Biomarkers, kits, methods of preparation and uses for early detection of alzheimer's disease

Abstract

The application provides a biomarker, a kit, a preparation method and application for early Alzheimer's disease detection. Mainly containing 7 capture antibodies and detection antibodies which can specifically bind with human blood metabolites, the application has the characteristics of high sensitivity, high specificity and high accuracy, and can not only distinguish blood samples of patients with amnestic mild cognitive impairment, but also identify patients with early Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to detection technology, in particular biomarkers, kits, preparation methods and applications for early Alzheimer's disease detection. BACKGROUND

[0002] Alzheimer's disease can be divided into early amnestic mild cognitive impairment and late dementia. Screening patients in the amnestic mild cognitive impairment stage is crucial for early detection and treatment of Alzheimer's disease. However, the current early Alzheimer's disease screening method has many limitations. The most commonly used traditional clinical diagnosis method of Alzheimer's disease is neuropsychological test, such as Alzheimer's Disease Assessment Scale (ADAS-Cog). However, it is very time-consuming (usually more than 30 minutes of testing), and is prone to practice effect and large measurement bias.

[0003] Most importantly, the accuracy and sensitivity of neuropsychological tests are low, and they are completely unable to diagnose early Alzheimer's disease. Other clinical diagnostic methods, such as medical imaging technology, can observe the changes in brain structure and function caused by Alzheimer's disease, but the high cost, long time-consuming and the need for professional equipment greatly limit the application of medical imaging technology in clinical and research fields. Although studies have shown that biomarkers such as Aβ42, Tau, pTau in cerebrospinal fluid can improve the accuracy of Alzheimer's disease diagnosis, the collection of cerebrospinal fluid involves lumbar puncture, which is highly traumatic and can pose a great risk to patients, especially the elderly. At the same time, the collection of cerebrospinal fluid is costly and requires high clinical skills and stringent aseptic methods.

[0004] In recent years, the application of biomarker joint detection in the early diagnosis and screening of various diseases has received much attention. With the deepening of research and the development of related detection technology, metabolites in blood have been continuously discovered as potential biomarkers for early diagnosis of Alzheimer's disease. Liquid chromatography-mass spectrometry (LC-MS / MS) technology, as the gold standard for metabolite detection, has excellent specificity and accuracy. However, its low throughput, complex pretreatment, and high requirements for equipment and personnel limit its widespread use in early screening of Alzheimer's disease.

[0005] Therefore, it is very important to develop a "blood metabolite" detection kit for early detection and screening of Alzheimer's disease. SUMMARY

[0006] The application aims to provide a biomarker and a kit for early Alzheimer's disease detection, so as to solve the above technical problems in the prior art.

[0007] The technical scheme, according to an aspect of the application, a kit for early Alzheimer's disease detection comprises a capture antibody and a detection antibody.

[0008] The capture antibody and the detection antibody comprise a llama-derived antibody against human guanidino arginine, a llama-derived antibody against human cycloheptanamide, a llama-derived antibody against human valine-proline, a llama-derived antibody against human glutathione trisulfide, a llama-derived antibody against human N-undecanoyl glycine, a llama-derived antibody against human trans-C75 carboxylic acid and a llama-derived antibody against human benzyl penicilloyl G.

[0009] The capture antibody and the detection antibody specifically bind to human blood metabolites, including guanidino arginine, cycloheptanamide, valine-proline, glutathione trisulfide, N-undecanoyl glycine, trans-C75 carboxylic acid and benzyl penicilloyl G.

[0010] According to an aspect of the application, the detection antibody is a monoclonal antibody or a polyclonal antibody; and the enzyme is horseradish peroxidase, alkaline phosphatase or beta-galactosidase.

[0011] According to an aspect of the application, the kit further comprises at least one of an enzyme-labeled plate, a standard, a dilution buffer, a coating antigen or antibody, a coating buffer, a blocking buffer, an enzyme-labeled secondary antibody, a substrate, a stop solution and a washing buffer.

