Kit for diagnosing transthyretin amyloidosis and preparation and purification method of substrate protein

Abstract

The invention relates to a kit for detecting transthyretin amyloidosis in vitro and a preparation and purification method of a substrate protein, the kit comprises the substrate protein, a sample extraction reagent of total protein in skin tissue, a reaction reagent for real-time oscillation induced conversion (RT-QuIC), a negative reference substance and a positive reference substance, the substrate protein comprises at least one of a WT-TTR recombinant protein and a D38A-TTR recombinant protein, the WT-TTR recombinant protein and the D38A-TTR recombinant protein correspond to a sequence SEQ ID NO 1 and a sequence SEQ ID NO 2 respectively, and accurate diagnosis of transthyretin amyloidosis is achieved through skin biopsy in combination with an RT-QuIC technology.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of in vitro diagnostic kits, and particularly relates to a kit for diagnosing transthyretin amyloidosis and a preparation and purification method of substrate protein. BACKGROUND

[0002] Transthyretin amyloidosis (ATTR) is a systemic fatal disease caused by abnormal conformation and misfolding of transthyretin (TTR), which is mainly divided into hereditary ATTR (hATTR) and wild-type ATTR (wtATTR). The clinical types include transthyretin cardiac amyloidosis (ATTR-CM) mainly with myocardial involvement and transthyretin amyloidosis polyneuropathy (ATTR-PN) mainly with peripheral nervous system involvement. Another common systemic amyloidosis disease is immunoglobulin light chain amyloidosis (AL). The etiology and pathogenesis of the two diseases are different, but the clinical manifestations (such as decreased cardiac function and peripheral nerve damage) are highly similar, and there is a certain cross between laboratory and imaging indicators. Therefore, in clinical practice, the differential diagnosis of ATTR and AL is a great challenge.

[0003] ATTR lacks specificity in clinical symptoms and is often misdiagnosed as other cardiovascular or nervous system diseases. Most patients are in the late stage when diagnosed, and the damage to organ structure and function is difficult to reverse, which adversely affects subsequent treatment options and prognosis. At present, myocardial biopsy is the recognized gold standard for ATTR diagnosis, with high sensitivity and specificity, but it is an invasive procedure that relies on experienced professional teams and carries a certain risk of complications, making it difficult to popularize.

[0004] Although 99Tcm-PYP myocardial scintigraphy is a non-invasive method, it requires high equipment conditions, has low popularization rate, and has radiation exposure risk. In addition, for hereditary ATTR, TTR gene mutation detection is feasible, but for wtATTR, gene detection cannot be effectively identified.

[0005] Studies have shown that there is a high consistency of TTR amyloid protein deposition in the skin tissue of ATTR patients with myocardial tissue, suggesting that skin biopsy is expected to become an ideal alternative to myocardial biopsy. This method has the advantages of small trauma, simple operation, safety, and can be quickly implemented in outpatient clinics, and has high clinical feasibility.

[0006] Real-Time Quaking-Induced Conversion (RT-QuIC) is a highly sensitive detection method based on the principle of protein misfolding "seed effect". It was first applied to the diagnosis of prion diseases and has been officially included in the relevant disease diagnosis guidelines of the United States, the European Union and China. In recent years, this technology has been successfully expanded to the detection of Parkinson's disease, Alzheimer's disease and other neurodegenerative diseases, further verifying its wide applicability and stability. The amyloid TTR protein also has a "seed effect" and is highly compatible with the RT-QuIC technology, which is expected to achieve real-time amplification and detection of extremely low abundance pathological TTR amyloid proteins.

[0007] Currently, there is no published or mature protocol worldwide that combines skin biopsy with RT-QuIC for precise diagnosis of ATTR and differential diagnosis of ATTR and AL. Therefore, the development of a minimally invasive detection kit based on skin biopsy and RT-QuIC technology is expected to break through the existing technical bottlenecks and achieve precise diagnosis of ATTR and effective differentiation of ATTR and AL, so as to help patients gain the best treatment opportunity. SUMMARY

[0008] The purpose of the present application is to overcome the following technical problems in the prior art:

[0009] (1) Invasive tests such as myocardial biopsy have a high risk of complications and low clinical acceptability;

[0010] (2) Nucleus imaging examination has high requirements for equipment conditions and professional operation, and the popularization rate is limited, and there is a risk of radiation exposure;

[0011] (3) ATTR lacks specific biomarkers, making diagnosis difficult;

[0012] (4) Although nuclear imaging has some value in differentiating ATTR and AL, there are still false positives or diagnostic uncertainties in some cases, especially with complex clinical backgrounds, making it difficult to accurately determine.

