Chromatographic test paper for combined detection of primary liver cancer markers and preparation method

Abstract

The invention belongs to the technical field of clinical medical examination, and relates to chromatographic test paper for combined detection of primary liver cancer markers and a preparation method, and the chromatographic test paper comprises a sample pad, a fluorescent microsphere combination pad, a nitrocellulose membrane and absorbent paper which are sequentially pasted on a bottom plate in the horizontal direction with overlapped parts; the fluorescent microsphere combination pad contains a fluorescent microsphere labeling mixed solution containing an AFP detection antibody and an FER detection antibody; and the nitrocellulose membrane is coated with a detection line of an FER capture antibody, a detection line of an AFP capture antibody and a quality control line of goat anti-mouse IgG. According to the chromatographic test paper, low-background and high-sensitivity near-infrared fluorescence is used as a detection signal, sensitive, accurate and rapid detection of a fluorescence immunochromatography platform is achieved, meanwhile, synchronous quantitative analysis of the primary liver cancer specific markers AFP and FER is achieved through one-time sampling and one-time sample adding, and therefore the precise diagnosis efficiency of the primary liver cancer is further improved; the patient examination cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of clinical medical examination, and particularly relates to a chromatographic test strip for combined detection of primary liver cancer markers and a preparation method thereof. Background Art

[0002] Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors globally, with relatively high morbidity and mortality rates. The onset of primary liver cancer is relatively concealed and the symptoms are not typical. The diagnosis mainly relies on methods such as imaging and liver tissue pathological biopsy. Its early imaging features and physical signs are not obvious. More than 70% of patients are found to be in the advanced stage when diagnosed, and the 5-year survival rate is only about 12%. The high mortality rate of primary liver cancer is closely related to the low early diagnosis rate. If early liver cancer is treated promptly and regularly, the survival rate over 5 years can reach 70%, but the diagnosis rate of early liver cancer patients is less than 30%. Early screening and accurate diagnosis of liver cancer are the keys to clinical diagnosis, treatment, and prognosis, and are a crucial step in improving the quality of life of patients.

[0003] Serum alpha-fetoprotein (AFP) is a commonly used and important indicator for the current diagnosis of HCC and efficacy monitoring. Its clinical normal reference value is 0 - 25 ng / mL. When liver cells become cancerous, AFP is secreted again by liver cancer cells. Approximately 70% of liver cancer patients have elevated AFP in their serum. When serum AFP ≥ 400 ng / mL for more than 1 month, excluding pregnancy, it highly suggests HCC. Therefore, AFP has become a specific clinical indicator for the diagnosis of HCC. However, an increase in AFP does not necessarily indicate the occurrence of liver cancer. AFP may also increase in non-HCC patients such as those with cirrhosis, chronic liver diseases, germ cell tumors, or pregnant women. Long-term clinical practice has shown that approximately 30% of liver cancer patients have normal AFP levels, and the sensitivity of its diagnosis of HCC is less than 60%. The specificity and sensitivity are not ideal. In order to improve the sensitivity and specificity of HCC diagnosis, better indicators are needed to improve the detection rate of HCC.

[0004] Ferritin (SF) is an important iron storage protein in the human body, mainly synthesized in the liver, and has the function of participating in the regulation of human hematopoiesis and immune system. The content of ferritin in the serum of normal people is very low. Generally, the normal average value is about 15 - 200 ng / ml for men and about 12 - 150 ng / ml for women. When liver cells become cancerous, cancer cells synthesize a large amount of acidic ferritin. In addition, due to damaged liver cells, the functions of ferritin clearance and iron transfer are affected, thus showing an abnormal increase in the content of serum ferritin. At present, it is considered that the determination of ferritin can be used as one of the means for the auxiliary diagnosis, efficacy monitoring, and prognosis judgment of HCC, especially meaningful for patients with negative AFP.

