Biomarker composition for detecting Alzheimer disease as well as detection method and application of biomarker composition

Abstract

The invention discloses a biomarker composition for detecting Alzheimer's disease as well as a detection method and application of the biomarker composition. The biomarker composition is prepared from at least three of the following albumin-metal conjugates: copper, iron, zinc and aluminum. The detection method comprises the following steps: providing a to-be-detected sample from peripheral blood of a detected object; the method comprises the following steps: extracting an albumin-metal conjugate in a to-be-detected sample, and then determining the concentration of metal ions copper, iron, zinc or aluminum in the albumin-metal conjugate by using ICP-MS (Inductively Coupled Plasma Mass Spectrometry); and comparing with a control group, and giving a possible judgment result. Compared with the prior art, the detection method using the peripheral blood biomarker composition can realize non-invasiveness, is simple in acquisition method, can detect and find the Alzheimer's disease earlier, is sensitive and reliable, and is a detection method which is high in specificity and sensitivity and can be used for early intervention of the Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly relates to a biomarker composition for Alzheimer's disease detection, a detection method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease is a highly prevalent age-related disease that is difficult to detect in advance and seriously threatens the health of the elderly. Currently, magnetic resonance imaging (MRI) and computed tomography (CT) examinations are mainly used clinically. In molecular imaging diagnosis, Aβ-PET is particularly important because it can achieve the distribution and semi-quantification of β-amyloid (Aβ), a key pathogenic protein in Alzheimer's disease, in the brain. Although the positive prediction of Aβ-PET for the progression of non-demented subjects to Alzheimer's disease has a relatively high sensitivity (reaching 95%), the specificity is insufficient (<60%). Clinicians mainly assist in judging whether a patient may have Alzheimer's disease by examining whether memory or thinking skills are impaired, whether there are changes in personality and behavior, and the degree of memory and thinking changes in the patient. Since it is difficult for doctors to distinguish Alzheimer's disease from other dementias or the physical conditions of patients with cognitive impairment, the diagnostic results are usually inaccurate.

[0003] The typical pathological changes in Alzheimer's disease are the deposition of amyloid protein outside nerve cells to form neuritic plaques, the aggregation of hyperphosphorylated tau protein inside nerve cells to form neurofibrillary tangles, neuron loss, and gliosis. Therefore, the amyloid cascade hypothesis and the tau protein hyperphosphorylation hypothesis are considered to be the classical pathogenesis of Alzheimer's disease. Existing research results show that abnormally elevated concentrations of iron, zinc, and aluminum have been found in brain neuritic plaques and neurofibrillary tangles, and the number of plaques is positively correlated with the concentrations of iron, zinc, and aluminum. Although the current research has not fully clarified the correlation between the level of copper in the body and Alzheimer's disease, the serum copper level of Alzheimer's disease patients is significantly higher than that of healthy people. In addition, there is also a certain correlation between the degree of cognitive function impairment in Alzheimer's disease patients and the serum copper level. Therefore, albumin-copper conjugate is also included as one of the reference biomarkers in this study.

[0004] Transgenic animal models are widely used in the study of the disease mechanism of Alzheimer's disease and drug screening, and the disease process is predicted through the animal model to better diagnose and intervene in Alzheimer's disease patients at an early stage.

[0005] A large number of physiological activities of biological organisms involve body fluid transportation. Transporting active or toxic components through the blood is an important process among them. Serum albumin is an important carrier involved in blood transportation and has the most significant function in transporting active or toxic components. Since the concentration levels of most disease molecules in the blood are closely related to those in the cerebrospinal fluid, using blood biomarker detection to replace cerebrospinal fluid detection is a non-invasive method that can track the progression of Alzheimer's disease.

