An immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres and its preparation method.

Abstract

This invention belongs to the field of fetoglobulin B detection technology, and discloses an immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres and its preparation method. The immunochromatographic test strip of this invention includes a sample pad, a release pad, and a nitrocellulose membrane; the release pad is coated with a fetoglobulin B monoclonal antibody labeled with quantum dot fluorescent microspheres and a mouse IgG antibody labeled with quantum dot fluorescent microspheres; the nitrocellulose membrane has a detection line and a control line, the detection line is coated with fetoglobulin B monoclonal antibody, and the control line is coated with goat anti-mouse IgG antibody. The immunochromatographic test strip of this invention reduces the detection time to 9-12 minutes, and the total detection time is only 20 minutes, achieving rapid and accurate quantitative detection of fetoglobulin B. It also features high detection sensitivity, a wide detection range, and simple and rapid operation, which is beneficial for the screening and monitoring of atherosclerotic coronary heart disease.

Description

Technical Field

[0001] This invention belongs to the field of fetoglobulin B detection technology, and specifically relates to an immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres and its preparation method. Background Technology

[0002] Fetoglobulin B (FGFB) is a new member of the cysteine ​​protease inhibitor protein family. It was first discovered in 1999 by Olivier et al. in their study of acute-phase reactive proteins in rats induced by inflammatory substances. Human FGFB is encoded by the FETUB gene, located on chromosome 3 (3q27.3), which consists of eight exons. FGFB is an acidic glycoprotein with a relative molecular weight of approximately 60,000, primarily synthesized by the liver and released into the peripheral blood circulation. The FGFB gene locus is the same as the genetic susceptibility locus for metabolic syndrome, and it plays an important regulatory role in disorders of glucose and lipid metabolism such as obesity and type 2 diabetes. Current research has found that FGFB levels are closely related to the occurrence and development of atherosclerosis, making it an ideal biomarker for reflecting atherosclerotic coronary heart disease. Therefore, rapidly and accurately understanding the levels of FGFB in human peripheral blood plays a crucial guiding role in the screening and auxiliary diagnosis of coronary heart disease.

[0003] Currently, the main in vitro detection method for fetoglobulin B is enzyme-linked immunosorbent assay (ELISA). This method has disadvantages such as low detection sensitivity, narrow linear range, many operation steps, long detection time (4-6 hours), and poor repeatability. These defects prevent it from providing accurate quantification and from helping clinicians to assess patients' conditions and guide further clinical treatment in a timely and effective manner.

[0004] Immunochromatography, as a point-of-care testing technique, boasts advantages such as simplicity, speed, low cost, and the absence of complex equipment, leading to its rapid adoption in clinical diagnostics and other fields. Among these techniques, immunochromatography using colloidal gold as a probe is the most mature and widely used. However, immunochromatography methods using colloidal gold as a probe can only be applied to quantitative or semi-quantitative detection of substances, and exhibit certain sensitivity differences compared to traditional ELISA methods.

[0005] Quantum dots, as a novel fluorescent labeling material, possess excellent properties such as a large Stokes shift, narrow and symmetrical emission spectrum, long fluorescence lifetime, and strong resistance to bleaching, leading to their widespread application in labeled immunological analysis. Quantum dot fluorescent microsphere technology significantly enhances fluorescence intensity by embedding a large number of individual fluorescent quantum dots within polymer microspheres, thereby amplifying the signal and improving detection sensitivity. Furthermore, because the quantum dots are encapsulated within the microspheres, they are less affected by the external environment, exhibiting more stable physicochemical properties and resistance to fluorescent bleaching.

[0006] The aforementioned characteristics of quantum dot fluorescent microspheres, when combined with immunochromatographic technology, can achieve functions such as reducing the sample volume required for the reaction, accelerating the reaction time, easy automation, and on-demand sample preparation. Currently, there are no reports or applications of immunochromatographic test strips based on quantum dot fluorescent microspheres for the quantitative detection of fetoglobulin B. This invention utilizes quantum dot technology for the highly sensitive immunochromatographic detection of fetoglobulin B, providing a powerful tool for the initial screening and disease monitoring of atherosclerotic coronary heart disease in the population. It is also suitable for widespread implementation in primary healthcare units, facilitating early diagnosis and treatment of coronary heart disease patients. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres.

[0008] The immunochromatographic test strip of the present invention can detect the level of fetoglobulin B in the sample and has the characteristics of high detection sensitivity, strong specificity and good repeatability.

