LU101972B1 - Test strip and method for detecting amyloid beta (Aß) in urine - Google Patents

Abstract

The present invention discloses a test strip and a method for detecting amyloid beta (Aß) in urine. The test strip for detecting Aß in urine includes a polyvinyl chloride (PVC) bottom plate. The PVC bottom plate is laid with a sample pad, a conjugation pad, a chromatography pad and an absorbent pad that are overlapped in sequence. The conjugation pad is coated with colloidal gold particles conjugated to a monoclonal antibody; the chromatography pad is provided with a test line on the side proximate to the conjugation pad, and is provided with a control line on the side proximate to the absorbent pad; the test line is coated with an Aß-binding polymer; and the control line is coated with a goat anti-mouse IgG polyclonal antibody. The method of the present invention can achieve high-sensitive, rapid and economical detection, with a detection limit of 40 picograms (pg). The method is suitable for: routine clinical pathological examination; general screening of a large number of people and self-screening of home end users; assisting the early diagnosis and prejudgment of mild cognitive impairment (MCI) clinically; and predicting and judging whether the patient has a high risk of developing MCI and Alzheimer's disease (AD).

Description

The present invention relates to a test strip and a method for detecting amyloid beta (AB) in ' urine. |

Alzheimer's disease (AD), the most common dementia, affects more than 26 million people worldwide.

It is found by the inventors and other investigators that a large proportion (30%) of the | elderly with normal cognition exhibits high retention of PET ligands in the brain and low level of AB1-42 in the cerebrospinal fluid (CSF), who are currently diagnosed as AD at the preclinical | latency stage.

Mild cognitive impairment (MCI) is considered to be the prodromal stage of AD, | and 40% to 60% of patients meeting the MCI criteria will eventually suffer from clinical AD, | about 5% to 25% per year.

Sporadic AD is mainly caused by the accumulation of AB and the | failure of AB clearance.

At present, the "amyloid cascade hypothesis" is generally supported in this | research field, that is, the metabolic imbalance of Aß in the brain is the root cause for ; neurodegeneration and cognitive decline of an AD patient.

Many animal and genetic studies have . shown that the innate phagocytosis of monocytes/macrophages/microglia may slow down the ; development of AD by promoting the clearance of AB and preventing the formation of senile | plaques. |

Biomarkers are promising to be an effective means for early diagnosis of AD, which includes | detecting the reduction of the size of the brain, and especially of the hippocampus (using magnetic | resonance imaging (MRI)); and detecting changes in functions of the brain (using functional MRI) | and changes in the concentrations of AB1-40, AB1-42 and tau in CSF.

However, the latter means is | invasive, with low patient compliance.

Another promising method is coordinated positron | emission tomography (PET)*. Neuroimaging (MRI, PET and CT) is expensive, and the number of | corresponding facilities available for clinical use is not adequate to meet the demand for | population screening.

Although studies have shown that the ratio of AB1-42/AB1-40 in plasma can be | used as an effective biomarker for identifying patients who are potentially at high risk of MCI or | AD, the detection of this biomarker requires special instruments and is not suitable for general | population screening.

Therefore, there is an urgent need to find a novel AD biomarker that is MOLE 101972 | practical, sensitive and specific. | AD is a systemic disease involving the whole body, and not just relates to the disorder and | damage of the brain.

It has been found that plasma-soluble Aß can be filtered out and excreted in | the urine, which may reflect the change of Aß in the brain circulation.

Since the extremely-low : concentration of AP in human urine is lower than the detection limit of the ELISA detection | method, there is no routine clinical diagnostic procedure for diagnosing MCI or AD by targeting | the AB in urine at present.

As the target antigen is too small, only of 4.3 kD, and tends to | non-specifically bind to other proteins, the commonly-used "sandwich ELISA" or "competitive : ELISA" is difficult to achieve high sensitivity and specificity. | The present invention is intended to overcome the shortcomings of the prior art, and to | provide a test strip and a method for detecting AP in urine. | In order to achieve the foregoing objective, the present invention provides the following | technical solution: | The present invention provides a test strip for detecting Aß in urine, including a polyvinyl | chloride (PVC) bottom plate (7). The PVC bottom plate (7) is laid with a sample pad (1), a | conjugation pad (2), a chromatography pad (3) and an absorbent pad (4) that are overlapped in | sequence.