[0012] According to an aspect of the application, the dilution buffer is phosphate buffered saline, Tris buffered saline, a BSA solution or PBS or TBS containing Tween-20; the blocking buffer is bovine serum albumin, non-fat milk, fetal bovine serum, collagen or polyethylene glycol; and the substrate is TMB, ABTS, OPD, PNPP, BCIP / NBT or X-gal.

[0013] According to another aspect of the application, a biomarker for early Alzheimer's disease detection is further provided, comprising guanidino arginine, cycloheptanamide, valine-proline, glutathione trisulfide, N-undecanoyl glycine, trans-C75 carboxylic acid and benzyl penicilloyl G.

[0014] According to another aspect of the application, the kit or the biomarker is further used for preparing an ELISA detection product related to early Alzheimer's disease.

[0015] According to another aspect of the application, a method for preparing the kit is further provided, comprising the following steps:

[0016] a) Seven metabolites, namely guanidino arginine, cycloheptanamide, valine-proline, glutathione trisulfide, N-undecanoyl glycine, trans C75 carboxylic acid and benzylpenicilloyl G, are coupled to carrier proteins by chemical cross-linking to prepare immunogens;

[0017] b) The alpacas are immunized by first immunization and multiple booster immunization to induce the alpacas to produce antibodies against the seven metabolites;

[0018] c) Peripheral blood is collected from the immunized alpacas, lymphocytes are separated and total RNA is extracted;

[0019] d) The coding sequence of variable region of heavy chain antibody (VHH) of alpaca is amplified by reverse transcription-polymerase chain reaction (RT-PCR);

[0020] e) A phage display library is constructed, and antibody clones specifically recognizing the seven metabolites are obtained by high-throughput screening;

[0021] f) The antibody sequences obtained by screening are cloned into an expression vector, and the antibody is expressed and purified;

[0022] g) The purified antibody is used as a capture antibody and a detection antibody, and is prepared into an early Alzheimer's disease-related blood metabolite detection kit together with other reagent components.

[0023] According to one aspect of the present application, step e) further comprises sequencing the positive clones obtained by screening and removing duplicate sequences; performing epitope analysis and cross-reactivity detection on the candidate clones; determining the affinity constant by surface plasmon resonance (SPR), and screening high-affinity antibodies with dissociation constant (KD) less than 10 -9 M.

[0024] According to one aspect of the present application, step f) further comprises: improving the affinity of the antibody by site-directed mutagenesis or CDR random mutagenesis; screening more stable antibody variants by introducing disulfide bonds or melting point scanning; adding different labels at the C-terminus and evaluating the best detection effect.

[0025] According to one aspect of the present application, step g) comprises:

[0026] The capture antibody, the detection antibody, the enzyme-labeled plate, the standard, the dilution buffer, the coating buffer, the blocking buffer, the enzyme-labeled secondary antibody, the substrate, the stop solution and the washing buffer;

[0027] The standard is a different concentration standard of the seven metabolites, which is freeze-dried for storage;

[0028] The coating buffer is carbonate buffer, phosphate buffered saline or Tris buffered saline;

[0029] The blocking buffer is bovine serum albumin, non-fat milk, fetal bovine serum, collagen or polyethylene glycol;

[0030] The substrate is TMB, ABTS, OPD, PNPP, BCIP / NBT or X-gal;

[0031] The stopping solution is sulfuric acid, hydrochloric acid or SDS solution;

[0032] The washing buffer is phosphate buffered saline or Tris buffered saline.

[0033] The kit provided by the present application has good accuracy and sensitivity in detecting early Alzheimer's disease related blood metabolites. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The coating concentration of the capture antibody and the detection titer of the detection antibody of the kit provided by the present application are optimized according to the condition optimization curve of optical density (OD value).

[0035] Figure 2 The difference in the levels of the combined marker in 30 patients with amnestic mild cognitive impairment and 30 healthy elderly people.

[0036] Figure 3 The ROC curve of the kit provided by the present application in 30 patients with amnestic mild cognitive impairment and 30 healthy elderly people. DETAILED DESCRIPTION

[0037] A kit for detecting early Alzheimer's disease related "blood metabolites" is provided, which mainly comprises a capture antibody and a detection antibody.

