Detection kit for amyloid transthyretin and application of detection kit

Abstract

The invention belongs to the technical field of protein detection, and provides an amyloid transthyretin detection kit and application thereof, the kit comprises the following reagents: normal recombinant TTR protein, a TTR-PFF standard substance, a fluorescent tracer and a buffer solution system; the TTR-PFF standard substance comprises a TTR substrate and an RT-QuIC reaction mixed solution, and the pH value of the RT-QuIC reaction mixed solution is 4.0 to 4.5; the buffer solution system comprises a PBS / N2 sample dilution buffer solution and a phosphate buffer solution. According to the detection kit, TTR-PFF is adopted as a reference, a subject sample is combined with normal recombinant human transthyretin, a fluorescent tracer is added, ATTR in a body can convert normal recombinant monomer TTR protein into amyloid fiber, and existence and activity of the ATTR are detected by detecting a fluorescence value of the ATTR. The detection has ultrahigh sensitivity and specificity, minimally invasive or non-invasive sampling is operated, the operation is simple and convenient, a key technical foundation is laid for developing a simple, convenient, efficient and clinical ATTR amyloidosis detection platform, especially an early detection platform, and the kit has a wide market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of protein detection technology, specifically relating to a detection kit for amyloid-to-thyroxine protein and its uses. Background Technology

[0002] ATTR amyloidosis is a systemic, progressive, and fatal disease caused by the misfolding of TTR. Misfolded TTR forms insoluble amyloid fibers that deposit in multiple organs, including the heart, nervous system, and gastrointestinal tract, leading to progressive multiple organ dysfunction. Based on protein origin, ATTR is divided into two main subtypes: hereditary (ATTRv) and wild-type (ATTRwt). ATTRv is caused by pathogenic mutations in the TTR gene and is inherited in an autosomal dominant pattern; more than 140 mutations have been identified. ATTRwt is associated with aging and is caused by the dissociation and deposition of wild-type TTR protein. Currently, existing cases are mainly identified through genetic testing or autopsy, with the most common mutation types being V30M and A97S. However, in the detection of confirmed cases, due to the high heterogeneity of clinical manifestations and insufficient clinical awareness, patients are often misdiagnosed with other common diseases.

[0003] The clinical manifestations of ATTR are highly heterogeneous, mainly depending on the dominant site of amyloid deposition: 1) Cardiac phenotype (ATTR-CM): characterized by progressive heart failure, atrial fibrillation, and conduction system disease, with echocardiographic features of ventricular wall thickening (≥12 mm) without ventricular cavity enlargement; 2) Neurological phenotype (ATTR-PN): characterized by sensorimotor peripheral neuropathy and autonomic neuropathy such as orthostatic hypotension and gastrointestinal dysfunction; 3) Mixed phenotype: with simultaneous involvement of both the cardiac and nervous systems.

[0004] In recent years, advancements in imaging technology have significantly improved the non-invasive detection capabilities of ATTR: 1) Radionuclide imaging: 99mTc-PYP scanning has become a core tool for ATTR-CM detection, with sensitivity and specificity both exceeding 90%, effectively differentiating ATTR from light chain amyloidosis (AL); 2) New cardiac MRI technologies: Extracellular volume (ECV) quantification technology can detect diffuse myocardial interstitial lesions at an early stage, identifying abnormalities even when amyloid protein load is <20%; 3) Artificial intelligence-assisted ultrasound: Deep learning-based analysis systems can automatically identify subtle changes in myocardial texture, improving the early lesion detection rate. The detection pathway follows the principle of "from non-invasive to invasive": PYP radionuclide scanning is the first choice for clinically suspected patients; when results are uncertain or contradictory, fat, salivary gland, or myocardial biopsy should be considered. Genetic testing should be a routine examination for all confirmed patients to clarify ATTRv or ATTRwt subtypes.

[0005] However, current detection methods still have limitations: imaging examinations such as PYP and MRI have high technical requirements, are expensive, involve the use of isotopes, lack standardization, and have limited value for early detection and prognostic prediction; endocardial biopsy is highly invasive and has low patient acceptance; genetic screening has low detection value for wild-type ATTRwt; and fat or salivary gland biopsy techniques used for ATTR detection have poor sensitivity and specificity.

[0006] Based on this, a detection kit for amyloid-transthyretin and its uses were studied and discussed. Summary of the Invention

[0007] Based on the aforementioned deficiencies in existing technologies, the present invention aims to provide a detection kit for amyloid-transthyretin (ATTR) and its applications. This technical solution addresses the problems of low sensitivity, limited specificity, relatively high invasiveness, and complex operation of existing ATTR amyloidosis detection technologies. The present application employs an ATTR detection kit, which prepares pre-formed transthyretin fibers (TTR-PFF) in vitro as a TTR-PFF standard. Utilizing the pathogenic prion-like seed amplification activity of misfolded TTR in vitro, it can transform normal recombinant TTR monomers to form pathological folded conformations and self-amplify to form TTR-PFF polymers. The fluorescence value of the TTR-PFF standard is detected using a fluorescent tracer. Subject samples are mixed with normal recombinant TTR protein, a fluorescent tracer is added, and the fluorescence value of the subject sample is compared with that of the TTR-PFF standard to detect whether the subject sample contains amyloid-transthyretin (ATTR). This detection method can detect ATTR in trace amounts in its early stages, serving as a preventative measure for related diseases. Furthermore, the detection method of the kit significantly improves the sensitivity and specificity of the test, providing key technical support for the clinical diagnosis and detection of ATTR amyloidosis. It also solves the technical problems of low sensitivity and low specificity in existing detection techniques.

