Aβ1-42 detection reagent based on fluorescence immuno-chromatography

Abstract

The present application relates to the A beta 1-42 detection reagent based on fluorescent immunochromatography. In one aspect, the present application relates to the detection reagent card for detecting A beta 1-42 by fluorescent immunochromatography, which comprises a card shell composed of two positive and negative pieces, and a detection reagent strip assembled in the two pieces of card shell. A sample hole is arranged on the front of the card shell at the sample pad, and the liquid to be detected is dropped from the sample hole. The liquid is chromatographed to the water absorption pad under the action of capillary. In addition, an observation window is arranged on the front of the card shell at the nitrocellulose membrane to allow observation of the detection line and the quality control line on the narrow strip. The detection reagent strip is made by the following steps: pretreatment of the sample pad, pretreatment of the fluorescently labeled conjugate pad, pretreatment of the nitrocellulose membrane, and assembly of the detection reagent strip. The reagent strip or reagent card of the present application can solve the problem of fluctuation of the results of fluorescent immunochromatographic determination, and the determination efficiency is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to an Aβ1-42 detection reagent based on fluorescence immunochromatography. More specifically, it relates to a detection reagent card for detecting Aβ1-42 using fluorescence immunochromatography. Furthermore, the invention also relates to a reagent strip for preparing this detection reagent card. In particular, the reagent strip or reagent card of this invention can solve the problem of fluctuations in fluorescence immunochromatographic assay results and significantly improve assay efficiency. Background Technology

[0002] As is well known, fluorescence immunoassay (FIA) is a protein immunoassay technique based on antibody-antigen specific recognition. It detects and quantifies target molecules moving in the chromatography paper using fluorescence signals. This technique combines immunology, fluorescence detection, and biochemistry, offering advantages such as high sensitivity, high specificity, speed, simplicity, convenience, ease of operation, and low cost. Fluorescence immunoassay has been widely used in point-of-care testing (POCT) applications.

[0003] The basic principle of fluorescence immunochromatography is typically as follows: Figure 1 As shown. Specifically, fluorescence immunochromatography uses a strip-shaped fiber chromatography material (often nitrocellulose membrane) with a detection line (T line, with capture antibody Ab2, also known as coated antibody) and a control line (C line, with capture antibody Ab3, also known as anti-antibody) as the stationary phase, and the test solution of the analyte as the mobile phase. The fluorescently labeled antibody Ab1 is immobilized in the conjugate pad, and the analyte moves on the chromatography strip through capillary action.

[0004] For analytes containing large molecular antigens (proteins, viruses, etc.) with multiple antigenic determinants, a "sandwich" type double-antibody immunochromatography method is typically used. In this method, the analyte solution, under the action of the mobile phase, first binds to the fluorescently labeled antibody. Upon reaching the detection line (T line), it then binds to the capture antibody Ab2, forming a double-antibody sandwich. During detection, the sample solution, under capillary action, will exhibit two different display results (depending on whether the analyte contains the antigen): If the result is negative (no antigen in the sample to be tested), the sample does not bind to the fluorescently labeled antibody. The fluorescently labeled antibody is chromatographically deposited along with the test solution to the T line, where it will not bind to the coating antibody. It continues to the C line and is captured by the anti-antibody. The instrument can detect fluorescence at the C line, while there is no fluorescence at the T line, and the result is negative. If the result is positive (the sample contains antigen), the antigen reacts with the fluorescently labeled antibody to form a fluorescently labeled antigen-antibody complex. This complex is chromatographically deposited with the test solution to the T line, where it binds to the coating antibody, forming a "sandwich" type double-antibody complex of fluorescently labeled antibody Ab1-antigen-capture antibody Ab2. Since the fluorescently labeled antibody immobilized on the binding pad is in excess, this excess fluorescently labeled antibody continues to chromatographically deposited with the test solution to the C line, where it binds to the capture antibody Ab3, forming a fluorescently labeled antibody Ab1-capture antibody Ab3 complex. The instrument can detect fluorescence at both the T line and the C line, indicating a positive result. Furthermore, in the case of a positive result, the antigen amount / concentration can be quantitatively calculated based on the fluorescence intensity ratio of the T line and the C line.

[0005] Those skilled in the art have designed fluorescence immunochromatographic assay strips, cards, or kits for various occasions and subjects based on the above principles. Of course, based on the above principles, those skilled in the art can also easily design assay strips, cards, or kits for other immunochromatographic modes.

[0006] Typically, labeled antibodies Ab1 are labeled using fluorescent microspheres. However, when designing reagent cards for detecting Aβ1-42, it has been regrettably found that reagent cards designed using this principle to detect Aβ1-42 have problems with large fluctuations in the measurement results, resulting in unreliable results. In addition, these reagent cards may also have problems with low measurement efficiency.

[0007] Several basic structures and preparation methods for reagent kits for detecting Aβ1-42 using fluorescence immunochromatography have been disclosed in this field. For example, CN115932241A (application number 202211735046.9) discloses a test kit for Aβ1-42 detection, its preparation method, and its application. The test kit includes a base plate, a sample pad, a fluorescently labeled conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a detection line and a control line; the detection line is coated with Aβ1-42 antibody, and the control line is coated with chicken IgY antibody. The fluorescently labeled conjugate pad is coated with fluorescently labeled anti-Aβ1-42 antibody and fluorescently labeled goat anti-chicken IgY. The preparation method of the above test kit includes pre-treating the fluorescently labeled conjugate pad and the nitrocellulose membrane, and assembling the sample pad, the pre-treated fluorescently labeled conjugate pad, the nitrocellulose membrane, and the absorbent pad onto the base plate to obtain the final test kit. This test kit can be used in the preparation of Aβ1-42 detection kits and in the detection of Aβ1-42. The invention is believed to have the advantage of enabling rapid detection of Aβ1-42, with simple operation and high sensitivity and accuracy. For example, CN116643055A (application number 202310653235.X) discloses a human β-amyloid protein Aβ1-42 detection kit, comprising calibrators, reagent A, reagent B, washing solution, acridil ester activation solution, and acridil ester pre-activation solution. The calibrators are a series of concentrations of Aβ1-42 antigen used to establish a standard curve. Reagent A is an Aβ1-42 antibody solution containing magnetically labeled microparticles, and reagent B is an Aβ1-42 antibody solution containing acridil ester. However, these disclosed Aβ1-42 detection strips / cards or kits still have some shortcomings, such as failing to address the problem of large fluctuations in measurement results after the antibody-labeled fluorescent microspheres are immobilized on the binding pad, leading to unreliable results, and / or low measurement efficiency.

