Immunochromatography test paper for rapid semi-quantitative detection of urine exosome soluble Fms-like tyrosine kinase receptor 1 and detection method thereof

Abstract

The invention relates to the technical field of biological detection, and particularly discloses immunochromatography detection test paper for rapid semi-quantitative detection of urine exosome soluble Fms-like tyrosine kinase receptor 1 and a detection method of the immunochromatography detection test paper. The immunochromatography detection test paper can rapidly detect the content of soluble Fms-like tyrosine kinase-1 in urine exosome; whether a patient suffers from preeclampsia or not can be judged through the test paper without depending on instruments and equipment. The immunochromatography test paper provided by the invention can be used for diagnosing and distinguishing the contents of soluble Fms-like tyrosine kinase-1 in pregnant women, namely 4ng / mL and 10ng / mL, and can be used for accurately judging high-risk groups. According to the application, the detection time is shortened, the detection accuracy is also improved, instant detection, mobile detection and household self-service detection are realized, the content and change of soluble Fms-like tyrosine kinase-1 can be automatically and timely mastered, and the risk of preeclampsia occurrence can be predicted in advance.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to an immunochromatographic test strip for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes and a detection method thereof. Background Art

[0002] Eclampsia and preeclampsia (PE) are acute and critical obstetric diseases that can cause unexplained convulsions in pregnant women and are one of the causes of perinatal and maternal death. Preeclampsia is a pregnancy-specific multisystem disease. The main clinical manifestations of patients are sudden high blood pressure, proteinuria and endothelial dysfunction after 20 weeks of pregnancy. The disease is the main cause of maternal death and is also very likely to cause premature birth in pregnant women. The pathogenesis of preeclampsia is still unclear, and there is currently no effective prevention and treatment. With the in-depth study of the pathogenesis of PE, more and more studies have begun to explore new treatment measures. In recent years, a large number of studies have suggested that it may be related to placental dysfunction and extensive vascular endothelial damage. The anti-angiogenic factor, soluble Fms-like tyrosine kinase receptor 1 (sFlt-1), can cause vascular endothelial damage and angiogenesis disorders. When pregnancy is abnormal, the level of soluble Fms-like tyrosine kinase receptor 1 in the blood is significantly increased, which can reflect the degree of endothelial cell function damage. Soluble Fms-like tyrosine kinase receptor 1 is expected to be one of the early prediction and evaluation indicators of preeclampsia.

[0003] Soluble Fms-like tyrosine kinase-1 in the blood of pregnant women belongs to the type III receptor tyrosine kinase family, with a molecular weight of approximately 63.6 kd; it is composed of 1 to 6 immunoglobulin-like domains in the extracellular region, without a tyrosine kinase domain, and the coding region is completely consistent with the extracellular region of Fms-like tyrosine kinase-1, which can compete with membrane surface receptors for binding to vascular endothelial growth factor. Soluble Fms-like tyrosine kinase-1 was initially discovered on umbilical vein-like cells as a direct inhibitor of vascular endothelial growth factor and placental growth factor. Elevated serum levels can lead to poor growth of placental trophoblasts and blood vessels, thereby causing endothelial cell damage. Soluble Fms-like tyrosine kinase-1 mainly affects the biological functions of vascular endothelial growth factor and placental growth factor by binding to vascular endothelial growth factor and placental growth factor, and participates in the formation, remodeling and replantation of blood vessels. Therefore, soluble Fms-like tyrosine kinase-1 and the prediction of preeclampsia have become a research focus. Changes in the concentration levels of placental growth factor and soluble Fms-like tyrosine kinase-1 in the blood circulation can distinguish normal pregnancy from preeclampsia before clinical symptoms appear. Soluble Fms-like tyrosine kinase-1 is the first biochemical marker that has been verified by large-scale clinical trials and can be used to predict preeclampsia in pregnant women. Studies have confirmed that the levels of soluble Fms-like tyrosine kinase-1 and placental growth factor in the serum and urine of pregnant women with preeclampsia are higher than those of normal pregnant women.

[0004] Exosomes can be used as markers for preeclampsia and other diseases. They are about 30-150nm in diameter and 1.13-1.21g / m1 in density. They are membrane vesicles actively secreted by cells and are formed through a series of regulatory processes such as "endocytosis-fusion-excretion". In recent years, a large number of domestic and foreign studies have reported that exosomes reflect the physiological and pathological state of the source cells and can be used as biomarkers for early prediction of diseases. Exosomes exist naturally in body fluids, including blood, saliva, urine, ascites and breast milk. Exosomes carry unique or key functional molecules of the source cells, resulting in different biological functions of exosomes from different sources. Therefore, it is possible to determine the changes of certain proteins and nucleic acids in certain cells based on the substances contained in the detection of exosomes. At present, there are many diseases, including acute and severe gynecological diseases, cardiovascular diseases, tumors, etc., which can detect exosomes in patient serum or plasma and liquids. The protein content in the samples is studied through certain experimental and data analysis methods, and the sample data of normal individuals are compared to achieve early prediction, providing an important basis for efficacy judgment and prognosis.

[0005] Exosomes can exist stably in urine, and urine samples are easy to obtain in large quantities and can avoid traumatic damage. Therefore, if the expression of soluble Fms-like tyrosine kinase-1 in urine exosomes can be detected, it is expected to be used for non-invasive prediction and evaluation of preeclampsia. Although differential centrifugation is considered the gold standard for exosome isolation, it also has some disadvantages: it is time-consuming and labor-intensive, highly dependent on manual labor, and has a low recovery rate. Repeated centrifugation operations are likely to damage the exosome vesicles, thereby reducing their quality, and they are easy to aggregate into blocks and cause contamination, which is not conducive to downstream analysis. At present, the methods of extracting and enriching exosomes using immunomagnetic beads, ultrafiltration, precipitation or kits have limitations, because each cell secretes at least 10 different types of nanovesicles, and it is difficult or impossible to distinguish these vesicles based on similar size, density and surface markers. Therefore, scholars at home and abroad are actively exploring an experimental method that can quickly and efficiently separate exosomes for early prediction of preeclampsia.

[0006] At present, patent CN202210182153.7 discloses a soluble Fms-like tyrosine kinase-1 assay kit and its preparation method, which uses a fluorescent immunoassay analyzer to detect the soluble Fms-like tyrosine kinase-1 concentration in serum and plasma samples. Based on the in vitro instant detection platform and its detection method of exosomes, exosomes are separated and extracted by air filtration membranes for semi-quantitative detection of specific tumor markers. However, this patent does not propose ideas and technologies for the separation, purification and detection of all exosomes, nor does it propose specific indicators and methods for the separation, purification and detection of soluble Fms-like tyrosine kinase-1 in urine exosomes.

[0007] In addition, the existing chemiluminescence technology, enzyme-linked immunosorbent assay technology, and fluorescent immunosorbent assay technology for detecting soluble Fms-like tyrosine kinase-1 all collect peripheral blood samples, and there are no reports on the rapid separation, extraction, and detection of urine exosomal soluble Fms-like tyrosine kinase-1 as a specific marker for preeclampsia.

