CN115144598A - Quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 and application - Google Patents
Quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 and application Download PDFInfo
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Abstract
The invention provides a quantum dot fluorescence detection method of fetal fibronectin and insulin growth factor binding protein-1 and application thereof, belonging to the technical field of analytical chemistry and detection. Based on a quantum dot immunofluorescence method, cervical and vaginal secretions or mucus of pregnant and lying-in women are taken as samples to be detected, and are subjected to pretreatment of sample diluent and optimization of detection process, so that the fFN and IGFBP-1 can be detected quickly, accurately and in a wide range, and the method has good practical application value.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and detection, and particularly relates to a quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 and application thereof.
Background
The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Preterm labor refers to labor from 28 to 37 weeks of gestation, with an incidence of 5% -10% of labor. Premature delivery is currently considered to be a syndrome caused by the involvement and interaction of multiple systems such as endocrine and immunity, and has a great influence on the healthy growth of the newborn. Premature rupture of the fetal membrane refers to rupture of the fetal membrane of a pregnant woman before delivery, wherein premature rupture of the fetal membrane refers to premature rupture of the fetal membrane of the pregnant woman after 28 to 37 weeks of pregnancy, which not only can lead to premature delivery of a fetus, but also can cause maternal and infant infection or postpartum umbilical cord prolapse and the like, so that the premature rupture of the fetal membrane is predicted in advance.
Fetal fibronectin (fn) is a stromal component outside the uterine chorion, is present between the chorion and decidua, and is produced primarily by trophoblast cells. Since fusion of chorion with decidua after 21 weeks of pregnancy prevents the release of fn, normal pregnant women have very low content at 22-35 weeks of pregnancy, and are only found in cervicovaginal secretions when chorion is separated from decidua, extracellular matrix at the interface of chorion and decidua is mechanically damaged (e.g. uterine contraction) or proteolytic enzymes are degraded. Thus, at weeks 22-35 of pregnancy, the level of cervicovaginal secretions is strongly correlated with the occurrence of preterm birth, i.e. the presence of excess fn in the cervicovaginal secretions in late gestation is a marker of preterm birth.
A fetal insulin-like growth factor binding protein-1 (insulin-like growth factor binding protein-1, IGFBP-1) belongs to a composition of human liver tissues and amniotic fluid decidua tissues, and is secreted after a pregnant woman is 12 weeks gestation, and is mostly present in the amniotic fluid of a body, so that the composition is difficult to find in serum tissues and difficult to detect in vaginal secretions and urine. When the fetal membrane is broken, the insulin-like growth factor binding protein-1 in the amniotic fluid can leak out of the cervix and vagina through the rupture of the fetal membrane to become a detection mark for premature rupture of the fetal membrane. The concentration of IGFBP-1 in amniotic fluid is 100-1000 times higher than that of maternal serum, and the concentration of IGFBP-1 in other body fluids is less, so cervical mucus, urine and semen have no influence on the experiment. Therefore, IGFBP-1 is an ideal marker for predicting premature rupture of membranes in amniotic fluid and becomes a specific index for diagnosing premature rupture of membranes.
In conclusion, fetal fibronectin (fFN) and insulin growth factor binding protein-1 (IGFBP-1) in cervical-vaginal secretions or mucous membranes can be used as important indexes for predicting preterm delivery/premature rupture of membranes.
The clinical prediction of preterm labor has biochemical detection, ultrasound examination and physical speculation. The physical conjecture of the premature birth is carried out according to the uterine contraction, the birth process, the pregnancy and the menstruation of the pregnant women, the clinical value is low, and the accuracy is low.