[0006] Currently, methods such as brain imaging detection and clinical diagnosis by clinicians are used to determine whether a patient has Alzheimer's disease. The specificity of the detection is relatively low, usually less than 60%, and the diagnostic results are not very reference-worthy. As another effective diagnostic method, cerebrospinal fluid detection mainly targets large molecular substances such as Aβ42, Aβ40, and Tau proteins. However, the requirements for cerebrospinal fluid collection are relatively high, and it is easily contaminated by blood during the collection process. In addition, the detection targets are easily affected by factors such as temperature, which can affect the detection results. Therefore, the cerebrospinal fluid detection diagnostic method is not only a detection method with relatively large interference factors, but also has a relatively small audience because it is an invasive detection method. ApoE risk gene detection is another diagnostic method with reference significance, but ApoE risk gene detection can only reflect the risk of a subject having Alzheimer's disease. In summary, the existing Alzheimer's disease diagnostic methods have very limited effects in early diagnosis, and these methods are highly invasive, expensive, and have a relatively small audience.

[0007] The prior art has disclosed Alzheimer's disease biomarker compositions and detection methods. For example, Patent CN115791987A discloses using inosine in feces as an Alzheimer's disease biomarker, Patent CN115791988A discloses using taurine in fecal samples as an Alzheimer's disease biomarker, and Patent CN115774059A discloses using the biomarker 11(Z),14(Z)-eicosadienoic acid as an Alzheimer's disease biomarker. However, the specificity and sensitivity of the detection are relatively limited and not ideal enough. There is an urgent need to propose a more optimized solution. Summary of the Invention

[0008] The purpose of the present invention is to provide a biomarker composition for Alzheimer's disease detection, its detection method, and application to solve the above problems.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A biomarker composition for Alzheimer's disease detection, which is a conjugate composed of albumin and at least three of the following metal ions: copper, iron, zinc, aluminum.

[0011] Preferably, for example, it can be metal ions such as copper, iron, and zinc; it can be metal ions such as copper, iron, and aluminum; it can be metal ions such as iron, zinc, and aluminum; it can be metal ions such as copper, zinc, and aluminum; or it can be metal ions such as copper, iron, zinc, and aluminum.

[0012] More preferably, they are metal ions of iron, zinc, and aluminum; even more preferably, they are metal ions of copper, iron, zinc, and aluminum.

[0013] Based on the important research result that the increase in the concentrations of iron, zinc, aluminum, and copper in serum is positively correlated with Alzheimer's disease, the inventor first proposed using the concentration levels of copper, iron, zinc, and aluminum metal ions bound to albumin in blood as a biomarker composition for the detection of Alzheimer's disease.

[0014] The application of the biomarker composition in the preparation of a reagent for the detection of Alzheimer's disease is to prepare the biomarker composition into a reagent for the detection of Alzheimer's disease, which is used to test the concentration change trend of the conjugates of albumin-bound copper, iron, zinc, aluminum, etc. in plasma samples to judge the disease status of Alzheimer's disease.

[0015] Furthermore, the biomarker composition is made into a reagent for the detection of Alzheimer's disease, which is used to test the concentration change trend of albumin-copper, iron, zinc, or aluminum conjugates in plasma samples to judge the disease status of Alzheimer's disease.

[0016] A detection method for a biomarker composition for the detection of Alzheimer's disease includes the following steps:

[0017] (1) Provide a sample to be tested, and the sample to be tested includes blood from a test subject;

[0018] (2) Extract the albumin-metal conjugates in the sample to be tested in step (1), and use ICP-MS to measure the concentrations of albumin-metal ion conjugates of copper, iron, zinc, or aluminum in plasma;

[0019] (3) Compare with a control group and give a judgment result.

[0020] Furthermore, in step (2), a purification column is prepared by using affinity filler - 001 to extract albumin-metal conjugates in plasma.

[0021] Furthermore, the specific method for extracting albumin-metal conjugates in plasma in step (2) is as follows:

[0022] An affinity filler - 001 is added to an empty purification column to prepare a purification column. After the purification column is pretreated, a sample is added. After the sample is incubated at room temperature, centrifuged, washed, eluted, etc., a purified sample is obtained. The purified sample is further dried and digested with nitric acid to obtain the sample to be tested.

[0023] Further, the purification column is composed of a purification empty column (product number: SLM001) produced by Tian Di Ren He and affinity packing - 001. The affinity packing - 001 is a packing composed of cyclic polypeptides. The main amino acid composition of the peptide segment of the cyclic polypeptide is Ac - ADGTGEFWFCEKLWAYPNHICFLDPEGGGGK - NH2 (C - C cyclization). Preferably, 400 μL of the affinity packing - 001 is loaded into the purification empty column to form the purification column.

