Pepsin detection card and preparation process thereof

Abstract

The invention provides a pepsin detection card and a preparation process thereof, and belongs to the technical field of microbiological detection. The kit comprises a test strip and a card shell, the test strip comprises a nitrocellulose membrane, a sample pad, absorbent paper, a combination pad and a bottom plate. The pepsase detection card is high in sensitivity, can accurately capture pepsase with extremely low concentration in a sample, effectively avoids missed diagnosis, strives for precious time for early intervention, is high in specificity, is only specifically combined with pepsase, is not interfered by other similar proteases or impurities in the sample, and can be used for detecting the pepsase in the sample. In a complex human body biological sample environment, the target pepsin can still be accurately recognized, the specificity reaches 98% or above, false positive results are greatly reduced, and a reliable basis is provided for clinical diagnosis.

Description

Technical Field

[0001] The invention relates to the technical field of microbial testing, and in particular to a pepsin detection card and a preparation process thereof. Background Art

[0002] Gastric diseases have always been a major problem that plagues human health worldwide, with the incidence rate rising year by year. From common gastritis and gastric ulcers to serious life-threatening gastric cancer, these diseases often have hidden symptoms in the early stages and are easily ignored by patients. According to statistics from the World Health Organization, there are more than tens of millions of new gastric disease patients worldwide each year, and most of them are in the middle and late stages when gastric cancer is diagnosed, with a low five-year survival rate. Early diagnosis has become a key breakthrough to improve cure rates and reduce mortality rates.

[0003] In the clinical diagnostic process, pepsin, as a key biomarker reflecting the physiological and pathological state of the stomach, plays a vital role in the screening, diagnostic staging and prognostic evaluation of gastric diseases. Pepsin is secreted by the chief cells of the gastric mucosa. Under normal physiological conditions, its content in gastric juice is maintained at a relatively stable level to assist in food digestion. However, once gastric tissue lesions occur, such as inflammation, tumor invasion, etc., gastric mucosal cells are dysfunctional, and the secretion, activity and molecular structure of pepsin may change significantly. Accurate monitoring of these changes can provide doctors with key diagnostic clues and timely formulate precise treatment strategies.

[0004] Traditional pepsin detection methods mainly include laboratory enzyme-linked immunosorbent assay (ELISA) technology, colorimetric detection, etc. ELISA has long been regarded as the "gold standard" for pepsin detection. It is based on the principle of antigen-antibody specific binding and quantitatively detects the concentration of pepsin by enzyme-catalyzed substrate color development. Although it has high accuracy, the operation process is extremely cumbersome and requires professional technicians to perform in a well-equipped laboratory environment. After sample collection, it usually undergoes complex pre-treatment steps, including multiple centrifugation, dilution, and sample addition. The entire detection process takes up to several hours, which is difficult to meet the needs of clinical rapid diagnosis, especially for emergency, primary medical institutions, and large-scale population screening scenarios; while the traditional colorimetric method uses the catalytic hydrolysis reaction of pepsin to specific substrates to qualitatively or semi-quantitatively judge the activity of pepsin by observing color changes. Although this method is relatively simple to operate and low in cost, it has poor sensitivity. It is difficult to accurately distinguish low-concentration pepsin samples, and is easily interfered by impurities and other proteases in the sample. The specificity is not strong, resulting in a high rate of misdiagnosis and missed diagnosis, and it cannot provide a reliable basis for early diagnosis of the disease.

[0005] Therefore, it is necessary to propose a pepsin detection card with high sensitivity and strong specificity and a preparation process thereof. Summary of the invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a pepsin detection card and its preparation process.

[0007] A pepsin detection card consists of a test strip and a card shell; The test strip includes: a nitrocellulose membrane, a sample pad, a blotting paper, a conjugate pad, and a base plate; In the nitrocellulose membrane, the test line T is coated with an antibody 1 against human pepsin with an effective content concentration of (0.2 - 0.8) μg / person - portion of mouse anti - human pepsin, and the control line C is coated with an antibody of dinitrophenyl - conjugated bovine serum albumin with an effective content concentration of (0.2 - 0.8) μg / person - portion; The conjugate pad includes fluorescent microspheres containing antibody 2 against human pepsin (the effective content of the labeled antibody is about 0.1 - 0.5 μg / person - portion) and fluorescent microspheres labeled with dinitrophenol (the effective content of the labeled antibody is about 0.1 - 0.5 μg / person - portion).