[0008] This invention is achieved through the following technical solution: A kit for detecting amyloid-to-thyroxine (TTR) protein, the kit being used to detect the presence of TTR protein in a subject sample by fluorescence value, the kit comprising the following reagents: normal recombinant TTR protein, TTR-PFF standard, fluorescent tracer, and buffer system; TTR-PFF standard, comprising TTR substrate and RT-QuIC reaction mixture, wherein the pH of the RT-QuIC reaction mixture is 4.0-4.5; The buffer system includes PBS / N2 sample dilution buffer and phosphate buffer.

[0009] The detection method of the kit in this technical solution is mainly based on the fact that ATTR amyloid protein has seeding activity similar to pathogenic prion proteins. It can continuously recruit and transform its normal isoform into misfolded protein in vitro. Specifically, misfolded TTR has pathogenic prion protein-like seed amplification activity, which can transform normal TTR protein to form a pathological folded conformation, and then form a larger polymer through self-amplification. This mechanism is discovered for the first time and has been applied in a crucial way.

[0010] In vitro prepared TTR-PFF, used as a standard, exhibits potent seed-seeding activity, specifically converting normal TTR protein into amyloid fibrils. These amyloid fibrils can be bound to and detected by fluorescent tracers. Based on this, a method for detecting amyloid-transferrogens (ATTRs) was established. This method can efficiently, minimally invasively, or even non-invasively detect pathological TTRs in minute amounts of peripheral tissue or body fluid samples from patients, providing a new approach for the early detection of ATTR amyloidosis.

[0011] The reaction mixture in which normal recombinant monomer TTR protein is added to the transthyretin protein pre-fiber TTR-PFF preparation process is a mixture containing 50 mM sodium acetate, 1 mM EDTA, 100 mM KCl, and 10 μM thiosulfate T (ThT), with a pH of 4.0-4.5. The pH is preferably 4.0 or 4.5, and the pH is adjusted with 2M HCl.

[0012] Furthermore, the step of using the test kit to detect the presence of amyloid-transthyretin in the subject sample is as follows: Step 1: Prepare TTR-PFF standard by adding the filtered TTR substrate to the RT-QuIC reaction mixture; Step 2: Prepare subject samples. After pretreatment, the peripheral tissue or body fluid samples of the subjects are mixed with the normal recombinant TTR protein prepared in vitro, and a fluorescent tracer is added. The fluorescent tracer binds to the ATTR-PFF polymer converted from the normal recombinant TTR protein and emits fluorescence. Step 3: Detect the fluorescence values ​​of TTR-PFF standard and subject samples. Add the prepared TTR-PFF standard and subject samples to 96-well plates respectively, and monitor them in real time using a fluorescence detector. The fluorescence measurement settings are: excitation wavelength of 450 nm, emission wavelength of 480 nm (bottom readout), and gain value of 1600. Compare the obtained fluorescence values ​​of TTR-PFF standard and subject samples to detect whether the subject samples contain amyloid-transferrothyroxine (ATTR).

[0013] TTR monomer was used as a negative control.

[0014] Subject samples are derived from the subject's peripheral tissues and / or body fluids. The misfolded TTR in these samples possesses pathogenic prion-like seed amplification activity, capable of transforming normal TTR monomers into pathological folded conformations and self-amplifying to form ATTR-PFF polymers. These polymers can be bound to and detected by a fluorescent tracer; the detection result is the fluorescence value of the subject sample. In vitro prepared transthyretin pre-fiber TTR-PFF is used as a TTR-PFF standard. The fluorescence value obtained from the TTR-PFF standard is used as a reference or standard and compared with the fluorescence value of the subject sample to determine whether the subject sample contains amyloid-transthyretin (ATTR). If the difference is within the expected range, such as when the subject sample fluorescence value is consistent with, the same as, or similar to that of the TTR-PFF standard, then the subject sample contains ATTR. If the fluorescence value of the subject sample is outside the expected range, then the subject sample does not contain ATTR. This detection method is beneficial for detecting ATTR in subject samples, significantly improves detection accuracy, has high detection sensitivity, and can play a role in the early prevention of amyloid-transthyretin ATTR.

[0015] Pretreatment refers to the pretreatment of tissue samples before ATTR detection of amyloidosis in the subject's tissue samples, such as homogenization, centrifugation, and filtration. Specifically, for skin biopsy samples, residual blood in frozen biopsy skin specimens is washed with PBS and then minced.

[0016] Further, in step two, subject samples are prepared. 2 μL of the pretreated subject sample is added to PBS / N2 sample dilution buffer, diluted, centrifuged, and the supernatant is added to a 96-well plate for microplate reading.

[0017] Further, in step one, the preparation method of the TTR-PFF standard is as follows: transthyretin pre-fiber TTR-PFF is prepared in vitro, the concentration of the TTR-PFF standard is 0.1 mg / mL, the concentration of the TTR substrate is 0.8 mg / mL, and the TTR substrate is dissolved in pH 7.4, 10 mM phosphate buffer, including 100 mM KCl and 1 mM EDTA.