The conjugation pad (2) is coated with colloidal gold particles conjugated to a | monoclonal antibody; the chromatography pad (3) is provided with a test line (5) on the side | proximate to the conjugation pad (2), and is provided with a control line (6) on the side proximate | to the absorbent pad (4); the test line (5) is coated with an AB-binding polymer; and the control | line (6) is coated with a goat anti-mouse IgG polyclonal antibody.

Preferably, the AB-binding polymer is a small peptide chain randomly synthesized from glutamic acid, lysine, alanine and tyrosine, with a molecular weight of 4 kD to 52 kD. | Preferably, the anti-A monoclonal antibody is a mouse anti-A monoclonal antibody prepared by a mouse hybridoma cell line; or is at least one of monoclonal antibody 1E8, monoclonal antibody 4G8, monoclonal antibody W0-2 and monoclonal antibody 6E10. Preferably, the sample pad (1) is coated with electrophoresis buffer at an amount of 10 uL/cm

3/13 \ to 16 uL/cm; the conjugation pad (2) is coated with colloidal gold particles conjugated to 2 101972 ! monoclonal antibody at an amount of 6 uL/cm to 9 ul/em; the test line (5) is coated with a | solution of the Aß-binding polymer in phosphate-buffered saline (PBS) at an amount of 1.5 uL/cm | to 2.5 uL/cm, which has a concentration of 2 mg/mL; and the control line (6) is coated with a : solution of the goat anti-mouse IgG polyclonal antibody in PBS at an amount of 1.5 uL/em to 2.5 | uL/cm, which has a concentration of 200 pg/mL. ,

The present invention provides a method for detecting AP in urine, including: capturing AB in | urine using colloidal gold particles conjugated to a monoclonal antibody to form a complex of ; colloidal gold particles and AP; and then using an AB-binding polymer to detect the complex of | colloidal gold particles and Aß; where, the colloidal gold particles have a particle diameter of 20 : nm to 50 nm; the monoclonal antibody is an anti-AB monoclonal antibody; and the AB-binding | polymer is a small peptide chain randomly synthesized from glutamic acid, lysine, alanine and : tyrosine.

Ë

Preferably, the small peptide chain has a molecular weight of 4 kD to 52 kD. ;

Preferably, the anti-AB monoclonal antibody is a mouse anti-AB monoclonal antibody | prepared by a mouse hybridoma cell line; or is at least one of monoclonal antibody 1E8, , monoclonal antibody 4G8, monoclonal antibody W0-2 and monoclonal antibody 6E10. )

The Aß-binding polymer can be used for preparing an Aß detection reagent, an MCI : diagnostic reagent and an AD diagnostic reagent. |

The present invention is further described below. |

In the present invention, colloidal gold particles conjugated to a monoclonal antibody are | adopted to capture AP in urine to form a complex of colloidal gold particles and AB, where the | colloidal gold particles have a particle diameter of 20 nm to 40 nm.

There is a group of | phagocytosis-promoting peptides (AB-binding polymers, known as PPPs), and it is found that the | group of PPPs includes some polymers and a small number of 15-mer peptides, and exhibits high | affinity to AB142. One polymer is a small peptide chain randomly synthesized from glutamic acid, | lysine, alanine and tyrosine, with a molecular weight of 4 kD to 52 kD (average: 6.5 kD). The | freshly-dissolved AB1-4 and the old AB14 have equilibrium dissociation constants (Kp) of 6.6 nM | and 25.4 nM, respectively.

This polymer is very promising to be used for detecting AB1-42, and | even can promote the clearance of AB within human body.

Therefore, an AB-binding polymer is |

4/13 f then used to detect the complex of colloidal gold particles and AP, with high sensitivity and 2101972 ; detection limit of 40 picograms (pg). The entire immunochromatographic assay is integrated in the | lateral flow chromatography test strip system, allowing rapid and economical detection.