[0009] Another objective of this invention is to provide a method for preparing the above-mentioned immunochromatographic test strip.

[0010] The objective of this invention is achieved through the following solution:

[0011] An immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres includes a sample pad, a release pad, and a nitrocellulose membrane;

[0012] The release pad is coated with a fetal protein B monoclonal antibody labeled with quantum dot fluorescent microspheres and a mouse IgG antibody labeled with quantum dot fluorescent microspheres.

[0013] The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with fetal globulin B monoclonal antibody, and the control line is coated with goat anti-mouse IgG antibody.

[0014] In the immunochromatographic test strip of the present invention, the quantum dot fluorescent microsphere-labeled fetal protein B monoclonal antibody and the quantum dot fluorescent microsphere-labeled mouse IgG antibody, whether the two are the same or different, are prepared by the following steps: Activated quantum dot fluorescent microspheres and antibodies are mixed uniformly at a mass ratio of 20:1-3:1, reacted at room temperature for 3-5 hours (A), and the reaction is terminated by adding ethanolamine; after centrifugation and washing, blocking solution is added, and the reaction is carried out at room temperature for 30-60 minutes (B) to obtain the quantum dot fluorescent microsphere-labeled antibody.

[0015] Furthermore, the quantum dot fluorescent microspheres are polystyrene microspheres with a particle size of 100-300 nm, which are internally encapsulated with oil-soluble core-shell structured CdSe / ZnS quantum dots, and the surface is modified with carboxyl, amino or aldehyde groups.

[0016] Furthermore, the quantum dot fluorescent microspheres have an excitation wavelength range of 300-450 nm and a fluorescence peak at 590-630 nm.

[0017] Furthermore, the activation is carried out by adding quantum dot fluorescent microspheres to carbodiimide (EDC) and N-hydroxythiosuccinimide (Sulfo-NHS); the activation can be carried out at room temperature; the activation time can be 20-40 min.

[0018] Furthermore, the blocking solution is formulated to contain 25 mM Tris-HCl, 2.5% BSA, 2.5% trehalose and 0.25% Tween-20, with a pH of 7.6.

[0019] Furthermore, after reaction B at room temperature, the product can be obtained by centrifugation and washing to discard the supernatant; the product can be resuspended using microsphere preservation buffer; the resuspended suspension can be stored at 4°C for later use.

[0020] Furthermore, the microsphere preservation buffer is formulated with 25 mM Tris-HC, 1.2% BSA, 5% trehalose, 0.15% Tween-20 and 0.02% Proclin-300, pH 9.0.

[0021] The sample pad in the immunochromatographic test strip of the present invention is prepared by the following steps: soaking the sample pad in sample pad buffer solution, and then taking it out and drying it for later use.

[0022] Furthermore, the sample pad buffer solution is formulated with 10 mM Na2B4O7·10H2O, 1% polyvinylpyrrolidone, 0.2% sodium caseinate, 1% Trition-X100, 1% S9 and 0.05% NaN3, pH 8.5.

[0023] Furthermore, the soaking time can be 3-6 hours.

[0024] Furthermore, the drying can be carried out at 37°C.

[0025] Furthermore, the dried sample pad is stored at 4°C for later use.

[0026] The release pad in the immunochromatographic test strip of the present invention is prepared by the following steps: immersing the release pad in an antibody mixture solution, and then removing and drying it for later use.

[0027] Furthermore, the antibody mixture solution is specifically obtained by mixing fetal protein B monoclonal antibody labeled with quantum dot fluorescent microspheres and mouse IgG antibody labeled with quantum dot fluorescent microspheres at a mass ratio of 1:0.5-1:1.5.

[0028] Furthermore, the total mass concentration of antibodies in the antibody mixture solution can be 3-8 mg / mL.

[0029] Furthermore, the soaking time can be 1-3 hours.

[0030] Furthermore, the drying can be carried out at 37°C.

[0031] Furthermore, the dried sample pad is stored at 4°C for later use.

[0032] The immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres of the present invention combines quantum dot fluorescent microspheres with immunochromatographic technology. It has a wider excitation spectrum and a narrower emission spectrum, improving sensitivity and offering a wider detection range and better specificity than traditional ELISA. At the same time, the present invention optimizes the immunochromatographic test strip, shortening the detection time and improving the detection efficiency. It only requires loading a small amount of serum or plasma and shortens the detection time to 9-12 minutes, enabling rapid and accurate quantitative detection of fetoglobulin B.