[0018] Furthermore, the preparation steps of the TTR-PFF standard are as follows: after filtering 0.8 mg / mL of TTR substrate, it is added to the RT-QuIC reaction mixture. During the preparation of the RT-QuIC reaction mixture, 10 μM thiosulfate T is added, and the pH value is adjusted with 2M HCl.

[0019] Furthermore, in step three, the fluorescence detector is an ELISA reader, and the real-time detection method of the ELISA reader is as follows: temperature is 50℃, dual-track oscillation at 700 rpm for 1 min, followed by a 1 min rest period, and the fluorescence value is detected every 45 min.

[0020] Furthermore, the peripheral tissue sample is any one of skin biopsy sample, muscle biopsy specimen, fat fine needle aspirate, or nasal mucosa, and the body fluid sample is any one of serum, cerebrospinal fluid, saliva, or urine.

[0021] Furthermore, the peripheral tissue sample is a muscle biopsy sample or a skin biopsy sample. The pretreatment method for the subject's sample is as follows: wash the frozen biopsy specimen with PBS to remove residual blood, cut it into small pieces, put the cut sample tissue into a test tube, weigh it, add lysis buffer, incubate in a shaker, then transfer it to a BeatsBeater and shake it, place it on dry ice for 5 minutes, repeat the shaking and freezing cycle twice, place it at -80°C overnight, thaw it on ice the next day, centrifuge it, and take the supernatant and store it at -80°C for later use.

[0022] Furthermore, the phosphate buffer is a pH 7.4, 10 mM phosphate buffer.

[0023] Another objective of this application is to provide a detection kit for amyloid-transthyretin (ATTR) in vitro for detecting ATTR in a subject sample, and the kit is used for detecting, preventing, or treating amyloid-transthyretin-related diseases.

[0024] The detection kit described in this technical solution is used to co-incubate pretreated peripheral tissue samples or body fluid samples with normal recombinant TTR protein, and enhance the results with a fluorescent tracer. The presence and activity of ATTR in the samples are then detected using an ELISA reader. The transthyretin pre-formed fibrils (TTR-PFF) formed in the incubation system are used as a TTR-PFF standard.

[0025] Compared with the prior art, the beneficial effects of the present invention are: This application's technical solution is based on the first discovery and application of the important mechanism by which ATTR amyloid protein exhibits seeding activity similar to pathogenic prion proteins, namely, its ability to continuously recruit and transform its normal isoform into misfolded proteins in vitro. Utilizing the potent seeding activity of pathological ATTR in the test specimen, the addition of normal recombinant human TTR protein can specifically convert it into amyloid fibrils, thereby achieving efficient in vitro amplification and detection of pathological ATTR seeding activity. The detection kit and method described in this application have the following significant advantages: 1) Ultra-high sensitivity and specificity: The seeding activity of TTR-PFFs amplifies the signal, suggesting that this method can significantly improve the detection ability of trace pathological ATTR in the test specimens and can effectively distinguish ATTR from other types of amyloidosis.

[0026] 2) Minimally invasive / non-invasive sampling: Directly applicable to peripheral tissues including but not limited to skin, muscle, nasal mucosa, fat, etc., or body fluids including but not limited to serum, plasma, cerebrospinal fluid, saliva, and urine, avoiding invasive sampling and reducing patient pain and risks.

[0027] 3) Great potential for clinical application: It lays a key technological foundation for the development of a simple, efficient and clinically applicable ATTR amyloidosis detection platform, especially for early detection, which is expected to significantly shorten detection delay and improve patient prognosis.

[0028] 4) Relatively simple to operate: Compared with some existing imaging techniques or biopsy pathology, the detection method of this kit has relatively low requirements for equipment dependence and operational complexity. Attached Figure Description

[0029] Figure 1 A schematic diagram illustrating the TTR synthesis, the pathogenesis of ATTR amyloidosis, and the principle of in vitro amplification detection. Figure 2 To provide real-time in vitro magnified monitoring of recombinant TTR-PFF formation; Figure 3 The image shows the seeding activity of recombinant TTR-PFFs added to serum for real-time in vitro magnification detection. A is the original RT-QuIC image, and B is the dynamic seeding activity image of different concentrations of TTR-PFFs detected by GraphPad in vitro magnification technology. Figure 4 The image shows the seeding activity of recombinant TTR-PFFs added to skin homogenate for real-time in vitro magnification detection. A is the original RT-QuIC image, and B is the dynamic seeding activity image of different concentrations of TTR-PFFs detected by GraphPad in vitro magnification technology. Figure 5 This image shows the real-time in vitro magnified detection of ATTR seeding activity in patient skin homogenate. A is the original RT-QuIC image, and B is the dynamic seeding activity image of different concentrations of ATTR detected by GraphPad in vitro magnification. Figure 6 RT-QuIC raw atlas for real-time in vitro magnified detection of ATTR seeding activity in patient muscle homogenate; Figure 7This image shows a real-time, in vitro magnified plot of ATTR seeding activity in patient muscle homogenate. Figure A is a violin plot of ATTR seeding activity, and Figure B is a ROC statistical analysis plot. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0031] Transthyretin amyloidosis (ATTR) is a progressive disease that can affect multiple organ systems, including: 1) the nervous system: downregulation of TTR expression promotes amyloid β (Aβ) deposition in Alzheimer's disease (AD), and TTR fiber infiltration of the perineurium can lead to sensorimotor dysfunction and autonomic failure; 2) the cardiovascular system: ATTR-induced myocardial amyloidosis causes ventricular wall thickening and diastolic dysfunction, and amyloid infiltration can lead to conduction block and arrhythmias; 3) the eye: manifesting as vitreous opacities and glaucoma; and 4) the kidneys: leading to nephrotic syndrome, characterized by proteinuria and progressive renal failure. A common molecular pathological feature of these diseases is the abnormal accumulation of misfolded TTR amyloid deposits in different organs and tissues. Because the symptoms of ATTR are often similar to those of other diseases, its identification and detection are challenging. However, untreated ATTR patients have a life expectancy of only 3 to 15 years after symptom onset, making early identification, detection, and treatment crucial.

