2013100645026A CN201310064502A CN103134935A CN 103134935 A CN103134935 A CN 103134935A CN 2013100645026 A CN2013100645026 A CN 2013100645026A CN 201310064502 A CN201310064502 A CN 201310064502A CN 103134935 A CN103134935 A CN 103134935A
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Abstract
The invention discloses a making method of immunochromatographic test paper, and concretely relates to a making method of immunochromatographic test paper for detecting the premature rupture of fetal membranes. The preparation method of colloidal gold used in the making method comprises the following steps: heating an aqueous solution of chloroauric acid to boil, adding 2-3mL of an aqueous solution of trisodium citrate having a concentration of 0.5-2g/100mL under stirring, boiling for 3-10min, cooling, adjusting the pH value to 6.0-7.0 by an aqueous K2CO3 solution to obtain the colloidal gold. The obtained colloidal gold and a mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody are used to make a colloidal gold bind pad. The sensitivity and the specificity of the immunochromatographic test paper for detecting premature rupture of fetal membranes obtained through the making method are obviously improved and reach above 97% and above 93% respectively, so the incidence rate of misdiagnosis is reduced.
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 who detects the premature rupture of fetal membranes immune chromatography test paper.
Background technology
Immune chromatography test paper is a kind ofly to utilize chromatographic theory that the target detection molecule in liquid to be detected or one-tenth are graded to flow to detection line (T line) and nature controlling line (C line) on test strips, when detection line generation change color illustrates that testing result is positive, otherwise negative, when nature controlling line generation change color, illustrate that testing result is effective, otherwise invalid.
Immune chromatography test paper has advantage very easily when detecting or diagnosing some disease, so it has fully been applied in the detection or diagnostic procedure of various diseases or symptom, for example the early pregnancy test paper.Generally, immune chromatography test paper includes following several formation: sample pad, collaurum pad, chromatographic film, adsorptive pads, offset plate, and described sample pad, collaurum pad, chromatographic film and adsorptive pads stick on offset plate successively; Be disposed with detection line and nature controlling line on described chromatographic film; Its principle of work is: drop to be measured is added on sample pad, and liquid to be measured utilizes chromatographic theory to flow to the collaurum pad, the collaurum bond in liquid to be measured dissolving collaurum pad, and jointly flow to successively detection line and nature controlling line on chromatographic film with it; Contain the target detection molecule in liquid to be measured, thereby target detection molecule, the collaurum bond antibody component that can comprise in detection line is combined and is formed immune complex, the detection line color changes simultaneously; When liquid to be measured flow to nature controlling line, the antibody component that comprises in nature controlling line can be combined with the collaurum bond, thereby the nature controlling line color changes.
Rupture of membranes (Rupture of Fetal Membrane is called for short ROM) may occur at any time in the pregnancy period, and the rupture of membranes that occurs before just before giving birth is called premature rupture of fetal membranes (PROM).After mature, the incidence of (after 37 pregnant weeks) PROM is about 10%, and the incidence of mature front (before 37 pregnant weeks) PROM is 2-3.5%.Premature rupture of fetal membranes is to cause that the obstetrical patient is antenatal, the principal element of postpartum complication, is also to cause the fetus premature labor and main cause that the neonate must move in the intensive care unit.Due to the medical worker have to prolong pregnancy week and in utero reach the risk of infection of pregnant women and Fetal Lung growth problem between ask balance, thereby high to the handling cost of Patients with Preterm Premature Rupture of Membranes, difficulty is large, accurately and timely diagnoses the pregnant woman whether premature rupture of fetal membranes to occur most important.
In the existing method that detects premature rupture of fetal membranes except the methods such as bed-side query medical history in the past, mainly the intercellular adhesion molecule-1 (being called for short ICAM-1) in gravid woman's vaginal fluid for the testing tool of detection index as newer method.Wherein played effect preferably take ICAM-1 as whether the detection premature rupture of fetal membranes immune chromatography test paper that detects index for the timely diagnosis of obstetrical patient, premature rupture of fetal membranes occurs; The detection line endoperidium of described immune chromatography test paper has goat-anti H-ICAM-1 polyclonal antibody working fluid, and the nature controlling line endoperidium has the sheep anti-mouse igg working fluid, contains mouse-anti H-ICAM-1 monoclonal antibody in the collaurum bond.