The gold standard for diagnosing premature rupture of the fetal membranes is to inject a dye (such as methylene blue) into the amniotic cavity, and if blue liquid flows out of the vagina, the diagnosis can be carried out. However, the method is difficult to operate clinically, and causes certain pain to patients, so the method is rarely applied clinically. The most common methods in the laboratory are the nitrazine test paper method and the ferula denticulation method, but the 2 methods are affected by urine, semen and blood, so that the accuracy and the sensitivity are poor, the requirements on the collection and the quantity of the samples are high, the human factors are more, and the tiny rupture membrane with less amniotic fluid outflow cannot be detected.
The inventor finds that the existing detection methods for fFN and IGFBP-1 mainly comprise enzyme-linked immunosorbent assay (ELISA), colloidal gold method and the like. The ELISA method has long operation time, and the detection result is easily influenced by blood, pH, protein, ions and the like of the puerpera; the colloidal gold method has high detection limit, cannot accurately quantify, and is easy to misjudge by visual observation, so that the colloidal gold method is limited in clinical use.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a quantum dot fluorescence detection method for fetal fiber binding protein and insulin growth factor binding protein-1 and application thereof. Based on a quantum dot immunofluorescence method, cervical and vaginal secretions or mucus of pregnant and lying-in women are taken as samples to be detected, and are subjected to pretreatment of sample diluent and optimization of detection processes, so that the fFN and IGFBP-1 can be detected quickly, accurately and in a wide range. The present invention has been completed based on the above results.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, a quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 is provided, and the method comprises the step of measuring the fetal fibronectin and insulin growth factor binding protein-1 in a solid-phase immunochromatography mode by using a double-antibody sandwich method.
Specifically, the detection method specifically comprises the following steps:
s1, adding a sample to be detected into a sample diluent for dilution and pretreatment, and eliminating the influence of changes such as protein, pH value and ionic strength on a detection result;
s2, adding the substance to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip after color development is completed.
The sample to be tested is preferably a cervicovaginal secretion (e.g. cervicovaginal mucus).
Wherein the sample diluent consists of 0.1-0.3M Tris-HCl buffer containing 0.15-0.5% EDTA, 0.5-2% PEG20000 and 0.4-1.2% S9 (TETRONIC 1307), wherein the Tris-HCl buffer helps to stabilize the pH of the sample to be tested, the EDTA is used to chelate divalent metal ions, and the PEG20000, S9 is used as a blocking agent and a surfactant, helps to improve the specificity of the detection and to eliminate non-specific adsorption.
The immunochromatographic test strip based on the quantum dots comprises a bottom plate, wherein a blood filtering membrane, a buffer pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and the adjacent pads are connected at the joint in an overlapping manner.
The detection pad takes a nitrocellulose membrane (NC membrane) as a base pad, and a marker line, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
When the test agent is fetal fibronectin (fFN),
the marker line is coated with a quantum dot-labeled anti-fFN monoclonal antibody (Q-Ab);
the detection line is coated with a fFN capture antibody;
when the test substance is insulin growth factor binding protein-1 (IGFBP-1),
the marker line is coated with an anti-IGFBP-1 monoclonal antibody marked by quantum dots;
the detection line is coated with an IGFBP-1 capture antibody;
the quality control line is coated with goat anti-chicken IgY;
the absorbent pad can be absorbent paper, and the bottom plate can be a PVC plate.
In a second aspect of the invention, a quantum dot fluorescence detection kit for fetal fibronectin and insulin growth factor binding protein-1 is provided, wherein the detection kit at least comprises an immunochromatographic test strip (or a detection card);
the immunochromatographic test strip based on quantum dots comprises a bottom plate, wherein a blood filtering film, a buffer pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and adjacent pads are connected at the joint in an overlapping manner.
The detection pad takes a nitrocellulose membrane (NC membrane) as a base pad, and a marker line, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
When the test agent is fetal fibronectin (fFN),
the marker line is coated with a quantum dot-labeled anti-fFN monoclonal antibody (Q-Ab);
the detection line is coated with an fFN capture antibody;
when the test substance is insulin growth factor binding protein-1 (IGFBP-1),
the marker line is coated with an anti-IGFBP-1 monoclonal antibody marked by quantum dots;
the detection line is coated with an IGFBP-1 capture antibody;
the quality control line is coated with goat anti-chicken IgY;
the absorbent pad can be absorbent paper, and the bottom plate can be a PVC plate.