[0008] Therefore, those skilled in the art expect to provide a test kit for detecting Aβ1-42 by fluorescence immunochromatography using antibody-labeled fluorescent microspheres, which has the advantages of small fluctuations in the test results and high detection efficiency. Summary of the Invention

[0009] The present invention aims to provide a test card or test strip for detecting Aβ1-42 using fluorescence immunochromatography, and / or another objective of the present invention is to provide a preservation solution for such a test card or test strip for detecting Aβ1-42 using fluorescence immunochromatography. It has been unexpectedly discovered that test cards or test strips for detecting Aβ1-42 prepared using the preservation solution of the present invention exhibit excellent characteristics of low fluctuation in measurement results and high measurement efficiency when measuring Aβ1-42. The present invention is based on such discoveries.

[0010] Therefore, in a first aspect, the present invention provides a detection strip for detecting Aβ1-42 by fluorescence immunochromatography, comprising: along the direction of chromatographic movement of the sample solution. The base plate, and the sample pad, fluorescent labeling pad, nitrocellulose membrane, and absorbent pad sequentially arranged on the base plate along its length; One end of the sample pad moves in the direction of chromatography and overlaps above one end of the fluorescently labeled conjugate pad. The other end of the fluorescently labeled conjugate pad overlaps above one end of the nitrocellulose membrane, and the other end of the nitrocellulose membrane overlaps below the absorbent pad. A detection line and a control line are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorbent pad. The detection line is coated with Aβ1-42 antibody, and the control line is coated with chicken IgY antibody. The fluorescently labeled binding pad is coated with fluorescently labeled anti-Aβ1-42 antibody and fluorescently labeled goat anti-chicken IgY. This test strip is prepared by a method including the following steps: (1) Pretreatment of sample pad: Immerse the sample pad in sample pad buffer solution, and dry it at room temperature after immersion to obtain the pretreated sample pad. (2) Pretreatment of fluorescently labeled conjugate pads: Using Na2SiO3 as a blocking agent, tetraethyl orthosilicate and fluorescein isothiocyanate were reacted, followed by treatment with p-ethyl-N,N-dimethylpropylcarbodiimide and nitrogen-hydroxysuccinimide to prepare a fluorescent microsphere suspension of fluorescein isothiocyanate-silica core-shell dual structure fluorescent microspheres. Desalted and purified anti-Aβ1-42 antibody was added to a biotin solution to obtain biotin-anti-Aβ1-42 antibody; fluorescent microsphere suspension was added to the desalted and purified biotin-anti-Aβ1-42 antibody and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-anti-Aβ1-42 antibody; Additionally... Desalted and purified sheep anti-chicken IgY was added to a biotin solution to obtain biotin-sheep anti-chicken IgY; fluorescent microsphere suspension was added to desalted and purified biotin-sheep anti-chicken IgY and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-sheep anti-chicken IgY. Next, bovine serum albumin was added to fluorescent microspheres-biotin-anti-Aβ1-42 antibody and fluorescent microspheres-biotin-goat anti-chicken IgY to a concentration of 1% for blocking treatment. Add 1 mL of preservation solution to the desalting column, and use the desalting column to separate and purify the blocked fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the blocked fluorescent microspheres-biotin-goat anti-chicken IgY; The desalted and purified fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the desalted and purified fluorescent microspheres-biotin-goat anti-chicken IgY present in the preservation solution were mixed and sprayed onto a glass cellulose membrane at a rate of 4 μL / cm. The mixture was dried at 30°C for 6 hours to obtain a fluorescently labeled binding pad. (3) Pretreatment of nitrocellulose membrane: Aβ1-42 antibody was diluted to 1 mg / mL with the first buffer, and chicken IgY antibody was diluted to 0.5 mg / mL with the second buffer. The two antibodies were sprayed onto the detection line and control line of the nitrocellulose membrane at a rate of 2 μL / cm and 6 cm / s, respectively. The membrane was dried at 30°C for 6 hours to obtain a nitrocellulose membrane containing the detection line and control line. (4) Assembly of test strips: Assemble the pretreated sample pad, pretreated fluorescent labeling pad, pretreated nitrocellulose membrane and absorbent pad on the base plate, and then cut them into narrow strips with a width of 2~8mm, which are the test strips for detecting Aβ1-42.

[0011] According to the first aspect of the present invention, in step (1), the sample pad is pretreated as follows: the sample pad is immersed in the sample pad buffer solution for 1 hour, and after immersion, it is dried at room temperature for 24 hours to obtain the sample pad.

[0012] According to the test strip of the first aspect of the present invention, in step (1), the composition of the sample pad buffer is: 1% bovine serum albumin, 0.5% S9, and PBS buffer.

[0013] According to the test strip of the first aspect of the present invention, in step (1), the PBS buffer refers to a phosphate buffer with pH 6.8, which is prepared as follows: take 500 ml of 0.2 mol / L potassium dihydrogen phosphate solution, dilute with water to about 900 ml, add sodium hydroxide test solution to adjust the pH to 6.8, add water to dilute to about 950 ml, then adjust the pH to 6.8 with sodium hydroxide test solution or phosphate test solution, add water to 1000 ml, shake well, and the test strip is obtained.

[0014] According to the first aspect of the present invention, in step (1), the sample pad is 10-30 mm wide and 100-400 mm long, and is arranged laterally in the reagent strip, and is cut to form a size of 10-30 mm long and 3-10 mm wide.