[0033] The immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres of the present invention has simple and rapid operation steps, high detection sensitivity, and wide detection range, which is beneficial for the screening and monitoring of atherosclerotic coronary heart disease.

[0034] This invention also provides a method for preparing an immunochromatographic test strip for quantitative detection of fetoglobulin B using the above-mentioned quantum dot fluorescent microspheres, specifically including the following steps:

[0035] (1) Quantum dot fluorescent microsphere-labeled fetal protein B monoclonal antibody and quantum dot fluorescent microsphere-labeled mouse IgG antibody were prepared by the following methods:

[0036] The activated quantum dot fluorescent microspheres and antibodies were mixed evenly at a mass ratio of 20:1-3:1 and reacted at room temperature for 3-5 hours. The reaction was terminated by adding ethanolamine. After centrifugation and washing, blocking solution was added and reacted at room temperature for 30-60 minutes to obtain the antibody labeled with quantum dot fluorescent microspheres.

[0037] (2) Preparation of release pad:

[0038] The fetal protein B monoclonal antibody and the mouse IgG antibody labeled with quantum dot fluorescent microspheres prepared in (1) above were coated on the release pad;

[0039] (3) Sample pad preparation:

[0040] The sample pad is immersed in the sample pad buffer solution and then removed and dried. The sample pad buffer solution is formulated with 10 mM Na2B4O7·10H2O, 1% polyvinylpyrrolidone, 0.2% sodium caseinate, 1% Tritium-X100, 1% S9 and 0.05% NaN3, pH 8.5.

[0041] (4) Nitrocellulose membrane

[0042] The nitrocellulose membrane is provided with a detection line and a control line along the chromatography direction, and the fetal globulin B monoclonal antibody and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane as the detection line and the control line, respectively.

[0043] (5) Assembly

[0044] The sample pad, release pad, nitrocellulose membrane, and absorbent pad are assembled onto a PVC base plate to obtain a test paper, which is then cut to obtain an immunochromatographic test strip.

[0045] In step (5), the sample pad, release pad, nitrocellulose membrane and absorbent pad overlap each other by 2-4 mm, whether they are the same or different.

[0046] In step (5), the width of the test strip can be 3-5 mm.

[0047] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0048] (1) This invention combines quantum dot fluorescent microspheres with immunochromatography, which has a wider excitation spectrum and a narrower emission spectrum, which helps to reduce background and improve sensitivity. It has a wider detection range and better specificity than traditional ELISA. At the same time, it also has the characteristics of short detection time and high efficiency, which can realize rapid and accurate quantitative detection of fetal protein B.

[0049] (2) The present invention optimizes the immunochromatographic test strip, requiring only a small amount of serum or plasma, and shortens the detection time to 9-12 minutes.

[0050] (3) The fetal protein B immunochromatographic reagent strip of the present invention has a simple and rapid operation procedure, high detection sensitivity, and wide detection range, which is beneficial for the screening and monitoring of atherosclerotic coronary heart disease. At the same time, as an economical and convenient detection method, it is more conducive to its promotion and application in grassroots units and has important significance in rapid detection. Attached Figure Description

[0051] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 This is a schematic diagram of the structure of the immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres, provided in an embodiment of the present invention. In the figure, 1 is the sample pad; 2 is the release pad; 3 is the detection line; 4 is the control line; 5 is the absorbent pad; 6 is the base plate; and 7 is the nitrocellulose membrane.

[0053] Figure 2 This is the standard curve for fluorescence testing.

[0054] Figure 3 and Figure 4 The graphs show the consistency analysis of detection results between two commercially available ELISA kits and the immunochromatographic test strip of this invention.

[0055] Figure 5 This is the ROC analysis curve of the test strip detection results of the present invention. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods. The amounts of each component are expressed in parts by mass and volume, in g and mL.

[0057] Example 1

[0058] like Figure 1 As shown, an immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres includes a PVC base plate and a sample pad, a release pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped and partially stacked on the base plate. The release pad is coated with a fetoglobulin B monoclonal antibody labeled with quantum dot fluorescent microspheres and a mouse IgG antibody labeled with quantum dot fluorescent microspheres. The nitrocellulose membrane has a detection line and a control line; the detection line is coated with fetoglobulin B monoclonal antibody, and the control line is coated with goat anti-mouse IgG antibody.