[0038] The capture antibody and the detection antibody comprise:

[0039] An antibody against human guanidino arginine from a llama,

[0040] An antibody against human cyheptamide from a llama,

[0041] An antibody against human valine-proline from a llama,

[0042] An antibody against human glutathione trisulfide from a llama,

[0043] An antibody against human N-undecanoylglycine from a llama,

[0044] Alpaca-derived antibody against human Trans-75C carboxylic acid; and

[0045] Alpaca-derived antibody against human Benzylpenicilloyl G.

[0046] The capture antibody and the detection antibody are specifically combined with the human blood metabolite;

[0047] The human blood metabolite is as shown in SerIVD NO: 1-7 metabolite name.

[0048] In this embodiment, by using the LC-MS / MS technology, the blood samples of the early Alzheimer's disease patients and the healthy elderly are detected by non-targeted metabolomics, the blood metabolites of the early Alzheimer's disease patients and the healthy elderly are compared, and the metabolites with abnormal levels in the blood of the early Alzheimer's disease patients are screened out; the metabolites are subjected to metabolic pathway analysis, and the metabolic pathways related to the occurrence and development of Alzheimer's disease are verified.

[0049] The artificial intelligence algorithm is used to analyze and optimize the combination of the blood metabolites with abnormal levels in the early Alzheimer's disease patients; the combined biomarker can be used as an early Alzheimer's disease detection marker.

[0050] As an example, the capture antibody is a monoclonal antibody or a polyclonal antibody.

[0051] As an example, the capture antibody is a monoclonal antibody.

[0052] As an example, the detection antibody is a monoclonal antibody or a polyclonal antibody.

[0053] As an example, the detection antibody is a monoclonal antibody.

[0054] As an example, the detection antibody is combined with a labeling substance.

[0055] As an example, the labeling substance is an enzyme.

[0056] As an example, the enzyme is horseradish peroxidase (HRP), alkaline phosphatase (AP) or beta-galactosidase.

[0057] As an example, the kit provided by the application further comprises an enzyme-labeled plate, a standard, a dilution buffer, a coated antigen or antibody, a coating buffer, a blocking buffer, an enzyme-labeled secondary antibody, a substrate, a stop solution and a washing buffer.

[0058] The capture antibodies in the kit of the present application can be packaged separately or coated on an enzyme-labeled plate. The present application does not limit this, and all implementations are within the scope of the present application.

[0059] As an example, the capture antibodies in the kit are coated on a solid carrier.

[0060] As an example, the solid carrier is an enzyme-labeled plate.

[0061] As an example, the coating concentration of the capture antibodies is 900 ng / mL.

[0062] As an example, the detection titer of the detection antibodies is 1:2000.

[0063] The dilution buffer in the present application is used to dilute antibodies, antigens, samples or other reagents to achieve a suitable concentration for experiments.

[0064] As an example, the dilution buffer is phosphate buffered saline (PBS), Tris buffered saline (TBS), BSA (bovine serum albumin) solution or PBS or TBS containing Tween-20.

[0065] As an example, the dilution buffer is phosphate buffered saline.

[0066] The coating buffer in the present application is used to stably adsorb antigens or antibodies to the bottom of the microwell plate well, ensuring that the antigens or antibodies can be effectively fixed on the microwell plate for subsequent immune reactions.

[0067] As an example, the coating buffer is carbonate buffer, phosphate buffered saline (PBS) or Tris buffered saline (TBS).

[0068] As an example, the coating buffer is phosphate buffered saline.

[0069] The blocking buffer in the present application prevents the adsorption of non-specific proteins or other molecules, thereby reducing background signals.

[0070] As an example, the blocking buffer is bovine serum albumin (BSA), non-fat dry milk, fetal bovine serum (FBS), gelatin or polyethylene glycol (PEG).

[0071] As an example, the blocking buffer is bovine serum albumin.

[0072] The substrate in the present application is a substrate of an enzyme, which produces a visible color change or luminescence under the action of the enzyme for signal detection.

[0073] As an example, the substrate is TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), OPD (o-phenylenediamine), PNPP (p-nitrophenylphosphate), BCIP / NBT (5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium) or X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside).

[0074] As an example, the substrate is TMB, ABTS or OPD.