[0015] According to the detection reagent strip of the first aspect of the present invention, the preparation of fluorescein isothiocyanate-silica core-shell dual-structure fluorescent microspheres in step (2) is as follows: 60 ml of anhydrous ethanol, 2 ml of ammonia, 3 ml of tetraethyl orthosilicate, 1 ml of ultrapure water and 1 mg of fluorescein isothiocyanate are mixed evenly and reacted in a constant temperature water bath for 8 hours. Then, 3% Na2SiO3 is added to block the microspheres. The size of the fluorescent microspheres is controlled by adjusting the pH value of the solution by adding hydrochloric acid. Active groups are modified on the surface of the fluorescent microspheres. The reaction solution is centrifuged at 1000×g for 15 min, and the precipitate is collected. The microspheres were suspended in 0.01M pH 4.8 borate buffer and the concentration of the microspheres was adjusted to OD450 nm = 0.2~0.25. Then, 100 μL of 50 mg / mL p-ethyl-N,N-dimethylpropylcarbodiimide and 150 μL of 5 mg / mL nitrogen-hydroxysuccinimide were added, the mixture was shaken and incubated at room temperature for 25 min. After centrifugation at 1000×g for 15 min, the precipitate was dried under vacuum and stabilized. The suspension was then resuspended in 0.01M pH 4.8 borate buffer and the concentration of the microspheres was adjusted to OD450 nm = 0.45~0.5 to obtain the fluorescent microsphere suspension.

[0016] According to the test strip of the first aspect of the present invention, in step (2), the biotin solution is prepared by using DMSO as a solvent to form a solution containing a biotin concentration of 10%.

[0017] According to the test strip of the first aspect of the present invention, in step (2), the addition ratio is: 0.06 mg biotin, 0.6 mg anti-Aβ1-42 antibody, and 0.25 mg fluorescent microspheres, or the addition ratio is: 0.06 mg biotin, 0.6 mg goat anti-chicken IgY, and 0.25 mg fluorescent microspheres.

[0018] According to the test strip of the first aspect of the present invention, in step (2), the preservation solution used is formulated as follows: 1% bovine serum albumin, 0.15%~0.25% Tetronic 1307, 2%~3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0019] According to the test strip of the first aspect of the present invention, in step (2), the preservation solution used is formulated as follows: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0020] According to the test strip of the first aspect of the present invention, in step (2), the preservation solution used is formulated as follows: 1% bovine serum albumin, 0.15% Tetronic 1307, 2% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0021] According to the test strip of the first aspect of the present invention, in step (2), the preservation solution used is formulated as follows: 1% bovine serum albumin, 0.25% Tetronic 1307, 3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0022] According to the test strip of the first aspect of the present invention, in step (2), the method for preparing pH 4.5 phosphate buffer is as follows: take 500 ml of 0.2 mol / L potassium dihydrogen phosphate solution, dilute with water to about 900 ml, add sodium hydroxide test solution to adjust the pH to 4.5, add water to dilute to about 950 ml, then adjust the pH to 4.5 with sodium hydroxide test solution or phosphate test solution, add water to 1000 ml, shake well, and the test strip is obtained.

[0023] According to the test strip of the first aspect of the present invention, in step (2), when the desalted purified fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the desalted purified fluorescent microspheres-biotin-goat anti-chicken IgY present in the preservation solution are mixed, the concentration of anti-Aβ1-42 antibody is 0.6 mg / ml and the concentration of goat anti-chicken IgY is 0.12 mg / ml.

[0024] According to the first aspect of the present invention, in step (2), the pad is 8 mm wide and 300 mm long, and is cut to form a length of 8 mm and a width of 4 mm.

[0025] According to the test strip of the first aspect of the present invention, in step (2), the conjugate pad is 5 to 20 mm wide and 100 to 400 mm long, and is arranged laterally in the test strip / card. After being cut, it is 5 to 20 mm long and 3 to 10 mm wide; for example, the conjugate pad is 8 mm wide and 300 mm long, and after being cut, it is 8 mm long and 4 mm wide.

[0026] According to the test strip of the first aspect of the present invention, in step (3), the distance between the test line and the control line is 3 to 10 mm, for example, 6 mm.

[0027] According to the test strip of the first aspect of the present invention, in step (3), the first buffer is a pH 6.8 phosphate buffer containing 1% trehalose, and the second buffer is a pH 6.8 phosphate buffer containing 0.5% trehalose.

[0028] According to the test strip of the first aspect of the present invention, in step (3), the nitrocellulose membrane is in the range of 10 to 50 mm in width and 100 to 400 mm in length, and is arranged laterally in the test strip / card, and is cut to form a length of 10 to 50 mm and a width of 3 to 10 mm; for example, the nitrocellulose membrane is 25 mm wide and 300 mm long, and is cut to form a size of 25 mm long and 4 mm wide.

[0029] According to the first aspect of the present invention, in step (4), the assembled test strip is cut into narrow strips with a width of 4 mm.

[0030] Furthermore, a second aspect of the present invention provides a test kit for detecting Aβ1-42 by fluorescence immunochromatography, comprising: a card case consisting of two pieces, a front and a back, and a test strip as described in any one of the first aspects of the present invention assembled within the two card cases; a sample application hole is provided on the front of the card case at the sample pad, through which the liquid to be tested is dripped, and the liquid is chromatographically deposited towards the absorbent pad under the action of a capillary; in addition, an observation window is provided on the front of the card case at the nitrocellulose membrane to allow observation of the test line and control line on the narrow strip.

[0031] Furthermore, a third aspect of the present invention provides a preservation solution for pretreating fluorescently labeled binding pads when preparing the test strips or test cards described in the first aspect of the present invention, the formulation of which is: 1% bovine serum albumin, 0.15%~0.25% Tetronic 1307, 2%~3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0032] According to the preservation solution of the third aspect of the present invention, the formulation is as follows: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% L-erythrose, and pH 4.5 phosphate buffer to 100%; or, the formulation is as follows: 1% bovine serum albumin, 0.15% Tetronic 1307, 2% L-erythrose, and pH 4.5 phosphate buffer to 100%; or, the formulation is as follows: 1% bovine serum albumin, 0.25% Tetronic 1307, 3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

[0033] The test strips or test cards of this invention exhibit excellent results, such as minimal fluctuation in measurement results, and can complete the test within a shorter time and a wider time window. Attached Figure Description

[0034] Figure 1 Schematic diagram of the basic principle of fluorescence immunochromatography.

[0035] Figure 2 Typical structure of a fluorescent immunochromatographic assay strip.

[0036] Figure 3 : Schematic diagram of the assembly of the test reagent card. Detailed Implementation

[0037] The present invention will be described in detail below through specific examples. Unless otherwise specified, all materials or reagents used herein are readily available from the market.