[0032] Currently, the detection of ATTR mainly relies on: 1) endocardial biopsy (gold standard: positive Congo red staining + positive TTR immunohistochemistry); 2) imaging examinations (such as late-contrast cardiac MRI showing subendocardial enhancement; non-invasive bone scintigraphy such as 99mTc-DPD / PYP for ATTR cardiomyopathy); 3) TTR gene mutation screening such as Val122Ile, Thr60Ala; and 4) biomarker detection, such as serum TTR levels <20 mg / dL indicating poor prognosis. However, the above detection methods still have certain limitations: cardiac biopsy is highly invasive; imaging examinations are expensive, involve isotopes, lack technical standardization, and lack early detection and prognostic value; gene screening has no detection value for sporadic cases; serum TTR level detection is neither suitable for early detection nor specific. It is worth noting that there are currently therapeutic drugs for ATTR amyloidosis, and early detection and intervention can significantly improve patient prognosis. Therefore, developing an early ATTR detection technology that is minimally invasive, low-cost, highly sensitive, and highly specific is of great clinical significance.

[0033] This application is based on a key discovery: misfolded ATTR in patients possesses pathogenic prion-like seed amplification activity, capable of transforming normal recombinant human TTR protein into a pathological folded conformation in vitro, and then self-amplifying to form larger polymers. These polymers can be bound to and detected by fluorescent tracers such as thioflavin-T (ThT). Figure 1 As shown.

[0034] TTR Synthesis: TTR is mainly synthesized by the liver and choroid plexus. ATTR Formation and Multi-Organ Damage: TTR tetramers dissociate, and monomers undergo misfolding. These misfolded TTR monomers further aggregate into insoluble amyloid fibrils (ATTR). ATTR abnormally accumulates and deposits in multiple organs such as the heart, nerves, eyes, and digestive tract, leading to systemic, progressive, and fatal ATTR amyloidosis. Based on the principle that misfolded TTR monomers or oligomers possess pathogenic prion-like seed amplification activity, this detection method collects skin and muscle or fat biopsy samples (minimally invasive) or various body fluids including serum, plasma, cerebrospinal fluid, saliva, or urine from patients. In vitro, the ATTR seeds contained in these samples are used to transform normal recombinant TTR protein to amplify its signal. This in vitro protein misfolding cyclic amplification technology can detect trace amounts of pathological ATTR seeds in samples with ultrasensitive sensitivity, thereby enabling the establishment of a novel ATTR detection method that is minimally invasive (e.g., for skin, muscle, fat) or non-invasive (e.g., for body fluids), low in cost, highly sensitive and specific, providing a window of opportunity for early intervention.

[0035] Example 1: This embodiment describes a detection method for preparing TTR-PFFs. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the field, and reagents not specifically described are all common commercially available reagents.

[0036] Experimental equipment: The microplate reader was manufactured by BMG LABTECH GmbH in Hamburg, Germany, model OMEGA series.

[0037] The experimental method is as follows: 1) Prepare TTR substrate: 0.8 mg / mL, dissolved in pH 7.4, 10 mM phosphate buffer containing 100 mM KCl and 1 mM EDTA; 2) Prepare RT-QuIC reaction mixture: containing 50 mM sodium acetate, 1 mM EDTA, 100 mM KCl, 10 μM thiosulfate T (ThT), with or without 10% AS (ammonium sulfate), and pH values ​​of 4.0, 4.5, 5.0 or 7.4 respectively.

[0038] 3) Prepare mixed solutions without 10% ammonium sulfate (AS): According to the above formula, prepare 2000 μL of each of the four different pH values ​​(pH 4.0, pH 4.5, pH 5.0 and pH 7.4) mixed solutions (without 10% AS), and adjust the pH value using 2M HCl; 4) Prepare a mixed solution containing 10% ammonium sulfate (AS): According to the above formula, prepare 2,000 μL of each of the four different pH values ​​(pH 4.0, pH 4.5, pH 5.0 and pH 7.4) mixed solutions (containing 10% AS), and adjust the pH value using 2M HCl; 5) Thaw and filter the TTR stock solution: Thaw the TTR stock solution (0.8 mg / mL) and filter; 6) Add TTR to the mixture: Add the filtered TTR to each mixture to achieve a final concentration of 0.1 mg / mL; 7) Add samples to 96-well plates: Add each mixture containing TTR substrate to the 96-well plates as follows, as shown in Table 1 below, experimental layout of 96-well plates with different pH values ​​and whether or not they contain AS mixtures; Table 1: Experimental Layout of 96-well Plate 8) Real-time monitoring with an ELISA reader: 50℃, 700 rpm dual-track oscillation for 1 min, followed by 1 min of rest, with fluorescence values ​​measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm, bottom readout, gain value set to 1,600.