Specifically, the immunochromatographic test strip is prepared by adopting the following method:
s1, preparing a coating film of a detection line and a quality control line region: taking an NC membrane and a PVC plate, pasting a non-sample application surface of the NC membrane on a set position of the PVC plate, taking a fetal fiber binding protein antibody, an insulin growth factor binding protein-1 antibody and goat anti-chicken IgY, scribing a membrane to coat on a detection line (comprising a first detection line and a second detection line) and a quality control line position of the NC membrane, and drying;
s2, preparing a coating film of the marker line region:
a: adding cadmium selenide quantum dots into MES buffer solution, and performing activation treatment;
b: taking the quantum dot marking solution prepared in the step a, sequentially adding EDC and NHS into the quantum dot marking solution, and adding a fetal fiber binding protein antibody for light-shielding reaction;
c: taking the marked mixed solution in the step b for centrifugation, and eluting by taking a borate buffer solution as an eluent;
d: c, diluting the marked solution obtained in the step c by using a compound solution, and uniformly mixing to obtain a fetal fibronectin antibody marked by a quantum dot; the compound solution comprises the following components: 0.1-0.3M HEPES buffer system containing 0.5-2% polyvinylpyrrolidone (PVP) K-30,1-3% casein, 3-8% hydroxypropyl β -cyclodextrin and 0.5-2% Tween20;
e: b, preparing a quantum dot labeled insulin growth factor binding protein-1 antibody and a quantum dot labeled chicken IgY antibody according to the same method of the steps a to d; then mixing the three labeled antibodies uniformly to prepare a binding pad coating solution, marking the binding pad coating solution on a marker line area of an NC membrane, and drying to obtain the antibody;
s3, preparing a blood filtering membrane;
s4, preparing the cushion pad, comprising:
a: preparing a buffer treatment solution containing 0.1M Tris-HCl buffer system (to stabilize the pH of a sample to be tested), 20-150 μ g/ml anti-red blood cell antibody (RBC antibody) for binding red blood cells in blood, 4-10% trehalose for protecting the stability of the antibody, and 0.2-0.8% Tween80 as a surfactant;
b: cutting the glass fiber film, and performing coating pretreatment; drying;
s5, assembling:
tearing off the sticky paper at the corresponding position on the bottom plate, sticking the absorbent paper above the NC film of the coating film, and covering the NC film; sticking a cushion pad below the NC membrane of the coating membrane to cover the NC membrane; and sticking the blood filtering membrane below the cushion pad, and covering the cushion pad to obtain the assembled large plate.
S6, slitting and casing: after the width of the test strip is set, cutting is carried out, and the prepared test strip is obtained;
s7, placing the test strip into a detection card shell in the forward direction, pressing the shell and packaging well to obtain the single human fetal fibronectin and insulin growth factor binding protein-1 combined detection card.
The detection kit also comprises a sample diluent;
wherein the sample diluent is composed of 0.1M Tris-HCl buffer containing EDTA, PEG20000 and S9 (TETRONIC 1307), and more specifically, the sample diluent is composed of 0.1-0.3M Tris-HCl buffer containing 0.15-0.5% EDTA, 0.5-2% PEG20000 and 0.4-1.2% S9 (TETRONIC 1307).
Wherein, buffer solution helps stabilizing the sample pH value to be measured, EDTA is used for chelating metal ion, and PEG20000, S9 use as blocking agent and surfactant active, help promoting the specificity of detection and can eliminate non-specific adsorption.
In a third aspect of the invention, there is provided the use of the detection method and/or kit described above in any one or more of:
1) Detecting fetal fibronectin;
2) Detecting insulin growth factor binding protein-1;
3) Preterm/premature rupture of membranes diagnosis or assisted diagnosis.