[0008] Considering that preeclampsia is an acute and critical obstetric disease involving the lives of both pregnant women and fetuses, and that gestational hypertension is a special clinical type, blood pressure rises rapidly and is difficult to control, and a large amount of proteinuria occurs, combined with target organ damage, which seriously endangers the health of mothers and infants. Early prediction and evaluation of preeclampsia can reduce adverse maternal and infant events. Therefore, it is necessary to establish a rapid semi-quantitative detection kit for soluble Fms-like tyrosine kinase-1 in urine exosomes, quickly predict the degree and severity of the disease in pregnant women and fetuses, realize instant detection, and facilitate clinical application to prevent the occurrence of adverse pregnancy outcomes. Summary of the invention

[0009] The purpose of the present application is to overcome the shortcomings of the above-mentioned prior art and provide an immunochromatographic test strip and its application for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes. The immunochromatographic test strip of the present application can quickly detect soluble Fms-like tyrosine kinase-1 in urine exosomes, and can determine whether a patient has preeclampsia by using the test strip without relying on instruments and equipment.

[0010] To achieve the above purpose, the technical solution adopted by this application is:

[0011] The present application provides an immunochromatographic test strip for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes, wherein the immunochromatographic test strip comprises a sample pad area, an antibody area, a gold-labeled pad area, a nitrocellulose membrane area, and a water-absorbing pad area connected in sequence;

[0012] The sample pad area is dripped with urine filtered by the filter head; the urine contains exosomes; the filter head is used to quickly separate and extract urine exosomes; the antibody area is coated with galectin-1 monoclonal antibody; the galectin-1 monoclonal antibody is used to further purify urine exosomes (fine product) on the basis of quickly separating and extracting urine exosomes (crude product) through the filter head; the gold label pad area is coated with soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold particles; the nitrocellulose membrane area is provided with a detection T line and a quality control C line;

[0013] The soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold particles binds to the soluble Fms-like tyrosine kinase-1 antigen in urine.

[0014] The labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 is the first antibody, which is the mouse anti-human soluble Fms-like tyrosine kinase-1 monoclonal antibody AB18A6 antibody subtype, and is used as the colloidal gold particle labeled antibody.

[0015] After extensive research and experiments, the inventors of the present application discovered that in the present application, urine filtered through a filter head is dripped onto the sample pad area, and then the urine is transferred to the antibody area through chromatography, where the glycoproteins on the surface of the exosomes in the urine (a large number of glycoproteins exist on the surface of the exosomes, including galectin-1 antigen) are combined with the galectin-1 monoclonal antibody coated on the antibody area, thereby further enriching, extracting and purifying the exosomes in the urine, thereby improving the quality of the extracted exosomes. At the same time, in order to make up for the deficiency that the filter head extracts exosomes from urine based on the size of the volume, it is inevitable that other miscellaneous protein molecules of the same volume are mixed, and the galectin-1 monoclonal antibody must be added to purify the exosomes to improve the quality.

[0016] This step has the advantages of low cost, simple operation, high yield and purity, and intact exosome structure; the urine is then chromatographed, and the soluble Fms-like tyrosine kinase-1 antigen in the urine is combined with the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1, and the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 (AB21D9 antibody subtype) coated on the nitrocellulose membrane is on the detection T line of the detection card. When the test urine flows to the detection T line, the antigen to be tested binds to the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold, and under the action of chromatography, binds to the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated in the detection area (T line) on the nitrocellulose membrane to form a double antibody sandwich complex (i.e., labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex), and then stays at the T line to form a red line T detection line (a positive result is shown here). The detection T line is set with detection concentrations of 4ng / mL and 10ng / mL respectively (there are also detection T line 1 group and detection T line 2 group respectively). When the detection concentration of the detection T line of 10ng / mL is reached, the detection T line 2 group will display a red line; if it only reaches the detection concentration of the detection T line of 4ng / mL, the detection T line 1 group will also display a red line. When the test urine flows through the quality control C line with goat anti-mouse IgG antibody fixed on the nitrocellulose membrane area, the excess (not intercepted by the detection T line) soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold will continue to flow to the quality control C line and combine with the goat anti-mouse antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Similarly, due to the large amount of colloidal gold gathered here, a red control line C is formed again, that is, the quality control C line appears (negative results are shown here), indicating that the experiment is effective. Therefore, the color changes of the detection T line and the quality control C line on the nitrocellulose membrane area (the T line on the nitrocellulose membrane area is used to detect the soluble Fms-like tyrosine kinase-1 antigen content on exosomes in urine, and the C line is used as a control line) can be observed to predict whether preeclampsia is present.

[0017] The immunochromatographic test strips of the present application can quickly detect soluble Fms-like tyrosine kinase-1 in urine excretion, and can predict whether a patient has preeclampsia through the test strips without relying on instruments and equipment. In addition, the immunochromatographic test strips of the present application can also distinguish between the soluble Fms-like tyrosine kinase-1 content of 4ng / mL and 10ng / mL in pregnant women while evaluating, and can accurately identify high-risk groups. The present application not only shortens the detection time, but also improves the accuracy of the detection, and realizes instant detection, mobile detection, and home self-service detection. The content and changes of soluble Fms-like tyrosine kinase-1 can be grasped in time by the patient, and the risk of preeclampsia can be predicted in advance.

[0018] The immunochromatographic test paper of the present application has the advantages of simple operation, convenient detection, rapid result acquisition, strong specificity, high sensitivity and low cost.

[0019] As a preferred embodiment of the immunochromatographic test strip described in the present application, the sample pad area is dripped with a filter head made of polytetrafluoroethylene, unsaturated polyester resin and epoxy resin to filter urine, and the filter head is used to quickly separate and extract urine exosomes.

[0020] This application uses a special filter head to quickly separate and extract exosomes (crude product) in urine, and then the filtered urine is chromatographed to the antibody area, and the glycoprotein (galactose lectin 1 antigen) in the urine is combined with the galactose lectin-1 monoclonal antibody coated on the antibody area to further purify the exosomes (fine product) in the urine and improve the quality of the extracted exosomes. At the same time, it makes up for the deficiency that the filter head extracts exosomes from urine based on the size of the volume, and inevitably mixes with other miscellaneous protein molecules of the same volume.

[0021] As a preferred embodiment of the immunochromatographic test paper described in the present application, the filter head includes the following components in parts by weight:

[0022] 10-40 parts of polytetrafluoroethylene, 50-75 parts of unsaturated polyester resin and 5-25 parts of epoxy resin.

[0023] The filter head has 6 circular holes, each with a diameter less than 300 nm.

[0024] The preparation method of the filter head comprises the following preparation steps:

[0025] A mixture of the above components (10-40 parts of polytetrafluoroethylene, 50-75 parts of unsaturated polyester resin and 5-25 parts of epoxy resin) by weight is prepared, mechanically stirred for 30-60 minutes, placed in a vacuum oven, and bubbles therein are removed by vacuuming. The mixture from which bubbles have been removed by vacuuming is poured into a polytetrafluoroethylene mold, cured at room temperature for 2-4 hours, and then cured at 150-300° C. for 6-10 hours to form.