[0075] The stop solution in the present application is used to terminate the reaction between the substrate and the enzyme, fix the color of the reaction product, and make it suitable for photometer reading.

[0076] As an example, the stop solution is sulfuric acid, hydrochloric acid or SDS (sodium dodecyl sulfate) solution.

[0077] As an example, the stop solution is sulfuric acid.

[0078] The washing buffer in the present application is used to clean the microwell plate between steps, remove unbound components, and reduce background signal.

[0079] As an example, the washing buffer is phosphate-buffered saline (PBS) or Tris-buffered saline (TBS).

[0080] As an example, the washing buffer is phosphate-buffered saline.

[0081] The detection kit provided by the present application is used in the preparation of an ELISA detection product related to early Alzheimer's disease.

[0082] The present application also provides a method for using the kit, comprising the following steps:

[0083] Step one: capture the combined marker in the sample to be tested with a capture antibody.

[0084] Step two: detect the above captured combined marker with a detection antibody.

[0085] Step three: draw a standard curve according to the detection value to obtain the level of the combined marker in the sample to be tested.

[0086] As an example, the specific use of the double antibody indirect ELISA kit provided by the present application includes the following steps:

[0087] Step 1: Preparation

[0088] Prepare all reagents according to the kit instructions, including dilution buffer, washing buffer, etc. Prepare the sample to be tested, and dilute as needed.

[0089] Step 2: Coating

[0090] Add the coating antigen or antibody to each well of the microplate, incubate at 4°C overnight or at room temperature for a few hours. Remove the unbound antigen or antibody, and wash the well plate with washing buffer.

[0091] Step 3: Blocking

[0092] Add blocking buffer to block the protein binding sites not occupied by the antigen or antibody, reducing non-specific binding. After incubation, wash again.

[0093] Step 4: Add sample and / or antibody

[0094] First add the sample, incubate and wash, then add the primary antibody, incubate and wash, then add the enzyme-labeled secondary antibody.

[0095] Step 5: Washing

[0096] Wash after each addition step to remove non-specifically bound molecules

[0097] Step 6: Substrate Addition

[0098] Add the appropriate enzyme-labeled substrate, and the enzyme acts on the substrate to produce a color change or luminescence.

[0099] Step 7: Stop the reaction

[0100] After a suitable time, add a stop solution to terminate the reaction.

[0101] Step 8: Result reading

[0102] Use the enzyme label meter to measure the absorbance (or luminescence) of each well at a specific wavelength, and calculate the concentration of antigen or antibody in the sample according to the standard curve.

[0103] Step 9: Data analysis

[0104] Analyze the absorbance (or luminescence) data to determine the concentration of the target molecule in the sample according to the standard curve.

[0105] The application provides a kit and application thereof, the kit comprising seven capture antibodies and detection antibodies capable of specifically binding to human blood metabolites, including a llama-derived antibody against human guanidino arginine, a llama-derived antibody against human cyheptamide, a llama-derived antibody against human valine-proline, a llama-derived antibody against human glutathione trisulfide, a llama-derived antibody against human N-undecanoylglycine, a llama-derived antibody against human trans-75C carboxylic acid, and a llama-derived antibody against human benzylpenicilloyl G.

[0106] The kit provided by the application has good accuracy and sensitivity in detecting blood metabolites related to early Alzheimer's disease.

[0107] Experiments show that the application compares blood samples of 30 diagnosed amnestic mild cognitive impairment patients and 30 healthy old people, and draws a receiver operating characteristic curve (ROC) and calculates the curve and an area under the curve (AUC).

[0108] Results show that the kit provided by the application has a sensitivity of 93.3%, a specificity of 90.0%, an accuracy of 91.7%, and an AUC of 0.909 in detecting blood samples of amnestic mild cognitive impairment patients and healthy old people.

[0109] In the following examples, the materials and reagents used, unless otherwise specified, can be obtained from commercial channels.

[0110] The blood metabolites detected by the early Alzheimer's disease-related blood metabolite detection kit provided by the application are shown in Table 1:

[0111] Table 1. Antibodies contained in the early Alzheimer's disease-related blood metabolite detection kit and blood metabolites detected

[0112]

[0113] Note: Up-regulation or down-regulation refers to up-regulation or down-regulation in the blood of patients with early Alzheimer's disease.