[0059] In this embodiment of the invention, the sample pad and the conjugation pad partially overlap, with an overlap length of 2 mm; the release pad and the nitrocellulose membrane partially overlap, with an overlap length of 2 mm; the nitrocellulose membrane and the absorbent pad partially overlap, with an overlap length of 3 mm; the width of the detection line and the control line is 1 mm; and the cutting width of the test strip is 4 mm.

[0060] In this embodiment, carboxylated quantum dot fluorescent microspheres with a particle size of 200 nm are preferably used for antibody labeling. The quantum dot fluorescent microspheres are excited at a wavelength of 340 nm and have a maximum emission wavelength of 605 nm. The fetoglobulin B monoclonal antibody and goat anti-mouse IgG antibody were purchased from Abcam and Sigma-Aldrich, respectively.

[0061] Specific preparation method:

[0062] (1) Preparation of quantum dot fluorescent microsphere-labeled fetal protein B monoclonal antibody and quantum dot fluorescent microsphere-labeled mouse IgG antibody

[0063] 1) Take 2 parts by weight of carboxylated quantum dot fluorescent microspheres into a centrifuge tube, add 550 parts by volume of activation buffer (25mM MES, pH 6.3) and sonicate to resuspend for 5 min;

[0064] 2) Carbodiimide (EDC) and N-hydroxythiosuccinimide (Sulfo-NHS) were prepared into solutions with a concentration of 25 mg / mL using activation buffer. 10 volumes of EDC and 90 volumes of Sulfo-NHS were added to the solutions in step 1), and the mixture was shaken at room temperature for 30 min. After centrifugation at 15000g and washing, the supernatant was discarded. The activated microspheres were resuspended by sonication in 600 volumes of labeling buffer (25 mM PB, pH 7.1) and aliquoted into two equal portions of the activated microsphere resuspension.

[0065] 3) Take 0.1 parts by weight of fetal globulin B monoclonal antibody and mouse IgG antibody, and add them to the above activated microsphere resuspension. Shake at room temperature for 4 hours.

[0066] 4) Add 2.5 parts by volume of ethanolamine to terminate the coupling reaction, centrifuge at 15000g, wash and discard the supernatant;

[0067] 5) Add 2000 volumes of blocking buffer (25mM Tris-HCl, 2.5% BSA, 2.5% trehalose and 0.25% Tween-20, pH 7.6) to each container, shake and react at room temperature for 1 hour, centrifuge at 15000g, wash and remove the supernatant to obtain quantum dot fluorescent microspheres labeled with fetal globulin B monoclonal antibody and quantum dot fluorescent microspheres labeled with mouse IgG antibody microspheres, respectively.

[0068] 6) Resuspend the microspheres separately in microsphere preservation buffer (25mM Tris-HCl, 1.2% BSA, 5% trehalose, 0.15% Tween-20 and 0.02% Proclin-300, pH 9.0) to obtain resuspensions, and store at 4℃ for later use.

[0069] (2) Sample pad treatment

[0070] After cutting the sample pad, immerse it in the sample pad buffer solution, let it stand at room temperature for 4 hours, then remove it and dry it at 37°C with a forced air dryer for 16 hours. Finally, seal and store it at 4°C for later use. The sample pad buffer solution is formulated with 10 mM Na2B4O7·10H2O, 1% polyvinylpyrrolidone, 0.2% sodium caseinate, 1% Trition-X100, 1% S9 and 0.05% NaN3, pH 8.5.

[0071] (3) Preparation of the release pad:

[0072] The resuspension of quantum dot fluorescent microsphere labeled fetal protein B monoclonal antibody and quantum dot fluorescent microsphere labeled mouse IgG antibody obtained in step (1) was mixed evenly at a volume ratio of 1:1. The release pad was completely immersed in the above mixture and allowed to stand at room temperature for 2 hours. Then it was placed at 37°C for constant temperature drying for 16 hours and sealed and stored at 4°C for later use.