[0026] The homemade filter head adopts polytetrafluoroethylene composite material. In the prior art, although there are many methods for preparing polytetrafluoroethylene porous materials, they are not suitable for use in the present application, and homemade polytetrafluoroethylene porous materials are required.

[0027] If other composite materials are to be filled in the preparation of polytetrafluoroethylene porous materials, a large amount of inhomogeneity will usually result in the final product, and these fillers may fall off or leach out when facing major external environmental challenges and various environmental stresses. This phenomenon is largely due to the poor dispersibility of the filler in the polytetrafluoroethylene matrix and the poor chemical and physical compatibility of the filler with the inert polytetrafluoroethylene. Therefore, after testing, the filter head obtained by the above formula and method is used to quickly separate and extract exosomes (exosome diameter is 30-200nm), and separate proteins and impurities larger than 300nm.

[0028] The present application adds unsaturated polyester resin and epoxy resin to the filter head material, which can separate the protein in urine and prevent the urine from coagulating due to excessive protein during filtration, resulting in clogging of the filter head holes.

[0029] As a preferred embodiment of the immunochromatographic test paper described in the present application, the Fc end of the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 coated with colloidal gold particles is modified by biotin ligase catalysis.

[0030] The Fc end of the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 of the present application is modified by biotin ligase catalysis, which can realize the glutaminase-catalyzed connection of glutamine with three amino acids, namely lysine, tryptophan and cysteine, and particle labeling of the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1.

[0031] As a preferred embodiment of the immunochromatographic test paper described in the present application, the marking antibody of the T line on the nitrocellulose membrane area is the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated on the detection area (detection T line) on the nitrocellulose membrane; the marking antibody of the quality control C line is the coated goat anti-mouse antibody. The coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 is the second antibody, which is the mouse anti-human soluble Fms-like tyrosine kinase-1 monoclonal antibody AB21D9 antibody subtype, and serves as the marking antibody of the T line.

[0032] That is, there are two groups of T lines, which can detect the soluble Fms-like tyrosine kinase-1 content on the exosomes in urine at 4 to 10 ng / mL. One group of T lines detects the soluble Fms-like tyrosine kinase-1 content on the exosomes in urine at 4 ng / mL (detection T line group 1); the other group of T lines detects the soluble Fms-like tyrosine kinase-1 content on the exosomes in urine at 10 ng / mL (detection T line group 2). When the detection concentration of the detection T line reaches 10 ng / mL, the detection T line group 2 will display a red line; if it only reaches the detection concentration of the detection T line 4 ng / mL, the detection T line group 1 will also display a red line.

[0033] The test line (T) of the test card is coated with a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 2, which is combined with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold particles coated on the gold label pad area to form an immune complex of labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 (i.e., labeled antibody 1-antigen-antibody 2 sandwich complex). The double antibody sandwich method is used to semi-quantitatively detect the content of soluble Fms-like tyrosine kinase-1 antigen in urine exosomes.

[0034] As a preferred embodiment of the immunochromatographic test strip described in the present application, the galectin-1 monoclonal antibody in the antibody region is glycosylated, which is O-glycosylation (O-GlcNA) modification.

[0035] The galactose aggregated protein-1 monoclonal antibody has a single sugar recognition structure region on the antibody region: namely, the O-glycosylation group (O-GlcNA), which can firmly bind to the sugar chain (O-sugar chain) on the surface of the exosome (there are a large number of sugar chains on the surface of the exosome).

[0036] Preferably, galectin-1 monoclonal antibody is used to further purify urine exosomes (fine product) based on the rapid separation and extraction of urine exosomes (crude product) through a filter.

[0037] The present application uses galectin-1 monoclonal antibody (Galectin-1) coated in the antibody region, which is helpful for separating and purifying exosomes in urine. Compared with the magnetic bead immunoassay for extracting urine exosomes (i.e., extracting urine exosomes based on specific markers on the surface of exosomes (such as CD9, CD81, CD63)), the present application uses a homemade filter head combined with galectin-1 monoclonal antibody to better extract and purify urine exosomes. The specific method of capturing exosome binding with galectin-1 monoclonal antibody greatly improves the separation, extraction and purification of exosomes, is suitable for extracting exosomes from large samples, and is not affected by nonspecific adsorption of the matrix. The exosome results can be specifically detected with high precision and accuracy, which is conducive to accurately detecting the soluble Fms-like tyrosine kinase-1 content in urine exosomes of patients with preeclampsia. The magnetic bead immunoassay extracts a small amount of urine exosomes and is affected by pH and salt concentration. The detection of soluble Fms-like tyrosine kinase-1 content is interfered, which is prone to missed diagnosis.

[0038] Efficient and complete extraction and purification of urine exosomes is a difficult point in current research. This application separates, extracts and purifies exosomes through galactose condensate-1 antibodies. It is through the binding of sugar chains outside the glycosylated vesicles of galactose condensate-1 antibodies that the exosomes that can be quickly obtained are complete in morphology and high in purity. Galactoglutinin-1 monoclonal antibodies are used to further purify urine exosomes (fine products) on the basis of rapid separation and extraction of urine exosomes (crude products) through a filter head. Since galactose condensate-1 antibodies separate, extract and purify exosomes, complete exosomes can be obtained without washing. It is significantly more accurate and concise than the traditional method of separating, extracting and purifying exosomes, and can leave room for further expansion to other inspections in the next step. In addition, the operating costs are greatly reduced and simplified, which is in line with national industrial policies and is a major progress.

[0039] As a preferred embodiment of the immunochromatographic test paper described in the present application, the method for preparing the gold label pad area comprises the following steps:

[0040] A colloidal gold particle solution with a diameter of 45 to 55 nm is taken, and the pH value of the colloidal gold particle solution is adjusted to 7.5 to 10.7 with K2CO3, and then a soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 is added to the colloidal gold particle solution and mixed, and then PEG20000 and BSA are added, and the precipitate is obtained by centrifugation, and the precipitate is re-dissolved with a re-solution to obtain a mixed solution, and then the mixed solution is sprayed on a polyester film, the reaction temperature is 33 to 40°C, and the reaction time is 50 to 250 minutes to form a gold label pad area.

[0041] Preferably, the pH value is 8.5, the reaction temperature is 37° C., and the reaction time is 100 min.