[0008] Furthermore, the preparation process of a pepsin detection card as described above includes the following steps: S1: Prepare the fluorescent microsphere labeling solution Prepare MES buffer solution, Sulfo - NHS solution, EDC solution, and latex blocking solution respectively; S2: Fluorescent microsphere labeling First, wash the fluorescent microspheres, then activate them with the above - mentioned Sulfo - NHS solution and EDC solution, then disperse them with the labeling antibody in the above - mentioned MES buffer solution, add the above - mentioned latex blocking solution, carry out rotary incubation, and finally mix with the gold storage solution to obtain labeled microspheres; S3: Prepare the coating dilution solutions for line C and line T Prepare Tris solution, bovine serum albumin solution, and sugar solution respectively, and then mix them with the antibody of dinitrophenyl - conjugated bovine serum albumin or antibody 1 against human pepsin to obtain the coating dilution solution for line C and the coating dilution solution for line T; S4: Prepare line C and line T Draw the above - mentioned coating dilution solutions for line C and line T on the nitrocellulose membrane at 1 μL / cm respectively, and dry at 50 - 60 °C for 20 - 24 h; S5: Prepare the sample pad Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium caseinate, PVP10, sodium cholate, surfactant S9, Tween 20 and purified water are stirred and mixed, and then a Proclin 300 preservative is added, and the stirring and mixing is continued to obtain a sample pad solution, and then a polyester film is immersed in the sample pad solution for 15-20 minutes, and then transferred to a drying room and dried at (37±3)°C for 12-24 hours to obtain a sample pad, wherein the sample pad solution includes: 2.9% disodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.2% sodium caseinate, 1% PVP10, 1% sodium cholate, 0.1% S9, 1% Tween 20 and 0.1% Proclin 300 preservative; S6: Finished product assembly Assemble the absorbent paper, nitrocellulose membrane, conjugate pad and sample pad, and cut them into test strips with a width of 4±0.5 mm. Then install the absorbent paper end of the test strip on the top of the bottom card position, and the absorbent paper side on the bottom of the bottom card position. Buckle the upper card cover, press it with a shell pressing machine, put the desiccant and test paper card in an aluminum foil bag, seal it, and pack it into a box to obtain a pepsin test card.

[0009] Furthermore, S1 specifically includes the following steps: S1.1: Add MES to purified water at a solid-liquid ratio of 1 g: (80-90) mL, stir thoroughly to dissolve, and then add 1-6 mol / L sodium hydroxide solution to adjust the pH to 6.0 to obtain MES buffer; S1.2: Add Sulfo-NHS to the above MES buffer at a solid-liquid ratio of 1 g: (90-100) mL, stir and mix well to obtain a Sulfo-NHS solution; S1.3: Add EDC to the above MES buffer at a solid-liquid ratio of 1 g: (90-100) mL, stir and mix well to obtain an EDC solution; S1.4: Add tris(hydroxymethyl)aminomethane) into purified water at a solid-liquid ratio of 1g:(130-140)mL, stir to mix evenly, then add bovine serum albumin, ethanolamine and Proclin300 preservative, stir to mix, and then add 1-6mol / L hydrochloric acid to adjust the pH to 9.0 to obtain a latex blocking solution.

[0010] Furthermore, S2 specifically includes the following steps: S2.1: Add the fluorescent microspheres to the MES buffer prepared in step S1.1, vortex mix, and prepare a 1% fluorescent microsphere solution, then vortex mix with the MES buffer prepared in step S1.1 at a volume ratio of 1:(6-8), and then centrifuge at 9000-10000 rpm for 10-20 min, remove the supernatant, and obtain the washed microspheres; S2.2: Add the MES buffer solution prepared in step S1.1 to the above-mentioned washed microspheres, ultrasonically treat for 10 - 20 min, then add the Sulfo-NHS solution prepared in step S1.2 and the EDC solution prepared in step S1.3. After vortex mixing, rotate and incubate at 1200 - 1300 rpm for 1 - 2 h. After removing the supernatant, activated microspheres are obtained. S2.3: Add the MES buffer solution prepared in step S1.1 to the above-mentioned activated microspheres, ultrasonically treat for 10 - 20 min, then centrifuge and remove the supernatant to obtain purified microspheres. S2.4: Add the MES buffer solution prepared in step S1.1 to the above-mentioned purified microspheres, ultrasonically treat for 10 - 20 min, then add the antibody for labeling. After vortex mixing, rotate and incubate for 2 - 6 h. Then add the latex blocking solution prepared in step S1.4, vortex mix and rotate and incubate for 1 - 2 h. After centrifuging to remove the supernatant, antibody-conjugated microspheres are obtained. S2.5: Add purified water to the above-mentioned antibody-conjugated microspheres, ultrasonically treat for 10 - 20 min, then centrifuge and remove the supernatant. Then add the gold storage solution and ultrasonically mix evenly to obtain labeled microspheres.

[0011] Furthermore, S3 specifically includes the following steps: S3.1: Add tris(hydroxymethyl)aminomethane to purified water according to the solid-liquid ratio of 1 g : (160 - 170) mL, stir and mix evenly, then add 1 - 6 mol / L hydrochloric acid to adjust the pH to 8.0 to obtain a Tris solution. S3.2: Add bovine serum albumin to purified water, stir and mix evenly to prepare a 5% bovine serum albumin solution. S3.3: Add sucrose and trehalose to purified water according to the mass ratio of (3.5 - 4.5) : 1, stir and mix evenly to prepare a 25% sugar solution. S3.4: Mix the dinitrophenyl-conjugated bovine serum albumin antibody, the above-mentioned sugar solution and the above-mentioned Tris solution evenly to obtain the C-line coating dilution solution. S3.5: Mix the antibody 1 against human pepsin, the above-mentioned sugar solution, the above-mentioned bovine serum albumin solution and the above-mentioned Tris solution to obtain the T-line coating dilution solution.