[0114] Example 1: Optimization of coating concentration of capture antibody and detection titer of detection antibody according to optical density (OD value)

[0115] 1. Dilute the capture antibody with coating buffer to 300 ng / mL, 600 ng / mL, 900 ng / mL, 1200 ng / mL, 1500 ng / mL and 1800 ng / mL respectively, and incubate at 100 μL / well overnight at 4°C.

[0116] 2. Remove the microplate from the kit and add 100 μL of 100 ng / mL standard to each well coated with capture antibody. Incubate at 37°C for 1.5 hours.

[0117] 3. Discard the solution, rinse each well five times with 300 μL of phosphate-buffered saline, and then pat dry.

[0118] 4. Dilute the labeled detection antibody with phosphate-buffered saline at concentrations of 1:500, 1:1000, 1:2000, 1:4000 and 1:8000, respectively.

[0119] 5. Add 100 μL of diluted detection antibody to each well and incubate at room temperature (37°C) for 40 minutes.

[0120] 6. Discard the solution, wash each well five times with 300 μL of phosphate-buffered saline, and then pat dry.

[0121] 7. Add 30 μL of substrate to each well and develop the color for 15 minutes in the dark at room temperature (37°C).

[0122] 8. 50 mL of sulfuric acid stop solution in each well.

[0123] 9. Measure the optical density OD450 value using an enzyme-linked immunosorbent assay (ELISA) reader.

[0124] 10. Plot a scatter plot with the coating concentration of different capture antibodies as the x-axis and the optical density OD450 value corresponding to the detection titer of different detection antibodies as the y-axis.

[0125] 11. The optimal antibody working conditions are the coating concentration of the capture antibody and the detection titer of the detection antibody corresponding to the obvious inflection point of optical density OD450 value of 1.5.

[0126] Depend on Figure 1 The inflection point marked by the circle indicates that the optimal coating concentration of the capture antibody is 900 ng / mL, and the optimal detection titer of the detection antibody is 1:2000.

[0127] Example 2: Use of the reagent kit

[0128] Using the kit prepared in Example 1, blood samples from patients with amnesic mild cognitive impairment were used as test samples, and blood samples from healthy elderly people were used as control samples to illustrate the method of using the kit provided by the present invention.

[0129] The experimental design is shown in Table 2:

[0130]

[0131] 1. Take each group of samples to be tested, and dilute each blood sample with the dilution buffer provided in the kit at a ratio of 1:20.

[0132] 2. Take out the enzyme-labeled plate in the kit, and add 100 μL of the diluted sample to be tested to each well coated with the capture antibody. Incubate at room temperature at 37°C for 1.5 hours.

[0133] 3. Discard the solution, wash each well with 300 μL of phosphate buffered saline for 5 times, and then pat dry.

[0134] 4. Add 100 μL of the diluted detection antibody to each well, and incubate at room temperature at 37°C for 40 minutes.

[0135] 5. Discard the solution, wash each well with 300 μL of phosphate buffered saline for 5 times, and then pat dry.

[0136] 6. Add 100 μL of the substrate to each well, and develop color for 15 minutes at room temperature at 37°C in the dark.

[0137] 7. Add 50 mL of sulfuric acid stop solution to each well.

[0138] 8. Measure the optical density OD450 value with an enzyme-linked detector.

[0139] 9. Determine the level of the combined marker in each blood sample.

[0140] The difference in the level of the combined marker in 30 patients with amnestic mild cognitive impairment and 30 healthy elderly people is shown in Table 3. Figure 2 The average detection level of the 30 patients with amnestic mild cognitive impairment is 0.474, the variance is 0.189, and the standard error is 0.034; while the average detection level of the 30 healthy elderly people is 0.832, the variance is 0.310, and the standard error is 0.057. The fold change of the detection value between the 30 patients with amnestic mild cognitive impairment and the 30 healthy elderly people is 1.754, and the corrected p value is 1.27417E-6, which is much lower than the p < 0.050 considered to be significant. It can be seen that the kit provided by the present application can distinguish between patients with amnestic mild cognitive impairment and healthy elderly people.