[0042] Colloidal gold is easier on common antigen labeling, but affected by the combination mode of antigen or antibody and colloidal gold, the two key links of colloidal gold labeling are the determination of pH during labeling and the optimal protein labeling amount. The present application, according to the principle of colloidal gold labeling, finds out and determines that only when pH is 7.5 to 10.7, the adsorption force of colloidal gold and soluble Fms sample tyrosine kinase-1 protein isoelectric point (8.1 to 9.6) of soluble Fms sample tyrosine kinase-1 protein is the strongest. pH is too high or too low and is not conducive to the combination of the two. For example, if pH is greater than 11, that is, higher than the isoelectric point of Fms sample tyrosine kinase-1, it is easy to dissociate Fms sample tyrosine kinase-1 antibody from colloidal gold. Along with the increase of pH, electrostatic repulsion increases, and hydrophobic force decreases due to the increase of protein and gold particles in spatial distance, and the protein attached to colloidal gold also decreases, and the labeling efficiency is low. When the pH value is lower than the isoelectric point of the protein (i.e., the pH value is less than 6.5), the electrostatic effect is the main force, the protein is positively charged, and the colloidal gold is negatively charged. Although the protein is easy to bind to the colloidal gold, the formed conjugates are easy to aggregate with each other, and the phenomenon of dead gold occurs, that is, the labeling efficiency is high, but the formed conjugates are poorly stable, and the colloidal gold labeled antibody is separated, which seriously affects the detection of the antibody content. Therefore, when labeling, a precise pH measuring instrument or test strip is selected, and different methods are used to repeat the calibration, and a gradient test is performed to find the best ratio of the labeled pH sol to the amount of the labeled protein, that is, colloidal gold labeling usually adjusts the pH value of the colloidal gold solution to about 0.5 to 1.0 higher than the isoelectric point of the protein to ensure the stability of the label state, which is an important factor for successful labeling. The present application overcomes the two major factors that affect the colloidal gold labeled antibody, namely: the colloidal gold labeling effect and the stability of the combination with the labeled antibody. The present application also found that too much protein labeling causes the test strip to carry over, while too little protein labeling results in incomplete colloidal gold labeling, thereby reducing the sensitivity of the test strip and causing false positives. The present application is further required to conduct a gradient test in the test, repeatedly comparing the stability of markers with different labeling amounts in the test, so as to determine the optimal labeling amount.

[0043] After exploration, the present application found that the particle size of colloidal gold is another important factor that affects whether the colloidal gold labeling protein is successful. If the particle size of colloidal gold is uneven (40 to 60nm), it is easy to cause errors in the preparation process of colloidal gold. The heterogeneity of colloidal gold particles may even affect the stability of protein binding. When the colloidal gold protein is labeled, the present application predicts that the molecular weight of soluble Fms-like tyrosine kinase-1 monoclonal antibody is 70 to 80KDa, requires the colloidal gold particle diameter to be 45 to 55nm, and also requires lysine, tryptophan and cysteine ​​to participate (to improve the labeling effect), and before each experiment, the soluble Fms-like tyrosine kinase-1 protein stability determination (including temperature, pH value, protein isoelectric point, salt concentration, glycosylation modification, disulfide bond, etc.) is carried out to ensure that the best labeling effect can be obtained.

[0044] As a preferred embodiment of the immunochromatographic test paper described in the present application, the reconstitution solution is composed of a final concentration of 0.01-0.2 mol / L Tris buffer, 1%-5% BSA buffer, 1%-3% trehalose, 1%-5% sucrose, 0.1%-2% Triton X-100, 0.1%-0.5% Tween-20, 0.1%-1% PEG 20000, 0.1%-2% sodium chloride or 0.1%-2% sodium hydroxide, and 0.05%-0.1% sodium azide.

[0045] The concentration of the soluble Fms-like tyrosine kinase-1 monoclonal antibody is 10-50 U / g, preferably 20 U / g.

[0046] The present application also provides an immunochromatographic detection method, comprising the following steps:

[0047] S1. The urine sample passes through the filter head to separate and collect the exosomes in the urine;

[0048] S2, dripping urine filtered through the filter head onto the sample pad, and then combining galactose agglutinin-1 on the antibody region with the exosome sugar chain in the urine through O-glycosylation to obtain exosomes with higher purity;

[0049] S3, the urine after step S2 reaches the gold-labeled pad area through chromatography, and the antigen to be detected in the urine combines with the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 to obtain a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be detected immune complex;

[0050] S4. The test urine continues to move forward. When the test urine flows to the detection T line, the test antigen combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold, and under the action of chromatography, combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated on the detection area T line on the nitrocellulose membrane to form a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-test antigen-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex, which is retained at the T line to form a red line T detection line, where a positive result is displayed. The detection T line is set with detection concentrations of 4ng / mL and 10ng / mL, respectively, and there are detection T line group 1 and detection T line group 2 respectively. When the detection concentration of the detection T line reaches 10ng / mL, the detection T line group 2 will display a red line; if it only reaches the detection concentration of the detection T line of 4ng / mL, the detection T line group 1 will also display a red line.

[0051] When the test urine flows through the quality control C line with goat anti-mouse IgG antibody fixed on the nitrocellulose membrane area, the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold that is not intercepted by the detection T line will continue to flow to the quality control C line, and combine with the goat anti-mouse IgG antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Similarly, due to the large amount of colloidal gold gathered here, a red control line C is formed again, that is, the quality control C line appears, and a negative result is displayed here. Therefore, the test result is judged by displaying a red color at the quality control C line or the detection T line.

[0052] In the technical solution of the present application, after the test urine sample is dropped into the sample pad area of ​​the immunochromatographic test paper, it reaches the galactose lectin-1 monoclonal antibody area through the capillary action of the absorbent fiber, and the test urine sample quickly soaks the gold-labeled pad, and the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 on the gold-labeled pad area moves forward along the chromatographic material with the test liquid. If the antigen to be tested exists in the test urine sample, it will react specifically with the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 to form a colloid. The gold-labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested immune complex combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated on the detection area (T line) of the nitrocellulose membrane on the T line to form a double antibody sandwich complex (labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex), which is retained at the T line to form a red line T detection line (a positive result is shown here). When the test urine flows through the quality control C line with goat anti-mouse IgG antibody fixed on the nitrocellulose membrane area, the excess (not intercepted by the detection T line) soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold will continue to flow to the quality control C line and combine with the goat anti-mouse antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Also, because colloidal gold gathers in large quantities here, a red control line C is formed again, that is, the quality control line C line appears (a negative result is shown here), indicating that the experiment is effective.

[0053] At this time, the T line is retained by the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex, that is, red can be observed at the T line, which means that the test result is positive, that is, there is antigen to be tested in the test urine; if color is only observed at the C line, and no color is observed at the T line, it means that the test result is negative, that is, there is no antigen to be tested in the test fluid; the content of soluble Fms-like tyrosine kinase-1 in the urine to be tested is different, and the detection T line is set with a detection concentration of 4ng / mL and 10ng / mL respectively, and there are also detection T line 1 group (T line 6.1) and detection T line 2 group (T line 6.2) respectively. When the detection concentration of the detection T line reaches 10ng / mL, the detection T line 2 group (T line 6.2) will display a red line; if the detection concentration of the detection T line is only 4ng / mL, the detection T line 1 group (T line 6.1) will also display a red line. When the test urine is dropped onto the sample pad of the test strip 5 to 10 minutes later, the appearance of the C and T lines can be directly observed by the naked eye to determine the semi-quantitative test results.