[0038] Example 1: Detection strip for Aβ1-42 This invention first provides a test strip for detecting Aβ1-42 with a basic structure that is essentially the same as that of existing technologies, as illustrated in the figure. Figure 2 As shown, along the direction of sample liquid chromatography movement, it includes: The base plate 1, and the sample pad 2, fluorescent labeling pad 3, nitrocellulose membrane 4, and absorbent pad 5 are sequentially arranged on the base plate 1 along its length; One end of the sample pad 2 in the direction of chromatographic movement overlaps above one end of the fluorescently labeled binding pad 3, the other end of the fluorescently labeled binding pad 3 overlaps above one end of the nitrocellulose membrane 4, and the other end of the nitrocellulose membrane 4 overlaps below the absorbent pad 5. A detection line 6 and a control line 7 are sequentially arranged on the nitrocellulose membrane 4 from the sample pad 2 to the absorbent pad 5. The detection line 6 is coated with Aβ1-42 antibody, and the control line 7 is coated with chicken IgY antibody. The fluorescently labeled binding pad 3 is coated with fluorescently labeled anti-Aβ1-42 antibody and fluorescently labeled goat anti-chicken IgY.

[0039] Example 2: Preparation of test strips and test cards for detecting Aβ1-42 This embodiment describes how to prepare the detection reagent strip for detecting Aβ1-42 as described in Embodiment 1 of CN115932241A.

[0040] In this invention, unless otherwise specified, the sample pad and fluorescent labeling pad are made of glass fiber membrane, and the absorbent pad is made of absorbent filter paper.

[0041] 1. Sample pad pretreatment: Soak the sample pad in sample pad buffer for 1 hour, and then dry it at room temperature for 24 hours after soaking.

[0042] Sample pad buffer composition: 1% bovine serum albumin, 0.5% S9, PBS buffer.

[0043] In this invention, unless otherwise specified, % refers to mass / mass percentage.

[0044] In this invention, unless otherwise specified, the PBS buffer mentioned refers to a phosphate buffer with a pH of 6.8. The preparation method is as follows: Take 500 ml of 0.2 mol / L potassium dihydrogen phosphate solution, dilute with water to about 900 ml, add sodium hydroxide test solution to adjust the pH to 6.8, add water to dilute to about 950 ml, then adjust the pH to 6.8 with sodium hydroxide test solution or phosphate test solution, add water to 1000 ml, and shake well to obtain the PBS buffer.

[0045] Sample pads typically range in width from 10 to 30 mm and length from 100 to 400 mm, or can be longer to accommodate manufacturing equipment. They are horizontally positioned within the reagent strip / card and, after being cut, are 10 to 30 mm long and 3 to 10 mm wide. In this embodiment, the sample pad is 25 mm wide and 300 mm long, and after being cut, it is 25 mm long and 4 mm wide.

[0046] 2. Pretreatment of fluorescently labeled conjugate pads: Desalted and purified anti-Aβ1-42 antibody was added to a biotin solution to obtain biotin-anti-Aβ1-42 antibody; R-phycoerythrin-labeled streptavidin (PE-SA) was added to the desalted and purified biotin-anti-Aβ1-42 antibody, and stirred at room temperature for 1 hour to obtain PE-SA-biotin-anti-Aβ1-42 antibody; Additionally... Desalted and purified goat anti-chicken IgY was added to a biotin solution to obtain biotin-goat anti-chicken IgY; R-phycoerythrin-labeled streptavidin was added to the desalted and purified biotin-goat anti-chicken IgY and stirred at room temperature for 1 hour to obtain PE-SA-biotin-goat anti-chicken IgY. [The above biotin solution was prepared using DMSO as the solvent to form a 10% biotin solution; the addition ratio was: 0.06 mg biotin, 0.6 mg anti-Aβ1-42 antibody, and 0.36 mg R-phycoerythrin-labeled streptavidin, or the addition ratio was: 0.06 mg biotin, 0.6 mg goat anti-chicken IgY, and 0.36 mg R-phycoerythrin-labeled streptavidin.] Next, bovine serum albumin was added to PE-SA-Biotin-anti-Aβ1-42 antibody and PE-SA-Biotin-goat anti-chicken IgY respectively to a concentration of 1%, and the mixture was then blocked. Add 1 mL of preservation solution to the desalting column, and use the desalting column to separate and purify the blocked PE-SA-Biotin-anti-Aβ1-42 antibody and the blocked PE-SA-Biotin-goat anti-chicken IgY; the preservation solution composition is: 1% bovine serum albumin, 0.1% Tween 20, and pH 4.5 phosphate buffer; [pH 4.5 phosphate buffer is prepared by taking 500 mL of 0.2 mol / L potassium dihydrogen phosphate solution, diluting it with water to about 900 mL, adjusting the pH to 4.5 with sodium hydroxide solution, adding water to dilute to about 950 mL, adjusting the pH to 4.5 with sodium hydroxide solution or phosphate solution, adding water to 1000 mL, and shaking well.] The desalted and purified PE-SA-Biotin-anti-Aβ1-42 antibody and the desalted and purified PE-SA-Biotin-goat anti-chicken IgY present in the preservation solution were mixed and sprayed onto a glass cellulose membrane at a rate of 4 μL / cm. The membrane was then dried at 30°C for 6 hours to obtain a fluorescently labeled binding pad. The concentration of anti-Aβ1-42 antibody was 0.6 mg / ml and the concentration of goat anti-chicken IgY was 0.12 mg / ml during the mixing process.

[0047] The conjugate pad typically has a width of 5-20 mm and a length of 100-400 mm, or it can be longer to fit manufacturing equipment. It is horizontally positioned in the reagent strip / card and is cut to a length of 5-20 mm and a width of 3-10 mm. In this embodiment, the conjugate pad is 8 mm wide and 300 mm long, and is cut to a length of 8 mm and a width of 4 mm.

[0048] In the pretreatment of fluorescently labeled binding pads and nitrocellulose membranes, the BioDot RR120 system is used for spraying the corresponding reagents. Of course, other brands of equipment can also be used for spraying in this field.