The beneficial technical effects of one or more of the above technical solutions are as follows:
the technical scheme optimizes the pretreatment and detection processes of the fetal fibronectin and insulin growth factor binding protein-1, so that the substances can be detected quickly (the detection can be completed within 3 min), accurately and widely, and the method has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a schematic diagram of the test strip of the present invention;
wherein, the kit comprises 1-blood filtering membrane, 2-buffer pad, 3-NC membrane, 4-water absorption pad, 5-PVC bottom plate, 6-detection line, 7-quality control line and 8-marker line; the arrow direction is the chromatography direction;
FIG. 2 is a schematic diagram of the external structure of the detection card of the present invention:
wherein, 9-sample adding hole, 10-observation window (window); the arrow direction is the direction of chromatography;
FIG. 3 is a graph of a fetal fibronectin standard curve of the invention;
FIG. 4 is a graph of a standard curve for insulin growth factor binding protein-1 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. Unless otherwise indicated, the experimental procedures of the specific conditions in the following detailed description are generally in accordance with conventional methods and conditions of biology within the skill of the art, which are fully explained in the literature.
After the premature rupture of the fetal membranes occurs, the uterus structure and the physicochemical environment of the pregnant women are changed, a large amount of protein, decidua cell fragments, blood cells (vaginal bleeding occurs) and the like are adhered to cervical/vaginal mucus, and the pH value is changed. All of these variations affect the detection accuracy of the prior art.
In view of the above, the present invention provides a method for detecting fetal fibronectin and insulin growth factor binding protein-1, and specifically, the principle of the method for detecting fetal fibronectin (fFN) based on quantum dot fluorescence technology of the present invention is as follows:
the kit adopts a double-antibody sandwich method, and a marker line, a detection line and a quality control line are sequentially arranged on a nitrocellulose membrane (NC membrane) of a detection pad: wherein the upper end of the NC membrane is marked with a mixture of quantum dot labeled fFN antibody (Q-Ab) and quantum dot labeled chicken IgY antibody as a marker line; scratching fFN capture antibody as a detection line, scratching goat anti-chicken IgY as a quality control line. After sample application, the antigen to be detected (fFN) in the sample is combined with the quantum dot labeled antibody respectively to form a quantum dot labeled antibody-antigen complex. And then, the sample passes through a detection line region solidified with the fFN capture antibody and is combined with the detection line antibody to form a quantum dot labeled antibody-antigen-antibody compound, the uncombined quantum dot labeled chicken IgY antibody continuously moves forwards and is combined with goat anti-chicken IgY antibody in a quality control line region, and the quality control calibration reaction is completed. The quantum dots are excited by a matched instrument to generate a fluorescence signal, a quantitative detection result is obtained, the content of the antigen to be detected (fFN) in the sample and the fluorescence signal intensity are in positive correlation in a certain range, and the concentration of the sample to be detected can be obtained through calculation of a standard curve.
The principle of the detection method of the insulin growth factor binding protein-1 (IGFBP-1) based on the quantum dot fluorescence technology is as follows:
the kit adopts a double-antibody sandwich method, and a marker line, a detection line and a quality control line are sequentially arranged on a nitrocellulose membrane (NC membrane) of a detection pad: wherein the upper end of the NC membrane is marked with a mixture of a quantum dot labeled IGFBP-1 antibody (Q-Ab) and a quantum dot labeled chicken IgY antibody as a marker line; and drawing an IGFBP-1 capture antibody as a detection line, and drawing sheep anti-chicken IgY as a quality control line. After sample addition, the antigen to be detected (IGFBP-1) in the sample is respectively combined with the quantum dot labeled antibody to form a quantum dot labeled antibody-antigen complex. And then, the sample passes through a detection line region solidified with an IGFBP-1 capture antibody and is combined with the detection line antibody to form a quantum dot labeled antibody-antigen-antibody compound, the uncombined quantum dot labeled chicken IgY antibody continuously moves forwards and is combined with the goat anti-chicken IgY antibody in a quality control line region, and the quality control calibration reaction is completed. The quantum dots are excited by a matched instrument to generate fluorescence signals, a quantitative detection result is obtained, the content of the antigen to be detected (IGFBP-1) in the sample and the fluorescence signal intensity are in positive correlation in a certain range, and the concentration of the sample to be detected can be obtained through calculation of a standard curve.