[0012] Furthermore, in step S2.2, the volume ratio of the Sulfo-NHS solution, the EDC solution to the MES buffer solution is 1:1:(20 - 24).

[0013] Furthermore, the antibody for labeling is the antibody 2 against human pepsin and dinitrophenol, and the labeled microspheres are the microspheres labeled with the antibody 2 against human pepsin and the microspheres labeled with dinitrophenol.

[0014] Further, after the fluorescent microspheres are labeled, the labeled mouse anti-human pepsin antibody 2 microspheres and the labeled dinitrophenol microspheres of equal mass are mixed evenly, and then sprayed on the conjugate pad at a spraying amount of 2.5 μL / cm.

[0015] Further, the C line is coated with (0.2 - 0.8) μg / person - portion of dinitrobenzene - conjugated bovine serum albumin antibody, the final concentration of the sugar solution is 2.5%, and the remaining volume is made up with Tris solution; the T line is coated with (0.2 - 0.8) μg / person - portion of mouse anti - human pepsin antibody 1, the final concentration of the sugar solution is 2.5%, the final concentration of the bovine serum albumin solution is 0.5%, and the remaining volume is made up with Tris solution.

[0016] Further, the gold storage solution includes: 0.6% tris (hydroxymethyl) aminomethane, 0.5% sodium caseinate, 10% sucrose, 2.5% trehalose, 0.5% pvp10, 0.5% S9, 1.5% mannitol, 1% glycine, 0.1% Proclin300 preservative and 0.01% Evans blue, and the volume ratio of the gold storage solution to the fluorescent microsphere solution is (7.5 - 8.5):1.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The pepsin detection card of the present invention not only has high sensitivity and can accurately capture extremely low - concentration pepsin in the sample. Even when there are only subtle changes in the pepsin in the patient's body, the detection card can keenly detect it, effectively avoiding missed diagnosis and winning precious time for early intervention. Moreover, it has strong specificity and specifically binds only to pepsin, without being interfered by other similar proteases or impurities in the sample. In a complex human biological sample environment, such as gastric juice containing various digestive enzymes, tissue fluid mixed with inflammatory factors, etc., it can still accurately identify the target pepsin, and the specificity reaches more than 98%, greatly reducing false - positive results and providing a reliable basis for clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings incorporated herein and constituting a part of the specification illustrate embodiments of the present disclosure and, together with the specification, are further used to explain the principles of the present disclosure and enable those skilled in the relevant art to implement and use the present disclosure.

[0019] Figure 1 It is a process flow chart of the preparation of the pepsin detection card adopted in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The following describes in detail a pepsin detection card and its preparation process provided by the present invention with reference to the drawings and specific embodiments.