[0054] Compared with the prior art, this application has the following beneficial effects:

[0055] The present application provides an immunochromatographic test strip and a detection method thereof for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes. The immunochromatographic test strip of the present application can quickly detect soluble Fms-like tyrosine kinase-1 in urine exosomes, and can predict whether the patient has preeclampsia through the test strip without relying on instruments and equipment. The immunochromatographic test strip of the present application can distinguish between the content of soluble Fms-like tyrosine kinase-1 of 4ng / mL and 10ng / mL in pregnant women's samples while evaluating, and can accurately judge high-risk groups. The present application not only shortens the detection time, but also improves the accuracy of the detection, realizes instant detection, mobile detection and home self-service detection, and can grasp the content and changes of soluble Fms-like tyrosine kinase-1 in time and predict the risk of preeclampsia in advance. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A front view of an immunochromatographic test strip for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes provided in an embodiment of the present invention (wherein: 1. filter head, 2. urine test tube with filter head, 3. sample pad area, 4. galactose-coated lectin 1 area, 5. gold-labeled pad area, 6. T-line area, first T-line: 6.1, second T-line: 6.2, 7. C-line area, 8. water-absorbent pad area, 9. bottom plate, 10. nitrocellulose membrane);

[0057] Figure 2 Electron microscopic images of urine exosomes enriched and purified by two different methods ( Figure 2 The left is an electron micrograph of the filter head enriching and separating urine exosomes. Figure 2 Right: Electron micrograph of urine exosomes purified by galectin-1 monoclonal antibody);

[0058] Figure 3 The particle size distribution of urine exosomes was measured by nanoparticle tracking analyzer;

[0059] Figure 4 Quantitative detection range diagram of the immunochromatographic test strip of the present application;

[0060] Figure 5 The sensitivity and specificity curve of the immunochromatographic test strip of Example 1 of the present application for detecting soluble Fms-like tyrosine kinase-1 in preeclampsia urine samples is shown.

[0061] Figure 6 The sensitivity and specificity curves of the immunochromatographic test strips of Examples 3 and 4 and Comparative Examples 3 and 4 of the present application for detecting soluble Fms-like tyrosine kinase-1 in preeclampsia urine samples are shown. DETAILED DESCRIPTION

[0062] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0063] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0064] The present invention firstly makes a filter head, which has 6 circular holes, each with a diameter of less than 300 nm, to remove larger impurities including macromolecules, large proteins and urine impurities. Figure 1 .

[0065] The filter head includes the following components in parts by weight:

[0066] 20 parts of polytetrafluoroethylene, 65 parts of unsaturated polyester resin and 15 parts of epoxy resin.

[0067] The filter head has 6 circular holes, each with a diameter less than 300 nm.

[0068] The preparation method of the filter head comprises the following preparation steps:

[0069] The mixture of the above components in parts by weight is prepared, mechanically stirred for 30 to 60 minutes, placed in a vacuum oven, and the bubbles therein are removed by vacuuming. The mixture from which the bubbles have been removed by vacuuming is poured into a polytetrafluoroethylene mold, cured at room temperature for 2 to 4 hours, and then cured at 150-300°C for 6 to 10 hours to form a mold.

[0070] The urine was then collected using the filter head obtained above. The urine passed through the homemade filter head and entered a test tube with a diameter of 15 mm. The urine that entered the test tube contained exosomes.

[0071] This application uses transmission electron microscopy to characterize the exosomes in the obtained urine. The specific characterization results are shown in Figure 2 Left: In the field of vision, the urine exosomes are clear and complete, with only a few small white spots (i.e., impurity proteins of equal volume) around them, which shows that the purity of urine exosomes obtained by this method is relatively good, and the exosome structure is intact. Figure 3 ,from Figure 3 It can be seen that the average particle size of the exosomes in urine obtained in this application is 109nm±28nm.

[0072] Example 1: An immunochromatographic test strip for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes and its preparation method

[0073] This embodiment provides an immunochromatographic test strip for rapid semi-quantitative detection of exosome soluble Fms-like tyrosine kinase receptor 1 in urine, comprising the following components:

[0074] 1. Sample pad area (1): 2.5 cm × 0.4 cm glass fiber;

[0075] 2. Antibody zone (2): glass fiber coated with galectin-1 monoclonal antibody;

[0076] 3. Gold label pad area (3): 0.4 cm × 0.4 cm polyester membrane containing soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold;

[0077] 4. Nitrocellulose membrane area (4): 2.5 cm × 0.4 cm in size, coated with soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 and goat anti-mouse IgG in sequence, with a spacing of 4 mm between them, forming detection line T1 (6.1), T2 (6.2), and quality control line C (7), respectively;

[0078] 5. Absorbent pad (5): 2.7 cm × 0.4 cm absorbent filter paper;

[0079] By connecting in sequence from (1) to (5), an immunochromatographic test strip for rapid detection of exosome soluble Fms-like tyrosine kinase receptor 1 in urine is formed. Before the urine is dripped onto the sample pad area, the urine is filtered using the filter head to quickly separate and extract urine exosomes. The urine enters a test tube with a diameter of 15 mm. The urine entering the test tube contains exosomes. The filtered urine (containing exosomes) is then dripped onto the sample pad area for reaction.

[0080] The immunochromatographic test paper of the present application includes the following specific preparation method:

[0081] S1. Preparation of sample pad and antibody area (coated with galectin-1 monoclonal antibody):

[0082] Preparation method of sample pad and antibody region coated with galectin 1: spray 100 μl of galectin-1 monoclonal antibody on 1.5 cm×0.4 cm glass fiber, dry at 37°C for 8 to 24 hours to obtain sample pad and antibody region coated with galectin 1. Seal in aluminum foil bag and store at room temperature for future use.

[0083] When the urine sample is combined with the coated galectin-1 monoclonal antibody, the exosomes in the urine are enriched and purified to obtain purified exosomes without impurity proteins and cells of equal volume, and the exosome structure is intact ( Figure 2Right). The galectin-1 of the present application has a single sugar recognition structure region: that is, O-glycosylation can bind to the sugar chains on the surface of exosomes and improve stability, and it does not separate even in a chromatographic environment. The galectin-1 monoclonal antibody is used to further purify urine exosomes (fine products) on the basis of rapid separation and extraction of urine exosomes (crude products) through a filter head.

[0084] S2. Make the gold label pad area:

[0085] The soluble Fms-like tyrosine kinase-1 monoclonal antibody in the gold-labeled pad region adopts mouse anti-human soluble Fms-like tyrosine kinase-1 monoclonal antibody 1, and the specific preparation method is as follows:

[0086] Take 10mL of the prepared colloidal gold solution, adjust the pH value to 8.2 with 1.0% K2CO3, and then take 120μL of 1mg / mL soluble Fms-like tyrosine kinase-1 monoclonal antibody 1, slowly add it to the colloidal gold solution, shake well and let it stand for 30min. Add 10μL of 1% PEG20000, shake well, then add 10μL of 10% BSA, shake well and let it stand, centrifuge, take the precipitate, and re-dissolve it with a re-solution according to 10% of the volume of the colloidal gold solution used for the original initial labeling. Spray 1ml of the re-dissolved colloidal gold marker on the polyester film of 0.4cm×0.4cm gold label pad, dry at 37℃ for 12 hours, seal in an aluminum foil bag, and store at room temperature for use, to obtain a soluble Fms-like tyrosine kinase-1 monoclonal antibody labeled with colloidal gold.

[0087] The experiment determined that the optimal binding pH value of the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold was 8.2, and the ratio of colloidal gold to antibody was 15 g antibody / ml colloidal gold.