The | method is suitable for: routine clinical pathological examination; general screening of a large | number of people and self-screening of home end users; assisting the early diagnosis and | prejudgment of MCI clinically; and predicting and judging whether the patient has a high risk of | developing MCI and AD.

Ë

FIG. 1 is a schematic diagram for the structure of a test strip for detecting AB in urine, where: : 1: sample pad; 2: conjugation pad; 3: chromatography pad; 4: absorbent pad; 5: test line; 6: control ! line; and 7: PVC bottom plate. |

FIG. 2 shows the direct binding of PPP to APi.42, with high affinity, where: Aßı.42 (in PBS, | with a final concentration of 80 nM) is labelled with HiLyte Fluor 488, then serially diluted at 1:1 | (volume), and mixed with PPP; and the measurement is conducted in a standard processing | capillary of NT.115 system, with 95% LED and 40% infrared laser power. :

FIG. 3 shows the interaction between AB1-42 and PPP, where, A: the secondary structure of AB | (0.2 mg/mL, 44 uM) is determined with circular dichroism (CD); B: the secondary structure of | PPP (44 uM); C: the structure of AB and PPP is determined at the jumping state; and the secondary | structure component is analyzed using the reference data set 3 compiled by Sreerama et al. with | computer program CDSSTR. |

FIG. 4 shows the tight binding of PPP to AB42, where: Aßaz (each 10 ug, 2.2 nmol) is mixed | with different amounts of PPP (0 nmol, 0.22 nmol, 1.1 nmol, 2.2 nmol, 6.6 nmol and 15.4 nmol, : columns 1 to 6) or 2.2 nmol of serum album ( column 7); the resulting mixtures (30 pL for each | sample) are diluted with 50 pM DTT and heated at 90°C for 5 min prior to SDS-PAGE (4% to | 12% NuPage gel, MES buffer, 100 V for 50 min); the protein is transferred and detected with an | anti-AB monoclonal antibody (clone W0-2); A: a western blotting image shows AB staining, and | each semi-quantitative 4.5 KD APs; monomer shows a bottom bar; B: Ponceau S staining is | conducted after the protein is transferred to a nitrocellulose membrane, a lot of PPP and bovine | serum albumin (BSA) are stained red, and the protein size is estimated by Seeblue + 2 standard pre-staining. LU101972 | FIG. 5 shows the detection results of test strips for the solutions of the synthesized AB42 in 100 | pL of ultrapure water (UPW) (left panel) and the solutions of the synthesized AB4 in100 uL of | urine (right panel), where: the solutions have a final concentration of 0 ng/mL to 5 ng/mL. | FIG. 6 shows the collection and detection of urine samples from healthy children by the Aß | test paper of the present invention, with 100 pL of urine sample for each test paper. | FIG. 7 shows examples of clinical sample testing. | FIG. 2 to FIG. 7 show the results in the examples of the present invention. |

1. Production and purification of anti-Aß monoclonal antibodies | A commercial mouse hybridoma cell line (BGM02) was purchased from Ab-Mart Pty Ltd. to | produce mouse anti-A monoclonal antibodies. Other commercial monoclonal antibodies, | including 1E8, 4G8, W0-2, 6E10, etc., could also be used alone or in combination. À vented | hydrophobic cell-culture flask was adopted. The hybridoma cells were thawed at 45°C. 1% of | glutamine, 1% of streptomycin/penicillin and 20% of fetal bovine serum (FBS) were added to a | serum-free hybridoma cell medium (% referred to the mass percentage of the component in the | serum-free hybridoma cell medium). | When the culture medium turned yellow, the cells were subcultured. The proportion of serum | in the medium for subculture decreased from 20% to 10%, 5%, 2% and 0%, and the cells could | survive in the final serum-free medium. This process usually takes one month. The supernatant | must not be pipetted until the serum is completely consumed. | When a medium was added to a bioreactor, the cell suspension was centrifuged, and the | resulting supernatant was removed. The cells were aggregated and collected in a syringe, and then | inoculated in the medium. The hybridoma cells were cultured in a serum-free medium in a | bioreactor for 10 d to 15 d, and the cell culture supernatant was collected. | The antibody was purified by affinity chromatography with the recombinant protein A binding | to resin. The pH of the culture supernatant for hybridoma cells was adjusted to 7.5 with IN NaOH or IN HCI. Then the culture supernatant was centrifuged at 3,600 rpm for 20 min with a desk