[0021] Example 1: A preparation process of a pepsin detection card, as Figure 1As shown, it includes the following steps: S1: Prepare a fluorescent microsphere labeling solution S1.1: Add MES to purified water at a solid-liquid ratio of 1 g: 80 mL, stir well to dissolve, then add 1 mol / L sodium hydroxide solution to adjust the pH to 6.0 to obtain a MES buffer solution; S1.2: Add Sulfo-NHS to the above MES buffer solution at a solid-liquid ratio of 1 g: 90 mL, stir and mix evenly to obtain a Sulfo-NHS solution; S1.3: Add EDC to the above MES buffer solution at a solid-liquid ratio of 1 g: 90 mL, stir and mix evenly to obtain an EDC solution; S1.4: Add tris(hydroxymethyl)aminomethane to purified water at a solid-liquid ratio of 1 g: 130 mL, stir and mix evenly, then add bovine serum albumin, ethanolamine and Proclin300 preservative, stir and mix, and then add 1 mol / L hydrochloric acid to adjust the pH to 9.0 to obtain a latex blocking solution; S2: Fluorescent microsphere labeling S2.1: Add fluorescent microspheres to the MES buffer solution prepared in step S1.1, vortex and mix well to prepare a 1% fluorescent microsphere solution, then vortex and mix well with the MES buffer solution prepared in step S1.1 at a volume ratio of 1:6, and then centrifuge at 9000 rpm for 10 min. After removing the supernatant, obtain washed microspheres; S2.2: Add the MES buffer solution prepared in step S1.1 to the above washed microspheres, ultrasonically treat for 10 min, then add the Sulfo-NHS solution prepared in step S1.2 and the EDC solution prepared in step S1.3, vortex and mix well, and then rotate and incubate at 1200 rpm for 1 h. After removing the supernatant, obtain activated microspheres, where the volume ratio of the Sulfo-NHS solution, the EDC solution to the MES buffer solution is 1:1:20; S2.3: Add the MES buffer solution prepared in step S1.1 to the above activated microspheres, ultrasonically treat for 10 min, then centrifuge and remove the supernatant to obtain purified microspheres; S2.4: Add the MES buffer solution prepared in step S1.1 to the above purified microspheres, ultrasonically treat for 10 min, then add the antibody for labeling, vortex and mix well, rotate and incubate for 2 h, then add the latex blocking solution prepared in step S1.4, vortex and mix well and rotate and incubate for 1 h, centrifuge and remove the supernatant to obtain antibody-conjugated microspheres, where the antibody for labeling is mouse anti-human pepsin antibody 2 and dinitrophenol; S2.5: Add purified water to the above antibody-conjugated microspheres. After ultrasonic treatment for 10 min, centrifuge and remove the supernatant. Then add the gold preservation solution and mix evenly by ultrasonic treatment to obtain labeled microspheres. Among them, the labeled microspheres are labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres. After the fluorescence microspheres are labeled, mix the labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres of equal mass evenly, and then spray them on the conjugate pad at a spraying volume of 2.5 μL / cm. In addition, the gold preservation solution includes: 0.6% tris(hydroxymethyl)aminomethane, 0.5% sodium caseinate, 10% sucrose, 2.5% trehalose, 0.5% pvp10, 0.5% S9, 1.5% mannitol, 1% glycine, 0.1% Proclin 300 preservative and 0.01% Evans blue, and the volume ratio of the gold preservation solution to the fluorescence microsphere solution is 7.5:1; S3: Prepare the coating diluent for the C line and the T line S3.1: Add tris(hydroxymethyl)aminomethane to purified water according to the solid-liquid ratio of 1 g:160 mL, stir and mix evenly, and then add 1 mol / L hydrochloric acid to adjust the pH to 8.0 to obtain a Tris solution; S3.2: Add bovine serum albumin to purified water, stir and mix evenly to prepare a 5% bovine serum albumin solution; S3.3: Add sucrose and trehalose to purified water according to the mass ratio of 3.5:1, stir and mix evenly to prepare a 25% sugar solution; S3.4: Mix the dinitrophenyl-conjugated bovine serum albumin antibody, the above sugar solution and the above Tris solution evenly to obtain the coating diluent for the C line. Among them, the C line is coated with 0.2 μg / person dinitrophenyl-conjugated bovine serum albumin antibody, the final concentration of the sugar solution is 2.5%, and the remaining volume is made up with the Tris solution; S3.5: Mix the mouse anti-human pepsin antibody 1, the above sugar solution, the above bovine serum albumin solution and the above Tris solution to obtain the coating diluent for the T line. Among them, the T line is coated with 0.2 μg / person mouse anti-human pepsin antibody 1, the final concentration of the sugar solution is 2.5%, the final concentration of the bovine serum albumin solution is 0.5%, and the remaining volume is made up with the Tris solution; S4: Prepare the C line and the T line Draw the above coating diluent for the C line and the T line on the nitrocellulose membrane at 1 μL / cm respectively, and dry at 50 °C for 20 h; S5: Prepare the sample pad Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium caseinate, PVP10, sodium cholate, surfactant S9, Tween 20 and purified water are stirred and mixed, and then Proclin300 preservative is added, and stirring and mixing are continued to obtain a sample pad solution, and then the polyester film is immersed in the sample pad solution for 15-20 minutes, and then transferred to a drying room and dried at 37°C for 12 hours to obtain a sample pad, wherein the sample pad solution includes: 2.9% disodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.2% sodium caseinate, 1% PVP10, 1% sodium cholate, 0.1% S9, 1% Tween 20 and 0.1% Proclin300 preservative; S6: Finished product assembly Assemble the absorbent paper, nitrocellulose membrane, conjugate pad and sample pad, and cut them into test strips with a width of 3.5 mm. Then install the absorbent paper end of the test strip on the top of the bottom card position, and the absorbent paper side on the bottom of the bottom card position. Buckle the upper card cover, press it with a shell pressing machine, put the desiccant and test paper card in an aluminum foil bag, seal it, and pack it into a box to obtain a pepsin test card.