[0049] 3. Pretreatment of nitrocellulose membrane: The Aβ1-42 antibody was diluted to 1 mg / mL with buffer 1, and the chicken IgY antibody was diluted to 0.5 mg / mL with buffer 2. The two solutions were sprayed onto the detection line and control line of the nitrocellulose membrane at a rate of 6 cm / s, respectively, at a rate of 2 μL / cm. (The spacing between the detection line and control line can be 3–10 mm; in this example, the spacing is 6 mm.) The membrane was then dried at 30°C for 6 hours to obtain the nitrocellulose membrane containing the detection line and control line. [The first buffer is a pH 6.8 phosphate buffer containing 1% trehalose, and the second buffer is a pH 6.8 phosphate buffer containing 0.5% trehalose.] Nitrocellulose membranes typically range in width from 10 to 50 mm and length from 100 to 400 mm, or can be longer to accommodate manufacturing equipment. They are horizontally positioned within reagent strips / cards and, after being cut, are configured to be 10 to 50 mm long and 3 to 10 mm wide. In this embodiment, the nitrocellulose membrane is 25 mm wide and 300 mm long, and after being cut, is configured to be 25 mm long and 4 mm wide.

[0050] 4. Assembling the test reagent cards: The pretreated sample pad, the pretreated fluorescently labeled binding pad, the pretreated nitrocellulose membrane, and the absorbent pad are assembled on a base plate (PVC material), and then cut into narrow strips with a width of 2~8mm (in this embodiment, they are cut into narrow strips with a width of 4mm) to obtain the test strip for detecting Aβ1-42 with the structure described in Example 1. The narrow strip (test strip) is then assembled into a card case to obtain a test card for detecting Aβ1-42.

[0051] like Figure 3 A schematic diagram of the assembly of the test kit is described. Narrow strips cut to a width of 2-8 mm are assembled into the inside of the kit shell 8. The kit shell 8 is assembled from two pieces, front and back. The front has a sample dispensing hole 9 located at the sample pad 2. The liquid to be tested is dripped into this sample dispensing hole 9, and the liquid is chromatographically separated towards the absorbent pad 5 under the action of capillary action. In addition, an observation window 10 is provided on the front of the kit shell 8 at the nitrocellulose membrane 4 to allow observation of the test line 6 and the control line 7 on the narrow strip.

[0052] After the test kit is assembled, it can be placed in an aluminum foil bag, with desiccant added if necessary, sealed, and stored in a dry environment at room temperature. It can usually be stored for at least 1.5 years.

[0053] Example 3: Preparation of test strips and test cards for detecting Aβ1-42 This embodiment mainly refers to the method of Embodiment 2 in this article to prepare a test strip for detecting Aβ1-42. The main difference is that fluorescent microspheres are used instead of streptavidin labeled with R-phycoerythrin when processing the fluorescently labeled conjugate pad.

[0054] 1. Sample pad pretreatment: Follow the corresponding steps in Example 2.

[0055] 2. Pretreatment of fluorescently labeled conjugate pads: Preparation of Fluorescein isothiocyanate-silica core-shell dual-structure fluorescent microspheres: 60 ml anhydrous ethanol, 2 ml ammonia, 3 ml tetraethyl orthosilicate, 1 ml ultrapure water, and 1 mg fluorescein isothiocyanate were mixed thoroughly and reacted in a constant-temperature water bath for 8 hours. Then, 3% Na₂SiO₃ was added to block the microspheres. The size of the fluorescent microspheres was controlled by adjusting the pH of the solution with hydrochloric acid. Active groups (-COOH) were added to the surface of the fluorescent microspheres for modification. The reaction solution was centrifuged at 1000 × g for 15 min, and the precipitate was collected and buffered with 0.01 M pH 4.8 borate solution. The microspheres were resuspended in a buffer solution, and the concentration was adjusted to OD450 nm = 0.2~0.25. Then, 100 μL of 50 mg / mL p-ethyl-N,N-dimethylpropylcarbodiimide (EDC) and 150 μL of 5 mg / mL N-hydroxysuccinimide (NHS) were added. The mixture was shaken and incubated at room temperature for 25 min. After centrifugation at 1000×g for 15 min, the precipitate was vacuum dried and stabilized. The microspheres were then resuspended in 0.01 M pH 4.8 borate buffer solution, and the concentration was adjusted to OD450 of 0.45~0.5 to obtain the fluorescent microsphere suspension.

[0056] Desalted and purified anti-Aβ1-42 antibody was added to a biotin solution to obtain biotin-anti-Aβ1-42 antibody; fluorescent microsphere suspension was added to the desalted and purified biotin-anti-Aβ1-42 antibody and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-anti-Aβ1-42 antibody; Additionally... Desalted and purified sheep anti-chicken IgY was added to a biotin solution to obtain biotin-sheep anti-chicken IgY; fluorescent microsphere suspension was added to desalted and purified biotin-sheep anti-chicken IgY and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-sheep anti-chicken IgY. [The above biotin solution was prepared using DMSO as the solvent to form a 10% biotin solution; the addition ratio was: 0.06 mg biotin, 0.6 mg anti-Aβ1-42 antibody, and 0.25 mg fluorescent microspheres, or the addition ratio was: 0.06 mg biotin, 0.6 mg goat anti-chicken IgY, and 0.25 mg fluorescent microspheres.] Next, bovine serum albumin was added to fluorescent microspheres-biotin-anti-Aβ1-42 antibody and fluorescent microspheres-biotin-goat anti-chicken IgY to a concentration of 1% for blocking treatment. Add 1 mL of preservation solution to the desalting column, and use the desalting column to separate and purify the blocked fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the blocked fluorescent microspheres-biotin-goat anti-chicken IgY; the preservation solution composition is: 1% bovine serum albumin, 0.1% Tween 20, and pH 4.5 phosphate buffer; [pH 4.5 phosphate buffer is prepared by taking 500 mL of 0.2 mol / L potassium dihydrogen phosphate solution, diluting it with water to about 900 mL, adjusting the pH to 4.5 with sodium hydroxide solution, adding water to dilute to about 950 mL, adjusting the pH to 4.5 with sodium hydroxide solution or phosphate solution, adding water to 1000 mL, and shaking well.] The desalted and purified fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the desalted and purified fluorescent microspheres-biotin-goat anti-chicken IgY present in the preservation solution were mixed and sprayed onto a glass cellulose membrane at a rate of 4 μL / cm. The membrane was then dried at 30°C for 6 hours to obtain a fluorescently labeled binding pad. The concentration of anti-Aβ1-42 antibody was 0.6 mg / ml and the concentration of goat anti-chicken IgY was 0.12 mg / ml during the mixing process.

[0057] The pad is 8mm wide and 300mm long, and after being cut, it is 8mm long and 4mm wide.