[0005] The main methods for detecting the above single indicators are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay (CLIA). Although they have advantages such as high sensitivity and strong specificity, they are cumbersome to operate, time-consuming, and require professional personnel to operate, and are suitable for large tertiary hospitals. Fluorescence immunochromatography assay (FLFA) has the advantages of simple operation, rapidity, relatively high sensitivity, and low cost, and is particularly suitable for rapid on-site disease diagnosis and population screening, and has broad application and development prospects in primary medical and health institutions. Among them, fluorescent microspheres are the key to affecting the performance of FLFA, and the development of high-performance fluorescent microspheres is a necessary condition for improving the performance of FLFA. Near-infrared fluorescent microspheres have significant advantages such as long fluorescence emission wavelength, high quantum efficiency, large Stokes shift, and small light scattering and autofluorescence interference, which can effectively reduce the problem of background fluorescence of reagent cards (such as nitrocellulose membranes) interfering with biological samples (such as blood). Selecting near-infrared fluorescence with excellent anti-interference ability as the detection signal can effectively improve the performance of FLFA, thereby further broadening its application scenarios and development potential.

[0006] At present, there are few applied studies on the combined quantitative rapid detection of HCC markers AFP and FER. Therefore, it has important development value and clinical application potential to develop high-performance fluorescence immunoassay reagents using the advantages of near-infrared fluorescence to achieve the combined quantitative detection of HCC specific markers AFP and FER. Summary of the Invention

[0007] In view of the above deficiencies in the existing technology, the present invention provides a chromatographic test strip for the combined detection of primary liver cancer markers and a preparation method thereof. The specific technical solutions are as follows:

[0008] The first object of the present invention is to provide a chromatographic test strip for the combined detection of primary liver cancer markers, including a bottom plate, a sample pad, a fluorescent microsphere conjugate pad, a nitrocellulose membrane, and a blotting paper;

[0009] The sample pad, the fluorescent microsphere conjugate pad, the nitrocellulose membrane, and the blotting paper are sequentially pasted on the bottom plate along the horizontal direction with overlapping parts between each other;

[0010] The fluorescent microsphere conjugate pad contains a fluorescent microsphere-labeled mixture, and the fluorescent microsphere-labeled mixture includes an AFP detection antibody and an FER detection antibody;

[0011] The nitrocellulose membrane is coated with a detection line of an FER capture antibody, a detection line of an AFP capture antibody, and a quality control line of goat anti-mouse IgG.

[0012] The chromatographic test strip for the combined detection of primary liver cancer markers of the present invention uses near-infrared fluorescence with low background and high sensitivity as the detection signal to achieve sensitive, accurate and rapid detection on the fluorescence immunochromatography platform. At the same time, the chromatographic test strip can realize the synchronous quantitative analysis of the specific markers AFP and FER of primary liver cancer through one sampling and one sample addition, thereby further improving the precision diagnosis efficiency of primary liver cancer and reducing the examination cost of patients.

[0013] Further, the preparation method of the fluorescent microsphere-labeled mixture includes the following steps:

[0014] A1. Activation treatment of fluorescent microspheres: Take carboxylated fluorescent microspheres, resuspend them by ultrasound, and discard the supernatant after centrifugation; add MES (2-morpholinoethanesulfonic acid) buffer solution to the precipitate, and ultrasonicate until dispersed; add the activator EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and the coupling agent Sulfo-NHS (N-hydroxysulfosuccinimide), after oscillating reaction, discard the supernatant after centrifugation, and then add PBS buffer solution and ultrasonicate until dispersed to obtain the activated solution of fluorescent microspheres;

[0015] A2. Labeling of AFP detection antibody on fluorescent microspheres: Take half of the activated solution of fluorescent microspheres obtained in step A1, add the AFP detection antibody, oscillate the reaction, then add BSA (bovine serum albumin) solution for blocking, and finally add ethanolamine solution to terminate the reaction; discard the supernatant after centrifugation, and then add microsphere washing solution and ultrasonicate until resuspended, repeat 2-3 times to obtain the first labeling solution; add microsphere protection solution and store at 4°C for later use;