[0013] The present application provides a kit for diagnosing transthyretin amyloidosis, which realizes high-precision diagnosis of ATTR through skin biopsy combined with RT-QuIC technology. Based on the detection results of misfolded TTR protein in the skin, an optimized differential diagnosis strategy for ATTR and AL is established. At the same time, this method has the advantages of simple operation, no need for expensive equipment, and no radiation risk, providing an innovative solution for precise diagnosis and differential diagnosis of ATTR.

[0014] The technical solution of the present application is as follows:

[0015] A kit for diagnosing transthyretin amyloidosis, comprising a substrate protein, a sample extraction reagent for extracting total protein in skin tissue, a reaction reagent for RT-QuIC reaction, a negative control and a positive control; wherein the substrate protein comprises at least one of WT-TTR recombinant protein and D38A-TTR recombinant protein, and the amino acid sequence of the WT-TTR recombinant protein is shown as SEQ ID NO 1, and the amino acid sequence of the D38A-TTR recombinant protein is shown as SEQ ID NO 2.

[0016] Further, the sample extraction reagent comprises a sample washing solution and a sample lysis solution.

[0017] Further, the sample lysis solution is 10 mM Tris-HCl, 133 mM NaCl, 2 mM , 0.25% (w / v) collagenase A, and a protease inhibitor.

[0018] Further, the protease inhibitor is a protease inhibitor cocktail tablet without EDTA.

[0019] Further, the reaction reagent comprises 50-200 mM sodium acetate, 50-200 mM KCl, 0.5-2 mM , and 10-50 μM thioflavin T, and the pH of the reaction reagent is 3.5-5.5.

[0020] Further, the negative control is a phosphate buffer.

[0021] Further, the positive control is a pre-prepared TTR amyloid fibril.

[0022] Further, the RT-QuIC reaction condition of the kit comprises: the shaking mode is double-orbit shaking, and the shaking frequency is 500-800 rpm; the reaction is carried out in cycles, and each cycle comprises shaking for 2-5 min and standing for 1-3 min; the fluorescence reading is recorded once every 6 cycles; the incubation temperature is 35-40℃, the excitation wavelength is 450 nm, and the emission wavelength is 480 nm; the above cycle and reading mode are continuously carried out until the total reaction time is 50-90 h.

[0023] Further, the kit further comprises a preparation and purification method of the substrate protein, for preparing and purifying the substrate protein in the kit, comprising the following steps:

[0024] S1, prokaryotic expression: construct pET-24a(+) expression plasmid containing WT-TTR or D38A-TTR gene, and transform into E. coli BL21(DE3) competent cells, after activation of the culture, inoculate into a large volume of medium in proportion, cultivate at 37℃ to the mid-log phase OD600=0.7-0.8, add 1 mM IPTG with a final concentration, induce overnight at 28℃;

[0025] S2, cell collection and lysis: collect the bacterial cells by low-temperature centrifugation, resuspend in a buffer system containing phosphate buffer, NaCl and imidazole, add protease inhibitors, break the bacterial cells by high-pressure homogenization, remove debris by high-speed low-temperature centrifugation and pre-filter through a 0.45 μm filter membrane, and then obtain the supernatant;

[0026] S3, nickel affinity chromatography: load the supernatant into a Ni-NTA column, use a low-concentration imidazole buffer to remove non-specifically bound proteins, and then elute the bound proteins with a high-concentration imidazole to obtain the elution product;

[0027] S4, dialysis and molecular sieve purification: dialyze the elution product into a low-salt buffer condition, and then separate the protein by Superdex200 molecular sieve chromatography;

[0028] S5, Q-Sepharose anion exchange chromatography: load the protein in a low-salt buffer condition, and elute by NaCl gradient;

[0029] S6, final processing: dialyze the protein into a storage buffer, then aliquot and freeze-dry for storage.

[0030] Compared with the prior art, the beneficial effects of the present application are:

[0031] 1. It can realize accurate detection of ATTR

[0032] In clinical verification, 30 ATTR patients (5 wild type and 25 mutant type) and 30 control samples (10 AL patients) were included. The results showed that the sensitivity was 93.3% and the specificity was 100% when using WT-TTR substrate; the sensitivity was 96.7% and the specificity was 90% when using D38A-TTR substrate. Especially in wild type ATTR patients, the detection results showed 5 positive cases, proving that the kit of the present application can realize the detection of wild type ATTR patients, breaking through the bottleneck that wild type ATTR is difficult to diagnose in the prior art.