6/13 ; centrifuge, or at 7,500 r/min for 10 min with a high-speed centrifuge (preferred) to remove all cell 01972 . debris and precipitate. | The culture supernatant was loaded into the recombinant protein A column at high flow. a 0.22 ; um pre-filter or a pre-filter with a glass fiber membrane could be adopted. The landing speed was : maintained below 2 mL/min. The column was washed with at least 50 ml of PBS. | Elution was conducted (at a rate of 1 mL/min) with 50 mL of 40 mM trisodium citrate (pH |

3.0), and the eluate was collected with a fraction collector. All collections were immediately | neutralized with NazHPO; buffer (0.5 M, pH 9.5). The column was immediately rinsed after Ë elution. The concentration was determined with a A280/A260 spectrophotometer or a protein | detection kit for the antibody solutions placed in centrifuge tubes together. The antibody was | dispensed and stored at -80°C. /

2. Preparation of 30 nm colloidal gold particles | 200 mL of 0.01% HAuCl4 solution was slowly stirred and boiled. The solution was boiled for : 2 min, then stirred at a higher speed, and immediately added with 2.0 mL of 1% trisodium citrate | rapidly. The resulting solution was continuously boiled until the solution turned red/purple, and : then further boiled for 5 min. The particles were checked under an electron microscope. The | expected particle size is 30 nm. The particle size can be adjusted by the volume of the 1% ' trisodium citrate, and the volumes of 8.0 mL, 6.0 mL, 4.0 mL and 1.5 mL correspond to particle | sizes of 10 nm, 15 nm, 20 nm and 50 nm, respectively. ; The solution was cooled to room temperature, and the pH was adjusted to 7.2 with 0.1 M | K>CO;. The cooled solution was centrifuged at 10,000 g for 30 min without braking, the | supernatant was reduced to about 20 mL, and the obtained particles were stored at 4°C. |

3. Conjugate coupling of colloidal gold particles to a monoclonal antibody | 100 pL of 5 mM boric acid buffer (pH 9.0) was added to tubes 1 to 10 separately; then 100 pL | of an antibody solution was added to tube 1, and the resulting mixture was well mixed; a serial | dilution was conducted at a certain ratio from tube 1 to tube 9 in sequence; and only 100 pL of | boric acid buffer was added to tube 10. 100 pL of colloidal gold solution (the pH was adjusted to |

9.0 with 0.1 M K2COs) was added to each tube. The resulting mixture was well mixed and | incubated for 10 min. 10 pL of 10% sodium chloride solution was added, and the resulting mixture | was well mixed, and stood for 60 min to 120 min. If the protein content is sufficient, the color will |

7/13 | not change. If the protein content is not sufficient, the color in tube 10 will turn dark blue. This 01972 : method can be used to find the minimum protein content (Xmin) required for 20 mL of colloidal | gold particle solution. / The colloidal gold particle solution (the pH was adjusted to 9.0 with 0.1 M K2COs) was added | with an antibody (in 5 mM borate buffer, pH 9.0) at an amount increased by 10% on the basis of Xmin. The resulting solution was placed in a shaker for 15 min at room temperature. | The saturation was checked by adding 5 uL of 10% NaCl to 50 uL of labelled colloidal gold. | The resulting mixture stood for 30 min. If the gold does not precipitate out by changing to a blue | colour, it is saturatedly labelled. | 1% polyethylene glycol (PEG) (20 kD, the pH was adjusted to 7.2 with 1% trisodium citrate | or 0.1 M K2COs3) was added to achieve the final concentration of 0.1%. Ë The resulting solution was centrifuged at 260 g for 20 min, and impurities (small particles) | were removed for the supernatant. | Then the supernatant was centrifuged at 10,000 g for 30 min without braking. 0.2% PEG was | added to 4 mM phosphate/citrate buffer (pH 6.8) to obtain a final volume of 10 mL. The particles | were resuspended and stored at 4°C. 10% of sucrose was added before use. |