[0058] 3. Pretreatment of nitrocellulose membrane: Follow the corresponding steps in Example 2.

[0059] 4. Assembling the test reagent cards: Follow the corresponding steps in Example 2.

[0060] Example 4: Detection using the Aβ1-42 test kit This embodiment examines the performance of the Aβ1-42 detection reagent card using methods described in existing literature.

[0061] The tests were conducted at room temperature (23~24°C) using a fully automated dry fluorescence immunoassay analyzer (LABSIM AFS4000, Lanbo Biotechnology) in accordance with the instruction manual.

[0062] Sample preparation: Venous blood from 8 clinically donated healthy volunteers (aged 68-76 years) was allowed to stand at room temperature for 30 minutes, centrifuged at 5000 rpm for 10 minutes, and the serum was collected. Eight serum aliquots were mixed in equal volumes to form serum sample A. Separately, venous blood from 7 clinically donated Parkinson's disease (PD) patients (aged 71-79 years) was allowed to stand at room temperature for 30 minutes, centrifuged at 5000 rpm for 10 minutes, and the serum was collected. Seven serum aliquots were mixed in equal volumes to form serum sample B. Both serum samples were stored at -18°C for later use.

[0063] 1. Repeatability of test results Using the test reagent cards obtained in Examples 2 and 3 respectively, the Aβ1-42 content of serum A and serum B was measured 10 minutes after the sample solution was added to the sample well. Each serum sample was measured 10 times using 10 cards of each reagent card, and the fluorescence intensity was read. The Aβ1-42 reference standard was also measured using the same method. The Aβ1-42 content in the serum samples was calculated using the fluorescence intensity and concentration data of the reference standard, and the repeatability of the results was calculated. The results are shown in Table 1.

[0064] Table 1: Results of serum Aβ1-42 levels (pg / ml) measured using the test kit

[0065] As can be seen from the results in the table, the results obtained by using the two Aβ1-42 test kits for each serum were basically the same. However, in terms of the coefficient of variation, the Aβ1-42 test kit of Example 2 was obviously superior to the Aβ1-42 test kit of Example 3. The former had less fluctuation in the test results and better repeatability.

[0066] 2. Time required for stability testing Take 6 test kits from Example 2, add the serum A sample to be tested, and read the fluorescence intensity every 1 minute starting from the 3rd minute after sample addition until 15 minutes, i.e., 3 min, 4 min, 5 min, ..., 15 min readings. Use the same method to measure Aβ1-42 reference standard. Calculate the Aβ1-42 content in the serum sample using the fluorescence intensity and concentration data of the reference standard. For each time point, calculate the mean of the 6 results. This gives the average measurement result (n=6) of the serum A sample measured by the test kit from Example 2 at each time point from 3 to 15 minutes.

[0067] Six test kits from Example 3 were used, and serum sample A was added. The procedure was repeated as above, and the average measurement results (n=6) at each time point from 3 to 15 minutes were obtained when using the test kits from Example 3 to measure serum A. Six test kits from Example 2 were used, and serum sample B was added. The procedure was repeated as above, and the average measurement results (n=6) at each time point from 3 to 15 minutes were obtained when using the test kits from Example 2 to measure serum B. Six test kits from Example 3 were used, and serum sample B was added. The procedure was repeated as above, and the average measurement results (n=6) at each time point from 3 to 15 minutes were obtained when using the test kits from Example 3 to measure serum B. The test results for the four experiments are shown in Table 2 below.

[0068] Table 2: Results of serum Aβ1-42 levels (pg / ml) measured at different time points after sample addition

[0069] As can be seen from the above results, the time required to reach stable measurement results for both serum A and serum B using the test kit obtained in Example 2 was 10-11 minutes, while the time required to reach stable measurement results for both serum A and serum B using the test kit obtained in Example 3 was only 5-6 minutes. The detection speed was significantly faster than that of the test kit in Example 2, and the results remained essentially stable until the end of 15 minutes. Therefore, the test kit in Example 3 can rapidly and efficiently determine the Aβ1-42 content, and after obtaining reliable results in a short time, the results remain unchanged for a longer period. This provides a wider measurement window for flexible scheduling of testing work. For example, when using the test kit in Example 3, fluorescence results can be read within a broad timeframe of 5-15 minutes, reducing the workload of staff. In other words, for pursuing higher work efficiency, such as requiring the completion of a sample from sample addition to accurate result reading within 15 minutes, the test kit in Example 3 allows for fluorescence value reading within a broad timeframe of 5-15 minutes, while the test kit in Example 2 requires fluorescence value reading within a short timeframe of 10-15 minutes.

[0070] Based on the above results, it is shown that the test strips and test cards for detecting Aβ1-42 prepared using fluorescent microspheres in Example 3 exhibit superior detection efficiency compared to test cards made using R-phycoerythrin-labeled streptavidin in the prior art, significantly reducing the workload of testing personnel. However, the test cards of Example 3 still require improvement in terms of the repeatability of test results. The inventors have unexpectedly discovered that the problem of poor repeatability of the test results of the test cards of Example 3 can be overcome by changing the composition of the preservation solution used in preparing the fluorescently labeled binding pad, as described in Example 5.

[0071] Example 5: Preparation of a test kit for detecting Aβ1-42 and evaluation of its performance. 1. Preparation of a test kit for detecting Aβ1-42 Example 51: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.2% Tetronic 1307 (MW18600, Braveds, USA), 2.5% L-erythrose (Kaiserlein Technologies), and pH 4.5 phosphate buffer to 100%. Example 52: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.15% Tetronic 1307, 2% L-erythrose, and pH 4.5 phosphate buffer to 100%. Example 53: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.25% Tetronic 1307, 3% L-erythrose, and pH 4.5 phosphate buffer to 100%. Example 54: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.1% Tween 20, 2.5% L-erythrose, and pH 4.5 phosphate buffer to 100%. Example 55: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% D-erythrose, and pH 4.5 phosphate buffer to 100%. Example 56: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% glucose, and pH 4.5 phosphate buffer to 100%. Example 57: A test kit for detecting Aβ1-42 was prepared according to the method of Example 3 of this document, except that the preservation solution used when processing the fluorescently labeled conjugate pad was changed to: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% fructose, and pH 4.5 phosphate buffer to 100%.