[0033] 2. Both substrate proteins have good detection performance

[0034] The application adopts two recombinant proteins, WT-TTR and D38A-TTR, as substrates, and both can achieve effective detection of ATTR patients (including genetic and wild type), and the sensitivity and specificity are not less than (>) 90%. Both substrates can be used for detection of ATTR, providing multiple feasible options for clinical application.

[0035] 3. High-efficiency identification of ATTR and AL

[0036] In 30 cases of control patients, 10 cases of AL patients were all detected as negative, verifying that the kit of the application can effectively distinguish ATTR and AL. Based on the seed effect mechanism of RT-QuIC, the positive or negative of the detection result can provide strong support for clinically identifying ATTR and AL, thereby reducing the diagnostic interference caused by the overlap of imaging or laboratory indicators.

[0037] 4. Significant advantages of minimally invasive and clinical accessibility

[0038] The application adopts skin biopsy instead of myocardial biopsy, which is minimally invasive, simple to operate and can be completed in outpatient service without significant complications. Compared with nuclide imaging, the method has no radiation risk and does not require expensive equipment, thereby reducing the cost of clinical popularization. Combined with the high sensitivity and specificity of RT-QuIC, the application realizes the detection goal of safety, precision and accessibility.

[0039] 5. High-quality protein substrate guarantees detection performance

[0040] WT-TTR and D38A-TTR recombinant proteins are efficiently prepared through a prokaryotic expression system (E. coli BL21 (DE3)), and the target proteins with a purity of ≥95% are obtained through multi-step chromatography purification (nickel affinity, molecular sieve, Q column anion exchange). Combined with freeze-drying preservation (about 1 mg per tube, low-temperature preservation) and ultrafiltration purification (100 kDa (MWCO), low-temperature centrifugation) strategies, the activity of the proteins is guaranteed, batch consistency is ensured, and stable and reliable substrate support is provided for RT-QuIC detection. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 : SDS-PAGE Coomassie blue staining analysis diagram of WT-TTR and D38A-TTR protein purification;

[0042] Figure 2 : ThT fluorescence kinetic curve diagram of RT-QuIC detection of skin samples of ATTR patients and control group;

[0043] Figure 3 : Maximum fluorescence intensity and diagnostic performance analysis diagram of RT-QuIC detection of ATTR patients and control group;

[0044] Figure 4Figure 1: Congo red staining of RT-QuIC products from skin samples of ATTR patients and controls;

[0045] Figure 5 Figure 2: ThS fluorescence microscopy observation of RT-QuIC products from skin samples of ATTR patients and controls;

[0046] Figure 6 Figure 3: Transmission electron microscopy (TEM) observation of RT-QuIC products from skin samples of ATTR patients and controls;

[0047] Figure 7 Figure 4: Atomic force microscopy (AFM) observation of RT-QuIC products from skin samples of ATTR patients and controls. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the protection scope of the present application.

[0049] Definitions and explanations of terms

[0050] Misfolded TTR protein: refers to amyloid fibril aggregation state protein of transthyretin (TTR) formed due to pathological factors (such as mutation or conformational abnormality), which is pathogenic.

[0051] RT-QuIC: Real-Time Quaking-Induced Conversion Assay, an in vitro amplification technique for detecting amyloid proteins.

[0052] 6His-TTR recombinant protein: a recombinant transthyretin protein containing 6 histidine tags, the amino acid sequence of which is shown in SEQ ID NO 1 (WT-TTR recombinant protein) and SEQ ID NO 2 (D38A-TTR recombinant protein).

[0053] Prepared TTR amyloid fibril body: TTR amyloid fibril prepared by TTR recombinant protein after 7 days of shaking at 37°C, used as a positive control for RT-QuIC reaction.

[0054] Thioflavin T (ThT): a dye that emits fluorescence after binding to β-sheet amyloid fibrils, used for real-time monitoring of amyloid fibril formation in RT-QuIC reaction.

[0055] Thioflavin S (ThS): a dye that produces fluorescence upon binding to β-pleated amyloid fibrils, used primarily for fluorescent microscopic staining to verify amyloid properties of the product.

[0056] Negative control: phosphate buffered saline (PBS) used in the present application as a negative control for the RT-QuIC reaction.