[0024] A large number of biomolecules contained in serum, such as salts, proteins, etc., these biomolecules are likely to increase the background signal, resulting in some elements not meeting the detection requirements. After metal elements enter the body, they first react with albumin and then are transported throughout the body through the blood to perform physiological functions. Therefore, in order to improve the specificity and sensitivity of detection, the present invention extracts the albumin - metal element conjugate in serum by the method of affinity purification. This effective method for extracting metal elements can significantly reduce the signal interference of substances such as salts and other proteins on metal elements.

[0025] In particular, by using a cyclic polypeptide packing containing a specific amino acid composition, the present invention has a high selectivity with the albumin - metal conjugate in the biological matrix, can specifically bind the albumin - metal conjugate in the biological matrix. Through this specific binding, the impurities in the biological matrix and the albumin - metal conjugate are effectively separated, greatly improving the specificity and sensitivity of detection.

[0026] Further, ultrapure water is added to the digested sample to be measured for dilution and volume fixation to obtain a sample to be measured. The sample to be measured is detected by ICP - MS to obtain the metal ion concentration.

[0027] Further, when there is a significant difference (p < 0.05) in the concentration of the albumin - metal conjugate in the sample to be measured and the control group, the concentration of the albumin - metal conjugate is regularly measured. The trend of concentration change can be used as one of the bases for judging Alzheimer's disease.

[0028] Compared with the prior art, the present invention has the following excellent effects:

[0029] Clinically, the diagnosis of Alzheimer's disease is mostly based on the cognitive function assessment of scales. However, scales are highly subjective, and the diagnostic results are not very reference-worthy. Cerebrospinal fluid diagnosis and the detection of the risk gene ApoE are another reference-worthy diagnostic methods. However, cerebrospinal fluid diagnosis is an invasive examination and quite expensive, so the target population is small. The detection of the ApoE risk gene can only reflect the risk of subjects suffering from Alzheimer's disease. Biomarkers based on peripheral blood are the current hotspots in clinical research, but the detection of Alzheimer's disease markers such as amyloid-β (Aβ) and phosphorylated tau (P-tau) in peripheral blood lacks sensitivity.

[0030] Compared with the existing technologies, the detection method of the present invention using albumin-metal conjugates in peripheral blood as a biomarker composition is a non-invasive method with a simple sampling method. In addition, albumin-metal conjugates are relatively stable in blood. Compared with the diagnostic methods of traditional imaging or cerebrospinal fluid detection, it can detect and discover the tendency of Alzheimer's disease earlier and the method is sensitive and reliable. The method of predicting the disease process of Alzheimer's disease by detecting albumin-metal conjugates as a biomarker composition is proposed by the present invention for the first time. Further, the method of peripheral blood detection is also helpful for monitoring biological efficacy, treatment effect, side effects and the safety of extending the drug administration interval. It is a diagnostic method with high specificity, high sensitivity and can be used for early intervention in the disease process of Alzheimer's disease.

[0031] The peripheral blood biomarker screened based on the animal model by the present invention is a non-invasive, economical and less harmful auxiliary detection method. The biomarker has a high correlation with Alzheimer's disease and the method is sensitive and reliable. The method of delaying or changing the disease process through early diagnosis, timely prevention and intervention strategies will greatly reduce the incidence of clinical Alzheimer's disease and has extremely important clinical value and social significance. Description of the Drawings

[0032] Figure 1 It is the electrophoresis pattern of commercially available standard albumin;

[0033] Figure 2 It is the electrophoresis pattern of albumin extracted from the plasma of the present invention;

[0034] Figure 3 It is the comparison of the albumin-copper conjugate concentrations between the model group and the control group;

[0035] Figure 4 It is the comparison of the albumin-iron conjugate concentrations between the model group and the control group;

[0036] Figure 5 It is the comparison of the albumin-zinc conjugate concentrations between the model group and the control group;

[0037] Figure 6 Comparison of albumin-aluminum conjugate concentrations between the model group and the control group. Specific implementation manners

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] Albumin in human plasma was extracted using affinity filler - 001, and its recovery rate and repeatability were investigated.