4. Assembly of a lateral flow chromatography test strip / As shown in FIG. 1, the test strip for detecting Aß in urine includes a PVC bottom plate 7. The | PVC bottom plate 7 is laid with a sample pad 1, a conjugation pad 2, a chromatography pad 3 and | an absorbent pad 4 that are overlapped in sequence. The conjugation pad 2 is coated with colloidal | gold particles conjugated to a monoclonal antibody; the chromatography pad 3 is provided with a | test line 5 on the side proximate to the conjugation pad 2, and is provided with a control line 6 on | the side proximate to the absorbent pad 4; the test line 5 is coated with an Aß-binding polymer; | and the control line 6 is coated with a goat anti-mouse IgG polyclonal antibody. | The specific assembly process is as follows. | The sample pad and conjugation pad were soaked in blocking buffer (20 mM sodium | tetraborate, pH 8.0, 2% BSA and 0.05% NaN3) at 4°C for 3 h or overnight. A liner was washed | once with distilled water (or UPW) at room temperature for 15 min, and then dried at 37°C. | The sample pad was cut into strips with a width of 15 mm, and sprayed with electrophoresis | buffer (20 mM sodium tetraborate, pH 8.0, 8% BSA, 0.05% Tween-20, and 0.05% NaNz3) at an |

8/13 | amount of 10 uL/cm. ii : The conjugation pad was cut into strips with a width of 10 mm. The labelled colloidal gold | was sprayed on the conjugation pad at an amount of 6 uL/em to 9 uL/em. | Two lines were scribed on the chromatography pad (nitrocellulose membrane) at a spacing of | 8 mm, namely, the test line and the control line. The test line was coated with a solution of the | polymer PPP in 10 mM PBS at an amount of 2.5 uL/cm, which had a concentration of 2mg/mL. | The control line was coated with a solution of the goat anti-mouse IgG polyclonal antibody in PBS | at an amount of 2.5 uL/cm, which had a concentration of 500 ug/mL. Each line was 1 mm wide |} and 4 mm long. | The absorbent pad was cut into strips with a width of 15 mm. Ë The above components were assembled on a 60 mm x 300 mm bottom plate, and the resulting | object was cut into 75 strips with a width of 4 mm. Fach test strip was assembled into a plastic | housing. :

5. Use of the lateral flow chromatography test strip for detecting AB in urine | Urine samples can be collected at any time of the day, but the determination is preferably : conducted on the same day. Urine samples can be freshly frozen at -80°C, but repeated freezing | and thawing are not allowed. | 100 pL of urine sample was slowly added dropwise via a sample-loading port of the kit housing. The kit stood for 20 min, and then the test line and control line were checked. | Optimally, the results were read visually or by the colloidal gold immunochromatographic | analyzer within 2 h. | Determination of results: | Negative (-): only the control line is colored. | Weak positive (+/-): the test line is slightly colored, while the control line is clearly colored. | Positive (+): the test line is colored at an intensity consistent with that of the control line | Strong positive (++): the test line is clearly colored, and the control line is normally or lightly | colored. | Extremely-strong positive (+++): the test line is clearly and deeply colored, and the control | line is extremely-lightly colored or may not be colored. |