[0141] Example 3: Sensitivity, specificity and accuracy of the kit detection

[0142] According to the detection results provided in Embodiment 2, the ROC curve is drawn, and the AUC is calculated, and the results show that:

[0143] Using the kit provided by the present application, 30 patients with amnestic mild cognitive impairment and 30 healthy old people are detected, 28 patients with amnestic mild cognitive impairment and 27 healthy old people can be correctly detected. As can be seen, the sensitivity of the kit provided by the present application for detecting amnestic mild cognitive impairment patient samples and healthy old person samples is 93.3%, the specificity is 90.0%, the accuracy is 91.7%, and the AUC is 0.909, as shown in Figure 3 .

[0144] In the detection of 30 patients with amnestic mild cognitive impairment and 30 healthy old people, the sensitivity, specificity and accuracy of the kit provided by the present application are all higher than 90%, indicating that it can significantly distinguish the blood samples of patients with amnestic mild cognitive impairment and the blood samples of healthy old people, and has high detection sensitivity, specificity and accuracy. It is shown that the kit provided by the present application has great diagnostic value for early Alzheimer's disease.

[0145] As an example, another method for preparing an early Alzheimer's disease-related blood metabolite detection kit mainly includes the following steps:

[0146] a) Metabolite-carrier protein coupling: seven metabolites, i.e., guanidino arginine, cycloheptanamide, valine-proline, glutathione trisulfide, N-undecanoyl glycine, trans C75 carboxylic acid and benzylpenicilloyl G, are coupled to carrier proteins by chemical cross-linking method respectively to prepare immunogens;

[0147] b) Immunization of alpaca: the alpaca is immunized by first immunization and multiple booster immunization to induce the alpaca to produce antibodies against the seven metabolites;

[0148] c) Collection of peripheral blood from the immunized alpaca, separation of lymphocytes and extraction of total RNA;

[0149] d) Amplification of the coding sequence of alpaca heavy chain antibody variable region (VHH) by reverse transcription-polymerase chain reaction (RT-PCR);

[0150] e) Construction of phage display library, and obtaining antibody clones specifically recognizing the seven metabolites by high-throughput screening;

[0151] f) Cloning of the screened antibody sequences into an expression vector, expression and purification of the antibodies;

[0152] g) Purification of the antibodies as capture antibodies and detection antibodies, and preparation of an early Alzheimer's disease-related blood metabolite detection kit together with other reagent components.

[0153] As an example, the carrier protein in step a) is KLH or BSA.

[0154] As an example, the chemical cross-linking method in step a) is EDC / NHS coupling method.

[0155] As an example, for metabolite molecules that are not easy to directly couple, a linker arm is introduced before coupling in step a).

[0156] As an example, the coupling ratio in step a) is 10-20 metabolite molecules / carrier protein molecules.

[0157] As an example, the immunogen prepared in step a) is purified and characterized, the purification method is dialysis, and the characterization method includes MALDI-TOF mass spectrometry and SDS-PAGE.

[0158] As an example, the immunization scheme in step b) includes:

[0159] First immunization: 500 μg of immunogen mixed with complete Freund's adjuvant, subcutaneous multiple point injection;

[0160] Boosting: 250 μg of immunogen mixed with incomplete Freund's adjuvant is injected every 2 weeks, a total of 4-6 times.

[0161] As an example, step e) includes:

[0162] Directly immobilize the target metabolite on the surface of an ELISA plate or magnetic beads;

[0163] Perform 3-5 rounds of screening, gradually increasing the selection pressure;

[0164] Add structural analogs in the last round for competitive screening to improve specificity.

[0165] As an example, step e) also includes sequencing the positive clones obtained by screening and removing duplicate sequences; epitope analysis and cross-reactivity detection of candidate clones; determination of affinity constant by surface plasmon resonance (SPR), screening of dissociation constant (KD) less than 10 -9 High-affinity antibodies of M.

[0166] As an example, step f) is an E. coli or yeast expression system.