[0041] Experimental materials and methods: Albumin, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium hydroxide, hydrochloric acid, affinity filler - 001 (composed of cyclic polypeptides, the main amino acid composition of its peptide segment is Ac - ADGTGEFWFCEKLWAYPNHICFLDPEGGGGK - NH2 (C - C cyclized)).

[0042] Solution preparation:

[0043] Loading and washing buffer: 10 mmol / L PBS buffer; Weigh 198.12 mg of sodium hydrogen phosphate, 1.232 g of sodium dihydrogen phosphate, and 1.66 g of sodium caprylate. The above powders are dissolved in 1000 mL of ultrapure water. After preparation, the buffer concentration in the solution is 10 mmol / L (pH = 6), and the sodium caprylate solution concentration is 10 mmol / L.

[0044] Elution buffer (200 mmol / L PBS containing 1 mol / L NaCl and 10 mmol / L sodium caprylate, pH = 8): Weigh 30.75 g of sodium hydrogen phosphate, 1.912 g of sodium dihydrogen phosphate, 58.5 g of sodium chloride, and 4.98 g of sodium caprylate. The above substances are dissolved in 1000 mL of ultrapure water. After preparation, the buffer concentration is 200 mmol / L (pH = 8), the sodium chloride concentration is 1 mol / L, and the sodium caprylate concentration is 30 mmol / L solution.

[0045] Experimental steps

[0046] 1. Add 400 μL of affinity filler - 001 to a purification column (a purification empty column produced by Tian Di Ren He, product number: SLM001);

[0047] 2. Take 400 μL of 10 mmol / L sodium chloride solution to wash the purification column (repeat 2 times);

[0048] 3. Take 50 μL of blank human plasma sample, dilute it with 450 μL of loading buffer, and take 50 μL of the solution and add it to the purification column;

[0049] 4. Incubate at room temperature for 10 minutes;

[0050] 5. Centrifuge at 3000 rpm for 5 seconds;

[0051] 6. Add 400 μL of washing buffer;

[0052] 7. Centrifuge at 3000 rpm for 5 seconds;

[0053] 8. Take 400 μL of elution buffer with pH = 6.95 and add it to the purification column;

[0054] 9. Centrifuge at 6000 rpm for 1 minute;

[0055] 10. Take 2 μL of the eluate and measure the ultraviolet absorption at 280 nm to determine the protein concentration (for the entire experiment, three batches of packing materials were selected and repeated three times to determine the difference between batches of packing materials).

[0056] The results are shown in Table 1.

[0057] Table 1

[0058]

[0059] The results show that for the affinity packing material - 001 to extract albumin from human plasma, on the same batch of resin packing material, the sample recovery rate and parallelism are both good, and the relative standard deviation is within the acceptable range (RSD < ±15%), indicating that the method of using the affinity packing material - 001 to extract albumin from plasma in the present invention is repeatable, and the extraction recovery rate of the three batches of packing materials is stable, with small differences between batches.

[0060] Experimental Example 2

[0061] Determination of the molecular weight of albumin. The molecular weight of the protein extracted by the affinity packing material - 001 was determined by capillary electrophoresis.

[0062] The experimental steps and methods refer to Experimental Example 1;

[0063] The extracted sample was treated with a commercial kit (high sensitivity protein 250 kit), and then the molecular weight of the extracted protein was measured using an Agilent Bioanalyzer 2100.

[0064] The test results of the protein molecular weight are shown in Figure 1 (HSA standard) and Figure 2 (human plasma sample).

[0065] The results showed that the molecular weight of the protein extracted by the affinity filler - 001 was consistent with that of commercially available albumin, indicating that the protein extracted by the affinity filler - 001 was albumin. The affinity filler - 001 had a high selectivity for albumin - metal conjugates in the biological matrix and could effectively separate the impurities and albumin - metal conjugates in the biological matrix, which was extremely helpful for improving the specificity and sensitivity of detection.

[0066] Experimental Example 3

[0067] Determine the concentration levels of albumin - bound metal ions (Cu, Fe, Zn, Al) in the plasma of normal mice and transgenic mice with Alzheimer's disease. After extracting albumin - metal conjugates from the plasma with the affinity filler - 001, the content was determined by ICP - MS (PerkinElmer NexION 350D). By comparing the concentration differences of albumin - metal conjugates between the plasma of normal mice and transgenic mice with Alzheimer's disease, the correlation between albumin - metal element conjugates and Alzheimer's disease was determined.