[0039] Experimental results are as follows Figure 2 As shown: In the 96-well plate, columns 1-3 are samples with pH 4.0, columns 4-6 are samples with pH 4.5, columns 7-9 are samples with pH 5.0, and columns 10-12 are samples with pH 7.2. The AD row contains samples with added ammonium sulfate (AS), while the EH row contains samples without added ammonium sulfate (AS).

[0040] Figure 2 The results showed that recombinant human TTR only showed an increase in ThT fluorescence value at pH 4.0-4.5, forming polymers. The pores without AS, E1-H1, E2-H2, G5, H5, H6, and the pores with AS, C1, A2-D2, B3 and D3, showed a weaker increase. Example 2: This embodiment describes an in vitro amplified detection method for TTR-PFFs added to serum. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the art, and reagents not specifically described are common commercially available reagents. The experimental method is as follows: 1) Add the recombinant TTR-PFFs prepared in vitro to normal serum and mix well. Then, extract 2 μL of serum containing TTR-PFFs and add sample dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)]. Analyze in a 10-fold series, starting from 10... -1 Up to 10 -12 dilution.

[0041] 2) Add 5 ml of H2O, sodium acetate, EDTA, KCl, and TTR monomer to a 15 ml centrifuge tube [labeled "mix"]. Gently rotate the centrifuge tube to mix 4-5 times. Adjust the pH to acidic pH 5.0 using 2M HCl, and check the pH value using a precision pH meter. Finally, add the remaining H2O and ThT (thiosulfate T).

[0042] 3) Add 2 μL of the above different dilutions to 98 μL of PBS / N2 sample dilution buffer, centrifuge at 5,000 g, 4℃ for 5 minutes, and then transfer the supernatant to a 96-well plate.

[0043] 4) Microplate reader detection: 50℃, 700 rpm dual-track oscillation for 1 min, followed by a 1 min rest period. Fluorescence values ​​are measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm (bottom reading), gain value set to 1,600.

[0044] Experimental results are as follows Figure 3 As shown. Figure 3 To enable real-time in vitro amplification detection of the seeding activity of recombinant TTR-PFFs added to serum.

[0045] in Figure 3 A in the image is the original RT-QuIC spectrum. To determine the detection sensitivity of this technique, recombinant TTR monomers and in vitro-formed recombinant ATTR-PFFs were analyzed from 10... -1 Dilute 10 times in series to 10 -12 Then, real-time oscillation-induced amplification (RT-QuIC) detection was performed. No positive reaction was observed at any concentration of TTR monomer. However, in vitro amplification experiments using TTR-PFFs as seeds showed a positive reaction related to the seed dose of TTR-PFFs. 10 -1 Up to 10 -3The strongest positive reactions were observed in four wells each of the diluted 1E to 1H (0.8 μg / mL) and 3E to 3H (8 ng / mL). 10 -4 Three-quarters of the wells in the diluted 3E to 3H wells (0.8 ng / mL) showed a positive reaction. -5 10 -6 and 10 -7 Positive reactions were observed in 1 / 4 wells of diluted 5E (80 pg / mL), 6F (8 pg / mL), and 7E (0.8 pg / mL).

[0046] These results suggest that our in vitro amplification technique can detect TTR-PFFs with a sensitivity of up to 0.8 pg / mL.

[0047] Figure 3 In Figure B, GraphPad was used to compare the dynamic seeding activity of different concentrations of TTR-PFFs. TTR monomer served as a negative control. Positive reactions were only observed at concentrations of 10. -1 Up to 10 -7 Diluted TTR-PFFs.

[0048] Example 3: This embodiment demonstrates the in vitro amplification of TTR-PFFs added to normal skin homogenate. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the art, and reagents not specifically described are common commercially available reagents. The experimental method is as follows: 1) The recombinant TTR-PFFs prepared in vitro were added to 10% normal skin homogenate and mixed well. Then, 2 μL of the skin homogenate containing TTR-PFFs was drawn and added to the sample dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)], in a 10-fold series, starting from 10... -1 Up to 10 -12 Dilution. As a control, the TTR monomer was also diluted in the same way.

[0049] 2) Add 5 ml of H2O, sodium acetate, EDTA, KCl, and TTR monomer to a 15 ml centrifuge tube [labeled "mix"]. Gently rotate the centrifuge tube to mix 4-5 times. Adjust the pH to acidic pH 5.0 using 2M HCl, and check the pH value using a precision pH meter. Finally, add the remaining H2O and ThT.

[0050] 3) Add 2 μL of the above different dilutions to 98 μL of PBS / N2 sample dilution buffer, centrifuge at 5,000 g, 4℃ for 5 minutes, and then transfer the supernatant to a 96-well plate.

[0051] 4) Microplate reader detection: 50℃, 700 rpm dual-track oscillation for 1 min, followed by a 1 min rest period. Fluorescence values ​​are measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm (bottom reading), gain value set to 1,600.