The invention treats the cervical and vaginal secretion (such as cervical and vaginal mucus) sample with sample diluent, directly uses a liquid transfer device to sample the sample and add the sample into a sample adding hole, samples, and takes out and detects after spreading the plate for 3 min. Particularly, in view of the wide concentration range of the object to be detected, the detection range of the product needs to be as large as possible, so that the marker is drawn on the NC membrane, the display board is accelerated, the detection range is increased, and a stable and accurate result can be obtained within 3 min.
1) The accuracy of the obtained experimental result is good, and the relative deviation with the standard substance is within +/-10%;
2) The obtained experimental result has good repeatability, and CV is not more than 10%;
3) The sample does not need special treatment, and the sample with little vaginal bleeding phenomenon can be used for detection;
4) The kit and the sample diluent do not need to be refrigerated, and can be transported and stored at normal temperature.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
The kit for combined detection of the fetal fibronectin and insulin growth factor binding protein-1 comprises a PVC (polyvinyl chloride) plate and a nitrocellulose membrane (NC membrane) arranged on the PVC plate, wherein a cushion pad made of a cellophane membrane is arranged at the upper end of the NC membrane, a blood filtering membrane is arranged at the upper end of the cushion pad, and a water absorbing pad (paper) is arranged at the other end of the NC membrane corresponding to the cushion pad and the blood filtering membrane. The marker line area on the nitrocellulose membrane (NC membrane) is coated with a solution of 1:1:1, quantum dot marking fetal fiber binding protein antibody, quantum dot marking insulin growth factor binding protein-1 antibody and quantum dot marking chicken IgY antibody; the NC membrane is sequentially provided with a detection line I, a detection line II and a quality control line, the detection line I is coated with a fetal fiber binding protein antibody, the detection line II is coated with an insulin growth factor binding protein-1 antibody, and the quality control line is coated with goat anti-chicken IgY; the quantum dots are cadmium selenide quantum dots, as shown in fig. 1.
The preparation method of the kit for the combined detection of the fetal fibronectin and insulin growth factor binding protein-1 comprises the following specific operation steps:
1) Preparing a coating film (detection line and quality control line area): taking an NC membrane and a PVC plate, pasting a non-sample application surface of the NC membrane on a set position of the PVC plate, taking a fetal fiber binding protein antibody, an insulin growth factor binding protein-1 antibody and goat anti-chicken IgY with the concentration of 1.2mg/ml, scratching a membrane by a membrane scratching gold spraying instrument according to 1 mu l/cm, coating on a detection line I, a detection line II and a quality control line position of the NC membrane, and drying for 8h at 37 ℃ to obtain the coating membrane.