[0088] The reconstitution solution is prepared from a final concentration of 0.01-0.2 mol / L Tris buffer, 1%-5% BSA, 1%-3% trehalose, 1%-5% sucrose, 0.1%-2% Triton X-100, 0.1%-0.5% Tween-20, 0.1%-1% PEG20000, 0.1%-2% sodium chloride or 0.1%-2% sodium hydroxide, and 0.05%-0.1% sodium azide. The pH value is adjusted to 7.0-8.0 with HCl, and the solution is stored at 4°C in the dark for later use.

[0089] The present application is to modify the Fc end of soluble Fms-like tyrosine kinase-1 by biotin ligase catalysis, realize the catalytic connection of glutamine with three amino acids, lysine, tryptophan and cysteine, by glutaminase, and perform colloidal gold labeling of soluble Fms-like tyrosine kinase-1. The optimal experimental conditions for glutaminase catalyzing the connection of glutamine with the above three amino acids are: enzyme concentration 20U / g, pH value 8.2, temperature 37 degrees, and reaction time for the connection of the three amino acids, lysine, tryptophan and cysteine, is 100 minutes.

[0090] S3. Preparation of nitrocellulose membrane area:

[0091] Soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 was diluted with antibody diluent to concentrations of 0.3-1.5 mg / mL, 0.5-3 mg / mL, and 1.0-5.0 mg / mL, respectively. The goat anti-mouse IgG on the quality control line was diluted to a concentration of 0.2-1.2 mg / mL and sprayed on the nitrocellulose membrane to form detection lines T: 6.1, 6.2, and quality control line C goat anti-mouse IgG (7). The T line and the C line were separated by 4 mm. Then, they were added to the T cup and C cup of the film spray gold instrument respectively; the film spray gold instrument was debugged and the spray volume was set to 1.0 μL / cm. After debugging, the film was coated to form detection lines T: 6.1, 6.2, corresponding to soluble Fms-like tyrosine kinase-1 contents of 4 ng / mL and 10 ng / mL and quality control line C, respectively, and dried at 37°C for 8-24 hours; after passing the inspection, the aluminum foil bag was sealed and stored at room temperature for use.

[0092] Thus, the immunochromatographic test strip for rapid semi-quantitative detection of urine exosome soluble Fms-like tyrosine kinase receptor 1 of the present application is prepared.

[0093] Example 2: An immunochromatographic detection method

[0094] This embodiment provides an immunochromatographic detection method, comprising the following steps:

[0095] S1. The urine sample passes through the filter head to separate and collect the exosomes in the urine;

[0096] S2. The urine (containing exosomes) filtered through the filter is dripped onto the sample pad, and then the galactose lectin-1 on the antibody area is combined with the exosomes in the urine to obtain exosomes with higher purity; the galactose lectin-1 monoclonal antibody is used to further purify the urine exosomes (fine product) on the basis of rapid separation and extraction of urine exosomes (crude product) through the filter.

[0097] S3, the urine after step S2 reaches the gold-labeled pad area through chromatography, and the antigen to be tested in the urine combines with the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody to obtain a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested immune complex;

[0098] S4. The test urine continues to move forward. When it flows to the detection T line, the antigen to be tested combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold to form an immune complex. Under the action of chromatography, it combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated in the detection area (T line) on the nitrocellulose membrane to form a double antibody sandwich complex (i.e., labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex), and then stays at the T line to form a red line T detection line (a positive result is shown here). The detection T line is set with detection concentrations of 4ng / mL and 10ng / mL respectively (there are also detection T line 1 group and detection T line 2 group respectively). When the detection concentration of the detection T line reaches 10ng / mL, the detection T line 2 group will display a red line; if it only reaches the detection concentration of the detection T line 4ng / mL, the detection T line 1 group will also display a red line.

[0099] When the test urine flows through the quality control C line with goat anti-mouse IgG antibody fixed on the nitrocellulose membrane area, the excess (not intercepted by the detection T line) soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold will continue to flow to the quality control C line and combine with the goat anti-mouse antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Similarly, due to the large amount of colloidal gold gathered here, a red control line C is formed again, that is, the quality control C line appears (negative results are shown here), indicating that the experiment is valid. Therefore, the test results are judged by showing red color at the quality control C line or the detection T line.

[0100] The present application uses the immunochromatographic test paper prepared in Example 1 to detect the content of salivary exosome soluble Fms-like tyrosine kinase-1. The technical principle uses a specific immune response double antibody sandwich method.

[0101] The specific principles are:

[0102] (1) After the urine sample filtered by the filter head is dropped into the sample pad area of ​​the test strip, the urine sample passes through the antibody area coated with galactose agglutinin 1 through the capillary action of the absorbent fiber, and quickly wets the gold-labeled pad area. The soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold on the gold-labeled pad area is dissolved and moves forward along the chromatographic material with the urine sample. (2) If the antigen to be tested exists in the test urine sample, it will react specifically with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold to form a soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested immune complex. (3) At the detection T line, the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 labeled with colloidal gold coated on the nitrocellulose membrane is combined to form a double antibody sandwich complex (i.e., labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be tested-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex), and the red T detection line (a positive result is shown here) is retained at the T line. The antigen content is semi-quantitatively detected by the double antibody sandwich method. That is, red can be observed at the T line, indicating that the test result is positive, that is, the test urine contains the antigen to be tested; (4) The test urine continues to move forward, and when it flows through the goat anti-mouse IgG antibody fixed on the nitrocellulose membrane to the quality control C line, the excess (not intercepted by the test T line) soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold will continue to flow to the quality control C line and combine with the goat anti-mouse antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Similarly, due to the large amount of colloidal gold gathered here, a red control line C is formed again, that is, the quality control C line (here showing a negative result) appears, indicating that the experiment is effective. If color is only observed at the quality control C line, but no color is observed at the test T line, it means that the test result is negative, that is, there is no antigen to be tested in the test fluid; (5) The content of soluble Fms-like tyrosine kinase-1 in the urine to be tested is different, and the test T line is set with a detection concentration of 4ng / mL and 10ng / mL, and there are also test T line 1 group (T line 6.1) and test T line 2 group (T line 6.2) respectively. When the detection concentration of the test T line reaches 10ng / mL, the test T line 2 group will display a red line; if the detection concentration of the test T line is only 4ng / mL, the test T line 1 group will also display a red line. (6) When the test urine is dripped into the sample pad of the test strip for 5 to 10 minutes, the appearance of the C and T lines can be directly observed by the naked eye, and the quantitative test results can be judged. Therefore, the test result is judged by displaying a red color at the C line or the test T line.

[0103] The linear detection range of the immunochromatographic test strip of the present application is 0.1 to 1000 ng / mL (see Figure 4). The test strip of the present application was used to detect 50 clinical samples of preeclampsia, and chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 was used as a control.