[0016] A3. Labeling of FER detection antibody on fluorescent microspheres: Take the other half of the activated solution of fluorescent microspheres obtained in step A1, add the FER detection antibody, oscillate the reaction, then add BSA solution for blocking, and finally add ethanolamine solution to terminate the reaction; discard the supernatant after centrifugation, and then add microsphere washing solution and ultrasonicate until resuspended, repeat 2-3 times to obtain the second labeling solution; add microsphere protection solution and store at 4°C for later use;

[0017] A4. Mixing of labeling solutions: Mix the first labeling solution in step A2 and the second labeling solution in step A3 in proportion for later use.

[0018] Further, another preparation method of the fluorescent microsphere-labeled mixture includes the following steps:

[0019] B1. Activation treatment of fluorescent microspheres: Take carboxylated fluorescent microspheres, resuspend them by ultrasound, and discard the supernatant after centrifugation; add MES buffer solution to the precipitate, and ultrasonicate until dispersed; add the activator EDC and the coupling agent Sulfo-NHS, after oscillating reaction, discard the supernatant after centrifugation, and then add PB buffer solution and ultrasonicate until dispersed to obtain the activated solution of fluorescent microspheres;

[0020] B2. Labeling mixture: Add the mixed solution of AFP detection antibody and FER detection antibody to the activated solution of fluorescent microspheres obtained in step B1, oscillate for reaction, then add BSA solution for blocking, and finally add ethanolamine solution to terminate the reaction; after centrifugation, discard the supernatant, add microsphere washing solution and ultrasonicate until resuspended, repeat 2 - 3 times; add microsphere protective solution and store at 4 °C for standby.

[0021] Further, in steps A1 and B1, the molar ratio of carboxylated fluorescent microspheres to EDC and Sulfo - NHS is 1:(3 - 6):(8 - 12).

[0022] Further, the concentration of AFP detection antibody in step A2 and FER detection antibody in step A3 is 0.05 - 0.5 mg / mL.

[0023] Further, in step A4, the volume ratio of labeling solution one to labeling solution two is 1:0.5 - 2.

[0024] Further, in step B2, the mass ratio of AFP detection antibody to FER detection antibody is 1:0.5 - 2.

[0025] Further, the fluorescent microspheres in the fluorescent microsphere labeling mixture are one of fluorescent dye microspheres, quantum dot fluorescent microspheres, and rare earth nanophosphor microspheres, preferably rare earth nanophosphor microspheres.

[0026] Preferably, the excitation wavelength of the rare earth nanophosphor microspheres is in the range of 700 - 1000 nm, and the fluorescence emission wavelength range is in the range of 700 - 1700 nm.

[0027] Preferably, the doping ion of the rare earth nanophosphor microspheres is Nd 3+ 、Yb 3+ 、Er 3+ 、Ho 3+ or Ce 3+ or any one or more of them.

[0028] Preferably, the rare earth nanophosphor microspheres are composed of a core and a shell structure.

[0029] Preferably, the core is an inorganic matrix doped with ions, including NaLnF4, LiLnF4 or LnF3, where Ln = Y, Yb, Gd or Lu; the shell is one or more layers of inorganic matrix coated outside the core, including NaLnF4, LiLnF4 or CaF2, where Ln = Y, Yb, Gd or Lu.

[0030] Preferably, the surface of the rare earth nano-fluorescent microspheres is carboxylated and modified with one or two or more of sodium citrate, polyacrylic acid, stearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl, polycaprolactone-polyethylene glycol-carboxyl, and polyethylene glycol-polylactic acid-carboxyl.

[0031] Preferably, the average particle size of the rare earth nano-fluorescent microspheres is 20 - 500 nm.

[0032] The second object of the present invention is to provide a preparation method of the chromatographic test strip for the combined detection of the above-mentioned primary liver cancer markers, including the following steps:

[0033] (1) Preparation of the sample pad: Select glass fiber or polyester fiber, soak it in the sample pad treatment solution, oscillate at room temperature for 2 - 3 h, and dry it in an oven.