[0057] A kit for diagnosing transthyretin amyloidosis and a method for preparing and purifying substrate protein, comprising the following contents:

[0058] I. Components:

[0059] 1. Substrate protein

[0060] At least one of the following 6×His-TTR recombinant proteins:

[0061] WT-TTR: having an amino acid sequence shown as SEQ ID NO 1;

[0062] The SEQ ID NO 1 is: MGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKELEHHHHHH.

[0063] D38A-TTR: having an amino acid sequence shown as SEQ ID NO 2;

[0064] The SEQ ID NO 2 is: MGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAAADTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKELEHHHHHH.

[0065] 2. Sample extraction reagent: for extracting total protein in skin tissue, comprising:

[0066] Sample washing solution: 10 mM Tris-HCl (pH 7.4), 133 mM NaCl.

[0067] Sample lysis solution: 10 mM Tris-HCl, 133 mM NaCl, 2 mM 0.25% (w / v) collagenase A, protease inhibitors, which are protease inhibitor cocktail tablets without EDTA.

[0068] 3. Reaction reagents: reaction solution for RT-QuIC, including 50-200 mM sodium acetate, 50-200 mM KCl, 0.5-2 mM and 10-50 μM thioflavin T, the pH of the reaction reagent being 3.5-5.5.

[0069] 4. Negative control: phosphate buffer (PBS).

[0070] 5. Positive control: pre-prepared TTR amyloid fibril, prepared from TTR recombinant protein after shaking at 37°C for 7 days.

[0071] 6. Consumables: black 96-well plate (adapted for fluorescence detection), zirconium oxide grinding beads (diameter 3 mm / 1 mm), centrifuge tubes, etc.

[0072] II. Preparation and purification of substrate protein:

[0073] 1. Prokaryotic expression: construct pET-24a(+) expression plasmid containing WT-TTR or D38A-TTR gene (kanamycin resistance), transform into E. coli BL21(DE3) competent cells, after activation culture inoculate into large volume medium in proportion, culture at 37°C to mid-log phase OD600=0.7-0.8, add IPTG (final concentration 1 mM) and induce overnight at 28°C.

[0074] 2. Bacterial cell collection and lysis: collect bacterial cells by low-temperature centrifugation, resuspend in a buffer system containing phosphate buffer (PBS), NaCl, imidazole, add protease inhibitors (PMSF), break the bacterial cells by high-pressure homogenization, remove debris by high-speed low-temperature centrifugation and pre-filter through a 0.45 μm filter membrane.

[0075] 3. Nickel affinity chromatography: load the supernatant onto a Ni-NTA column, use a low-concentration imidazole buffer (10-100 mM) to remove non-specifically bound proteins, and then elute the bound proteins with a high-concentration imidazole (500 mM).

[0076] 4. Dialysis and molecular sieve purification: dialyze the eluted product to a low-salt buffer condition, and then separate the protein and remove aggregates by Superdex200 molecular sieve chromatography.

[0077] 5. Q-Sepharose anion exchange chromatography: load the protein in a low-salt buffer condition, and further improve the purity by eluting with a NaCl gradient (0-1 M).

[0078] 6. Final processing: Dialyze the product to storage buffer (50 mM). 100mM NaCl, pH 7.4), dispensed (1mg per tube), lyophilized (−20℃ or −80℃), stored at low temperature, avoid repeated freeze-thaw cycles.

[0079] like Figure 1 As shown, both WT-TTR and D38A-TTR purified products, after SDS-PAGE staining with Coomassie Brilliant Blue, exhibited a single, clear target band at 15 kDa with minimal background bands, indicating effective removal of contaminating proteins. These results demonstrate that target proteins with a purity ≥95% can be obtained through Ni-NTA affinity chromatography, molecular sieve chromatography, and Q-column anion exchange, providing a stable and reliable substrate for subsequent RT-QuIC detection. The substrate protein was reconstituted before use, and aggregates were removed using a 100 kDa ultrafiltration tube to adjust the concentration to 0.5 mg / mL.

[0080] III. Detection Methods

[0081] The method of using the kit includes the following steps:

[0082] 1. Sample processing steps

[0083] ① Obtain peripheral skin biopsy tissue from the subject (3mm in diameter, full-thickness skin, including epidermis, dermis and subcutaneous fat tissue).

[0084] ②Ice washing: Place the peripheral skin biopsy tissue on ice and wash 2–4 times with 1 mL TBS solution (10 mM Tris-HCl buffer).

[0085] ③ Weigh the tissue net weight: Weigh the empty weight of the grinding tube and the weight after adding the sample. The difference is the tissue net weight (mg).