Although existingly provide convenience take sICAM-1 as the detection premature rupture of fetal membranes immune chromatography test paper that detects index as the obstetrical patient, but its sensitivity, specificity and accuracy are all not ideal enough, thereby easily cause mistaken diagnosis, can bring inconvenience equally concerning the obstetrical patient.
Summary of the invention
In view of this, the invention provides a kind of preparation method who detects the premature rupture of fetal membranes immune chromatography test paper, sensitivity and specificity that this preparation method prepares the detection premature rupture of fetal membranes immune chromatography test paper of gained have obvious raising, can reach respectively more than 97% and more than 93%, thereby reduce the incidence of mistaken diagnosis.
For solving above technical matters, technical scheme of the present invention is to adopt a kind of preparation method who detects the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.005g-0.03g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2-3mL of 0.5-2g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 6.0-7.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate.
Preferably, in described B step, the concentration of aqueous solution of chloraurate is 0.01g/100mL.
The concentration of the trisodium citrate aqueous solution that preferably, adds in described B step is 2g/100mL.
The amount of the trisodium citrate aqueous solution that preferably, adds in described B step is 2.5mL.
Preferably, K in described B step
2CO
3The concentration of aqueous solution is 0.1-0.3mol/L.
Preferably, after adding mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min in described B step gained collaurum, add centrifuging after bovine serum albumin(BSA) sealing 20-50min, the centrifugal product of gained is the collaurum bond; Gained collaurum bond is dissolved in the phosphate buffer of pH7.2-7.4, contains the Sodium azide of 10-30mg/100mL in described phosphate buffer.
Preferably, the Sodium azide that contains 20mg/100mL in described phosphate buffer.
Preferably, to account for the ratio of collaurum be 150-180 μ g/100mL to the amount of the described mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody that adds.
Preferably, to account for the ratio of collaurum be 150-200mg/100mL to the amount of the described bovine serum albumin(BSA) that adds.
Preferably, in described C step, sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2-6 hour, in 36-38 ℃ of dry 8-12 hour sample pad.
Preferably, also contain the Triton-X100 of 0.5-3.0mL/100mL and the BSA of 0.5-2g/100mL in phosphate buffer in described C step.
The present invention compared with prior art, it is described in detail as follows:
The technical solution used in the present invention is: detect the preparation method of premature rupture of fetal membranes immune chromatography test paper, 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, aqueous solution of chloraurate is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2-3mL of 0.5-2g/mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 6.0-7.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate.
At first, ICAIU (intercellular adhesive molecule1, be called for short ICAM-1) be a kind ofly to participate between cell and cell and the molecule of cell and extracellular matrix interaction is referred to as, include the soluble intercellular adhesion molecule (soluble intercellular adhesive molecule1 is called for short sICAM-1) that is present in body fluid and the membranous type ICAIU (being called for short membranous type ICAM-1) that is present in cell surface.Wherein, sICAM-1 has not only kept the biological characteristics of membranous type ICAM-1, itself or the soluble form of membranous type ICAM-1 in body fluid.The inventor finds through research fully, the gravid woman that premature rupture of fetal membranes occurs is subjected to the expression of ICAM-1 on monocyte in fetal membrane and amniotic fluid and the impact of neutrophil leucocyte, ICAM-1 content in its vaginal fluid obviously raises, and comprises the content of membranous type ICAM-1 and sICAM-1.Now existing is the testing tool that target molecule detects premature rupture of fetal membranes with ICAM-1, but in actual application, its sensitivity and specificity are all lower, easily produce mistaken diagnosis.
The inventor is through research discovery fully, and the reason that causes ICAM-1 content to raise in the gravid woman's that premature rupture of fetal membranes occurs vaginal fluid mainly is the variation of sICAM-1 content, rather than the less membranous type ICAM-1 of content; The inventor found through experiments, and sICAM-1 appears at one of main protein in amniotic fluid third trimester of pregnancy, and content is generally the 14ng/mL left and right; And when premature rupture of fetal membranes occured, the membranous type ICAM-1 on the fetal membrane superficial cell can flow to vagina with amniotic fluid, and is main or take amniotic fluid as main, in amniotic fluid content more sICAM-1 can appear in vaginal fluid.Therefore, the sICAM-1 in employing gravid woman vaginal fluid is as detecting target molecule with respect to as the detection target molecule detects premature rupture of fetal membranes, better sensitivity and specificity being arranged take ICAM-1.