[0068] Experimental materials and methods:

[0069] Blood of normal mice, blood of 3Tg transgenic mice with Alzheimer's disease, nitric acid, ultrapure water;

[0070] The method for extracting albumin - metal conjugates was referred to Experimental Example 1;

[0071] The extracted albumin - metal conjugate solution was freeze - dried into powder by the method of freeze - drying. Weighed and took 10 mg of the powder, dissolved it with 1 mL of ultrapure water, transferred 500 μL of the sample solution to a test tube, added 1 mL of nitric acid to the test tube for digestion. Added ultrapure water to the digested sample to make the volume up to 10 mL and mixed well; diluted the sample 2 times with ultrapure water according to 1:1 (v / v), took 300 μL of the diluted sample, and detected it by ICP - MS (PerkinElmer NexION 350D).

[0072] The experimental results are shown in Table 2 and Figures 3 to 6 .

[0073] Table 2 Concentration table of albumin - metal conjugates in the transgenic mouse model group and the control group (μg / dL)

[0074]

[0075] The results showed that there were statistically significant differences in the concentrations of copper, iron, zinc, and aluminum in the plasma between the model group and the blank control group (P < 0.05), suggesting that the concentration levels of copper, iron, zinc, and aluminum in the plasma were positively correlated with Alzheimer's disease.

[0076] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should be within the protection scope of the present invention.

Claims

1. A biomarker composition for Alzheimer's disease detection, characterized in that, The biomarker composition is composed of at least three of the following albumin-metal conjugates: copper, iron, zinc, and aluminum.

2. Use of the biomarker composition for Alzheimer's disease detection according to claim 1 in the preparation of a reagent for Alzheimer's disease detection.

3. The use according to claim 2, characterized in that, The biomarker composition is made into an Alzheimer's disease detection reagent, which is used to test the concentrations of albumin-bound copper, iron, zinc, or aluminum in plasma samples to determine the status of Alzheimer's disease.

4. A detection method for the biomarker composition for Alzheimer's disease detection according to claim 1, characterized in that, It includes the following steps: (1) Provide a sample to be tested, and the sample to be tested includes peripheral blood from a test subject. (2) Extract the albumin-metal conjugate in the sample to be tested in step (1), and use ICP-MS to measure the concentrations of metal ions copper, iron, zinc, or aluminum in the albumin-metal conjugate. (3) Compare with a control group and give a judgment result.

5. The detection method for the biomarker composition for Alzheimer's disease detection according to claim 4, characterized in that, In step (2), the albumin-metal conjugate in plasma is extracted by affinity filler - 001.

6. The detection method for the biomarker composition for Alzheimer's disease detection according to claim 5, characterized in that, The specific method for extracting the albumin-metal conjugate in plasma in step (2) is as follows: Prepare a purification column by adding affinity filler - 001 to a purified empty column. After pretreatment of the purification column, add the sample. After the sample is incubated, centrifuged, washed, eluted, etc., a purified sample is obtained. The purified sample is further dried and digested with nitric acid to obtain the sample to be tested.

7. The detection method for the biomarker composition for Alzheimer's disease detection according to claim 5 or 6, characterized in that, The affinity filler - 001 is a filler composed of cyclic polypeptides, and the main amino acid composition of the peptide segment of the cyclic polypeptide is Ac-ADGTGEFWFCEKLWAYPNHICFLDPEGGGGK-NH2.

8. The detection method for the biomarker composition for Alzheimer's disease detection according to claim 6, characterized in that, Add ultrapure water to the digested sample to be tested for dilution and volume determination to obtain a sample to be measured. The sample to be measured is detected by ICP-MS to obtain the concentration of metal ions in the albumin-metal conjugate.

9. The detection method for the biomarker composition for Alzheimer's disease detection according to claim 5, characterized in that, When there is a significant difference in the concentration of the albumin-metal conjugate in the sample to be tested and the control group, regularly measure the concentration of the albumin-metal conjugate, and use the concentration change trend as the basis for judging Alzheimer's disease.

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