CN113884683A - Double-antibody sandwich ELISA kit for rapidly detecting Abeta 42 - Google Patents

Abstract

The invention provides a double-antibody sandwich ELISA kit for rapidly detecting Abeta 42, which specifically comprises: coating antibody: monoclonal antibody BJ-1; detecting an antibody: monoclonal antibody 6E 10; the monoclonal antibody BJ-1 is preserved in China center for type culture Collection from 2011, 7 months and 1 days, the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO: c201155 hybridoma cell strain BJNA is obtained by secretion; the A beta 42 detection kit can be used for efficiently, sensitively and stably determining the content of the A beta 42 in cerebrospinal fluid.

Description

Double-antibody sandwich ELISA kit for rapidly detecting Abeta 42

Technical Field

The invention belongs to the technical field of molecular biological detection, and particularly relates to a double-antibody sandwich ELISA kit for rapidly detecting Abeta 42.

Background

Alzheimer's Disease (AD), commonly known as senile dementia, is a neurodegenerative disease and one of the most common types of dementia in the elderly. According to the annual report of Alzheimer's disease of 2018, about 5000 million dementia patients exist around the world; the number of patients suffering from Alzheimer's disease in China currently exceeds 1000 million, and is the first in the world. Alzheimer's disease seriously affects the lives of patients and their family members, and particularly, in the case of an aging tendency, imposes a heavy economic burden on families and society.

The onset of AD is more hidden, the disease is famous in the early stage of the disease, and no effective method exists for early detection and early diagnosis, so that early diagnosis and early treatment of AD are particularly important. Because AD is clinically lack of a specific diagnosis method for a long time and the standard and the basis are not unified, the clinical misdiagnosis rate reaches 27 to 57 percent. Some have attempted to make a prenatal correct diagnosis using brain biopsy, and it is clear that this invasive diagnostic method is not welcomed by physicians and patients. Needless to say, the 'standard matching' of the aging society, AD, has become a great challenge to achieve the goal of healthy china, and the blank of the early diagnosis method of AD is the biggest bottleneck to solve the problem.

The main pathological features of AD include Senile Plaques (SPs) formed by extracellular a β deposition, neurofibrillary tangles (NFTs) formed by intracellular hyperphosphorylated tau protein (p-tau), and neuronal cell loss. Biomarkers for AD can be divided into two categories: one is that pathophysiological markers that indirectly reflect AD-specific pathological changes, such as abeta 42 and tau in cerebrospinal fluid (CSF); and the second is a morphological marker capable of reflecting brain morphology, structure and metabolic changes of AD patients. Currently clinically used methods for objective diagnosis of AD mainly include Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), and cerebrospinal fluid biomarker detection. Given that the pathological course of AD usually precedes clinical symptoms for years, early diagnosis and recognition of disease progression can play a critical role. A great deal of research at present shows that the detection of a β 42 in cerebrospinal fluid and peripheral blood is closely related to the early diagnosis of AD, and particularly, the detection of a β 42 in cerebrospinal fluid is incorporated into a new clinical diagnosis standard of AD (see fig. 1), and is a core index for AD diagnosis. Early diagnosis of AD is thus accomplished, and recognition of disease progression can play a critical role.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a double-antibody sandwich ELISA kit for rapidly detecting Abeta 42.

Compared with the existing kit, the kit for detecting the Abeta 42 provided by the invention has the advantages that through the antibody pair proportion design, the using amount of antibodies is reduced, the antibody incubation time is shortened, the working process is greatly simplified, the content of the Abeta 42 in cerebrospinal fluid can be efficiently and stably determined, the long-time detection waiting is effectively avoided, the broad-spectrum property and the detectability are realized, and the test repeatability is good.

Based on the preparation, property and function identification of a specific anti-human amyloid protein (beta-amyloid peptide, Abeta) oligomer monoclonal antibody completed in the previous period in a laboratory, the invention establishes a double-antibody sandwich ELISA method for analyzing the most suitable coating antibody and concentration by using oligomerized Abeta 42 as an antigen, and establishes a simple, effective, rapid, sensitive and strong-specificity ELISA detection kit by using a horseradish peroxidase-labeled detection antibody as a detection system to detect the content of Abeta 42 in a cerebrospinal fluid clinical sample. Provides a technical basis for early diagnosis, effective prevention and control of AD, and brings great social and economic benefits.