2) Preparation of envelope film (marker line region):
a: adding 30 μ l of cadmium selenide quantum dots into 3ml of MES buffer (pH 6.5) with concentration of 50mM, activating for 15min, and storing at 4 deg.C in dark place;
b: taking the quantum dot marking solution prepared in the step a, sequentially adding 5 mul of each of 20mg/ml EDC and 20mg/ml NHS, adding 200 mul of fetal fiber binding protein antibody of 5mg/ml, and reacting for 2h in a dark place;
c: b, transferring the marked mixed solution in the step b to a 100KD ultrafiltration tube for centrifugation, and centrifuging for 15min at 4000g/min to ensure that the volume of the centrifuged liquid is less than 1/10 of the original volume; eluting 5 times with 0.1M borate buffer (pH8.0) to ensure that the volume of the liquid after each centrifugation is less than 1/10 of the original volume;
d: c, diluting the marked solution obtained in the step c by using a compound solution, and uniformly mixing to obtain a fetal fibronectin antibody marked by a quantum dot; the formula of the compound solution is as follows: 0.2M HEPES buffer system, 1% PVP K-30,2% casein, 5% hydroxypropyl β -cyclodextrin, 1% Tween20;
e: preparing a quantum dot labeled insulin growth factor binding protein-1 antibody and a quantum dot labeled chicken IgY antibody according to the same method of the steps a to d; the three labeled antibodies were then mixed at a volume ratio of 1:1:1, uniformly mixing to prepare a bonding pad coating solution, scribing on a marker line area of an NC membrane by using a membrane scribing metal spraying instrument according to 1.5 mu l/cm, and drying for 4 hours at 37 ℃ to obtain a coating membrane;
3) Preparing a blood filtering membrane: the glass cellulose membrane was cut as a blood filter.
4) Preparing a cushion pad:
a: preparing a cushion pad treating fluid, wherein the formula is as follows: 0.1M Tris-HCl buffer, 100. Mu.g/ml anti-erythrocyte antibody (RBC antibody), 5% trehalose, 0.5% Tween80;
b: cutting the glass fiber film, and applying a cushion pad treatment solution to carry out coating pretreatment on the glass fiber film;
c: putting into an oven, and drying at 37 ℃ for 6h.
5) Assembling:
tearing off the sticky paper at the corresponding position on the bottom plate, and sticking the absorbent paper above the NC film of the coating film to cover the NC film by 1.5mm; sticking a cushion pad below the NC membrane of the coating membrane, and covering the NC membrane by 1.5mm; and (3) sticking the blood filtering membrane below the cushion pad, and covering the cushion pad for 2mm to obtain the assembled large plate.
6) Slitting and casing: a microcomputer cutting machine is used, the width of the test strip is set, and then the test strip is cut to obtain the prepared test strip; the test paper is placed into a detection card shell in the forward direction, and the detection card is well encapsulated by pressing the shell, so that the single human fetal fibronectin and insulin growth factor binding protein-1 combined detection card is obtained, as shown in figure 2.
In addition, it should be noted that, provided in the above examples is a combined detection card of fetal fibronectin and insulin growth factor binding protein-1. Of course, a test strip for detecting fetal fibronectin alone and a test strip for detecting insulin growth factor binding protein-1 alone can be prepared, and the difference is only that when the test strip for detecting fetal fibronectin antibody alone is prepared, the mass ratio of the coating on the binding pad is 1:1, a mixture of a quantum dot labeled fetal fibronectin antibody and a quantum dot labeled chicken IgY antibody; the nitrocellulose membrane (NC membrane) is only provided with a detection line and a quality control line, the detection line is coated with a fetal fiber binding protein antibody antigen, and the quality control line is coated with goat anti-chicken IgY; when the test strip for separately detecting the insulin growth factor binding protein-1 is prepared, the binding pad is coated with the test strip with the mass ratio of 1:1 quantum dot labeling insulin growth factor binding protein-1 antibody and quantum dot labeling chicken IgY antibody; the nitrocellulose membrane (NC membrane) is only provided with a detection line and a quality control line, the detection line is coated with an insulin growth factor binding protein-1 antigen, and the quality control line is coated with goat anti-chicken IgY. The specific preparation method is adjusted correspondingly.
Furthermore, the kit also contains a sample diluent consisting of 0.2M Tris-HCl buffer containing 0.2% EDTA, 1% PEG20000 and 1% S9.