Abundant research in view of above-mentioned experimental result and the inventor, adopt goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody as the detection line working fluid in the preparation method of the described detection premature rupture of fetal membranes of technical solution of the present invention immune chromatography test paper, the sheep anti-mouse igg polyclonal antibody is as the nature controlling line working fluid.
The sensitivity of immune chromatography test paper during for detection of disease is the number percent that the sample that draws positive detection in ill crowd accounts for patient's sum, and specificity is to draw the number percent that the negative sample that detects accounts for the Healthy People sum in healthy population.Affect sensitivity and specific many factors, include the difference of manufacturing process of test paper itself and individual and whether operate standard etc., but basic or take the manufacturing process of test paper itself as main.Include several committed steps in the manufacturing process of immune chromatography test paper, be mainly the preparation of chromatographic film, collaurum, collaurum pad and sample pad.
The present invention finds through the protein chip shaker test, the premature rupture of fetal membranes puerpera who makes a definite diagnosis compares (two groups of puerperas' age and coupling in age in gestation week with the non-premature rupture of fetal membranes puerpera who makes a definite diagnosis, the foundation-free disease is 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 of making a definite diagnosis the concentration of sICAM-1 more than 90% all more than 2ng/mL, and in the non-premature rupture of fetal membranes puerpera vaginal fluid sample of making a definite diagnosis the concentration of sICAM-1 more than 90% all below 1ng/mL.
In view of above-mentioned experimental result, whether premature rupture of fetal membranes occurs in order to allow immune chromatography test paper can correctly detect the gravid woman, the preparation method of immune chromatography test paper just needs through certain adjustment, and the present invention adjusts sensitivity and the specificity that the preparation method of collaurum wherein improves immune chromatography test paper.
Gold chloride of the present invention is tetra chlorauric acid, and chemical formula is AuCl
3HCl4H
2O。
Collaurum of the present invention can adopt existing known preparation method to obtain, and can also adopt the mode of buying commercially available collaurum to obtain; The existing known method for preparing collaurum includes aqueous solution of chloraurate under any one or the multiple effect such as reductive agent such as white phosphorus, ascorbic acid, trisodium citrate, tannic acid, reductive polymerization becomes a certain size gold grain, the gained gold grain is stable colloidal state under electrostatic interaction, be also collaurum.Wherein, add the concentration of reductive agent and addition all can adopt existing preparation condition and method to carry out.
The present invention finds through research fully, in preparation method of the present invention for make in gained collaurum pad mouse-anti human soluble ICAIU monoclonal antibody can with the better combination of collaurum, the present invention preferably adopts the preparation method of collaurum to be: aqueous solution of chloraurate is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution of 0.5-2g/100mL, boiled 3-10 minute, and used K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 6.0-7.0.
Adopt trisodium citrate as the reductive agent of gold chloride in preparation method of the present invention, its concentration and addition are by a large amount of experimental study gained.Trisodium citrate is as the reductive agent of gold chloride, and the gold grain degree of being uniformly dispersed of gained collaurum depends primarily on the reaction conditions of aqueous solution of chloraurate and reductive agent, wherein take the concentration of the trisodium citrate aqueous solution that adds and addition as main.The joining day of trisodium citrate of the present invention is that aqueous solution of chloraurate is when being heated to boil, this mode can make the trisodium citrate that adds can rapid dispersion under the condition of sufficient temp in aqueous solution of chloraurate, thereby the gold grain of gained collaurum size evenly.
When the excessive concentration of trisodium citrate aqueous solution, it adds, and easily to produce local concentration in aqueous solution of chloraurate too high, just is difficult for reaching rapid dispersion in purpose wherein, thereby causes the gold grain size of gained collaurum even not; When the concentration of trisodium citrate aqueous solution was less than normal, it added the amount of aqueous solution of chloraurate just inadequate, at this moment just need to repeatedly repeat to add trisodium citrate aqueous solution, and in the gained collaurum, the grain size of gold grain still can be even not.
Same, the addition of trisodium citrate aqueous solution also has certain impact to the uniform particles degree of prepared collaurum.In view of the foregoing, preferably in preparation method of the present invention adopt that to add the concentration of the reductive agent trisodium citrate aqueous solution in aqueous solution of chloraurate be 0.5-2.0g/100mL, addition is 2mL-3mL.The concentration of trisodium citrate aqueous solution and the combination of addition are not simple combination, need just can obtain better effect through a large amount of experiments.