[0072] 2. Performance evaluation of the repeatability of test results from the test reagent card Next, following the method described in Section 1, "Reproducibility of Detection Results," of Example 4, the Aβ1-42 content in serum A and serum B was determined using the test reagent cards from Examples 51-57. Each serum sample was tested 10 times using 10 cards of each reagent card, resulting in 10 results from the 10 tests. The mean (AV value), standard deviation (SD), and coefficient of variation (COP) of the 10 results were then calculated. CV The results of measuring the Aβ1-42 content (pg / ml) in serum A of the test subjects using 7 test reagent cards are shown in Table 3.

[0073] Table 3: Results of Aβ1-42 content (pg / ml) in serum A of the test kit

[0074] In addition, the results of measuring the content (pg / ml) of 10 Aβ1-42 in serum B using the test kit in Example 51 were as follows: 1628.4, 1538.7, 1765.4, 1842.1, 1519.2, 1683.2, 1517.6, 1684.2, 1713.8, and 1692.7, with a mean of 1658.53, an SD of 102.54, and a CV of 6.18%. Example 52: The mean (n=10) result (for the test kit) of Aβ1-42 content (pg / ml) in serum B was 1627.68, with an SD of 110.37 and a CV of 6.78%. Example 53: The mean (n=10) result (for the test kit) of Aβ1-42 content (pg / ml) in serum B was 1646.53, with an SD of 115.84 and a CV of 7.04%. Example 54: The mean (n=10) result (for the test kit) of Aβ1-42 content (pg / ml) in serum B was 1662.14, with an SD of 212.46 and a CV of 12.78%. The mean (n=10) result (for the test kit in Example 55) of the Aβ1-42 content (pg / ml) of serum B was 1637.48, with an SD of 216.83 and a CV of 13.24%. The mean (n=10) result (for the test kit in Example 56) of the Aβ1-42 content (pg / ml) of serum B was 1651.36, with an SD of 215.78 and a CV of 13.07%. The mean (n=10) result (for the test kit in Example 57) of the Aβ1-42 content (pg / ml) of serum B was 1642.28, with an SD of 207.36 and a CV of 12.63%.

[0075] These results show that the test results obtained by the methods of Examples 51-57 are basically consistent with the test results obtained by the methods of Examples 2 and 3. However, only the test results of the test cards of Examples 51-53, which are made by using Tetronic 1307 and L-erythrose at the same time, have significantly better repeatability than the other test cards. This finding is something that the prior art could not have predicted.

[0076] 3. Performance evaluation of the assay efficiency of the test reagent card Referring to "2. Time required for stable detection" in Example 4 of this invention, the performance of the detection efficiency of the test reagent cards in Examples 51-53 was examined.

[0077] The mean serum Aβ1-42 content (pg / ml) at each time point (n=6) from 3 min to 15 min, as determined by the test reagent card in Example 51, was as follows: 853.6, 958.3, 1118.4、1153.2、 The average serum Aβ1-42 content (pg / ml) at each time point (n=6) from 3 min to 15 min, measured by the test reagent card in Example 51, is as follows: 1037.2, 1437.8, 1158.3, 1124.3, 1141.4, 1162.4, 1125.4, 1138.7, 1164.3, 1137.6; 1636.4、1652.4、 The results, 1644.7, 1638.6, 1656.2, 1643.8, 1648.5, 1662.8, 1628.2, 1639.6, and 1647.1, indicate that the test kit of Example 51 achieved stable results within 5-6 minutes when measuring two serum samples. Similarly, the test kits of Examples 52 and 53 were examined, and the results showed that both kits achieved stable results within 5-6 minutes when measuring two serum samples.

[0078] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A test strip for detecting Aβ1-42 by fluorescence immunochromatography, comprising: The base plate, and the sample pad, fluorescent labeling pad, nitrocellulose membrane, and absorbent pad sequentially arranged on the base plate along its length; The downstream end of the sample pad along the direction of chromatographic movement overlaps above one end of the fluorescently labeled binding pad, the other end of the fluorescently labeled binding pad overlaps above one end of the nitrocellulose membrane, and the other end of the nitrocellulose membrane overlaps below the absorbent pad. A detection line and a control line are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorbent pad. The detection line is coated with Aβ1-42 antibody, and the control line is coated with chicken IgY antibody. The fluorescently labeled binding pad is coated with fluorescently labeled anti-Aβ1-42 antibody and fluorescently labeled goat anti-chicken IgY. This test strip is prepared by a method including the following steps: (1) Pretreatment of sample pad: Immerse the sample pad in sample pad buffer solution, and dry it at room temperature after immersion to obtain the pretreated sample pad. (2) Pretreatment of fluorescently labeled conjugate pads: Tetraethyl orthosilicate and fluorescein isothiocyanate were reacted, and the resulting microspheres were then blocked with the blocking agent Na2SiO3. Subsequently, the microspheres were treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to prepare a suspension of fluorescein isothiocyanate-silica core-shell dual-structure fluorescent microspheres. Desalted and purified anti-Aβ1-42 antibody was added to a biotin solution to obtain biotin-anti-Aβ1-42 antibody; fluorescent microsphere suspension was added to the biotin-anti-Aβ1-42 antibody and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-anti-Aβ1-42 antibody; Additionally... Desalted and purified goat anti-chicken IgY was added to a biotin solution to obtain biotin-goat anti-chicken IgY; fluorescent microsphere suspension was added to biotin-goat anti-chicken IgY and stirred at room temperature for 1 hour to obtain fluorescent microsphere-biotin-goat anti-chicken IgY. Next, bovine serum albumin was added to fluorescent microspheres-biotin-anti-Aβ1-42 antibody and fluorescent microspheres-biotin-goat anti-chicken IgY to a concentration of 1% for blocking treatment. Add 1 mL of preservation solution to the desalting column, and use the desalting column to separate and purify the blocked fluorescent microspheres-biotin-anti-Aβ1-42 antibody and the blocked fluorescent microspheres-biotin-goat anti-chicken IgY; Fluorescent microspheres-biotin-anti-Aβ1-42 antibody and fluorescent microspheres-biotin-goat anti-chicken IgY present in the preservation solution were mixed, and the mixture was sprayed onto a glass cellulose membrane at a rate of 4 μL / cm and dried at 30°C for 6 hours to obtain a fluorescently labeled binding pad. (3) Pretreatment of nitrocellulose membrane: Aβ1-42 antibody was diluted to 1 mg / mL with the first buffer and chicken IgY antibody was diluted to 0.5 mg / mL with the second buffer. The two antibodies were sprayed onto the detection line and control line of the nitrocellulose membrane at a rate of 2 μL / cm and 6 cm / s, respectively. The membrane was dried at 30°C for 6 hours to obtain the nitrocellulose membrane containing the detection line and control line. (4) Assembly of test strips: Assemble the pretreated sample pad, pretreated fluorescent labeling pad, pretreated nitrocellulose membrane and absorbent pad on the base plate, and then cut them into narrow strips with a width of 2~8mm, which are the test strips for detecting Aβ1-42. The preservation solution in step (2) is formulated as follows: 1% bovine serum albumin, 0.15%~0.25% Tetronic 1307, 2%~3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