[0073] (4) Preparation of nitrocellulose membrane:

[0074] Fetoglobulin B monoclonal antibody and goat anti-mouse IgG antibody were taken separately and added to phosphate coating buffer (10mM Na2HPO4·12H2O, 0.9% NaCl, 2.5% BSA, 0.5% trehalose and 0.01% NaN3, pH 7.6) to adjust the concentration to 2mg / mL. The antibodies were uniformly sprayed onto nitrocellulose membranes using a Shanghai Gold Standard gold spraying instrument. Fetoglobulin B monoclonal antibody was sprayed onto the detection line, and goat anti-mouse IgG antibody was sprayed onto the quality control line. The membranes were then dried at 37℃ for 12 hours and stored in a sealed container at 4℃ for later use.

[0075] (5) Assembly of immunochromatographic test strips:

[0076] The assembly of the immunochromatographic test strip in this embodiment of the invention is carried out in an environment with an ambient humidity of 35% and a temperature of 24-26°C, and the operation is performed in a clean bench. The sample pad, release pad, nitrocellulose membrane, and absorbent pad prepared through the above steps are sequentially overlapped and assembled on an adhesive PVC base plate, as follows:

[0077] 1) Peel off the protective film on the surface of the PVC base plate, and then stick the nitrocellulose membrane to the middle part of the base plate. The nitrocellulose membrane is coated with a detection line of fetal globulin B monoclonal antibody and a quality control line coated with goat anti-mouse IgG antibody.

[0078] 2) Attach release pads and absorbent pads to the top of both ends of the nitrocellulose membrane, respectively; wherein, the release pads partially overlap the nitrocellulose membrane at the end near the test line, with an overlap length of 2mm; the absorbent pads partially overlap the nitrocellulose membrane at the end near the control line, with an overlap length of 3mm.

[0079] 3) Overlap the sample pad portion onto the release pad, with an overlap length of 2mm;

[0080] 4) After each part is assembled, press it gently again to make it flat and adhered. Then, use a strip cutter to cut the test paper into 4mm wide strips, add desiccant, and seal and store at 4℃ for later use.

[0081] Example 2: Detection Method

[0082] The quantitative detection of fetoglobulin B in a sample using the immunochromatographic test strip of the present invention includes the following steps:

[0083] (1) Establishing a standard curve: Fetoglobulin B antigen was diluted to 0, 5, 10, 50, 100, and 200 ng / mL using standard buffer (containing 50 mM Tris-HCl, 0.9% NaCl, 1.5% BSA, 0.02% Tween-200, 0.02% Proclin-3000, 0.01% NaN3, pH 7.6) to serve as a series of reference standard solutions. Each concentration was measured in triplicate. A standard curve was plotted using the ratio of concentration to fluorescence value. The results are shown below. Figure 2 As shown, the linear equation of the standard curve is: log Y = 0.30414logX - 0.72646, R 2 =0.99668. Using zero reference standard as the sample, the measurement was repeated 20 times. The mean fluorescence value and standard deviation were calculated. The fluorescence value obtained by adding twice the standard deviation to the mean was substituted into the standard curve equation to calculate that the sensitivity of the self-made test strip reached 0.299 ng / mL.

[0084] (2) Detection: Take 15 μL of the sample to be tested and add 85 μL of standard buffer. After mixing evenly, drop it onto the sample pad of the above immunochromatographic test strip. After standing at room temperature for 9 min, detect it on the fluorescence immunoassay analyzer to obtain the fluorescence signal value of the detection line and the fluorescence signal value of the control line. Substitute the ratio of the fluorescence signal value of the detection line and the fluorescence signal value of the control line into the standard curve to obtain the content of fetoglobulin B in the sample to be tested.

[0085] Example 3: Precision Experiment

[0086] The precision of the immunochromatographic test strips of this invention was evaluated by measuring reference standards at low, medium, and high concentrations. Intra-analytical precision: 10 replicates of each sample were performed on the same day using the same batch of test strips. Inter-analytical precision: 10 replicates of the same batch of test strips were performed on three consecutive days for different experiments. The results are shown in Table 1. The intra-assay coefficient of variation was 6.99-8.70%, and the inter-assay coefficient of variation was 4.59-9.69%, indicating that the immunochromatographic test strips of this invention have good precision.

[0087] Table 1. Precision test results

[0088]

[0089] Example 6: Recovery Rate Experiment

[0090] Serum samples with known fetoglobulin B concentrations were divided into three aliquots, and reference standards of low, medium, and high concentrations were added to each aliquot. Ten replicates were performed for each concentration. The results are shown in Table 1. Table 3 shows the recoveries for the three concentrations, which ranged from 100.90% to 109.58%, indicating good accuracy.