6. Polymer identifying AB |

9/13 ; It was tested whether PPP could interact with AB. Microscale thermophoresis (MST) of Nang | 101972 | Temperor was adopted to determine the affinity between PPP and AB1-4. Aßı.42 (in PBS, 80 nM) ; was labelled with HiLyte Fluor 488. PPP was serially diluted at 1:1 (volume), with an average | molecular weight of 6.5 KD. The measurement was conducted in a standard processing capillary : under the Monolith NT.115 system, with 95% LED and 40% infrared laser power. The result | showed a high affinity binding between PPP and AB42 with Kp = 6.6 nM. The stoichiometric ratio ; of PPP to AP1-42 monomer is 3. Even if AB1-æ is placed at 4°C for one week to allow more | oligomers and fiber structures to be precipitated, the effective Kp value is still as low as 25.4 nM | (FIG. 2), which is similar to the affinity of an anti-Aß monoclonal antibody. | Circular dichroism (CD) is a practical method for studying the peptide-peptide interaction in a | solution. The CD in the far ultraviolet region (178 nm to 260 nm) is derived from the amide of the | protein backbone, and is sensitive to the protein conformation. Therefore, CD can be used to ; determine whether the conformations of polypeptides change during the interaction of | polypeptides. CD can be used to detect Aßı.42 and PPP and obtain the related results (FIG. 3). CD i is a quantitative analysis technique. Once the interaction between AB1-42 and PPP occurs, the | change in the CD spectrum is proportional to the amount of the peptide-peptide complex formed. | The results indicate that the B-pleated sheet is preferably formed in AB1-4, while no distinct ' B-pleated sheet can be seen in PPP. This complex exhibits a distinctly different structure, which | indicates the interaction between Aßı-42 and PPP (FIG. 3). ; The interaction between AP14 and PPP was further studied using western blotting. The | synthesized AB1-42 was mixed with different amounts of PPP at different ratios, and then the | mixture was subjected to the simplified electrophoresis of SDS-PAGE. By reducing the dosage for | electrophoresis, it can be found that PPP can significantly retain AB14, even under the | simplification condition of decrement (FIG. 4), indicating the tight binding between AB14 and | Therefore, unlike the traditional lateral flow immunochromatography using a polyclonal | antibody, the present invention uses PPP as a capture molecule to detect AB binding to conjugated | colloidal gold particles. Compared with ELISA, this method greatly improves the detection | sensitivity. |

7. The clinical use of the lateral flow chromatography test strip for detecting AP in urine |

/ 13 ; First, 100 uL of MilliQ UPW was used to prepare the solutions of the synthesized AB42 with 01972 / different concentrations, from 0 ng/mL to 100 ng/mL, and then the resulting solutions were tested | with test strips separately. The results show that there is an excellent correlation from 0 ng/mL to 5 | ng/mL, and the results at high concentrations do not further increase the intensity of the T line, | which may be caused by excessive antigens (FIG. 5). | Then ABs; was added to a negative urine sample, and then detected with a test strip. The | results are consistent with test results for ABs; samples prepared with UPW. When urine samples | with 0.5 ng/mL to 5 ng/mL of APs were detected, the T-line was developed, and the excessive | antigens (10 pg/mL) also did not exhibit detection results ascending proportionally (FIG. 5). | The test strip of the present invention was tested with urine samples from healthy children | under 10 years old (FIG. 6). More than 90% of urine samples from healthy children have negative |} results. ; These test strips were tested with urine samples from 152 Han Chinese (FIG. 7). The subjects | of the study were outpatients recruited from the Department of Neurology at Huashan Hospital of : Fudan University in Shanghai, China. There were 55 males, with an average age of 68.5; and 97 Ë females, with an average age of 68.1. The subjects had an age ranging from 42 to 89, and were ;

68.3 years old on average. | The subjects include the following: ; (1) Normal cognition control group (n = 30): none develops with a known age-related disease; | the score of Mini-Mental State Examination (MMSE) is not less than 27; and 22 in this group | complains of memory loss at the outpatient department recently, and the other 8 are family | members of the patients. | (2) MCI group (n = 47): clinical evaluation (MMSE score: 22 to 30), early AD patients are | included. | (3) Mild AD group (n = 37): MMSE score: 10 to 20. | (4) Severe AD group (n = 6): MMSE < 10. | (5) Other types of dementia group (n = 21): there are 15 vascular dementia patients, 3 | frontotemporal dementia patients, and 3 Lewy body dementia patients. | (6) Parkinson's disease group (n = 3). | Routine urine test was conducted for each urine sample. High protein (2+ or higher) urine | samples were excluded. | The overall results (Table 1) show that a higher detection rate is exhibited for the MCI and | early/mild AD, which are accompanied with other types of dementia, indicating that these test | strips have a higher potential for clinical diagnosis of MCI and AD. | Table 1: Test results for AB by test strips .