[0086] ④ Lysis incubation: Add sample extraction reagent at a ratio of tissue weight (mg): lysis buffer volume (μL) = 1:10, and incubate at 37℃ and 350rpm for 4 hours to obtain lysed tissue.

[0087] 2. Tissue fragmentation steps

[0088] ① Add fragmentation medium: Add 6 zirconium oxide grinding beads (yttrium-stabilized zirconium oxide material) to the pyrolyzed tissue, 3 beads each of 3mm and 1mm in diameter.

[0089] ② Grinding process: Grind using a tissue homogenizer with the following parameters: oscillation frequency 65Hz, 45 seconds per oscillation, 10-second interval (operate on ice to prevent overheating), repeat 2 times; if the tissue is not completely homogenized, the number of grinding times can be increased appropriately.

[0090] ③Centrifugal collection of supernatant: After grinding, centrifuge the tube at 4°C, 500xg for 5 minutes, collect the supernatant for use.

[0091] 3. RT-QuIC reaction steps

[0092] ①Reaction system preparation: Add substrate protein and reaction reagents to the reaction plate, then add 2 μL of centrifugally collected supernatant (remove impurities by centrifugation at 4°C, 2000xg for 5 minutes, take the supernatant and dilute for use), total system 100 μL.

[0093] ②Negative control: PBS; Positive control: Pre-made TTR amyloid fiber body.

[0094] ③Cycling reaction:

[0095] Place the reaction plate at 35-40°C, and perform "500-800 rpm double-track shaking for 2-5 minutes → stand for 1-3 minutes" cycling, total reaction time 50-90 h.

[0096] ④Fluorescence detection: Detect the fluorescence signal (excitation wavelength 450 nm, emission wavelength 480 nm) every 6 cycles; set 4 replicate wells for each sample, and take the arithmetic mean as the final detection value.

[0097] ⑤Result interpretation: Draw the ROC curve according to the detection data, and take the RFU corresponding to the maximum Youden index as the threshold value. When the final fluorescence value of the sample is higher than the threshold value, it is determined to be positive for abnormal conformation TTR protein.

[0098] 4. Clinical verification results

[0099] This example includes 30 ATTR patients (5 wild type, 25 mutant) and 30 control samples (10 AL patients).

[0100] As shown in Figure 2 , when using WT-TTR substrate and D38A-TTR substrate for RT-QuIC detection, the ThT fluorescence of ATTR patient samples increases in an S-shaped manner over time, and the control group maintains the baseline. The horizontal line is the interpretation threshold value based on the maximum Youden index: WT threshold RFU = 96201, D38A threshold RFU = 95282. Most ATTR curves cross the corresponding threshold within the specified reaction time, while the control group curves do not cross the threshold.

[0101] As shown in Figure 3As shown, the maximum fluorescence intensity Imax of ATTR patients was significantly higher than that of the control group under two substrate conditions (****, p<0.0001). The threshold determined by the maximum Youden index was interpreted, with a sensitivity of 93.3% and a specificity of 100% under WT-TTR conditions; a sensitivity of 96.7% and a specificity of 90% under D38A-TTR conditions. The results show that both substrates have the accuracy required for clinical interpretation, and D38A-TTR is faster in reaction kinetics.

[0102] As shown in Figure 4 After the RT-QuIC product was stained with Congo red, under the conditions of WT-TTR and D38A-TTR, a large number of brick red deposits were observed in ATTR patient samples; under polarized light, typical apple green birefringence was observed, indicating the formation of amyloid fibers with beta-sheet structure. No characteristic coloration and birefringence signals were observed in the control samples, indicating negative results.

[0103] As shown in Figure 5 After the RT-QuIC product was stained with ThS, ATTR patient samples under the conditions of WT-TTR and D38A-TTR showed dense green fluorescent point / floc signals, indicating the presence of ThS-bound amyloid fibers; the background of the control samples was almost black, with no obvious fluorescent signals, and were determined to be negative. This result is consistent with the kinetic curve, indicating that the amplification product is a ThS-positive amyloid structure.

[0104] As shown in Figure 6 The RT-QuIC product of ATTR patient samples showed typical elongated filamentous, bundled or reticular fiber morphology under transmission electron microscopy, and continuous bundled fiber aggregation was observed under the conditions of WT-TTR and D38A-TTR; only sparse background particles were observed in the control sample field of view, and no shaped fibers were observed.