In addition, in the preparation process of collaurum, need to regulate the pH value to 6.0-7.0 after the aqueous solution of chloraurate after reduction is cooling in preparation method of the present invention, thereby obtain collaurum.In this preparation process, pH can have certain impact to sensitivity and the specificity of obtained test paper equally.
Further, preferably to adopt the concentration of the aqueous solution of chloraurate that adds in described B step be 0.01g/100mL in the present invention.
Further, preferably to adopt the concentration of the described trisodium citrate aqueous solution that adds be 0.5-2g/100mL in the present invention.The present invention preferably adopts when the concentration of described trisodium citrate aqueous solution is 0.5-2g/100mL, and its addition is 2.5mL; The present invention preferably adopts aforesaid way can make the gold grain size in the gained collaurum more even.
Further, the present invention preferably adopts described K
2CO
3The concentration of aqueous solution is 0.1-0.3mol/L.The present invention preferably adopts the aforesaid way can be so that the pH value of regulator solution; The K of excessive concentration
2CO
3Excessive situation easily appears in solution in the process of regulator solution pH, and the too low K of concentration
2CO
3Solution is oversize in the meeting consuming time of regulator solution pH value, so preparation method of the present invention preferably adopts K
2CO
3The concentration of solution is 0.1-0.3mol/L.
Further, after the present invention preferably adopts and adds mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min in described B step gained collaurum, add centrifuging after bovine serum albumin(BSA) sealing 20-50min, the centrifugal product of gained is the collaurum bond; Gained collaurum bond is dissolved in the phosphate buffer of pH7.2-7.4, contains the Sodium azide of 10-30mg/100mL in described phosphate buffer.
Collaurum bond of the present invention adopts above-mentioned preparation method, the holding time of gained collaurum bond is in the time of comparatively desirable, the chromatography effect of the collaurum pad of its preparation is also comparatively desirable, thereby the sensitivity of gained immune chromatography test paper and specific performance access comparatively significantly raising.
Collaurum bond of the present invention is the bond of mouse-anti human soluble ICAIU monoclonal antibody and collaurum.Usually can add sodium chloride solution to extend its pot-life in the preparation method of existing collaurum bond, can add simultaneously Sodium azide better to play the purpose that extends the pot-life in phosphate buffer.Although adding the pot-life that can extend the collaurum bond of sodium chloride and Sodium azide, both add the chromatography ability that membership reduces gained collaurum pad to a certain extent, thereby reduce sensitivity and the specificity of immune chromatography test paper.
Do not add sodium chloride solution in the preparation method of collaurum bond of the present invention, and the concentration of Sodium azide is adjusted into 10-30mg/100mL, this preparation condition is less to the chromatography capacity of gained collaurum pad, sensitivity and the specificity of gained immune chromatography test paper are effectively improved, meanwhile, the pot-life of gained collaurum bond can not be affected.
Further, the present invention preferably adopts the Sodium azide that contains 20mg/100mL in described phosphate buffer.The present invention can further adjust the concentration of Sodium azide in phosphate buffer on the basis of above-mentioned preparation condition, specifically is preferably 20mg/mL.
Further, the present invention's ratio of preferably adopting the amount of the described mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody that adds to account for collaurum is 150-180 μ g/100mL.Can be further in the preparation process of collaurum bond of the present invention preferred mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody the amount ratio that accounts for collaurum be 150-180mg/100mL, the chromatography ability of gained collaurum pad is better under described preferred implementation, and sensitivity and the specificity of gained immune chromatography test paper improve.Further, the present invention's ratio of preferably adopting the amount of the described bovine serum albumin(BSA) that adds to account for collaurum is 150-200mg/100mL.
Further, the present invention preferably adopts in described C step sample pad for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2-6 hour, in 36-38 ℃ of dry 8-12 hour sample pad.Sample pad of the present invention 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 for adopting above-mentioned optimal way to prepare gained, and this method gained sample pad is for sensitivity and the specificity effect of improving of gained test paper.
Further, the present invention preferably adopts and also contains the Triton-X100 of 0.5-3.0mL/100mL and the BSA of 0.5-2g/100mL in described C step in phosphate buffer.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 jointly be formulated in above-mentioned two kinds of materials in phosphate buffer and be used for soaking glass fibre, the sample pad of its preparation gained is better for the buffer action effect of testing sample, thereby sensitivity and the specificity of test paper is significantly improved.