[0167] As an example, the purification method in step f) includes metal affinity chromatography, ion exchange and gel filtration chromatography.

[0168] As an example, step f) further comprises: improving antibody affinity by site-directed mutagenesis or CDR random mutagenesis; screening more stable antibody variants by introducing disulfide bonds or melting point scanning; adding different labels at the C-terminus and evaluating the best detection effect.

[0169] As an example, the detection kit in step g) is a sandwich ELISA detection system or a competitive ELISA system.

[0170] As an example, the coating concentration of the capture antibody in the sandwich ELISA detection system is 900 ng / mL, and the dilution ratio of the detection antibody is 1:2000.

[0171] As an example, step g) further comprises methodological validation of the prepared detection kit, including specificity, accuracy, precision, sensitivity, and linear range.

[0172] As an example, specificity verification is less than 5% cross-reactivity with similar structural metabolites; accuracy verification is that the spiked recovery rate is within the range of 80-120%; precision verification is that the coefficient of variation within and between batches is less than 15%; sensitivity verification is that the minimum detection limit reaches the nanomolar level; linear range verification covers the expected concentration range of clinical samples, reaching about 3 orders of magnitude.

[0173] As an example, step g) further comprises clinical validation of the prepared detection kit, including: correlation analysis of the developed ELISA kit with the LC-MS / MS method, with a correlation coefficient r greater than 0.9; evaluating clinical performance using known early Alzheimer's disease patient samples and healthy control samples; determining the optimal diagnostic threshold according to the ROC curve.

[0174] As an example, the detection kit in step g) includes: capture antibody, detection antibody, enzyme-labeled plate, standard, dilution buffer, coating buffer, blocking buffer, enzyme-labeled secondary antibody, substrate, stop solution, and washing buffer.

[0175] As an example, the standard is a different concentration standard of seven metabolites preserved by freeze-drying; the coating buffer is carbonate buffer, phosphate buffered saline, or Tris buffered saline; the blocking buffer is bovine serum albumin, non-fat milk, fetal bovine serum, collagen, or polyethylene glycol; the substrate is TMB, ABTS, OPD, PNPP, BCIP / NBT, or X-gal; the stop solution is sulfuric acid, hydrochloric acid, or SDS solution; the washing buffer is phosphate buffered saline or Tris buffered saline.

[0176] As an example, step g) further comprises performing stability studies on the prepared test kit, the stability studies comprising accelerated stability testing, real-time stability monitoring, and freeze-thaw stability evaluation.

[0177] As an example, the kit is used for detecting the levels of the seven metabolites of guanidinoarginine, cycloheptanamide, valine-proline, glutathione trisulfide, N-undecanoylglycine, trans-C75 carboxylic acid, and benzylpenicilloyl G in a blood sample.

[0178] As an example, the test kit has a sensitivity of at least 90% and a specificity of at least 90% in the detection of early Alzheimer's disease.

[0179] As an example, the test kit is used to distinguish amnestic mild cognitive impairment patients from healthy elderly people.

[0180] As an example, the test kit is used in the preparation of an ELISA detection product related to early Alzheimer's disease.

[0181] In this embodiment, the kit provided by the present application detects a brand-new combination of blood metabolite biomarkers closely related to early Alzheimer's disease, and is simple and fast to operate, conducive to high-throughput clinical detection, and can reduce the cost of detection. The detection method of the present application provides an important reference for early detection and early treatment of Alzheimer's disease in clinical practice.

[0182] The above describes preferred embodiments of the present application, but the present application is not limited to the specific details of the above-described embodiments. Within the technical concept of the present application, various equivalent transformations of the technical solutions of the present application can be made, and these equivalent transformations all belong to the protection scope of the present application.

Claims

1. A kit for early Alzheimer's disease detection, characterized by, The capture antibody and the detection antibody; The capture antibody and the detection antibody are: a llama-derived nanobody against human guanidinoarginine, a llama-derived nanobody against human cycloheptanamide, a llama-derived nanobody against human valine-proline, a llama-derived nanobody against human glutathione tri-sulfide, a llama-derived nanobody against human N-undecanoylglycine, a llama-derived nanobody against human trans-C75 carboxylic acid, and a llama-derived nanobody against human benzylpenicilloyl G; The capture antibody and the detection antibody specifically bind to human blood metabolites, which are guanidinoarginine, cycloheptanamide, valine-proline, glutathione tri-sulfide, N-undecanoylglycine, trans-C75 carboxylic acid, and benzylpenicilloyl G.