Test example:
diluting fetal fibronectin to 20ng/ml, 50ng/ml, 200ng/ml, 500ng/ml, 1500ng/ml, 4000ng/ml; the insulin growth factor-binding protein-1 was diluted to 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 400ng/ml, 800ng/ml, 1200ng/ml as standard solutions, and blank solutions were prepared at the same time.
Taking 70 mu l of standard substance solution with each concentration, respectively adding the standard substance solution with each concentration to the sample pad of the detection card prepared in the embodiment 1, putting the detection card with the display plate after 3min into a fluorescence immunoassay analyzer (Shandong Zifeng Biotech limited) for interpretation, obtaining the fluorescence values of detection lines and quality control lines with different concentrations, and recording the corresponding relation between the fluorescence ratio (T1/C, T/C) of the standard substance with each concentration and the concentration of the standard substance. And (4) establishing a standard curve equation according to the fluorescence signal-calibration concentration value of the measurement result. As shown in fig. 3-4, the standard curve regression equation for fetal fibronectin is: LOG (Y-0.0027) =1.1373 × LOG (X) -3.3496; the standard curve equation for insulin growth factor binding protein-1 is: LOG (Y-0.0415) =1.2175 XLOG (X) -2.5062. And burning the standard curve into the ID card to prepare a fetal fibronectin and insulin growth factor binding protein-1 calibration card.
In addition, a blank solution was added to the test card prepared in example 1, and the reverse concentration of fetal fibronectin was 0ng/ml, and the reverse concentration of insulin growth factor-binding protein-1 was 0ng/ml.
100 random clinical samples are taken, the kit prepared in the embodiment 1 of the invention is adopted to detect the concentrations of fetal fibronectin and insulin growth factor binding protein-1, and the detection method comprises the following steps: inserting the calibration card into a fluorescence immunoassay analyzer (Shandong Zifeng Biotechnology Co., ltd.) to calibrate the apparatus; adding 10 μ l sample into 90 μ l sample diluent, mixing, adding 70 μ l sample solution dropwise into the sample adding hole, placing the display plate in a fluorescence immunoassay analyzer (Shandong Zifeng Biotechnology Co., ltd.) for 3min, and measuring and recording the detection results of fetal fibronectin and insulin growth factor binding protein-1. The results are shown in table 1 below:
TABLE 1 detection results of fetal fibronectin, insulin growth factor binding protein-1
Therefore, the kit can directly and quantitatively detect the concentrations of the fetal fibronectin and the insulin growth factor binding protein-1, is convenient and quick, can obtain results in 3min, and is suitable for popularization and use in obstetrical hospitals.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (9)
1. A quantum dot fluorescence detection method for fetal fibronectin and insulin growth factor binding protein-1 comprises the steps of determining the fetal fibronectin and insulin growth factor binding protein-1 in a solid-phase immunochromatography mode by using a double-antibody sandwich method;
the detection method specifically comprises the following steps:
s1, adding a sample to be detected into a sample diluent for dilution and pretreatment;
s2, adding the object to be detected to an immunochromatography test strip based on quantum dots, and carrying out fluorescence detection on the test strip subjected to color development;
the sample to be detected is cervical and vaginal secretion.
2. The method of fluorescence detection of quantum dots according to claim 1, wherein the sample diluent is composed of 0.1-0.3M Tris-HCl buffer containing 0.15-0.5% EDTA, 0.5-2% PEG20000 and 0.4-1.2% S9.
3. The quantum dot fluorescence detection method of claim 1, wherein the immunochromatographic test strip based on quantum dots comprises a bottom plate, a blood filtering membrane, a buffer pad, a detection pad and a water absorption pad are sequentially arranged on the bottom plate, and adjacent pads are connected in an overlapping manner at the connection position.
4. The quantum dot fluorescence detection method of claim 3, wherein the detection pad uses a nitrocellulose membrane as a base pad, and a marker line, a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane.