2. The test strip according to claim 1, wherein in step (1), the sample pad is pretreated as follows: the sample pad is soaked in the sample pad buffer solution for 1 hour, and after soaking, it is dried at room temperature for 24 hours to obtain the test strip.

3. The test strip according to claim 1, wherein in step (2), the preparation of fluorescein isothiocyanate-silica core-shell dual-structure fluorescent microspheres is as follows: Take 60 mL of anhydrous ethanol, 2 mL of ammonia, 3 mL of tetraethyl orthosilicate, 1 mL of ultrapure water and 1 mg of fluorescein isothiocyanate, mix them evenly, react in a constant temperature water bath for 8 hours, add 3% Na2SiO3 to block the microspheres, adjust the pH value of the solution by adding hydrochloric acid to control the size of the fluorescent microspheres, modify the surface of the fluorescent microspheres with active groups, centrifuge the reaction solution at 1000×g for 15 min, collect the precipitate, suspend it in 0.01M pH 4.8 borate buffer, and adjust the concentration of the microspheres to OD. 450nm =0.2~0.25, then add 100 μL of 50 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 150 μL of 5 mg / mL N-hydroxysuccinimide, vortex to mix, incubate at room temperature for 25 min, centrifuge at 1000×g for 15 min, vacuum dry the precipitate, accurately weigh it, then resuspend it in 0.01 M pH 4.8 borate buffer, and adjust the microsphere concentration to OD. 450nm The concentration was 0.45~0.5, resulting in a fluorescent microsphere suspension.

4. The test strip according to claim 1, wherein the biotin solution in step (2) is prepared by using DMSO as solvent to form a solution containing 10% biotin.

5. The test strip according to claim 1, wherein in step (2), when preparing fluorescent microspheres-biotin-anti-Aβ1-42 antibody, the feeding ratio is: biotin 0.06 mg, anti-Aβ1-42 antibody 0.6 mg, and fluorescent microspheres 0.25 mg.

6. The test strip according to claim 1, wherein in step (2), when preparing fluorescent microspheres-biotin-goat anti-chicken IgY, the feeding ratio is: biotin 0.06 mg, goat anti-chicken IgY 0.6 mg, and fluorescent microspheres 0.25 mg.

7. The test strip according to claim 1, wherein the preservation solution in step (2) is formulated as follows: 1% bovine serum albumin, 0.2% Tetronic 1307, 2.5% L-erythrose, and pH 4.5 phosphate buffer to 100%.

8. The test strip according to claim 1, wherein the preservation solution in step (2) is formulated as follows: 1% bovine serum albumin, 0.15% Tetronic 1307, 2% L-erythrose, and pH 4.5 phosphate buffer to 100%.

9. The test strip according to claim 1, wherein the preservation solution in step (2) is formulated as follows: 1% bovine serum albumin, 0.25% Tetronic 1307, 3% L-erythrose, and pH 4.5 phosphate buffer to 100%.

10. The test strip according to claim 1, wherein the pH 4.5 phosphate buffer solution is prepared as follows: Take 500 mL of 0.2 mol / L potassium dihydrogen phosphate solution, dilute with water to 900 mL, add sodium hydroxide test solution to adjust the pH to 4.5, add water to 950 mL, add sodium hydroxide test solution or phosphate test solution to adjust the pH to 4.5, add water to 1000 mL, and shake well to obtain the solution.

11. The test strip according to claim 1, wherein in step (2), when fluorescent microspheres-biotin-anti-Aβ1-42 antibody and fluorescent microspheres-biotin-goat anti-chicken IgY present in the preservation solution are mixed, the concentration of anti-Aβ1-42 antibody is 0.6 mg / mL and the concentration of goat anti-chicken IgY is 0.12 mg / mL.

12. The test strip according to claim 1, wherein in step (3), the distance between the test line and the control line is 3~10 mm.

13. The test strip according to claim 1, wherein in step (3), the distance between the test line and the control line is 6 mm.

14. The test strip according to claim 1, wherein in step (3), the first buffer is a pH 6.8 phosphate buffer containing 1% trehalose, and the second buffer is a pH 6.8 phosphate buffer containing 0.5% trehalose.

15. The test strip according to claim 1, wherein in step (4), the assembled test strip is cut into narrow strips with a width of 4 mm.

16. A test kit for detecting Aβ1-42 by fluorescence immunochromatography, comprising a card case consisting of two parts, a front and a back, and a test strip as described in any one of claims 1 to 15 assembled within the two card cases. A sample application hole is provided on the front of the card case at the sample pad, through which the liquid to be tested is dripped, and the liquid is chromatographically deposited towards the absorbent pad under the action of a capillary. In addition, an observation window is provided on the front of the card case at the nitrocellulose membrane to allow observation of the test line and control line on the narrow strip.

17. A preservation solution for pretreating fluorescently labeled conjugate pads when preparing the test strips according to any one of claims 1 to 15, the formulation of which is: 1% bovine serum albumin, 0.15% to 0.25% Tetronic 1307, 2% to 3% L-erythrose, and pH 4.5 phosphate buffer to 100%; wherein the pH 4.5 phosphate buffer is prepared by: taking 500 mL of 0.2 mol / L potassium dihydrogen phosphate solution, diluting it with water to 900 mL, adjusting the pH to 4.5 with sodium hydroxide solution, adding water to 950 mL, then adjusting the pH to 4.5 with sodium hydroxide solution or phosphate solution, adding water to 1000 mL, and shaking well.

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