The double-antibody sandwich ELISA kit for detecting Abeta 42 provided by the invention has the advantages of good repeatability, good stability and detectability, and can be used for early clinical diagnosis of AD.

The technical scheme of the invention

A double-antibody sandwich ELISA detection kit of human amyloid A beta 42 comprises the following components:

coating antibody: monoclonal antibody BJ-1;

detecting an antibody: monoclonal antibody 6E 10;

the monoclonal antibody BJ-1 is preserved in China center for type culture Collection from 2011, 7 months and 1 days, the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO: and C201155 hybridoma cell strain BJNA is obtained by secretion.

Preferably, the detection kit further comprises an antigen standard: the amino acid sequence of the antigen is shown as SEQ ID NO. 1.

Preferably, according to the present invention, the detection kit further comprises: an ELISA plate, a washing solution, a diluent, a stop solution and a horseradish peroxidase display substrate.

Preferably according to the invention, the detection antibody is: HRP-labeled monoclonal antibody 6E 10.

According to the invention, the enzyme label plate is a detachable 96-hole enzyme label plate.

According to the invention, the washing solution is obtained by adding Tween-20 into PBS solution according to the proportion of 0.1 percent by volume and mixing, and the pH value of the PBS solution is 7.4.

Preferably, according to the invention, the dilution is a 5% FBS-PBST solution.

Preferably, according to the invention, the antibody pair incubation reaction time is 1.5 hours.

According to the invention, the stop solution is preferably 0.5mol/L H₂SO₄And (3) solution.

According to the invention, the horse radish peroxidase chromogenic substrate is TMB solution.

According to the invention, the antibody BJ-1 is coated on an enzyme label plate.

Preferably, according to the invention, the antibody BJ-1 is coated in 2000ng per well and the detecting antibody 6E10-HRP dilution ratio is 1: 1000.

The preparation method of the kit comprises the following steps:

(1) preparing human amyloid A beta 42 with an amino acid sequence shown as SEQ ID NO. 1;

(2) preparing an antibody BJ-1;

(3) coating the antibody BJ-1 prepared in the step (2) on an enzyme label plate;

(4) and (4) subpackaging the rest reagents according to the conventional requirements, and then assembling the kit.

According to the invention, the coating method of the step (3) preferably comprises the following steps:

adopting a direct adsorption method, wrapping a capture antibody BJ-1 by using PBS (phosphate buffer solution) at 2000ng per well, and standing overnight at 2-8 ℃; PBST is washed for 3 times, each time is 3min, then the PBST is sealed by 10% FBS-PBST sealing liquid, 200 mu L/hole is kept at 37 ℃ for 2h, PBS is washed for 3 times, each time is 3min, the liquid is discarded, then the PBST is put into an incubator at 37 ℃ for drying, the PBST is put into a tinfoil bag after being dried, 2-3 bags of solid drying agents are added at the same time, and then the PBST is vacuumized by a vacuum sealing machine and stored at 2-8 ℃.

Introduction to the principle

The double-antibody sandwich ELISA is an ELISA method which combines a specific antibody on a solid phase carrier to form an antigen-antibody immune complex with an antigen in a sample to be detected, and completes detection by detecting the reaction of the antibody and the antigen in the complex, and has the characteristics of high sensitivity, economy, convenience and quantifiability.

The kit adopts a double-antibody sandwich ELISA method. When the ELISA plate is coated with the capture antibody, A beta 1-42 in the standard substance is combined with the capture antibody, and free components are washed away. Horseradish peroxidase (HRP) labeled detection antibody is added to form an antibody-antigen-antibody immune complex, and free components are washed away. Adding chromogenic substrate 3,3',5,5' - tetramethyllbenzidine 3,3',5,5' -Tetramethyllbenzidine (TMB), wherein the TMB turns blue under the catalysis of HRP, and turns yellow after adding a stop solution. Measuring OD value at 450nm wavelength with enzyme labeling instrument, drawing amyloid standard curve, calculating Abeta 1-42 concentration by substituting formula, and making double antibody sandwich ELISA schematic diagram shown in figure 2.

Advantageous effects

The double-antibody sandwich ELISA is an ELISA method which combines a specific antibody on a solid phase carrier to form an antigen-antibody immune complex with an antigen in a sample to be detected, and completes detection by detecting the reaction of the antibody and the antigen in the complex, and has the characteristics of high sensitivity, economy, convenience and quantifiability. Can be used for large-scale screening.