Description of drawings
Fig. 1 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of premature rupture of fetal membranes group vaginal fluid sample;
Fig. 2 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of normal healthy controls group vaginal fluid sample;
Fig. 3 is the cell factor/chemotactic factor (CF) antibody chip testing result figure of healthy gravid woman's amniotic fluid sample;
Fig. 4 is the testing result figure of the enzyme linked immunosorbent assay of sICAM-1.
Embodiment
In order to make those skilled in the art understand better technical scheme of the present invention, the present invention is described in further detail below in conjunction with specific embodiment.
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 30 mature front prematures rupture of fetal membranes of example);
Normal healthy controls group: 110 routine prepartal intact fetal membranes gravid woman;
Amniotic fluid sample group: 30 customary caesarean delivered intact fetal membranes gravid woman.
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) is not observed amniotic fluid before just before giving birth and is spilt;
(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 mature front 31 years old premature rupture of fetal membranes mean age of example (the range of age 19-45 year), 30 years old normal healthy controls group mean age (the range of age 18-43 year).
The sample collection method: the gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), after peeping out vagina, stretches into the posterior fornix place with disposable sterilized cotton swab, and rotation 5 circles are taken a sample.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:
The film (RayBiotech, the U.S.) that 1) will be marked with in advance 174 cytokine antibodies is put into the detection box, then adds the 2ml confining liquid, room temperature sealing 6 hours;
2) discard confining liquid, add 1 part, 1.2ml sample to be checked, 4 ℃ of night incubation;
3) discard sample, clean 5 times with the 2ml lavation buffer solution;
4) add the detection antibody of 1ml biotin coupling, incubated at room 2 hours;
5) discard antibody, clean 5 times with the 2ml lavation buffer solution;
6) add the streptomysin Avidin of 2ml horseradish peroxidase (HRP) coupling, incubated at room 2 hours;
7) discard liquid, clean 5 times with the 2ml lavation buffer solution;
8) add chemical illuminating reagent 500 μ l, lucifuge effect 2 minutes, observations on film.
Each sample to be tested of collecting all adopts above-mentioned from premature rupture of fetal membranes group, normal healthy controls group and amniotic fluid sample group
Experiment condition detects, acquired results such as Fig. 1, Fig. 2 and shown in Figure 3.
B, experimental technique: enzyme linked immunosorbent assay quantitatively detects
Experiment condition:
Experiment material: sICAM-1ELISA detection kit (R﹠amp; D company)
1. the preparation of reagent
Lavation buffer solution (Wash Buffer)
Substrate solution (Substrate Solution): before use, the developer A in kit and developer B equivalent, lucifuge were mixed 15 minutes, each hole needs the potpourri 200 μ l of two kinds of developers.
SICAM-1 standard items: the sICAM-1 standard items are diluted with the 1ml deionized water.Standard items original liquid concentration after dilution is 250ng/ml.For guaranteeing the abundant mixing of standard items, before further diluting, standard items are put in rock gently mixing on shaking table more than at least 15 minutes.
Standard items with 250ng/ml are configured to the sICAM-1 examination criteria product that concentration is 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, 1.56ng/ml respectively; RD5-7 calibration thinning agent is as blank (0ng/ml)
2. trace routine
Add 100 μ l sICAM-1Conjugate in each micropore; The addition of standard items, tester, sample is 100 μ l, and joins in corresponding micropore.Micropore is built with the adhesive tape that kit provides.Microwell plate is placed on horizontal shaking table 500 ± 50 rev/mins at ambient temperature to be cultivated 1.5 hours.Wash plate; Every hole adds substrate solution (Substrate Solution) 200 μ l, under room temperature condition, lucifuge, keeps flat and cultivates 30 minutes; Every hole adds 50 μ l stop buffers (Stop Solution).In the hole, the color of the solution will become yellow by blueness.If in the hole, solution colour is that green or change color are inconsistent, chucked is dull and stereotyped, to guarantee the abundant mixing of solution; Survey the 450nm light absorption value in 30 minutes.
3. the production standard curve, calculate sample content.
4. will calculate the content of each sample to be tested of gained and add up and be depicted as chart, the gained chart is seen accompanying drawing 4.