[0022] Example 2: A preparation process of a pepsin detection card, such as Figure 1 As shown, the following steps are included: S1: Prepare fluorescent microsphere labeling solution S1.1: Add MES to purified water at a solid-liquid ratio of 1 g:85 mL, stir thoroughly to dissolve, and then add 3 mol / L sodium hydroxide solution to adjust the pH to 6.0 to obtain MES buffer; S1.2: Add Sulfo-NHS to the above MES buffer at a solid-liquid ratio of 1 g:95 mL, stir and mix well to obtain a Sulfo-NHS solution; S1.3: Add EDC to the above MES buffer at a solid-liquid ratio of 1 g:95 mL, stir and mix well to obtain an EDC solution; S1.4: Add tris(hydroxymethyl)aminomethane to purified water at a solid-liquid ratio of 1 g:135 mL, stir and mix evenly, then add bovine serum albumin, ethanolamine and Proclin 300 preservative, stir and mix, and then add 3 mol / L hydrochloric acid to adjust the pH to 9.0 to obtain a latex blocking solution; S2: Fluorescent microsphere labeling S2.1: Add the fluorescent microspheres to the MES buffer prepared in step S1.1, vortex mix, prepare a 1% fluorescent microsphere solution, and then vortex mix with the MES buffer prepared in step S1.1 at a volume ratio of 1:7, and then centrifuge at 9500 rpm for 15 minutes. After removing the supernatant, the washed microspheres are obtained; S2.2: Add the MES buffer solution prepared in step S1.1 to the above-mentioned washed microspheres, ultrasonically treat for 15 min, then add the Sulfo-NHS solution prepared in step S1.2 and the EDC solution prepared in step S1.3, vortex and mix well, then rotate and incubate at 1250 rpm for 1.5 h. After removing the supernatant, activated microspheres are obtained, wherein the volume ratio of the Sulfo-NHS solution, the EDC solution to the MES buffer solution is 1:1:22; S2.3: Add the MES buffer solution prepared in step S1.1 to the above-mentioned activated microspheres, ultrasonically treat for 15 min, then centrifuge and remove the supernatant to obtain purified microspheres; S2.4: Add the MES buffer solution prepared in step S1.1 to the above-mentioned purified microspheres, ultrasonically treat for 15 min, then add the antibody for labeling, vortex and mix well, then rotate and incubate for 4 h. Then add the latex blocking solution prepared in step S1.4, vortex and mix well and rotate and incubate for 1.5 h. After centrifuging and removing the supernatant, antibody-conjugated microspheres are obtained, wherein the antibody for labeling is mouse anti-human pepsin antibody 2 and dinitrophenol; S2.5: Add purified water to the above-mentioned antibody-conjugated microspheres, ultrasonically treat for 15 min, then centrifuge and remove the supernatant, and then add the gold storage solution, ultrasonically mix evenly to obtain labeled microspheres. The labeled microspheres are labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres. After the labeling of the fluorescent microspheres is completed, the labeled mouse anti-human pepsin antibody 2 microspheres and the labeled dinitrophenol microspheres of equal mass are mixed evenly, and then sprayed on the conjugate pad at a spraying amount of 2.5 μL / cm. In addition, the gold storage solution includes: 0.6% tris(hydroxymethyl)aminomethane, 0.5% sodium caseinate, 10% sucrose, 2.5% trehalose, 0.5% pvp10, 0.5% S9, 1.5% mannitol, 1% glycine, 0.1% Proclin 300 preservative and 0.01% Evans blue, and the volume ratio of the gold storage solution to the fluorescent microsphere solution is 8:1; S3: Prepare the coating diluent for the C line and the T line S3.1: Add tris(hydroxymethyl)aminomethane to purified water according to the solid-liquid ratio of 1 g:165 mL, stir and mix evenly, then add 3 mol / L hydrochloric acid to adjust the pH to 8.0 to obtain a Tris solution; S3.2: Add bovine serum albumin to purified water, stir and mix evenly to prepare a 5% bovine serum albumin solution; S3.3: Add sucrose and trehalose to purified water according to the mass ratio of 4:1, stir and mix evenly to prepare a 25% sugar solution; S3.4: Evenly mix the dinitrobenzene-coupled bovine serum albumin antibody, the sugar solution and the Tris solution to obtain a C-line coating dilution solution, wherein the C-line is coated with 0.2 μg / person dinitrobenzene-coupled bovine serum albumin antibody, the final concentration of the sugar solution is 2.5%, and the remaining volume is filled with Tris solution; S3.5: Mix the mouse anti-human pepsin antibody 1, the sugar solution, the bovine serum albumin solution and the Tris solution to obtain a T-line coating diluent, wherein the T-line is coated with 0.2 μg / person of the mouse anti-human pepsin antibody 1, the final concentration of the sugar solution is 2.5%, the final concentration of the bovine serum albumin solution is 0.5%, and the remaining volume is filled with the Tris solution; S4: Preparation of C-line and T-line The C-line coating dilution and T-line coating dilution were streaked onto nitrocellulose membranes at 1 μL / cm and dried at 55°C for 22 h. S5: Prepare the sample pad Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium caseinate, PVP10, sodium cholate, surfactant S9, Tween 20 and purified water were stirred and mixed, and then Proclin 300 preservative was added, and stirring and mixing were continued to obtain a sample pad solution, and then the polyester film was immersed in the sample pad solution for 17 minutes, and then transferred to a drying room and dried at 34°C for 18 hours to obtain a sample pad, wherein the sample pad solution includes: 2.9% disodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.2% sodium caseinate, 1% PVP10, 1% sodium cholate, 0.1% S9, 1% Tween 20 and 0.1% Proclin 300 preservative; S6: Finished product assembly Assemble the absorbent paper, nitrocellulose membrane, conjugate pad and sample pad, and cut them into test strips with a width of 4 mm. Then install the absorbent paper end of the test strip on the top of the bottom card position, and the absorbent paper side on the bottom of the bottom card position. Buckle the upper card cover, press it with a shell pressing machine, put the desiccant and test paper card in an aluminum foil bag, seal it, and pack it into a box to obtain a pepsin test card.