[0091] Table 2 Results of Recovery Rate Experiment

[0092]

[0093] Recovery rate calculation formula: Recovery rate (%) = Measured concentration / Original concentration.

[0094] Example 7: Comparison of Clinical Blood Sample Measurement Methods

[0095] After testing some serum or plasma samples using the Fetuin B Human ELISA Kit manufactured by Thermofisher, the test strip of this invention was used to test the samples, and the consistency of the results from the two methods was analyzed. The test results are as follows: Figure 3 As shown, the linear correlation equation between the detection results of the two methods is: y = 0.121 + 0.928x, with a correlation coefficient r = 0.985. It can be seen that the test strip of this invention has a good correlation with the detection results of Invitrogen's Fetuin B Human ELISA Kit for fetuin B in human serum or plasma samples.

[0096] After testing some serum or plasma samples using the Human Fetuin B ELISA Kit manufactured by Abcam, the test strip of this invention was used to test the samples, and the consistency of the results from the two methods was analyzed. The test results are as follows: Figure 4 As shown, the linear correlation equation for the detection results of the two methods is: y = 1.041x - 0.079, and the correlation coefficient r = 0.950. It can be seen that the test strip of this invention has a good correlation with the detection results of human fetuin B in human serum or plasma samples using Abcam's Human Fetuin B ELISA Kit.

[0097] Example 8: Performance Comparison of Different Commercial Reagent Kits

[0098] Furthermore, the immunochromatographic test strip of this invention was compared with existing fetoglobulin B detection kits from different manufacturers, focusing on parameters such as detection sensitivity, linear range, and detection time. The results are shown in Table 3. As can be seen from the table, compared with existing detection kits, which suffer from low detection sensitivity, narrow linear range, numerous operation steps, long detection time (4-6 hours), and poor repeatability, the immunochromatographic test strip of this invention reduces the detection time to 9-12 minutes, achieving rapid and accurate quantitative detection of fetoglobulin B in a total detection time of only 20 minutes. It also boasts high detection sensitivity, a wide detection range, and simple and rapid operation, which is beneficial for the screening and monitoring of atherosclerotic coronary heart disease.

[0099] Table 3 Performance Comparison of Reagent Kits from Different Manufacturers

[0100] factory Item number Detection sensitivity Linear range Total detection time Thermofisher EHFETUB 4ng / mL 4.39-50 ng / mL 4h45min Abcam ab240684 12.01 pg / mL 93.75-6000 pg / mL 1 hour 30 minutes RayBiotech ELH-FetuinB-1 4ng / mL 4-5 ng / mL 4h45min Cloud-Clone SEB860Hu 59 pg / mL 125-8000 pg / mL 2h45min Jianglai Biotechnology JL31327-96T 0.11 ng / mL 0.31-20 ng / mL 2h The test strip of this invention - 0.299 ng / mL 1-200 ng / mL 20mim

[0101] Example 9: Verifying the accuracy of plasma fetoglobulin B in diagnosing coronary heart disease

[0102] To verify the diagnostic accuracy of plasma fetoglobulin B in atherosclerotic coronary heart disease, plasma samples were collected from 51 patients diagnosed with coronary heart disease by coronary angiography. Plasma from 34 patients with normal coronary angiography results served as a normal control group. All peripheral blood samples were stored in standard EDTA anticoagulant tubes. Within 4 hours of fasting blood collection, plasma was separated by centrifugation at 1600g for 15 min at 4°C and then at 12000g for 10 min at 4°C. The samples were then rapidly flash-frozen in liquid nitrogen and stored at -80°C. The test strips of this invention were used to test the above samples, and the predictive value of plasma fetoglobulin B in the diagnosis of coronary heart disease was evaluated using receiver operating characteristic (ROC) curve analysis. The results are as follows: Figure 5As shown in the figure, the area under the ROC curve (AUC) for plasma fetoprotein B in diagnosing coronary heart disease patients was 0.708, P = 0.001. Therefore, plasma fetoprotein B, as a biomarker, can effectively distinguish between coronary heart disease patients and normal individuals.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres, characterized in that... Includes sample pads, release pads, and nitrocellulose membranes; The release pad is coated with a fetal protein B monoclonal antibody labeled with quantum dot fluorescent microspheres and a mouse IgG antibody labeled with quantum dot fluorescent microspheres. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with fetal globulin B monoclonal antibody, and the control line is coated with goat anti-mouse IgG antibody.