[0034] (2) Preparation of the fluorescent microsphere conjugate pad: Add the microsphere diluent to the fluorescent microsphere labeling mixture, and then evenly coat it on the glass fiber or polyester fiber, and dry it thoroughly in an oven.

[0035] (3) Coating treatment of the nitrocellulose membrane: Prepare the AFP capture antibody solution, FER capture antibody solution, and goat anti-mouse IgG solution respectively with PBS buffer, and use a three-dimensional membrane scribing instrument to spray the detection line of the AFP monoclonal capture antibody, the detection line of the FER capture antibody, and the quality control line of the goat anti-mouse IgG on the nitrocellulose membrane in parallel, and dry it thoroughly in an oven.

[0036] (4) Preparation of the absorbent paper: Cut the absorbent paper into appropriate specifications.

[0037] (5) Assembly and card making: Paste the prepared sample pad, fluorescent microsphere conjugate pad, nitrocellulose membrane, and absorbent paper on the bottom plate in sequence with overlapping parts, then cut it into test strips, put it into a card shell, make a test reagent card, and store it in low-temperature and dry conditions.

[0038] Further, in the step (3), the concentrations of the AFP capture antibody solution, FER capture antibody solution, and goat anti-mouse IgG solution are 0.2 - 3.0 mg / mL.

[0039] Further, in the step (3), the overlapping part is 1.5 - 3 mm.

[0040] The beneficial effects of the present invention are:

[0041] 1) The chromatographic test strip for the combined detection of primary liver cancer markers provided by the present invention can simultaneously achieve the quantitative detection of two primary liver cancer markers, alpha-fetoprotein (AFP) and ferritin (FER), through one sampling and one sample addition, greatly improving the detection rate of primary liver cancer and facilitating the improvement of its early accurate diagnosis rate; 2) The present invention uses near-infrared fluorescence with low background and high sensitivity as the detection signal, which can effectively improve the detection sensitivity and accuracy of the immunochromatographic platform; 3) When the chromatographic test strip of the present invention is used for detection, the required sample amount is small, which can reduce the pain of blood sampling for patients and also reduce the inspection cost. Brief Description of the Drawings

[0042] Figure 1 It is a schematic structural diagram of the chromatographic test strip for the combined detection of primary liver cancer markers prepared by the present invention;

[0043] Reference numerals: 1, bottom plate; 2, sample pad; 3, fluorescence microsphere binding pad; 4, nitrocellulose membrane; 5, absorbent paper; 6, detection line 1; 7, detection line 2; 8, quality control line.

[0044] Figure 2 It is a transmission electron microscope image of the rare earth nanophosphor microspheres prepared in the embodiment of the present invention;

[0045] Figure 3 It is a particle size distribution diagram of the rare earth nanophosphor microspheres prepared in the embodiment of the present invention;

[0046] Figure 4 It is an excitation spectrum diagram of the rare earth nanophosphor microspheres prepared in the embodiment of the present invention;

[0047] Figure 5 It is a fluorescence emission spectrum diagram of the rare earth nanophosphor microspheres prepared in the embodiment of the present invention;

[0048] Figure 6 It is a standard curve diagram for detecting the AFP concentration by the near-infrared fluorescence immunochromatographic test strip prepared in the embodiment of the present invention;

[0049] Figure 7 It is a standard curve diagram for detecting the FER concentration by the near-infrared fluorescence immunochromatographic test strip prepared in the embodiment of the present invention. Detailed Description of the Invention

[0050] The principles and features of the present invention will be described below in conjunction with examples. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0051] Example:

[0052] As Figure 1As shown in the figure, a chromatographic test strip for the combined detection of primary liver cancer markers includes a bottom plate 1, a sample pad 2, a fluorescent microsphere binding pad 3, a nitrocellulose membrane 4, and a blotting paper 5. The sample pad 2, the fluorescent microsphere binding pad 3, the nitrocellulose membrane 4, and the blotting paper 5 are sequentially pasted on the bottom plate 1 in the horizontal direction, and adjacent parts overlap each other by 2 mm. The fluorescent microsphere binding pad 3 is fixed with a fluorescent microsphere-coupled antibody complex, and the fluorescent microsphere-coupled antibody complex is a mixture obtained by adding a microsphere diluent to a fluorescent microsphere labeling mixture, then uniformly coating it on glass fiber, and fully drying it in an oven. The nitrocellulose membrane 4 is respectively coated with a detection line 6 for FER capture antibody, a detection line 7 for AFP capture antibody, and a quality control line 8 for goat anti-mouse IgG.

[0053] The fluorescent microspheres are rare earth nanophosphors (RENPs: NaYbF4 with a fluorescence emission peak in the second near-infrared region, NaYF4:Nd 70% for the chromatographic test strip for the combined detection of primary liver cancer markers, NaYF4) that have a fluorescence emission peak in the second near-infrared region; the excitation wavelength of the rare earth nanophosphors is 808 nm, and the fluorescence emission wavelength is 980 nm; the particle size is 180 nm.

[0054] The preparation method of the chromatographic test strip for the combined detection of primary liver cancer markers includes the following steps:

[0055] (1) Preparation of the sample pad:

[0056] Soak a 300 mm long and 10 mm wide glass fiber in the sample pad treatment solution, oscillate it at room temperature for 2 h, and dry it in an oven at 45 °C for 12 hours; the sample pad treatment solution is a 20 mM Tris buffer solution containing 0.5% S9, 0.05% BSA, 0.05% Tween-20, 0.3% PVP-K30, and 0.05% Proclin-300.

[0057] (2) Preparation of the fluorescent microsphere binding pad:

[0058] Add a microsphere diluent (10 mM sodium citrate solution containing 1% sucrose) to the fluorescent microsphere labeling mixture, dilute it to 40% of the original concentration, and then uniformly spread it on glass fiber or polyester fiber at 10 μL / cm, and dry it in an oven at 45 °C for 12 h.

[0059] The fluorescent microsphere labeling mixture is a rare earth nanophosphor (RENPs) labeling mixture, and its preparation method includes the following steps:

[0060] ①Activation treatment of RENPs: Take 200 μL of RENPs solution modified with polyacrylic acid (PAA) (concentration 0.05 mM), ultrasonicate for 5 min, centrifuge at 14000 rpm for 15 min, and discard the supernatant; add 10 mM MES buffer (pH = 6.0) to the precipitate, ultrasonicate until dispersed; sequentially add 10 mg of coupling agent Sulfo-NHS and 5 mg of activator EDC, oscillate and react for 30 min; after completion, centrifuge at 14000 rpm for 15 min, discard the supernatant, and then add 10 mM PB buffer (pH = 7.4) and ultrasonicate until dispersed to obtain the RENPs activation solution. Divide the RENPs activation solution into two equal parts;

[0061] ②Labeling of RENPs with AFP detection antibody: Take one part of the RENPs activation solution, add 100 μg of AFP detection antibody, oscillate and react for 120 min, then add 10% BSA solution to block for 60 min, and finally add 40 mM ethanolamine solution to terminate the reaction; centrifuge at 14000 rpm for 15 min, discard the supernatant, add microsphere washing solution and ultrasonicate until resuspended, repeat 2 times to obtain Labeling Solution 1; finally, add microsphere protection solution and store at 4 °C for standby;

[0062] ③Labeling of RENPs with FER detection antibody: Take the other part of the RENPs activation solution, add 100 μg of FER detection antibody, oscillate and react for 120 min, then add 10% BSA solution to block for 60 min, and finally add 40 mM ethanolamine solution to terminate the reaction; centrifuge at 14000 rpm for 15 min, discard the supernatant, add microsphere washing solution and ultrasonicate until resuspended, repeat 2 times to obtain Labeling Solution 2; finally, add microsphere protection solution and store at 4 °C for standby;

[0063] ④Mixing of labeling solutions: Mix Labeling Solution 1 from step ② and Labeling Solution 2 from step ③ in an equal volume ratio for standby.