[0052] Experimental results are as follows Figure 4 As shown. Figure 4 To enable real-time in vitro magnification detection of the seeding activity of recombinant TTR-PFFs added to skin homogenate. Figure 4 Image A shows the original RT-QuIC spectrum. To determine the sensitivity of this technique in detecting ATTR in skin, recombinant TTR monomers and in vitro-formed recombinant TTR-PFFs were first diluted in skin homogenate, and then further diluted from 10... -1 Dilute 10 times in series to 10 -12 Then, RT-QuIC detection was performed. No positive reaction was observed at any concentration of TTR monomer. However, in vitro scale-up experiments using TTR-PFFs as seeds showed a positive reaction that was dose-dependent on the TTR-PFF seed concentration. 10 -1 Up to 10 -3 The strongest positive reactions were observed in four wells each of the diluted 1E to 1H (0.8 μg / mL) and 3E to 3H (8 ng / mL). 10 -4 Three-quarters of the wells in the diluted 3E to 3H wells (0.8 ng / mL) showed a positive reaction. -5 and 10 -6 Diluted 5E / 5G (80 pg / mL) and 6E / 6G (8 pg / mL) also showed positive reactions. Although 10 -7 No positive reaction was observed at dilution, 10 -8 One-quarter of the wells (8H) (80 fg / mL) showed a positive reaction at dilution. This result suggests that our in vitro amplification technique has a sensitivity of at least approximately 8 pg / mL for detecting TTR-PFFs in the skin. Figure 4 Figure B shows the dynamic seeding activity of different concentrations of TTR-PFFs detected by GraphPad in vitro amplification technology. TTR monomers served as a negative control. Positive reactions were clearly observed at 10. -1 Up to 10 -6 Diluted TTR-PFFs.

[0053] Example 4: This embodiment describes a method for in vitro magnified detection of ATTR in skin homogenates. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the art, and reagents not specifically described are common commercially available reagents.

[0054] The experimental method is as follows: 1) Preparation of skin biopsy specimens: Wash the frozen skin biopsy specimens with PBS to remove residual blood, and then mince them. Adjust a clean 1.5 mL EP tube to zero on an analytical balance, place the minced skin tissue into the tube, and weigh it. Add skin lysis buffer, incubate at 37°C for 4 hours at 300 rpm, then transfer to a BeatsBeater and shake for 1 minute. Place on dry ice for 5 minutes, repeating the shaking and freezing cycle twice. Incubate overnight at -80°C. Thaw on ice the next day, centrifuge at 500 g for 5 min at 4°C, and store the supernatant at -80°C for later use.

[0055] 2) Take 2 μL of the skin homogenate prepared above, add specimen dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)], and perform serial dilution in 10-fold series, starting from 10... -1 Up to 10 -4 dilution.

[0056] 3) In another group, 5 μL of the skin homogenate prepared above was drawn and added to the sample dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)], and serialized in 10-fold series, starting from 10... -1 Up to 10 -4 Dilution. As a control, the TTR monomer was also diluted in the same way.

[0057] 4) Add 5 mL of H₂O, sodium acetate, EDTA, KCl, and TTR monomer to a 15 mL centrifuge tube [labeled "mix"]. Gently rotate the tube to mix 4-5 times. Adjust the pH to approximately 5.0 with 2M HCl (about 70 μL). Due to the small sample volume, use pH paper to check the pH. Finally, add the remaining H₂O and ThT (thiosulfate T).

[0058] 5) Add 2 μL or 5 μL of the above different dilutions to 98 μL or 95 μL of PBS / N2 sample dilution buffer, centrifuge at 5,000 g, 4°C for 5 minutes, and then transfer the supernatant to a 96-well plate.

[0059] 6) Microplate reader detection: 50℃, 700 rpm dual-track oscillation for 1 min, followed by a 1 min rest period. Fluorescence values ​​are measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm (bottom reading), gain value set to 1,600.

[0060] Test results as follows Figure 5 As shown, Figure 5 To enable real-time in vitro magnification detection of ATTR seeding activity in patient skin homogenates.Figure 5 Image A is the original RT-QuIC pattern. The top four rows of the 96-well microplate (AD): columns 1-3 show the TTR monomer at 10... -3 Up to 10 -5 Diluted, columns 4-7 are 2μL of normal control skin at 10 -1 Up to 10 -4 Diluted, columns 8-12 were 5 μL of normal control skin at 10 -1 Up to 10 -5 dilution.

[0061] The bottom four rows of the 96-well EH microplate: Columns 1-3 are TTR and PFF respectively, at 10... -3 Up to 10 -5 Dilute, columns 4-7 are 2 μL ATTR patient skin at 10 -1 Up to 10 -4 Dilute, columns 8-12, 5 μL ATTR patient skin at 10 -1 Up to 10 -5 dilution.

[0062] Figure 5 Figure B shows the dynamic seeding activity of ATTR in patient skin at different concentrations detected by GraphPad in vitro magnification. TTR monomers served as a negative control. Positive reactions were clearly observed at 10. -3 Up to 10 -4 Diluted TTR-PFFs. 2 μL of ATTR patient skin diluted 10 -1 Up to 10 -4 A positive reaction was observed, but no positive reaction was observed in 5 μL of the sample or in the skin of the control patient.