N - + + + ++ Positive rate % | Normal cognition level group 30 4 7 17 2 0 86.6 | MCIearly AD group 47 2 13 30 2 0 95.7 | Mild AD 37 2 8 19 7 1 94.5 | Advanced AD 6 1 0 4 1 0 83.3 | Other types of dementia 21 0 5 13 1 0 100 . Parkinson's disease 3 0 2 1 0 0 100 |

Claims (10)

What is claimed is: LU101972 |

1. A test strip for detecting amyloid beta (AP) in urine, comprising a polyvinyl chloride (PVC) . bottom plate (7), wherein, the PVC bottom plate (7) is laid with a sample pad (1), a conjugation | pad (2), a chromatography pad (3) and an absorbent pad (4) that are overlapped in sequence; the : conjugation pad (2) is coated with colloidal gold particles conjugated to a monoclonal antibody; | the chromatography pad (3) is provided with a test line (5) on the side proximate to the ; conjugation pad (2), and is provided with a control line (6) on the side proximate to the absorbent | pad (4); the test line (5) is coated with an Aß-binding polymer; and the control line (6) is coated | with a goat anti-mouse IgG polyclonal antibody. |

2. The test strip according to claim 1, wherein, the Aß-binding polymer is a small peptide | chain randomly synthesized from glutamic acid, lysine, alanine and tyrosine, with a molecular / weight of 4 kD to 52 kD. /

3. The test strip according to claim 1, wherein, the anti-Aß monoclonal antibody is a mouse | anti-Aß monoclonal antibody prepared by a mouse hybridoma cell line; or is at least one of | monoclonal antibody 1E8, monoclonal antibody 4G8, monoclonal antibody W0-2 and monoclonal | antibody 6E10.

4. The test strip according to claim 1, wherein, the sample pad (1) is coated with ; electrophoresis buffer at an amount of 10 uL/cm to 16 uL/cm; the conjugation pad (2) is coated | with colloidal gold particles conjugated to a monoclonal antibody at an amount of 6 uL/cm to 9 ) uL/cm; the test line (5) is coated with a solution of the Aß-binding polymer in phosphate-buffered | saline (PBS) at an amount of 1.5 uL/cm to 2.5 uL/cm, which has a concentration of 2 mg/mL; and | the control line (6) is coated with a solution of the goat anti-mouse IgG polyclonal antibody in | PBS at an amount of 1.5 uL/em to 2.5 uL/cm, which has a concentration of 200 pg/mL. |

5. A method for detecting AB in urine, comprising: capturing AP in urine using colloidal gold | particles conjugated to a monoclonal antibody to form a complex of colloidal gold particles and | Aß; and then using an Aß-binding polymer to detect the complex of colloidal gold particles and | AB; wherein, the colloidal gold particles have a particle diameter of 20 nm to 50 nm; the | monoclonal antibody is an anti-AB monoclonal antibody; and the AB-binding polymer is a small | peptide chain randomly synthesized from glutamic acid, lysine, alanine and tyrosine. |

6. The method according to claim 3, wherein the small peptide chain has a molecular weight | of 4 kD to 52 kD. |

7. The method according to claim 3, wherein, the anti-AB monoclonal antibody is a mouse | anti-AB monoclonal antibody prepared by a mouse hybridoma cell line; or is at least one of | monoclonal antibody 1E8, monoclonal antibody 4G8, monoclonal antibody W0-2 and monoclonal | antibody 6E10. |

8. An AB-binding polymer, comprising a small peptide chain randomly synthesized from | glutamic acid, lysine, alanine and tyrosine. |

9. The AB-binding polymer according to claim 8, wherein the small peptide chain has a | molecular weight of 4 kD to 52 kD. |

10. Use of the AB-binding polymer according to claim 8 or 9 in the preparation of an AB | detection reagent, a mild cognitive impairment (MCI) diagnostic reagent and a (Alzheimer's | disease) AD diagnostic reagent. |