2. The immunochromatographic test strip according to claim 1, characterized in that: The quantum dot fluorescent microsphere-labeled fetoglobulin B monoclonal antibody and the quantum dot fluorescent microsphere-labeled mouse IgG antibody, whether identical or different, are prepared by the following steps: Activated quantum dot fluorescent microspheres and antibodies are mixed uniformly at a mass ratio of 20:1-3:1, reacted at room temperature for 3-5 hours (A), and the reaction is terminated by adding ethanolamine; after centrifugation and washing, blocking buffer is added, and the reaction is carried out at room temperature for 30-60 minutes (B) to obtain the quantum dot fluorescent microsphere-labeled antibody.

3. The immunochromatographic test strip according to claim 2, characterized in that: The quantum dot fluorescent microspheres are polystyrene microspheres with a particle size of 100-300 nm, which are encapsulated with oil-soluble core-shell CdSe / ZnS quantum dots and have carboxyl, amino or aldehyde groups modified on their surface.

4. The immunochromatographic test strip according to claim 2, characterized in that: The quantum dot fluorescent microspheres have an excitation wavelength range of 300-450 nm and a fluorescence peak at 590-630 nm.

5. The immunochromatographic test strip according to claim 2, characterized in that: The activation is performed by adding quantum dot fluorescent microspheres to carbodiimide and N-hydroxythiosuccinimide.

6. The immunochromatographic test strip according to claim 2, characterized in that: The blocking solution is formulated with 25 mM Tris-HCl, 2.5% BSA, 2.5% trehalose and 0.25% Tween-20, pH 7.

6.

7. The immunochromatographic test strip according to claim 1, characterized in that: The sample pad is prepared by the following steps: immersing the sample pad in a sample pad buffer solution, then removing and drying it for later use; the sample pad buffer solution is formulated with 10 mM Na2B4O7·10H2O, 1% polyvinylpyrrolidone, 0.2% sodium caseinate, 1% Tritium-X100, 1% S9 and 0.05% NaN3, pH 8.

5.

8. The immunochromatographic test strip according to claim 1, characterized in that: The release pad is prepared by the following steps: immersing the release pad in an antibody mixture solution, then removing it and drying it for later use; the antibody mixture solution is specifically obtained by mixing fetal globulin B monoclonal antibody labeled with quantum dot fluorescent microspheres and mouse IgG antibody labeled with quantum dot fluorescent microspheres at a mass ratio of 1:0.5-1:1.

5.

9. The immunochromatographic test strip according to claim 8, characterized in that: The total mass concentration of antibodies in the antibody mixture is 3-10 mg / mL.

10. A method for preparing an immunochromatographic test strip for quantitative detection of fetoglobulin B using quantum dot fluorescent microspheres according to any one of claims 1-9, characterized in that... Specifically, the following steps are included: (1) Quantum dot fluorescent microsphere-labeled fetal protein B monoclonal antibody and quantum dot fluorescent microsphere-labeled mouse IgG antibody were prepared by the following methods: The activated quantum dot fluorescent microspheres and antibodies were mixed evenly at a mass ratio of 20:1-3:1 and reacted at room temperature for 3-5 hours. The reaction was terminated by adding ethanolamine. After centrifugation and washing, blocking solution was added and reacted at room temperature for 30-60 minutes to obtain the antibody labeled with quantum dot fluorescent microspheres. (2) Preparation of release pad: The quantum dot fluorescent microsphere-labeled fetal protein B monoclonal antibody and the quantum dot fluorescent microsphere-labeled mouse IgG antibody prepared above (1) were coated on the release pad; (3) Sample pad preparation: The sample pad is immersed in the sample pad buffer solution and then removed and dried. The sample pad buffer solution is formulated with 10 mM Na2B4O7·10H2O, 1% polyvinylpyrrolidone, 0.2% sodium caseinate, 1% Tritium-X100, 1% S9 and 0.05% NaN3, pH 8.

5. (4) Nitrocellulose membrane The nitrocellulose membrane is provided with a detection line and a control line along the chromatography direction, and the fetal globulin B monoclonal antibody and goat anti-mouse IgG antibody are coated on the nitrocellulose membrane as the detection line and the control line, respectively. (5) Assembly The sample pad, release pad, nitrocellulose membrane, and absorbent pad are assembled onto a PVC base plate to obtain a test paper, which is then cut to obtain an immunochromatographic test strip.

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