Compared with the existing kit, the A beta 42 detection kit provided by the invention has the advantages that through the antibody pair ratio design, the number of antibodies is reduced, the antibody incubation time is shortened, the work flow is greatly simplified, the content of the A beta 42 in cerebrospinal fluid can be efficiently and stably determined, long-time detection waiting is effectively avoided, the broad-spectrum and detectability are realized, and the test repeatability is good.

Drawings

FIG. 1IWG-2 typical diagnostic criteria for AD.

FIG. 2 schematic diagram of a double antibody sandwich ELISA.

FIG. 3 silver staining test antigen Abeta 42 standard;

m: protein molecular weight standards; 1-7: the standard substance of the A beta 42 with different concentrations is 200 mu mol/L, 100 mu mol/L, 50 mu mol/L, 25 mu mol/L, 12.5 mu mol/L, 6.25 mu mol/L and 1 mu mol/L in sequence.

FIG. 4 antibody peak of monoclonal antibody BJ-1 after purification by affinity chromatography.

FIG. 5 is an SDS-PAGE pattern of the BJ-1 monoclonal antibody denaturing gel.

FIG. 6A β 42 double sandwich ELISA assay system standard curve.

FIG. 7 is a schematic diagram of the standard dilution method.

FIG. 8 is an analysis chart comparing results of biotin and HRP detection systems;

in the figure: a.P/N value comparison, no significant difference; a450nm value comparison, with significant difference, p < 0.05.

FIG. 9 is a graph showing comparative analysis of P/N values of the results of detection at different reaction times;

in the figure: p <0.05, p <0.01, p < 0.001.

FIG. 10(BJ-1)/(6E10-HRP) checkerboard titration.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following examples and the drawings of the specification, but the scope of the present invention is not limited thereto.

The details not described in the examples are according to the state of the art.

Sources of reagents

96-well enzyme label plate: brand Corning.

The A beta 42 antigen standard is artificially synthesized by Suzhou Qiangyao biotechnology, Inc.

Detecting an antibody: 6E10-HRP was purchased from Biotech, Inc., Beijing Dake.

Sample source: the human cerebrospinal fluid specimens come from Tianjin Huanhu Hospital and Shandong provincial Hospital, and all conform to the principles of medical ethics and informed consent.

TMB color development liquid: from ThermoFisher.

Tris-HCl was purchased from Boototta technologies, Inc., China; tween-20 was purchased from Amresco, USA. Bovine Serum Albumin (BSA) was purchased from shanghai bioengineering, ltd, china. PBS, FBS, 1640 medium, DMEM/F12 medium were purchased from ThermoFisher.

Wash (1 × PBST): 0.5ml of Tween-20 was added to 1L of PBS (pH 7.4).

Diluent (5% FBS-PBST): 5mL FBS plus wash solution to 100 mL.

Blocking solution (10% FBS-PBST): 10mL FBS plus washing solution to 100 mL.

Stop solution (0.5mol/L H)₂SO 4): 178.3mL of deionized water, 21.7mL of concentrated sulfuric acid (98%) was added dropwise.

Biological material preservation information:

culture name: a hybridoma cell line BJNA;

the preservation number is: CCTCC NO: c201155;

the preservation unit: china center for type culture Collection;

and (4) storage address: china, wuhan university;

preservation time: 7/1/2011.

Example 1

A preparation method of a human amyloid A beta 42ELISA early detection kit comprises the following steps:

(1) preparation of antigen standard Abeta 42:

the antigen Abeta 42 has an amino acid sequence shown in SEQ ID NO.1 and is synthesized by Qianzhou biotechnology, Inc. in Suzhou province.

The antigen a β 42 after lysis and centrifugation was placed on ice. Incubate to colorless at room temperature with shaking. Diluting with DMEM/F12 solution to 200 μmol/L, standing in 37 deg.C constant temperature incubator for 6h, shaking every 1h, mixing, packaging, and storing at-80 deg.C. mu.L of 200. mu. mol/L of the above antigen was diluted to 50. mu. mol/L, 4.5. mu.L was diluted to 50ng/mL with 5% FBS-PBST solution, and 100. mu.L/bottle was dispensed into An-dissected bottles and pre-frozen at-80 ℃. The lyophilizer was opened and the lyophilization procedure was started. After about 3h the sample was lyophilized to a powder with no liquid in the vial. And (4) sealing the ampoule cover by a cover, and storing at 2-8 ℃.

The silver staining pattern of the antigen A beta 42 standard prepared above is shown in FIG. 3.