[0063] Example 5: This embodiment describes a method for in vitro amplification and detection of pathological TTR polymers in muscle homogenates. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the art, and reagents not specifically described are common commercially available reagents.

[0064] The experimental method is as follows: 1) Preparation of muscle biopsy specimens: Wash the frozen muscle biopsy specimens with PBS to remove residual blood, and mince them. Zero-1.5 mL of EP tube on an analytical balance, place the minced muscle tissue into the tube, and weigh. Add lysis buffer, incubate at 37°C for 4 hours at 300 rpm, then transfer to a BeatsBeater and shake for 1 minute. Place on dry ice for 5 minutes, repeating the shaking and freezing cycle twice. Incubate overnight at -80°C. Thaw on ice the next day, centrifuge at 500 g for 5 min at 4°C, and store the supernatant at -80°C for later use.

[0065] 2) Take 2 μL of the muscle homogenate prepared above, add specimen dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)], and perform serial dilution in 10-fold series, starting from 10... -1 Up to 10 -4 dilution.

[0066] 3) Add 5 mL of H₂O, sodium acetate, EDTA, KCl, and TTR monomer to a 15 mL centrifuge tube [labeled "mix"]. Gently rotate the tube to mix 4-5 times. Adjust the pH to approximately 5.0 with 2M HCl (approximately 70 μL). Due to the small sample volume, use pH paper to check the pH value. Finally, add the remaining H₂O and ThT (thiosulfate T).

[0067] 4) Add 2 μL of the above different dilutions to 98 μL of PBS / N2 sample dilution buffer, centrifuge at 5,000 g, 4℃ for 5 minutes, and then transfer the supernatant to a 96-well plate.

[0068] 5) Microplate reader (BMG, OMG, Hamburg, Germany) detection: 50℃, 700 rpm dual-track oscillation for 1 min, followed by a 1 min rest period, with fluorescence values ​​measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm, bottom readout, gain value set to 1,600.

[0069] Experimental results are as follows Figure 6 As shown. Figure 6 To enable real-time in vitro magnification detection of ATTR seeding activity in patient muscle homogenates, i.e., the raw RT-QuIC spectrum.

[0070] Figure 6 The top 4 rows of the 96-well microplate AD: columns 1-3 are TTR monomers in 10... -1 Up to 10 -3 Diluted, columns 4-6 each contain 2 μL of normal control #1 muscle homogenate diluted at 10... -1 Up to 10 -3 Diluted, columns 7-9 each contain 2 μL of normal control #2 muscle homogenate diluted at 10... -1 Up to 10 -3 Dilution. Columns 10-12 are 2 μL of normal control #3 muscle homogenate diluted at 10... -1 Up to 10 -3 dilution.

[0071] The bottom four rows of the 96-well microplate EH: columns 1-3 are TTR and PFF respectively, with 10 -1 Up to 10 -3Dilute, columns 4-7 are 2 μL ATTR muscle homogenate at 10 -1 Up to 10 -4 Dilute, columns 8-12 were each 5 μL ATTR muscle homogenate at 10 -1 Up to 10 -5 Dilution. Columns 4-6 are 2 μL of ATTR patient #1 muscle homogenate diluted at 10... -1 Up to 10 -3 Dilute, columns 7-9 are 2 μL ATTR patient #2 muscle homogenate at 10 -1 Up to 10 -3 Dilution. Columns 10-12 are 2 μL of ATTR patient #3 muscle homogenate diluted at 10... -1 Up to 10 -3 Dilution. The above shows that all TTR monomers and muscle homogenates from control patients were negative, while all ATTR polymers and muscle homogenates from ATTR patients were positive, with significantly increased ThT fluorescence values, indicating positive seeding activity of ATTR.

[0072] Example 6: This embodiment describes a method for in vitro amplification and detection of pathological TTR polymers in muscle homogenates. The experiment can be performed as follows, where parts not specifically described refer to conventional procedures in the art, and reagents not specifically described are common commercially available reagents.

[0073] The experimental method is as follows: 1) Preparation of muscle biopsy specimens: Wash the frozen muscle biopsy specimens with PBS to remove residual blood, and mince them. Zero-1.5 mL of EP tube on an analytical balance, place the minced muscle tissue into the tube, and weigh it. Add lysis buffer, incubate at 37°C for 4 hours at 300 rpm, then transfer to a BeatsBeater and shake for 1 minute, place on dry ice for 5 minutes, and repeat the shaking and freezing cycle twice. Incubate overnight at -80°C. Thaw on ice the next day, centrifuge at 500 g for 5 min at 4°C, and store the supernatant at -80°C for later use.

[0074] 2) Take 2 μL of the muscle homogenate prepared above, add specimen dilution solution [1 x PBS, 1 x N2 additive (Gibco#17502048)], and perform serial dilution in 10-fold series, starting from 10... -1 Up to 10 -4 dilution.

[0075] 3) Add 5 mL of H₂O, sodium acetate, EDTA, KCl, and TTR monomer to a 15 mL centrifuge tube [labeled "mix"]. Gently rotate the tube to mix 4-5 times. Adjust the pH to 5.0 with 2M HCl (approximately 70 μL). Due to the small sample volume, use pH paper to check the pH value. Finally, add the remaining H₂O and ThT (thiosulfate T).