5. The quantum dot fluorescence detection method of claim 3,
when the test substance is a fetal fibronectin,
the marker line is coated with a quantum dot marker anti-fFN monoclonal antibody;
the detection line is coated with a fFN capture antibody;
when the test substance is insulin growth factor binding protein-1,
the marker line is coated with an anti-IGFBP-1 monoclonal antibody marked by quantum dots;
the detection line is coated with an IGFBP-1 capture antibody;
the quality control line is coated with goat anti-chicken IgY;
the water absorption pad is made of water absorption paper, and the bottom plate is made of a PVC plate.
6. A quantum dot fluorescence detection kit for fetal fibronectin and insulin growth factor binding protein-1 is characterized by at least comprising an immunochromatography test strip;
the immunochromatography test strip is prepared by adopting the following method:
s1, preparing a coating film in a detection line and quality control line area: taking an NC membrane and a PVC plate, pasting a non-sample application surface of the NC membrane on a set position of the PVC plate, taking a fetal fiber binding protein antibody, an insulin growth factor binding protein-1 antibody and goat anti-chicken IgY, scribing a membrane, coating the membrane on a detection line and a quality control line of the NC membrane, and drying;
s2, preparing a coating film of the marker line region:
a: adding cadmium selenide quantum dots into MES buffer solution for activation treatment;
b: taking the quantum dot marking solution prepared in the step a, sequentially adding EDC and NHS into the quantum dot marking solution, and adding a fetal fiber binding protein antibody into the quantum dot marking solution to perform a light-proof reaction;
c: b, centrifuging the marked mixed solution in the step b, and eluting by using a borate buffer solution as an eluent;
d: c, diluting the marked solution obtained in the step c by using a compound solution, and uniformly mixing to obtain a fetal fibronectin antibody marked by a quantum dot; the compound solution comprises the following components: 0.1-0.3M HEPES buffer system containing 0.5-2% polyvinylpyrrolidone K-30,1-3% casein, 3-8% hydroxypropyl β -cyclodextrin and 0.5-2% Tween20;
e: b, preparing a quantum dot labeled insulin growth factor binding protein-1 antibody and a quantum dot labeled chicken IgY antibody according to the same method of the steps a to d; then mixing the three labeled antibodies uniformly to prepare a binding pad coating solution, marking the binding pad coating solution on a marker line area of an NC membrane, and drying to obtain the antibody;
s3, preparing a blood filtering membrane;
s4, preparing the cushion pad, comprising:
a: preparing a buffer treatment solution, wherein the components of the buffer treatment solution comprise 0.1M Tris-HCl buffer system, 20-150 μ g/ml anti-erythrocyte antibody, 4-10% trehalose, 0.2-0.8% Tween80;
b: cutting the glass fiber film, and performing coating pretreatment; drying;
s5, assembling:
tearing off the sticky paper at the corresponding position on the bottom plate, sticking the absorbent paper above the NC film of the coating film, and covering the NC film; sticking a cushion pad below the NC membrane of the coating film to cover the NC membrane; sticking a buffer film below the combining pad, and covering the buffer pad to obtain an assembled large plate;
s6, slitting and casing: and after the width of the test strip is set, cutting to obtain the prepared test strip.
7. The quantum dot fluorescence detection kit of claim 6, wherein the immunochromatographic test strip is prepared by a method further comprising:
s7, placing the test strip into a detection card shell in the forward direction, pressing the shell and packaging well to obtain the single human fetal fibronectin and insulin growth factor binding protein-1 combined detection card.
8. The quantum dot fluorescence detection kit of claim 6, wherein the detection kit further comprises a sample diluent; the sample diluent consists of 0.1-0.3M Tris-HCl buffer containing 0.15-0.5% of EDTA, 0.5-2% of PEG20000 and 0.4-1.2% of S9.
9. Use of the assay method of any one of claims 1 to 5 or the kit of any one of claims 6 to 8 in any one or more of:
1) Detecting fetal fibronectin;
2) Insulin growth factor binding protein-1 assay.
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