(2) Preparation of monoclonal antibody BJ-1:

1) cell recovery: and taking out the hybridoma cell strain BJNA frozen in liquid nitrogen, quickly putting the hybridoma cell strain BJNA into warm water at 42 ℃, and slightly shaking until the hybridoma cell strain BJNA is thawed for 1-2 min. The cells were aseptically transferred into 15mL centrifuge tubes and centrifuged at 1000rpm for 5 min. The supernatant was discarded, and 1mL of 1640 medium containing 20% by volume of FBS was added thereto, and the cells were gently pipetted to resuspend the cells. Transferring to a T25 cell culture bottle, adding 4mL of 1640 culture medium containing 20% FBS by volume fraction, mixing well, and culturing in a 5% CO2 incubator at 37 ℃ under saturated humidity.

2) Cell passage: the hybridoma cells grow in a semi-adherent manner, and when the confluence degree of the cells reaches 70% -80%, passage is carried out. The cells were pipetted with a 10mL disposable pipette, avoiding as much as possible the generation of air bubbles. When cells were detached as observed under a microscope, the culture medium containing the cells was transferred to a 15mL centrifuge tube and centrifuged at 1000rpm for 5 min. The supernatant was discarded, the cells were resuspended, and subcultured at a volume ratio of 1: 3.

3) Ascites production and purification: injecting the cultured hybridoma cells into the abdominal cavity of a mouse according to a conventional method, producing ascites after 1-2 weeks, purifying the ascites by protein A/G, and purifying to obtain a monoclonal antibody BJ-1;

4) and (3) detecting the antibody titer after purification: the monoclonal antibody BJ-1 is qualified by using an indirect ELISA method to detect the titer, if the titer is more than 1:64000, otherwise, the cell strain corresponding to the antibody is cultured again, and the cells are injected into a mouse to produce the ascites purified antibody.

The peak of the monoclonal antibody BJ-1 after purification is shown in FIG. 4.

FIG. 5 shows SDS-PAGE of denatured gel of monoclonal antibody BJ-1.

Coating a 96-well enzyme label plate:

and (3) coating the coated antibody BJ-1 by adopting a direct adsorption method according to 2000ng per hole, and standing overnight at 2-8 ℃. PBST washing 3 times, each time 3 min. Then blocking with 10% FBS-PBST blocking solution, 200 u L/hole, 37 degrees C2 h, PBS 3 times, each time 3 min. The liquid is discarded and then placed into an incubator at 37 ℃ for drying. And (3) after drying, putting the mixture into a tinfoil bag, adding 2-3 bags of solid drying agents, vacuumizing the mixture by using a vacuum sealing machine, and storing the mixture at 2-8 ℃.

And assembling the rest components of the kit into the human amyloid A beta 42ELISA early detection kit according to a conventional mode.

Example 2

The procedure for using the kit described in example 1 was as follows:

please remove the kit from the refrigerator 20 minutes earlier and equilibrate to room temperature.

Taking out the washing solution from refrigerator, if crystallization occurs, it is normal phenomenon, heating with 40 deg.C water bath to dissolve the crystal completely, and using at the same day (heating temperature is not more than 50 deg.C, and washing solution should be room temperature when using).

Antigen standard: and (3) balancing to room temperature, slightly opening the bottle cap, adding 1mL of diluent into the antigen standard substance Abeta 42, covering the tube cap, standing for 10min, reversing for several times, and after the diluent is fully dissolved, slightly and uniformly mixing the diluent with a pipettor, wherein the concentration of the standard substance solution is 5 ng/mL. Then, the sample is diluted in a multiple ratio according to the needs (note: the sample is not directly diluted in a reaction hole, the following concentration gradient is suggested to be prepared, wherein the concentration gradient is 1000, 500, 250, 125, 62.5, 31.25, 15.6 and 0pg/mL, 200 mu L of 5ng/mL standard substance is added into an EP tube containing 800 mu L of diluent liquid, the standard substance is uniformly mixed to prepare 1000pg/mL standard substance, and the rest concentrations are sequentially diluted in a multiple ratio according to the figure 6.