[0023] Example 3: A preparation process of a pepsin detection card, such as Figure 1 As shown, the following steps are included: S1: Prepare fluorescent microsphere labeling solution S1.1: Add MES to purified water at a solid-liquid ratio of 1 g:90 mL, stir thoroughly to dissolve, and then add 6 mol / L sodium hydroxide solution to adjust the pH to 6.0 to obtain MES buffer; S1.2: Add Sulfo-NHS to the above MES buffer at a solid-liquid ratio of 1 g:100 mL, stir and mix well to obtain a Sulfo-NHS solution; S1.3: Add EDC to the above MES buffer solution according to a solid-liquid ratio of 1 g: 100 mL, stir and mix evenly to obtain an EDC solution; S1.4: Add tris (hydroxymethyl) aminomethane to purified water according to a solid-liquid ratio of 1 g: 140 mL, stir and mix evenly, then add bovine serum albumin, ethanolamine and Proclin300 preservative, stir and mix, and then add 6 mol / L hydrochloric acid to adjust the pH to 9.0 to obtain a latex blocking solution; S2: Fluorescent microsphere labeling S2.1: Add the fluorescent microspheres to the MES buffer solution prepared in step S1.1, vortex and mix evenly to prepare a 1% fluorescent microsphere solution, then vortex and mix evenly with the MES buffer solution prepared in step S1.1 according to a volume ratio of 1:8, and then centrifuge at 10000 rpm for 20 min. After removing the supernatant, the washed microspheres are obtained; S2.2: Add the MES buffer solution prepared in step S1.1 to the above washed microspheres, ultrasonically treat for 20 min, then add the Sulfo-NHS solution prepared in step S1.2 and the EDC solution prepared in step S1.3, vortex and mix evenly, and then rotate and incubate at 1300 rpm for 2 h. After removing the supernatant, the activated microspheres are obtained, where the volume ratio of the Sulfo-NHS solution, the EDC solution to the MES buffer solution is 1:1:24; S2.3: Add the MES buffer solution prepared in step S1.1 to the above activated microspheres, ultrasonically treat for 20 min, then centrifuge and remove the supernatant to obtain purified microspheres; S2.4: Add the MES buffer solution prepared in step S1.1 to the above purified microspheres, ultrasonically treat for 20 min, then add the antibody for labeling, vortex and mix evenly, rotate and incubate for 6 h, then add the latex blocking solution prepared in step S1.4, vortex and mix evenly and rotate and incubate for 2 h. After centrifuging and removing the supernatant, the antibody-conjugated microspheres are obtained, where the antibody for labeling is mouse anti-human pepsin antibody 2 and dinitrophenol; S2.5: Add purified water to the above antibody-conjugated microspheres, ultrasonically treat for 20 min, then centrifuge and remove the supernatant, and then add the gold storage solution, ultrasonically mix evenly to obtain labeled microspheres, where the labeled microspheres are labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres. After the fluorescent microsphere labeling is completed, the labeled mouse anti-human pepsin antibody 2 microspheres and the labeled dinitrophenol microspheres of equal mass are mixed evenly, and then sprayed on the conjugate pad according to a spraying amount of 2.5 μL / cm. In addition, the gold storage solution includes: 0.6% tris (hydroxymethyl) aminomethane, 0.5% sodium caseinate, 10% sucrose, 2.5% trehalose, 0.5% pvp10, 0.5% S9, 1.5% mannitol, 1% glycine, 0.1% Proclin300 preservative and 0.01% Evans blue, and the volume ratio of the gold storage solution to the fluorescent microsphere solution is 8.5:1; S3: Prepare the coating diluent for the C line and T line S3.1: Add tris(hydroxymethyl)aminomethane to purified water according to the solid-liquid ratio of 1 g: 170 mL, stir and mix evenly, then add 6 mol / L hydrochloric acid to adjust the pH to 8.0 to obtain a Tris solution; S3.2: Add bovine serum albumin to purified water, stir and mix evenly to prepare a 5% bovine serum albumin solution; S3.3: Add sucrose and trehalose to purified water according to the mass ratio of 4.5: 1, stir and mix evenly to prepare a 25% sugar solution; S3.4: Mix the dinitrophenyl-coupled bovine serum albumin antibody, the above sugar solution and the above Tris solution evenly to obtain the coating diluent for the C line. Among them, 0.8 μg / person-dose of dinitrophenyl-coupled bovine serum albumin antibody is coated on the C line, the final concentration of the sugar solution is 2.5%, and the remaining volume is made up with the Tris solution; S3.5: Mix the antibody 1 against human pepsin, the above sugar solution, the above bovine serum albumin solution and the above Tris solution to obtain the coating diluent for the T line. Among them, 0.8 μg / person-dose of the antibody 1 against human pepsin is coated on the T line, the final concentration of the sugar solution is 2.5%, the final concentration of the bovine serum albumin solution is 0.5%, and the remaining volume is made up with the Tris solution; S4: Prepare the C line and T line Draw the above coating diluent for the C line and T line on the nitrocellulose membrane at 1 μL / cm respectively, and dry at 60 °C for 24 h; S5: Prepare the sample pad Stir and mix disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium caseinate, PVP10, sodium cholate, surfactant S9, Tween 20 and purified water, then add the Proclin300 preservative and continue to stir and mix to obtain the sample pad solution. Then immerse the polyester membrane in the sample pad solution for 20 min, and then transfer it to the drying room and dry at 40 °C for 24 h to obtain the sample pad. Among them, the sample pad solution includes: 2.9% disodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.2% sodium caseinate, 1% PVP10, 1% sodium cholate, 0.1% S9, 1% Tween 20 and 0.1% Proclin300 preservative; S6: Final product assembly Assemble the absorbent paper, nitrocellulose membrane, conjugate pad and sample pad, and cut them into test strips with a width of 4.5 mm. Then install the absorbent paper end of the test strip at the top of the bottom card slot, the absorbent paper side against the bottom of the bottom card slot, fasten the upper card cover, press it well with a shell pressing machine, put the desiccant and the test strip card into an aluminum foil bag, seal it, and put it into a box to obtain the pepsin detection card.