[0104] The present application detected 50 cases of preeclampsia patients with urine exosome soluble Fms-like tyrosine kinase-1, all of which showed abnormal results, and the consistency rate with the clinical diagnosis of preeclampsia was 100%. The results showed that the sensitivity of the immunochromatographic test strip of the present application was 89.5% and the specificity was 97.0% (see Figure 5 ), the above is the result of testing 50 clinical samples of preeclampsia using a test strip with a pH of 8.2 in the gold standard pad area. The results of chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 were compared: among the 50 patients with preeclampsia, 6 chemiluminescence test results showed normal, and the consistency rate with the clinical diagnosis of preeclampsia was 88%). The sensitivity of chemiluminescence detection of soluble Fms-like tyrosine kinase-1 was 78.1%, and the specificity was 86.9% (see Figure 5 ).

[0105] Example 3

[0106] Compared with Example 1, the difference is that the pH used in the process of preparing the gold label pad area in step S2 is 7.5, and the remaining components and preparation method of the immunochromatographic test strip are the same as those in Example 1.

[0107] Thirty clinical samples of preeclampsia were tested with a test strip with a pH of 7.5 in the gold-labeled pad area, and chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 was used as a control.

[0108] like Figure 6 , the present application detected soluble Fms-like tyrosine kinase-1 in urine of 30 patients with preeclampsia, and only 1 case showed a normal result, with a sensitivity of 85.6% and a specificity of 92.3% (see Figure 6 A), the consistency rate with the clinical diagnosis of preeclampsia was 96.67%. The chemiluminescence detection results showed that there were 4 normal results. The sensitivity of chemiluminescence detection of soluble Fms-like tyrosine kinase-1 was 78.1% and the specificity was 86.9%. Figure 6 A), the consistency rate with the clinical diagnosis of preeclampsia was 86.67%.

[0109] Example 4

[0110] Compared with Example 1, the difference is that the pH used in the process of preparing the gold label pad area in step S2 is 10.7, and the remaining components and preparation method of the immunochromatographic test strip are the same as those in Example 1.

[0111] A test strip with a pH of 10.7 was used to test 30 clinical samples of preeclampsia in the preparation of the gold-labeled pad, and chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 was used as a control.

[0112] like Figure 6 , the present application detected soluble Fms-like tyrosine kinase-1 in urine exosomes of 30 patients with preeclampsia, and showed 2 normal results, with a sensitivity of 82.7% and a specificity of 90.4% (see Figure 6 B), the consistency rate with the clinical diagnosis of preeclampsia was 93.33%. The chemiluminescence detection results showed that there were 4 normal results. The sensitivity of chemiluminescence detection of soluble Fms-like tyrosine kinase-1 was 78.1% and the specificity was 86.9%. Figure 6 B), the consistency rate with the clinical diagnosis of preeclampsia was 86.67%.

[0113] Comparative Example 1

[0114] Compared with Example 1, the difference of Comparative Example 1 is that the immunochromatographic test strip uses magnetic bead immunoassay to extract urine exosomes, does not use a filter to filter urine, and the antibody area is not coated with galactose condensate-1 antibody. The remaining components and preparation method of the immunochromatographic test strip are the same as those of Example 1.

[0115] The specific differences are:

[0116] The immunochromatographic test strips prepared in Example 1 and Comparative Example 1 were used to detect the soluble Fms-like tyrosine kinase-1 content in the urine exosomes of 10 patients with preeclampsia. The magnetic bead immunoassay (CD9, CD81, CD63) was used to extract urine exosomes. Only 6 patients with preeclampsia showed positive results, that is, the soluble Fms-like tyrosine kinase-1 content in the urine exosomes was in the range of 4-10 ng / mL. There were 4 cases with negative results, which were false negatives and missed diagnosis. However, the present application used a filter head combined with galactose condensate-1 antibody to extract urine exosomes. The results of 10 patients with preeclampsia were all positive, that is, the soluble Fms-like tyrosine kinase-1 content was in the range of 4-10 ng / mL, which was consistent with the diagnosis of preeclampsia.

[0117] Comparative Example 2

[0118] Compared with Example 1, the antibody area in the immunochromatographic test paper of Comparative Example 2 is not coated with galactose aggregate-1 antibody, and the remaining components and preparation method of the immunochromatographic test paper are the same as those of Example 1.

[0119] In comparative example 2, only the filter head was used to enrich the exosomes in urine, and the galactose condensate-1 antibody was not used to enrich the exosomes. The exosomes obtained were cystic bodies with a diameter of 30 to 150 nm. Although larger than general proteins, this method would be mixed with interference from other vesicles of similar size to exosomes, and even false positive results would appear in the detection of soluble Fms-like tyrosine kinase-1 in urine exosomes. For example, among 10 normal pregnant women selected as the control group, 3 cases had increased detection values ​​of soluble Fms-like tyrosine kinase-1 in urine exosomes, 2 cases had soluble Fms-like tyrosine kinase-1 of 4 ng / mL, and another case had 10 ng / mL. In the group using the filter head combined with galactose condensate-1 antibody to capture exosomes, the soluble Fms-like tyrosine kinase-1 content of urine exosomes in the same 10 normal pregnant women was detected to be normal. The method of capturing exosomes by using a filter head combined with galactose condensate-1 antibody avoids interference from other vesicles of the same size as exosomes and does not affect the detection results. In addition, the strong combination of enriched exosomes and galactose condensate-1 antibodies in this application lays a solid foundation for the next step of integrated separation, enrichment and detection of exosome soluble Fms-like tyrosine kinase-1 monoclonal antibody complexes.

[0120] Comparative Example 3

[0121] Compared with Example 1, the difference is that the pH used in the process of preparing the gold label pad area in step S2 is 11, and the remaining components and preparation method of the immunochromatographic test strip are the same as those in Example 1.

[0122] A test strip with pH 11 was used to test 30 clinical samples of preeclampsia in the preparation area of ​​the gold label pad, and chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 was used as a control.

[0123] like Figure 6 , the present application detected 30 cases of preeclampsia patients with urine exosome soluble Fms-like tyrosine kinase-1, and all showed 3 normal results, with a sensitivity of 81.7% and a specificity of 87.5% (see Figure 6 C), and the coincidence rate with the clinical diagnosis of preeclampsia was 90.00%. The chemiluminescence detection results showed that there were 4 normal results. The sensitivity of chemiluminescence detection of soluble Fms-like tyrosine kinase-1 was 78.1% and the specificity was 86.9% (see Figure 6 C), the consistency rate with the clinical diagnosis of preeclampsia was 86.67%.

[0124] If compared with the test strip with a pH of 8.2 used in the production of the gold standard pad area, which has a sensitivity of 89.5%, a specificity of 97.0%, and a 100% compliance rate for the clinical diagnosis of preeclampsia, the test strip with a pH of 11 used in the production of the gold standard pad area has certain false negative or false positive results.

[0125] Comparative Example 4

[0126] Compared with Example 1, the difference is that the pH used in the process of preparing the gold label pad area in step S2 is 6.5, and the remaining components and preparation method of the immunochromatographic test strip are the same as those in Example 1.

[0127] A test strip with a pH of 6.5 was used to test 30 clinical samples of preeclampsia in the preparation area of ​​the gold label pad, and chemiluminescence quantitative detection of soluble Fms-like tyrosine kinase-1 was used as a control.