The specific operation steps are as follows:

a. respectively setting standard hole, blank hole and sample hole to be tested. And adding 50 mu L of diluent into the blank hole, respectively adding 50 mu L of antigen standard substance diluted in a gradient manner into the standard hole, and adding 50 mu L of sample to be detected into the sample hole to be detected.

b. 50 μ L of the diluent is added into the blank hole, and 50 μ L of the detection antibody 6E10-HRP (1:1000 dilution) is added into each of the rest standard holes and the sample holes to be detected.

c. Gently shaking and mixing, covering a film on the ELISA plate, and incubating at room temperature for 1.5 h.

d. The liquid in the wells was discarded, spin-dried, and the plate was washed 5 times with washing liquid.

e. 100. mu.L of chromogenic substrate TMB was added to each well.

f. Incubation for 15-30min at room temperature in the dark, and stopping when a blue color gradient appears in the standard wells.

g. 100 μ L of stop solution was added to each well. The reaction was terminated, at which point the blue color turned immediately yellow.

h. The optical density (OD value) of each well was immediately measured at a wavelength of 450nm with a microplate reader.

3. And (3) detection results:

within 15min after the stop solution is added, an enzyme-linked analyzer is used for detecting the optical density OD value of each detection hole under the condition of the wavelength of 450nm, through detection, the CV value of the variation coefficient of the coated 96-hole enzyme label plate prepared by the invention is less than 10%, test tests are carried out on detection kits of the same batch and different batches, and the test results show that the CV values of the kits in batches and among batches are less than 10%, which indicates that the double-antibody sandwich ELISA kit has high precision, and is a standard curve of the kit as shown in FIG. 7.

Experimental example 1

In order to further improve the sensitivity of the detection system, different labeled detection antibodies are selected, the detection systems of (BJ-1)/(6E10-Bio) and (BJ-1)/(6E10-HRP) are respectively adopted, the dosage of each level of antibody is optimized by adopting a chessboard titration method, and the sensitive detection system is selected, and the results are as follows.

Comparing the P/N values of the optimal antibody combinations for the 6E10-Bio detection system and the 6E10-HRP detection system, there was no significant difference between the two groups (P >0.05), but the a450nm signal value for the positive value of the 6E10-HRP system was higher (P <0.05), see fig. 8. And the reaction step of the detection system (BJ-1)/(6E10-HRP) is simpler and more convenient than that of the detection system (BJ-1)/(6E10-Bio), and the reaction time of the antibody can be shortened by 1.5h (see Table 1), so that the detection system (BJ-1)/(6E10-HRP) is selected subsequently.

TABLE 1 comparison of reaction procedures for different detection systems

Experimental example 2

Selection of confining liquids

In order to obtain the best blocking effect, we compared various blocking solutions, and kept the other conditions unchanged, and performed the double sandwich ELISA experiment, and the results are shown in Table 2, when selecting the 10% FBS-PBST group, the positive OD value is the highest and the negative OD value is the lowest at A450 nm. Therefore, 10% FBS-PBST was selected as a blocking solution for the detection system.

TABLE 2 detection results of different confining liquids

Note: abeta 42 was used at 1000pg/mL and coated with 200ng antibody per well.

Experimental example 3

Optimization of reaction time

To obtain optimal reaction time for antigen antibody, comparison was performed by double sandwich ELISA, as shown in figure 9: under the condition of reaction for 1.5h, the P/N value is the highest (P is less than 0.05), so 1.5h is selected as the antigen-antibody reaction time of the detection system.

Experimental example 4

Optimizing the use amount of the antibody:

the optimal amount of antibody pairs used was optimized by a checkerboard titration method. As shown in Table 3, when the same concentration of standard was measured, the positive A450nm values of 6E10-HRP 1:500 and 1:1000 in each well were 1.70 or more, as shown in FIG. 10: the highest P/N values were obtained at BJ-12000 ng per well for 6E10-HRP 1:1000 and 1: 2000, with no significant difference (P >0.05) as shown by differential analysis, but the positive A450nm values of the 6E10-HRP 1:1000 group were larger and relatively lower cost when the same concentration of standard was tested, so the final selected antibody dosage was BJ-1 per well 2000ng of the coated plate and the detection antibody 6E10-HRP dilution ratio was 1: 1000.

TABLE 3(BJ-1)/(6E10-HRP) checkerboard titration

Note: the concentration of the standard substance is 500 pg/mL; the reaction condition is 22-24 ℃ for 1.5 h; the blocking solution is 10% FBS-PBST; the results are shown in the table as "a 450nm values"; ". indicates antibody combinations with higher P/N values; "bold shows" indicates the optimal combination of antibodies selected.