[0064] (3) Coating treatment of nitrocellulose membrane:

[0065] Coat the detection line 1 for FER capture antibody, detection line 2 for AFP capture antibody, and quality control line 8 for goat anti-mouse IgG with an interval of 4 mm from each other on the nitrocellulose membrane 4;

[0066] Preparation of the detection line 1 for FER capture antibody: Prepare a 2 mg / mL solution of FER capture antibody (Fitebio Co., Ltd.) with 50 mM PBS buffer (pH = 7.4), and use a three-dimensional membrane scribing instrument to spray parallelly on the nitrocellulose membrane 4 at a rate of 1.2 μL / cm and a scribing speed of 50 mm / s;

[0067] Preparation of the test line 7 of the AFP capture antibody: Prepare a 2 mg / mL solution of the AFP capture antibody (Fitzgerald Industries International, Inc.) with 50 mM PBS buffer (pH = 7.4), and spray it parallelly on the nitrocellulose membrane 4 with a three-dimensional membrane scribing instrument at a rate of 1.2 μL / cm and a scribing speed of 50 mm / s;

[0068] Preparation of the quality control line 8 of goat anti-mouse IgG: Prepare a 1 mg / mL solution of goat anti-mouse IgG (Fitzgerald Industries International, Inc.) with 50 mM PBS buffer (pH = 7.4), and spray it parallelly on the nitrocellulose membrane 4 with a three-dimensional membrane scribing instrument at a rate of 1.2 μL / cm and a scribing speed of 50 mm / s.

[0069] Place the sprayed nitrocellulose membrane in an oven at 45 °C and dry it for 12 h. Store it dry for later use.

[0070] (4) Preparation of the absorbent paper: Cut the absorbent paper into strips with a specification of 30 * 2.5 cm each;

[0071] (5) Assembly and card making:

[0072] Attach the prepared sample pad, fluorescent microsphere conjugate pad, nitrocellulose membrane and absorbent paper to the PVC bottom plate 1 in sequence, with an overlap of 2 mm each other, cut them into test strips with a width of 4.0 mm using an automatic cutting machine, and install them into the card shell to make a test reagent card. Store it in a low-temperature and dry place for later use.

[0073] See Figure 2 The transmission electron microscope image of the RENPs prepared in this example. The RENPs prepared in this example have regular shapes and good dispersibility, and can be used for subsequent antibody conjugation. See Figure 3 The particle size distribution diagram of the RENPs prepared in this example. The RENPs prepared in this example have a moderate particle size (340 nm) and a low dispersity index (PDI = 0.293), indicating that the particle sizes are relatively uniform. See Figure 4 The absorption spectrum diagram of the RENPs prepared in this example. The RENPs have an obvious absorption near 808 nm, indicating successful preparation. See Figure 5 The fluorescence emission spectrum diagram of the RENPs prepared in this example. The RENPs have a strong fluorescence emission peak at 980 nm, which is helpful for achieving highly sensitive detection.

[0074] Application example

[0075] Use the reagent card of the example to detect serum alpha-fetoprotein (AFP)

[0076] Restore the AFP serum quality control product and the reagent card prepared in the example to room temperature. Add 40 μL of AFP serum quality control products with concentrations of 1.0, 3.0, 16.0, 45.0, 90.0, and 200.0 ng / mL to the sample addition holes of the reagent card respectively, and then add 40 μL of sample diluent respectively. React for 15 min, and then place the reagent card in a NIR-II fluorescence immunoassay analyzer for testing. Record the fluorescence intensities of the T line and the C line, calculate the T / C ratio, and establish a standard curve of concentration and fluorescence ratio (see Figure 6 ). It can be seen that the linear correlation is good (R = 0.9923), which can make the test results more accurate. Each concentration point is tested in parallel 3 times. The results of the AFP detection precision experiment are shown in Table 1. The within-batch coefficient of variation (CV) is lower than 10%, and the between-batch CV is lower than 14%, indicating good detection precision and proving that the test strip has good reproducibility and high reliability in detecting AFP.