[0024] Test example: pepsin, trypsin and chymotrypsin were added to the buffer at 1 mg / mL to prepare sample A, sample B and sample C, and an equal amount of blank buffer was used as sample D, and then the samples were dripped onto the pepsin detection cards prepared in Example 1, Example 2 and Example 3, respectively. After waiting for a specified time, the reading was performed. The results showed that the pepsin detection cards prepared in Examples 1-3 were all positive for sample A, and negative for samples B, C and D. It can be seen that the pepsin detection card of the present invention is not only highly sensitive, but can accurately capture extremely low concentrations of pepsin in the sample. Even when there is only a slight change in pepsin in the patient's body, the detection card can also be keenly detected, effectively avoiding missed diagnosis and gaining precious time for early intervention. Moreover, it has strong specificity and only specifically binds to pepsin, and is not interfered by other similar proteases or impurities in the sample. In a complex human biological sample environment, such as gastric juice containing a variety of digestive enzymes, tissue fluid mixed with inflammatory factors, etc., it can still accurately identify the target pepsin, with a specificity of more than 98%, greatly reducing false positive results, and providing a reliable basis for clinical diagnosis.

[0025] The above embodiments are only for explaining the principle and effect of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A preparation process of a pepsin detection card, characterized in that: The steps include: S1: Prepare fluorescent microsphere labeling solution Prepare MES buffer, Sulfo-NHS solution, EDC solution and latex blocking solution respectively; S2: Fluorescent microsphere labeling The fluorescent microspheres are first washed, activated with the above-mentioned Sulfo-NHS solution and EDC solution, dispersed in the above-mentioned MES buffer together with the labeled antibody, added with the above-mentioned latex blocking solution, incubated by rotation, and finally mixed with the gold preservation solution to obtain labeled microspheres; S3: Prepare C-line and T-line coating diluent Prepare Tris solution, bovine serum albumin solution and sugar solution respectively, and then mix with dinitrobenzene-coupled bovine serum albumin antibody and mouse anti-human pepsin antibody 1 respectively to obtain C-line coating diluent and T-line coating diluent; S4: Preparation of C-line and T-line Streak the C-line coating dilution and T-line coating dilution on the nitrocellulose membrane at 1 μL / cm, and dry at 50-60°C for 20-24h; S5: Prepare the sample pad Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium caseinate, PVP10, sodium cholate, surfactant S9, Tween 20 and purified water are stirred and mixed, and then Proclin 300 preservative is added, and stirring and mixing are continued to obtain a sample pad solution, and then the polyester film is immersed in the sample pad solution for 15-20 minutes, and then transferred to a drying room and dried at (37±3)°C for 12-24 hours to obtain a sample pad, wherein the sample pad solution includes: 2.9% disodium hydrogen phosphate, 0.3% sodium dihydrogen phosphate, 0.2% sodium caseinate, 1% PVP10, 1% sodium cholate, 0.1% surfactant S9, 1% Tween 20 and 0.1% Proclin 300 preservative; S6: Finished product assembly Assemble the absorbent paper, nitrocellulose membrane, conjugate pad and sample pad, and cut them into test strips with a width of 4±0.5 mm. Then install the absorbent paper end of the test strip on the top of the bottom card position, and the absorbent paper side on the bottom of the bottom card position. Buckle the upper card cover, press it with a shell pressing machine, put the desiccant and test paper card in an aluminum foil bag, seal it, and pack it into a box to obtain a pepsin test card.

2. The preparation process of a pepsin detection card according to claim 1, characterized in that: S1 specifically includes the following steps: S1.1: Add MES to purified water at a solid-liquid ratio of 1 g: (80-90) mL, stir thoroughly to dissolve, and then add 1-6 mol / L sodium hydroxide solution to adjust the pH to 6.0 to obtain MES buffer; S1.2: Add Sulfo-NHS to the above MES buffer at a solid-liquid ratio of 1 g: (90-100) mL, stir and mix well to obtain a Sulfo-NHS solution; S1.3: Add EDC to the above MES buffer at a solid-liquid ratio of 1 g: (90-100) mL, stir and mix well to obtain an EDC solution; S1.4: Add tris(hydroxymethyl)aminomethane) into purified water at a solid-liquid ratio of 1g:(130-140)mL, stir to mix evenly, then add bovine serum albumin, ethanolamine and Proclin300 preservative, stir to mix, and then add 1-6mol / L hydrochloric acid to adjust the pH to 9.0 to obtain a latex blocking solution.