2. A kit according to claim 1, characterised in that The detection antibody is a monoclonal antibody or a polyclonal antibody; the labeled enzyme on the detection antibody is horseradish peroxidase, alkaline phosphatase, or β-galactosidase.

3. The kit according to claim 1, characterized in that Further comprising at least one of an enzyme-labeled plate, a standard, a dilution buffer, a coating buffer, a blocking buffer, a substrate, a stop solution, and a washing buffer.

4. Kit according to claim 3, characterized in that The dilution buffer is phosphate buffered saline, Tris buffered saline, a BSA solution, PBS containing Tween-20, or TBS containing Tween-20; the blocking buffer is bovine serum albumin, non-fat milk, fetal bovine serum, collagen, or polyethylene glycol; and the substrate is TMB, ABTS, OPD, PNPP, BCIP / NBT, or X-gal.

5. Biomarkers for early Alzheimer's disease detection, characterized in that, The biomarker is a combination of guanidinoarginine, cycloheptanamide, valine-proline, glutathione tri-sulfide, N-undecanoylglycine, trans-C75 carboxylic acid, and benzylpenicilloyl G.

6. Use of the kit of any one of claims 1 to 4 or the reagent for detecting the biomarker of claim 5 in the preparation of an early Alzheimer's disease ELISA detection product.

7. A method of preparing the kit of any one of claims 1 to 4, characterized in that, The kit comprises the following steps: a) Metabolite-carrier protein coupling: coupling the seven metabolites, guanidinoarginine, cycloheptanamide, valine-proline, glutathione tri-sulfide, N-undecanoylglycine, trans-C75 carboxylic acid, and benzylpenicilloyl G, to carrier proteins respectively by chemical cross-linking to prepare immunogens; b) Immunization of llamas: using a first immunization and multiple booster immunizations to induce llamas to produce antibodies against the seven metabolites; c) Collecting peripheral blood from the immunized llamas, isolating lymphocytes, and extracting total RNA; d) Amplifying the coding sequence of llama heavy chain antibody variable region (VHH) using reverse transcription-polymerase chain reaction (RT-PCR); e) Constructing a phage display library and obtaining antibody clones that specifically recognize the seven metabolites through high-throughput screening; f) Cloning the screened antibody sequences into an expression vector, expressing and purifying the antibodies; g) Using the purified antibodies as capture antibodies and detection antibodies, together with other reagent components, to prepare an early Alzheimer's disease-related blood metabolite detection kit.

8. The method according to claim 7, characterized in that, In step e) also includes sequencing the positive clones obtained by screening, removing duplicate sequences; epitope analysis and cross-reactivity detection of candidate clones; affinity constant determination by surface plasmon resonance (SPR), screening the dissociation constant (KD) less than 10⁻ 9 high affinity antibodies of M.

9. The method according to claim 7, characterized in that, Step f) further comprises: improving the affinity of the antibody by site-directed mutation or CDR random mutation; screening more stable antibody variants by introducing disulfide bonds or melting point scanning; adding different labels at the C-terminus and evaluating the best detection effect.

10. The method of claim 7, wherein, The detection kit in step g) comprises: capture antibody, detection antibody, enzyme-labeled plate, standard, dilution buffer, coating buffer, blocking buffer, substrate, stop solution and washing buffer; The standard is a different concentration standard of seven metabolites, which is freeze-dried; The coating buffer is carbonate buffer, phosphate buffered saline or Tris buffered saline; The blocking buffer is bovine serum albumin, non-fat milk, fetal bovine serum, collagen or polyethylene glycol; The substrate is TMB, ABTS, OPD, PNPP, BCIP / NBT or X-gal; The stop solution is sulfuric acid, hydrochloric acid or SDS solution; The washing buffer is phosphate buffered saline or Tris buffered saline.

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