Experimental result: in Fig. 4, AF of control is the amniotic fluid sample, and CVF of PROM is the vaginal fluid of premature rupture of fetal membranes group, and CVF of control is the vaginal fluid of normal healthy controls group.As can be seen from Figure 4, the concentration of sICAM-1 in healthy gravid woman's amniotic fluid sample is the highest, on average can reach 80ng/mL; More than the concentration of sICAM-1 in premature rupture of fetal membranes group vaginal fluid sample can reach 2ng/mL more than 90%; The concentration of sICAM-1 in normal healthy controls group vaginal fluid sample more than 90% below 1ng/mL.
From the experimental result of embodiment 1 as can be known, occur that main in premature rupture of fetal membranes gravid woman's vaginal fluid that one of content occurs is soluble intercellular adhesion molecule sICAM-1, and sICAM-1 comes from mainly in the amniotic fluid of rupture of membranes.
Be below that sICAM-1 as detecting target molecule, and prepares the reference examples of the immune chromatography test paper of gained according to different preparation methods in gravid woman's vaginal fluid, 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.03g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 1.2mL of 9g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into reference examples 1-on offset plate and detect the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.003g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 1.2mL of 10g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into reference examples 2-on offset plate and detect the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.005g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 1.5mL of 9g/100mL, boils 3-10 minute, coolingly regulates the pH value with the K2CO3 aqueous solution and gets collaurum to 7.0-8.0 afterwards; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into reference examples 3-on offset plate and detect the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.005g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2mL of 0.5g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into reference examples 4-on offset plate and detect the premature rupture of fetal membranes immune chromatography test paper; Described sample pad, adsorptive pads are for buying 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into reference examples 5-on offset plate and detect the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.02g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 3mL of 0.5g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2mL of 2g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 7.0-8.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.0 and is got collaurum; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 7.0 and is got collaurum; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20min of 200 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 20min of 140mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 10mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 120 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 30mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 20min of 150mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 10mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 180 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 150mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 30mg/100mL in described phosphate buffer.
Reference examples 15
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 20min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad, adsorptive pads are buys the commercially available prod.
Reference examples 17
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2 hours, in 36 ℃ of dryings 8 hours sample pad; Described adsorptive pads is for buying the commercially available prod.
Reference examples 18
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 6 hours, in 38 ℃ of dryings 12 hours sample pad; Described adsorptive pads is for buying the commercially available prod.
Reference examples 19
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2-6 hour, in 36-38 ℃ of dry 8-12 hour sample pad; Contain the Triton-X100 of 0.5mL/100mL and the BSA of 0.5g/100mL in described phosphate buffer; Described adsorptive pads is for buying the commercially available prod.
Reference examples 20
A, goat-anti human soluble intercellular adhesion molecule-1 polyclonal antibody are as the working fluid of detection line, and the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.01g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2.5mL of 2g/100mL, boils 3-10 minute, cooling rear K with 0.1-0.3mol/L
2CO
3Aqueous solution is regulated pH value to 6.5 and is got collaurum; After in the gained collaurum, every 100mL adds the mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 50min of 150 μ g in proportion, and every 100mL adds centrifuging after the bovine serum albumin(BSA) sealing 50min of 200mg in proportion, and the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 20mg/100mL in described phosphate buffer.
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate; Described sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2-6 hour, in 36-38 ℃ of dry 8-12 hour sample pad; Contain the Triton-X100 of 3.0mL/100mL and the BSA of 2g/100mL in described phosphate buffer; Described adsorptive pads is for buying the 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: available from R﹠amp; D company;
Sheep anti-mouse igg polyclonal antibody: available from the female bioengineering of upper Shanghai's style company limited;
Mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody: available from R﹠amp; D company;
Sample pad (commercially available): available from Ahlstrom company
Adsorptive pads (commercially available): available from the outstanding biological Science and Technology Ltd. in Shanghai
Collaurum (commercially available): available from the prompt peaceful bio tech ltd in Shanghai
Bovine serum albumin(BSA) (BSA): Roche company
The reference examples 1-20 immune chromatography test paper that distinct methods is prepared from is respectively used to detect the clinical sample of same gravid woman's vaginal fluid, include premature rupture of fetal membranes group and normal healthy controls group, wherein the premature rupture of fetal membranes group is 240 examples, the 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: the gravid woman gets lithotomy position (lie down with horizontal position, two lower limb separate flexing, expose vulva), after peeping out vagina, stretches into the posterior fornix place with disposable sterilized cotton swab, and rotation 5 circles are taken a sample.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.