Examples of effects

The competitive analysis with similar technologies or products (other similar products sold in the market can be used for replacing) at home and abroad is shown in Table 4, and the kit provided by the invention has the following characteristics:

1. the detection sensitivity is superior to that of the domestic kit, the experimental steps are simple, and the time is shorter;

2. the detection sensitivity of the foreign kit can be achieved, but the experimental steps are simple and the time is shorter;

3. the coated monoclonal antibody BJ-1, the monoclonal antibody marked by HRP is high-efficient, the stability is better, the cost is lower, and the operation is simpler.

TABLE 4 COMPARATIVE TABLE WITH HOUSEHOLD AND OUTDOOR PRODUCTS OF THE SAME TYPE

Effect example 2

Sample detection using the Α β 42 detection kit of the invention:

20 cases of cerebrospinal fluid samples are detected by using the detection kit, the detection results are shown in table 5, the lowest detection value is 20.47pg/ml and is within the detection range of the kit (15.6 pg/ml) according to the detection results, and the diagnosis of doctors can be assisted by combining clinic according to the diagnosis standard (NIAA) for reducing the level of Abeta 42 in AD cerebrospinal fluid.

TABLE 5 cerebrospinal fluid sample test results

SEQUENCE LISTING

<110> Beijing university of transportation of Shandong Jingji Biotechnology Ltd

Shandong Xinchuang Biotechnology Co.,Ltd.

<120> double-antibody sandwich ELISA kit for rapidly detecting Abeta 42

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 42

<212> PRT

<213> Artificial sequence

<400> 1

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

Claims (10)

1. A double-antibody sandwich ELISA detection kit of human amyloid A beta 42 is characterized by comprising the following components:

coating antibody: monoclonal antibody BJ-1;

detecting an antibody: monoclonal antibody 6E 10;

the monoclonal antibody BJ-1 is preserved in China center for type culture Collection from 2011, 7 months and 1 days, the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO: and C201155 hybridoma cell strain BJNA is obtained by secretion.

2. The test kit of claim 1, further comprising an antigen standard: the amino acid sequence of the antigen is shown as SEQ ID NO. 1.

3. The test kit of claim 2, wherein the test antibody is: HRP-labeled monoclonal antibody 6E 10.

4. The test kit of claim 2, further comprising: an ELISA plate, a washing solution, a diluent, a stop solution and a horseradish peroxidase display substrate.

5. The detection kit as claimed in claim 4, wherein the ELISA plate is a detachable 96-well ELISA plate;

preferably, the washing solution is obtained by adding Tween-20 into a PBS solution according to the proportion of 0.1 percent by volume and mixing, and the pH value of the PBS solution is 7.4;

preferably, the diluent is a 5% FBS-PBST solution;

preferably, the antibody pair incubation reaction time is 1.5 hours;

preferably, the stop solution is 0.5mol/L H₂SO₄And (3) solution.

6. The detection kit of claim 4, wherein the horse radish peroxidase chromogenic substrate is TMB solution.

7. The detection kit of claim 4, wherein the antibody BJ-1 is coated on an enzyme label plate.

8. The assay kit of claim 4, wherein the antibody BJ-1 is coated at 2000ng per well and the dilution ratio of the detection antibody 6E10-HRP is 1: 1000.

9. The method for preparing the detection kit according to any one of claims 1 to 8, characterized by comprising the steps of:

(1) preparing human amyloid A beta 42 with an amino acid sequence shown as SEQ ID NO. 1;

(2) preparing an antibody BJ-1;

(3) coating the antibody BJ-1 prepared in the step (2) on an enzyme label plate;

(4) and (4) subpackaging the rest reagents according to the conventional requirements, and then assembling the kit.

10. The method for preparing a test kit according to claim 9, wherein the coating method of step (3) comprises the steps of:

adopting a direct adsorption method, wrapping a capture antibody BJ-1 by using PBS (phosphate buffer solution) at 2000ng per well, and standing overnight at 2-8 ℃; PBST is washed for 3 times, each time is 3min, then the PBST is sealed by 10% FBS-PBST sealing liquid, 200 mu L/hole is kept at 37 ℃ for 2h, PBS is washed for 3 times, each time is 3min, the liquid is discarded, then the PBST is put into an incubator at 37 ℃ for drying, the PBST is put into a tinfoil bag after being dried, 2-3 bags of solid drying agents are added at the same time, and then the PBST is vacuumized by a vacuum sealing machine and stored at 2-8 ℃.

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