[0105] As shown in Figure 7 AFM showed that ATTR patient samples formed obvious fiber / reticular structures under the conditions of WT-TTR and D38A-TTR; the control sample surface was flat with a few background particles.

[0106] In summary, the sensitivity was 93.3% and the specificity was 100% when using WT-TTR substrate protein; the sensitivity was 96.7% and the specificity was 90% when using D38A-TTR substrate protein. The results of Congo red, ThT, TEM and AFM confirm each other, proving that the amplification product of ATTR patients is a fibrous amyloid structure, and the control sample is not.

[0107] The above results show that the kit of the present application can detect both genetic and wild-type ATTR patients, and can effectively distinguish ATTR from AL, and has high clinical application value.

[0108] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified, or some of the technical features can be replaced by equivalent features, by those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A kit for diagnosing transthyretin amyloidosis, characterized in that: The product includes a substrate protein, a sample extraction reagent for extracting total protein from skin tissue, a reaction reagent for RT-QuIC reaction, a negative control, and a positive control; wherein the substrate protein includes at least one of WT-TTR recombinant protein and D38A-TTR recombinant protein, and the amino acid sequence of the WT-TTR recombinant protein is shown in SEQ ID NO 1, and the amino acid sequence of the D38A-TTR recombinant protein is shown in SEQ ID NO 2.

2. The kit for diagnosing transthyretin amyloidosis according to claim 1, characterized in that: The sample extraction reagents include sample washing solution and sample lysis solution.

3. The kit for diagnosing transthyretin amyloidosis according to claim 2, characterized in that: Sample lysis buffer: 10 mM Tris-HCl, 133 mM NaCl, 2 mM 0.25% (w / v) collagenase A, protease inhibitor.

4. The kit for diagnosing transthyretin amyloidosis according to claim 3, characterized in that: The protease inhibitor is a mixture of protease inhibitor tablets that do not contain EDTA.

5. The kit for diagnosing transthyretin amyloidosis according to claim 1, characterized in that: The reaction reagents include 50-200 mM sodium acetate, 50-200 mM KCl, and 0.5-2 mM sodium acetate. And 10-50 μM thioflavone T, wherein the pH of the reaction reagent is 3.5-5.

5.

6. The kit for diagnosing transthyretin amyloidosis according to claim 1, characterized in that: The negative control was phosphate buffer.

7. The kit for diagnosing transthyretin amyloidosis according to claim 1, characterized in that: The positive control was a pre-prepared TTR amyloid fibrous material.

8. The kit for diagnosing transthyretin amyloidosis according to claim 1, characterized in that: The RT-QuIC reaction conditions described in the kit include: dual-track oscillation at a frequency of 500–800 rpm; the reaction is performed in cycles, with each cycle consisting of oscillation for 2–5 min followed by a resting period of 1–3 min; fluorescence readings are recorded after every 6 cycles; the incubation temperature is 35–40 °C, the excitation wavelength is 450 nm, and the emission wavelength is 480 nm; the above cyclic and reading procedures are continued until the total reaction time is 50–90 h.

9. A method for preparing and purifying the substrate protein according to any one of claims 1-8, characterized in that: Includes the following steps: S1. Prokaryotic expression: pET-24a(+) expression plasmid containing WT-TTR or D38A-TTR gene was constructed and transformed into Escherichia coli BL21(DE3) competent cells. After activation culture, the cells were seeded into a large volume of culture medium at a ratio and cultured at 37℃ until the mid-logarithmic growth phase OD600=0.7–0.

8. Then, 1 mM IPTG was added and induced overnight at 28℃. S2. Cell collection and lysis: Cells were collected by low-temperature centrifugation, resuspended in a buffer system containing phosphate buffer, NaCl, and imidazole, protease inhibitors were added, and cells were lysed by high-pressure homogenization. Fragments were removed by high-speed low-temperature centrifugation and pre-filtered through a 0.45μm filter membrane to obtain the supernatant. S3, Nickel affinity chromatography: Load the supernatant onto a Ni-NTA column, use a low concentration of imidazole buffer to remove non-specific binding proteins, and then elute the binding proteins with a high concentration of imidazole to obtain the elution product; S4. Dialysis and molecular sieve purification: The elution product was dialyzed to a low-salt buffer and then separated by Superdex 200 molecular sieve chromatography. S5, Q-Sepharose anion exchange chromatography: Proteins are loaded into a low-salt buffer and eluted by a NaCl gradient. S6. Final processing: After dialyzing the protein into storage buffer, aliquot and freeze-dry for storage.

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