The age distribution of sampling test object:
Utilize reference examples 1-20 respectively 500 collected routine samples to be detected, the feminine gender of gained testing result and positive number of cases statistics see Table one:
Table one
In upper table one, reference examples 1-3 is the existing prepared immune chromatography test paper of preparation method; Reference examples 4-7 is only to adjust the preparation method's of collaurum chromatographic test paper with respect to reference examples 1-3; Reference examples 8-10 is main preparation methods of the present invention; Reference examples 11-20 is the further improved preparation method of the present invention, and wherein reference examples 11-16 has mainly further improved the preparation method of collaurum bond, and reference examples 17-20 has mainly further improved 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 obviously improve sensitivity and the specificity of gained immune chromatography test paper; Same, sensitivity and the specificity of the immune chromatography test paper of the further improved preparation method's gained of the present invention also have obvious raising.
Be below only the preferred embodiment of the present invention, 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 the 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 (10)
1. preparation method who detects the 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 the 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, and described detection line working fluid and nature controlling line working fluid are packaged as chromatographic film on nitrocellulose filter;
B, the aqueous solution of chloraurate of 0.005g-0.03g/100mL is heated to boil, adding concentration under stirring is the trisodium citrate aqueous solution 2-3mL of 0.5-2g/100mL, boils 3-10 minute, uses K after cooling
2CO
3Aqueous solution is regulated the pH value and is got collaurum to 6.0-7.0; Gained collaurum and mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody are prepared into the collaurum pad;
C, the chromatographic film of A and B step gained and collaurum pad are sticked on successively according to the order of sample pad, collaurum pad, chromatographic film, adsorptive pads be prepared into described detection premature rupture of fetal membranes immune chromatography test paper on offset plate.
2. preparation method according to claim 1, it is characterized in that: the concentration of the trisodium citrate aqueous solution that adds in described B step is 2g/100mL.
3. preparation method according to claim 1, it is characterized in that: the amount of the trisodium citrate aqueous solution that adds in described B step is 2.5mL.
4. preparation method according to claim 1, is characterized in that: K in described B step
2CO
3The concentration of aqueous solution is 0.1-0.3mol/L.
5. preparation method according to claim 1, it is characterized in that: after adding mouse-anti human soluble intercellular adhesion molecule-1 labeling of monoclonal antibody 20-50min in described B step gained collaurum, add centrifuging after bovine serum albumin(BSA) sealing 20-50min, the centrifugal product of gained is the collaurum bond; Make the collaurum pad after gained collaurum bond being dissolved in the phosphate buffer of pH7.2-7.4, contain the Sodium azide of 10-30mg/100mL in described phosphate buffer.
6. preparation method according to claim 5, is characterized in that: contain the 20mg/100mL Sodium azide in described phosphate buffer.
7. preparation method according to claim 5, it is characterized in that: the ratio that the amount of the described mouse-anti human soluble intercellular adhesion molecule-1 monoclonal antibody that adds accounts for collaurum is 150-180 μ g/100mL.
8. preparation method according to claim 5, it is characterized in that: the ratio that the amount of the described bovine serum albumin(BSA) that adds accounts for collaurum is 150-200mg/100mL.
9. preparation method according to claim 1, it is characterized in that: in described C step, sample pad is for adopting following methods to be prepared from: glass fibre was soaked in the phosphate buffer of pH7.2-7.4 after 2-6 hour, in 36-38 ℃ of dry 8-12 hour sample pad.
10. preparation method according to claim 9, is characterized in that: also contain the Triton-X100 of 0.5-3.0mL/100mL and the BSA of 0.5-2g/100mL in described C step in phosphate buffer.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014131359A1 (en) * | 2013-02-28 | 2014-09-04 | 成都创宜生物科技有限公司 | Method for preparing immunochromatographic test strip that detects premature rupture of fetal membrane |
| CN108398567A (en) * | 2017-12-31 | 2018-08-14 | 深圳市金乐智能健康科技有限公司 | A kind of multifunctional household electromedical equipment control method and system |
| CN108700587A (en) * | 2016-02-24 | 2018-10-23 | 积水医疗株式会社 | The method for detecting cancer embryo fibronectin using immunochromatography |
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