[0128] like Figure 6 , the present application detected 5 normal results in 30 patients with preeclampsia for urine exosome soluble Fms-like tyrosine kinase-1, with a sensitivity of 76.2% and a specificity of 84.7% (see Figure 6 D), and the coincidence rate with the clinical diagnosis of preeclampsia was 83.33%. The chemiluminescence detection results showed that there were 4 normal results. The sensitivity of chemiluminescence detection of soluble Fms-like tyrosine kinase-1 was 78.1% and the specificity was 86.9% (see Figure 6 D), the consistency rate with the clinical diagnosis of preeclampsia was 86.67%.

[0129] If compared with the test strip with a pH of 8.2 used in the production of the gold standard pad area, which has a sensitivity of 89.5%, a specificity of 97.0%, and a 100% compliance rate for the clinical diagnosis of preeclampsia, the test strip with a pH of 6.5 used in the production of the gold standard pad area has certain false negative or false positive results.

[0130] The above results show that the immunochromatographic test strip provided by the present application uses human urine as the test sample to effectively monitor the soluble Fms-like tyrosine kinase-1 in exosomes in urine, and the minimum detection amount reaches 0.1ng / mL. The soluble Fms-like tyrosine kinase-1 test strip of the present application can distinguish the content of soluble Fms-like tyrosine kinase-1 in the population at 4ng / mL and 10ng / mL while predicting quickly, and can accurately evaluate or judge high-risk populations. The test strip is simple to operate, intuitive, rapid, high detection efficiency, simple method, pollution-free, low cost, high sensitivity, and strong specificity. It does not require special instruments and equipment, does not require professional training, and the results are clear and easy to identify. It is easy to promote and is widely used in clinical field testing (POCT), field sites and family self-testing. You can grasp the content and changes of soluble Fms-like tyrosine kinase-1 in time and predict the risk of preeclampsia in advance.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. An immunochromatographic test strip for rapid semi-quantitative detection of soluble Fms-like tyrosine kinase receptor 1 in urine exosomes, characterized in that: The immunochromatographic test paper comprises a sample pad area, an antibody area, a gold label pad area, a nitrocellulose membrane area, and a water-absorbing pad area connected in sequence; The sample pad area is dripped with urine filtered by a filter head; the urine contains exosomes; the antibody area is coated with galactose agglutinin 1 monoclonal antibody; the gold label pad area is coated with soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold particles; the nitrocellulose membrane area is provided with a detection T line and a quality control C line; The labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 binds to the soluble Fms-like tyrosine kinase-1 antigen in urine.

2. The immunochromatographic test paper according to claim 1, characterized in that: The sample pad area is dripped with a filter head made of polytetrafluoroethylene, unsaturated polyester resin and epoxy resin to filter urine; the filter head is used for quickly separating and extracting urine exosomes.

3. The immunochromatographic test paper according to claim 2, characterized in that: The filter head comprises the following components in parts by weight: 10-40 parts of polytetrafluoroethylene, 50-75 parts of unsaturated polyester resin and 5-25 parts of epoxy resin.

4. The immunochromatographic test paper according to claim 3, wherein: The preparation method of the filter head comprises the following steps: The mixture of the above-mentioned components in parts by weight is prepared and mechanically stirred for 30 to 60 minutes, placed in a vacuum oven, and the bubbles therein are removed by vacuuming. The mixture from which the bubbles have been removed by vacuuming is poured into a polytetrafluoroethylene mold, cured at room temperature for 2 to 4 hours, and then cured at 150 to 300° C. for 6 to 10 hours to form a mold.

5. The immunochromatographic test paper according to claim 1, characterized in that: The Fc end of the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 coated with colloidal gold particles is modified by biotin ligase catalysis.

6. The immunochromatographic test paper according to claim 1, characterized in that: The marking antibody for detecting the T line on the nitrocellulose membrane area is the coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2; the marking antibody for the quality control C line is the coated goat anti-mouse antibody.

7. The immunochromatographic test paper according to claim 1, characterized in that: On the antibody region, the galectin-1 monoclonal antibody is glycosylated.

8. The immunochromatographic test paper according to claim 1, wherein: The method for preparing the gold label pad area comprises the following steps: A colloidal gold particle solution with a diameter of 45 to 55 nm is taken, and the pH value of the colloidal gold particle solution is adjusted to 7.5 to 10.7 with K2CO3, and then a soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 is added to the colloidal gold particle solution and mixed, and then PEG20000 and BSA are added, and the precipitate is obtained by centrifugation, and the precipitate is re-dissolved with a re-solution to obtain a mixed solution, and then the mixed solution is sprayed on a polyester film, the reaction temperature is 33 to 40°C, and the reaction time is 50 to 250 minutes to form a gold label pad area.

9. The immunochromatographic test paper according to claim 8, characterized in that: The reconstitution solution is composed of a final concentration of 0.01-0.2 mol / L Tris buffer, 1%-5% BSA buffer, 1%-3% trehalose, 1%-5% sucrose, 0.1%-2% Triton X-100, 0.1%-0.5% Tween-20, 0.1%-1% PEG 20000, 0.1%-2% sodium chloride or 0.1%-2% sodium hydroxide, and 0.05%-0.1% sodium azide.

10. An immunochromatographic detection method, characterized in that: The following steps are involved: S1. The urine sample passes through the filter head to separate and collect the exosomes in the urine; S2, dripping urine filtered through the filter head onto the sample pad, and then combining galactose agglutinin-1 on the antibody region with the exosome sugar chain in the urine through O-glycosylation to obtain exosomes with higher purity; S3, the urine after step S2 reaches the gold-labeled pad area through chromatography, and the antigen to be detected in the urine combines with the labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 to obtain a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-antigen to be detected immune complex; S4, the test urine continues to move forward. When the test urine flows to the detection T line, the test antigen combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold, and under the action of chromatography, combines with the soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 pre-coated on the detection area T line of the nitrocellulose membrane to form a labeled soluble Fms-like tyrosine kinase-1 monoclonal antibody 1-test antigen-coated soluble Fms-like tyrosine kinase-1 monoclonal antibody 2 immune complex, which is retained at the T line to form a red line T detection line, where a positive result is displayed. The detection T line is respectively provided with detection concentrations of 4ng / mL and 10ng / mL, and is respectively provided with detection T line group 1 and detection T line group 2. When the detection concentration of the detection T line of 10ng / mL is reached, the detection T line group 2 will display a red line; if it only reaches the detection concentration of the detection T line of 4ng / mL, the detection T line group 1 will also display a red line; When the test urine flows through the quality control C line with goat anti-mouse IgG antibody fixed on the nitrocellulose membrane area, the soluble Fms-like tyrosine kinase-1 monoclonal antibody 1 labeled with colloidal gold that is not intercepted by the detection T line will continue to flow to the quality control C line, and combine with the goat anti-mouse antibody pre-coated on the nitrocellulose membrane at the C line to form a gold-labeled antibody-C line antibody complex. Similarly, due to the large amount of colloidal gold gathered here, a red control line C is formed again, that is, the quality control C line appears, and a negative result is displayed here. Therefore, the test result is judged by displaying a red color at the quality control C line or the detection T line.

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