[0076] 4) Add 2 μL of the above different dilutions to 98 μL of PBS / N2 sample dilution buffer, centrifuge at 5,000 g, 4°C for 5 minutes, and then transfer the supernatant to a 96-well plate.

[0077] 5) Microplate reader (BMG, OMG, Hamburg, Germany) detection: 50℃, 700 rpm dual-track oscillation for 1 min, followed by a 1 min rest period, with fluorescence values ​​measured every 45 min. Fluorescence measurement settings: excitation wavelength 450 nm, emission wavelength 480 nm, bottom readout, gain value set to 1,600.

[0078] Experimental results are as follows Figure 7 As shown. Figure 7 In the middle A, there are violin plots of in vitro magnified fluorescence values ​​of muscle tissue biopsies from non-ATTR controls (n = 16) and ATTR patients (n = 30). Figure 7 Figure B shows the receiver operating characteristic (ROC) curve. The area under the curve is greater than 0.9, indicating that this method can reliably distinguish between non-ATTR and ATTR. Moreover, the sensitivity and specificity are both 100% when the threshold is greater than 1900 fluorescence units.

[0079] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A detection kit for amyloid-to-thyroxine protein, characterized in that: The kit is used to detect the presence of amyloid-transthyretin protein in subject samples by fluorescence value. The kit includes the following reagents: normal recombinant TTR protein, TTR-PFF standard, fluorescent tracer and buffer system. TTR-PFF standard, comprising TTR substrate and RT-QuIC reaction mixture, wherein the pH of the RT-QuIC reaction mixture is 4.0-4.5; The buffer system includes PBS / N2 sample dilution buffer and phosphate buffer.

2. The detection kit for amyloid-to-thyroxine protein according to claim 1, characterized in that: The steps for using the detection kit to detect the presence of amyloid-transthyretin in subject samples are as follows: Step 1: Prepare TTR-PFF standard by adding the filtered TTR substrate to the RT-QuIC reaction mixture; Step 2: Prepare subject samples. After pretreatment, the peripheral tissue or body fluid samples of the subjects are mixed with the normal recombinant TTR protein prepared in vitro, and a fluorescent tracer is added. The fluorescent tracer binds to the ATTR-PFF polymer converted from the normal recombinant TTR protein and emits fluorescence. Step 3: Detect the fluorescence values ​​of TTR-PFF standard and subject samples. Add the prepared TTR-PFF standard and subject samples to 96-well plates respectively, and monitor them in real time using a fluorescence detector. The fluorescence measurement settings are: excitation wavelength of 450 nm, emission wavelength of 480 nm (bottom readout), and gain value of 1600. Compare the fluorescence values ​​of the obtained TTR-PFF standard with those of the subject samples to detect whether the subject samples contain amyloid-transferrothyroxine (ATTR).

3. The detection kit for amyloid-to-thyroxine protein according to claim 2, characterized in that: In step two, subject samples are prepared. 2 μL of the pretreated subject sample is added to PBS / N2 sample dilution buffer, diluted, centrifuged, and the supernatant is added to a 96-well plate.

4. The detection kit for amyloid-to-thyroxine protein according to claim 2, characterized in that: In step one, the TTR-PFF standard is prepared in vitro, the concentration of the TTR-PFF standard is 0.1 mg / ml, and the concentration of the TTR substrate is 0.8 mg / mL.

5. The detection kit for amyloid-to-thyroxine protein according to claim 4, characterized in that: The preparation steps of the TTR-PFF standard are as follows: after filtering 0.8 mg / mL of TTR substrate, it is added to the RT-QuIC reaction mixture. During the preparation of the RT-QuIC reaction mixture, 10 μM thiosulfate T is added, and the pH value is adjusted with 2M HCl.

6. The detection kit for amyloid-to-thyroxine protein according to claim 2, characterized in that: In step three, the fluorescence detector is an ELISA reader. The real-time monitoring method of the ELISA reader is as follows: the temperature is 50℃, the dual-track oscillation is 700 rpm for 1 min, then it is allowed to stand for 1 min, and the fluorescence value is detected every 45 min.

7. The detection kit for amyloid-to-thyroxine protein according to claim 2, characterized in that: The peripheral tissue sample is any one of skin biopsy sample, muscle biopsy specimen, fat fine needle aspiration, or nasal mucosa, and the body fluid sample is any one of serum, cerebrospinal fluid, saliva, or urine.

8. The detection kit for amyloid-to-thyroxine protein according to claim 7, characterized in that: The peripheral tissue samples were muscle biopsy samples or skin biopsy samples. The pretreatment method for the subject samples was as follows: wash the frozen biopsy specimens with PBS to remove residual blood, cut them into small pieces, put the cut sample tissue into a test tube, weigh it, add lysis buffer, incubate in a shaker, then transfer it to a BeatsBeater and shake it, place it on dry ice for 5 minutes, repeat the shaking and freezing cycle twice, place it at -80°C overnight, thaw it on ice the next day, centrifuge it, and take the supernatant and store it at -80°C for later use.

9. The detection kit for amyloid-to-thyroxine protein according to claim 1, characterized in that: The phosphate buffer solution is a pH 7.4, 10 mM phosphate buffer solution.

10. Use of a test kit according to any one of claims 1-9 for in vitro detection of amyloid-transthyretin.

Images