3. The preparation process of a pepsin detection card according to claim 2, characterized in that: S2 specifically includes the following steps: S2.1: Add the fluorescent microspheres to the MES buffer prepared in step S1.1, vortex mix, and prepare a 1% fluorescent microsphere solution, then vortex mix with the MES buffer prepared in step S1.1 at a volume ratio of 1:(6-8), and then centrifuge at 9000-10000 rpm for 10-20 min, remove the supernatant, and obtain the washed microspheres; S2.2: Add the MES buffer prepared in step S1.1 to the above-mentioned cleaned microspheres, ultrasonically treat for 10-20 minutes, then add the Sulfo-NHS solution prepared in step S1.2 and the EDC solution prepared in step S1.3, vortex mix, rotate and incubate at 1200-1300 rpm for 1-2 hours, remove the supernatant, and obtain activated microspheres; S2.3: Add the MES buffer prepared in step S1.1 to the activated microspheres, sonicate for 10-20 min, centrifuge and remove the supernatant to obtain purified microspheres; S2.4: Add the MES buffer prepared in step S1.1 to the purified microspheres, ultrasonically treat for 10-20 min, add the labeled antibody, vortex mix, rotate and incubate for 2-6 h, then add the latex blocking solution prepared in step S1.4, vortex mix and rotate and incubate for 1-2 h, centrifuge and remove the supernatant to obtain the antibody-coupled microspheres; S2.5: Add purified water to the above antibody-coupled microspheres, ultrasonically treat for 10-20 minutes, centrifuge and remove the supernatant, then add gold preservation solution, ultrasonically mix evenly, and obtain labeled microspheres.

4. The preparation process of a pepsin detection card according to claim 3, characterized in that: S3 specifically includes the following steps: S3.1: Add tris(hydroxymethyl)aminomethane into purified water at a solid-liquid ratio of 1 g: (160-170) mL, stir and mix evenly, then add 1-6 mol / L hydrochloric acid to adjust the pH to 8.0 to obtain a Tris solution; S3.2: Add bovine serum albumin to purified water, stir and mix well to prepare a 5% bovine serum albumin solution; S3.3: Add sucrose and trehalose into purified water in a mass ratio of (3.5-4.5):1, stir and mix well to prepare a 25% sugar solution; S3.4: Evenly mix the dinitrobenzene-conjugated bovine serum albumin antibody, the sugar solution and the Tris solution to obtain a C-line coating diluent; S3.5: Mix the mouse anti-human pepsin antibody 1, the sugar solution, the bovine serum albumin solution and the Tris solution to obtain a T-line coating diluent.

5. The preparation process of a pepsin detection card according to claim 3, characterized in that: The volume ratio of Sulfo-NHS solution, EDC solution and MES buffer in step S2.2 is 1:1:(20-24).

6. The preparation process of a pepsin detection card according to claim 3, characterized in that: The labeled antibodies are mouse anti-human pepsin antibody 2 and dinitrophenol, and the labeled microspheres are labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres.

7. The preparation process of a pepsin detection card according to claim 3, characterized in that: After the fluorescent microspheres are labeled, equal masses of labeled mouse anti-human pepsin antibody 2 microspheres and labeled dinitrophenol microspheres are mixed evenly and then sprayed on the conjugate pad at a spray volume of 2.5 μL / cm.

8. The preparation process of a pepsin detection card according to claim 4, characterized in that: The C line was coated with (0.2-0.8) μg / person dinitrobenzene-coupled bovine serum albumin antibody, the final concentration of the sugar solution was 2.5%, and the remaining volume was made up with Tris solution; the T line was coated with (0.2-0.8) μg / person mouse anti-human pepsin antibody 1, the final concentration of the sugar solution was 2.5%, the final concentration of the bovine serum albumin solution was 0.5%, and the remaining volume was made up with Tris solution.

9. The preparation process of a pepsin detection card according to claim 3, characterized in that: The gold preservation solution includes: 0.6% tris(hydroxymethyl)aminomethane, 0.5% casein sodium salt, 10% sucrose, 2.5% trehalose, 0.5% pvp10, 0.5% S9, 1.5% mannitol, 1% glycine, 0.1% Proclin300 preservative and 0.01% Evans blue, and the volume ratio of gold preservation solution to fluorescent microsphere solution is (7.5-8.5):

1.

10. A pepsin detection card prepared by the preparation process of the pepsin detection card according to any one of claims 1 to 9, characterized in that: It is composed of a test strip and a card; The test strips include: nitrocellulose membrane, sample pad, absorbent paper, conjugate pad and base plate; The detection line T in the nitrocellulose membrane is the coating effective content concentration of (0.2-0.8) μg / person of mouse anti-human pepsin antibody 1, and the quality control line C is the coating effective content concentration of (0.2-0.8) μg / person of dinitrobenzene-coupled bovine serum albumin antibody; The conjugate pad includes fluorescent microspheres containing mouse anti-human pepsin antibody 2 and fluorescent microspheres labeled with dinitrophenol.

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