[0077] Table 1 Results of the AFP detection precision experiment

[0078]

[0079] Use the reagent card of the example to detect serum ferritin (FER)

[0080] Restore the FER serum quality control product and the reagent card prepared in the example to room temperature. Add 40 μL of FER serum quality control products with concentrations of 1.0, 10.0, 45.0, 170.0, 290.0, 480.0, 600.0, 800.0, and 1000.0 ng / mL to the sample addition holes of the reagent card respectively, and then add 40 μL of sample diluent respectively. React for 15 min, and then place the reagent card in a NIR-II fluorescence immunoassay analyzer for testing. Record the fluorescence intensities of the T line and the C line, calculate the T / C ratio, and establish a standard curve of concentration and fluorescence ratio (see Figure 7 ). It can be seen that the linear range is wide (1 - 1000 ng / mL) and the linear correlation is good (R = 0.9937), which can make the test results more accurate. Each concentration point is tested in parallel 3 times. The results of the FER detection precision experiment are shown in Table 2. The within-batch coefficient of variation (CV) is lower than 11%, and the between-batch CV is lower than 10%, indicating good detection precision and proving that the test strip has good reproducibility and high reliability in detecting FER.

[0081] Table 2 Results of the FER detection precision experiment

[0082]

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A chromatographic test strip for the combined detection of primary liver cancer markers, characterized in that, It includes a bottom plate, a sample pad, a fluorescent microsphere binding pad, a nitrocellulose membrane and a blotting paper; The sample pad, the fluorescent microsphere binding pad, the nitrocellulose membrane and the blotting paper are horizontally pasted on the bottom plate in sequence with overlapping parts between each other; The fluorescent microsphere binding pad contains a fluorescent microsphere labeled mixture, and the fluorescent microsphere labeled mixture includes an AFP detection antibody and a FER detection antibody; The nitrocellulose membrane is coated with a detection line of a FER capture antibody, a detection line of an AFP capture antibody and a quality control line of a goat anti-mouse IgG.

2. The chromatographic test strip for the combined detection of primary liver cancer markers according to claim 1, wherein The preparation method of the fluorescent microsphere labeled mixture includes the following steps: A1. Activation treatment of fluorescent microspheres: Take carboxylated fluorescent microspheres, resuspend them by ultrasonic treatment, discard the supernatant after centrifugation; add MES buffer to the precipitate, ultrasonicate until dispersed; add an activator EDC and a coupling agent Sulfo-NHS, react by oscillation, discard the supernatant after centrifugation, and then add PBS buffer and ultrasonicate until dispersed to obtain an activated fluorescent microsphere solution; A2. Labeling of AFP detection antibody on fluorescent microspheres: Take half of the activated fluorescent microsphere solution obtained in step A1, add the AFP detection antibody, react by oscillation, then add a BSA solution for blocking, and finally add an ethanolamine solution to terminate the reaction; discard the supernatant after centrifugation, and then add a microsphere washing solution and ultrasonicate until resuspended, repeat 2 - 3 times to obtain labeling solution one; add a microsphere protection solution and store at 4°C for standby; A3. Labeling of FER detection antibody on fluorescent microspheres: Take the other half of the activated fluorescent microsphere solution obtained in step A1, add the FER detection antibody, react by oscillation, then add a BSA solution for blocking, and finally add an ethanolamine solution to terminate the reaction; discard the supernatant after centrifugation, and then add a microsphere washing solution and ultrasonicate until resuspended, repeat 2 - 3 times to obtain labeling solution two; add a microsphere protection solution and store at 4°C for standby; A4. Mixing of labeling solutions: Mix labeling solution one in step A2 and labeling solution two in step A3 for standby.

3. The chromatographic test strip for combined detection of primary liver cancer markers according to claim 1, characterized in that The preparation method of the fluorescent microsphere labeled mixture includes the following steps: B1. Activation treatment of fluorescent microspheres: Take carboxylated fluorescent microspheres, resuspend them by ultrasonic treatment, discard the supernatant after centrifugation; add MES buffer to the precipitate, ultrasonicate until dispersed; add an activator EDC and a coupling agent Sulfo-NHS, react by oscillation, discard the supernatant after centrifugation, and then add PB buffer and ultrasonicate until dispersed to obtain an activated fluorescent microsphere solution; B2. Labeling mixture: Add a mixture of an AFP detection antibody and a FER detection antibody to the activated fluorescent microsphere solution obtained in step B1, react by oscillation, then add a BSA solution for blocking, and finally add an ethanolamine solution to terminate the reaction; discard the supernatant after centrifugation, and then add a microsphere washing solution and ultrasonicate until resuspended, repeat 2 - 3 times; add a microsphere protection solution and store at 4°C for standby.

4. The chromatographic test strip for combined detection of primary liver cancer markers according to claim 2 or 3, characterized in that The molar ratio of the carboxylated fluorescent microspheres to EDC and Sulfo-NHS is 1:5:

10.

5. The chromatographic test strip for the combined detection of primary liver cancer markers according to claim 2, wherein The concentrations of the AFP detection antibody in step A2 and the FER detection antibody in step A3 are respectively 0.05 - 0.5 mg / mL.

6. The chromatographic test strip for the combined detection of primary liver cancer markers according to claim 2, wherein In step A4, the volume ratio of labeling solution one to labeling solution two is 1:0.5 - 2.

7. The chromatographic test strip for the combined detection of primary liver cancer markers according to claim 3, wherein, In the step B2, the mass ratio of the AFP detection antibody to the FER detection antibody is 1:0.5 - 2.

8. The chromatographic test strip for the combined detection of primary liver cancer markers according to claim 1, characterized in that, The fluorescent microspheres in the fluorescent microsphere labeling mixture are one of fluorescent dye microspheres, quantum dot fluorescent microspheres, and rare earth nanophosphor microspheres.

9. A method for preparing a chromatographic test strip for the combined detection of primary liver cancer markers as described in any one of claims 1-8, characterized in that, It includes: (1) Preparation of the sample pad: Select glass fiber or polyester fiber, soak it in the sample pad treatment solution, oscillate at room temperature for 2 - 3 h, and dry it in an oven at 45°C. (2) Preparation of the fluorescent microsphere conjugate pad: Add the microsphere diluent to the fluorescent microsphere labeling mixture, then evenly coat it on the glass fiber or polyester fiber, and dry it thoroughly in an oven at 45°C. (3) Coating treatment of the nitrocellulose membrane: Prepare the AFP capture antibody solution, FER capture antibody solution, and goat anti-mouse IgG solution with PBS buffer respectively, and use a three-dimensional membrane scribing instrument to spray the detection line of the AFP capture antibody, the detection line of the FER capture antibody, and the quality control line of the goat anti-mouse IgG on the nitrocellulose membrane in parallel, and dry it thoroughly in an oven at 45°C. (4) Preparation of the absorbent paper: Cut the absorbent paper into strips with a specification of 30 * 2.5 cm each. (5) Assembly and card making: Paste the prepared sample pad, fluorescent microsphere conjugate pad, nitrocellulose membrane, and absorbent paper on the bottom plate in sequence with overlapping parts between each other, then cut it into test strips, put them into the card shell, make a reagent card for detection, and store it in low-temperature and dry conditions.

10. The preparation method of the chromatographic test strip for the combined detection of primary liver cancer markers according to claim 9, wherein, In the step (3), the concentrations of the AFP capture antibody solution, FER capture antibody solution, and goat anti-mouse IgG solution are 0.2 - 3.0 mg / mL respectively.

Images