CN117741154B - Biomarker combination for cognitive impairment detection and application - Google Patents

Abstract

The present invention provides a biomarker combination for the detection of cognitive impairment, wherein the marker is selected from beta-amyloid protein 1-40 (Aβ1-40), beta-amyloid protein 1-42 (Aβ1-42), tau protein (t-Tau), and phosphorylated tau181 protein (pTau-181). The protein marker combination of the present invention has high sensitivity and specificity as a diagnostic marker for cognitive impairment. In the training queue, the combined sensitivity of Aβ1-42, Aβ1-42/40, pTau-181, and tTau can reach 85.58%, the specificity can reach 91.11%, and the area under the ROC curve (AUC) can reach 0.913. The combination of four protein markers Aβ42+Aβ42/Aβ40+pTau181+tTau is used as a biomarker for detecting different cognitive impairments, and its sensitivity is more than 80%, which has a high clinical detection significance.

Description

Biomarker combinations and applications for cognitive impairment detection

Technical Field

The present invention relates to the fields of biotechnology and medical diagnosis, and in particular to a combination of biomarkers for cognitive impairment detection and their application in cognitive impairment detection.

Background Art

Cognitive impairment, also known as neurocognitive impairment, is a disease in which the brain function declines due to neuronal lesions. It is a disease that mainly affects the patient's cognitive abilities such as memory, comprehension, language, learning, calculation, and judgment. Some patients also experience changes in emotions, behavior, and perception. According to the degree of cognitive impairment, it can be divided into mild cognitive impairment and dementia (mainly including Alzheimer's disease, vascular dementia, etc.).

Cognitive impairment is a degenerative disease of the central nervous system that progresses slowly, worsens over time, and is irreversible. Patients usually develop from early short-term memory loss to loss of physical function, and eventually die. Cognitive impairment is not only life-threatening, but also causes a serious decline in quality of life, and the associated loss of labor and care costs are immeasurable. Early diagnosis and early treatment can significantly delay the onset of the disease and maintain the patient's normal living standards.

At present, the diagnosis of cognitive impairment requires a large number of tests including: medical history, mental status assessment, neuropsychological assessment, brain MRI, and cerebrospinal fluid examination. Medical history, mental status assessment, and neuropsychological assessment are greatly affected by the patient's subjective factors, and are difficult to identify in the early stages and are easily misdiagnosed. Brain MRI and cerebrospinal fluid examinations are expensive, and brain MRI cannot be used as a routine examination method due to its certain radiation hazards, while cerebrospinal fluid examinations require invasive sampling methods and are limited in clinical application. Therefore, it is necessary to develop a method suitable for early screening of patients with early cognitive impairment and normal people in vitro. At present, some studies have proposed the use of blood biomarker detection for the diagnosis of cognitive impairment. However, due to the diverse influencing factors leading to cognitive impairment, the cause has not yet been clearly studied, and the immune system responses of different individuals are different, the sensitivity of a single biomarker in the diagnosis of cognitive impairment is relatively low. Therefore, the combined use of multiple different biomarker combinations can increase the sensitivity of detection.

Summary of the invention

The purpose of the present invention is to provide a biomarker combination and application for the detection of cognitive impairment in order to address the problems in the prior art of insufficient means for the detection of cognitive impairment and low diagnostic specificity and sensitivity.

The purpose of the present invention is to provide a combination of protein markers in the blood for diagnosing cognitive impairment patients. At the same time, based on the combination of protein markers as biomarkers, a detection reagent for detecting the corresponding protein markers is provided, aiming to solve the problem that the existing detection methods have certain subjectivity, low sensitivity, and are prone to detection delays or misdiagnosis.

In order to solve the above technical problems, the present invention adopts the following technical solutions:

The present invention discloses a biomarker combination for detecting cognitive impairment, wherein the biomarkers include tau protein (t-Tau), phosphorylated tau181 protein (pTau-181) and amyloid beta protein 1-42 (Aβ1-42).

Preferably, the marker further includes amyloid β 1-40 (Aβ1-40).

The present invention discloses a biomarker combination for detecting cognitive impairment, wherein the markers are selected from 3, 4, 5 or 6 of amyloid β-protein 1-40 (Aβ1-40), amyloid β-protein 1-42 (Aβ1-42), tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), phosphorylated tau217 protein (pTau-217) and phosphorylated tau231 protein (pTau-231).

Preferably, a biomarker combination for detecting cognitive impairment is characterized in that the markers include tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), and one or two proteins selected from β-amyloid protein 1-40 (Aβ1-40) and β-amyloid protein 1-42 (Aβ1-42).

Preferably, the markers include tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), and amyloid β-protein 1-40 (Aβ1-40).

Preferably, the markers include Aβ1-42, Aβ1-42/Aβ1-40, and pTau181.

Preferably, the markers include Aβ1-42, Aβ1-42/Aβ1-40, and tTau.

Preferably, the markers include Aβ1-42/Aβ1-40, tTau, and pTau181.

Preferably, the markers include Aβ1-42, Aβ1-42/Aβ1-40, pTau181, and tTau.

Preferably, the markers include tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), and amyloid β-protein 1-42 (Aβ1-42).

Preferably, the markers include tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), and amyloid β 1-40 (Aβ1-40) and amyloid β 1-42 (Aβ1-42).

Preferably, the markers are selected from three or four of amyloid β-protein 1-40 (Aβ1-40), amyloid β-protein 1-42 (Aβ1-42), tau protein (t-Tau), and phosphorylated tau181 protein (pTau-181).

Preferably, the marker combination is amyloid β-protein 1-42 (Aβ1-42), tau protein (t-Tau) and phosphorylated tau181 protein (pTau-181).

Preferably, the marker combination is amyloid β-protein 1-40 (Aβ1-40), tau protein (t-Tau) and phosphorylated tau181 protein (pTau-181).

Preferably, the marker combination is amyloid β 1-40 (Aβ1-40), amyloid β 1-42 (Aβ1-42) and phosphorylated tau181 protein (pTau-181).

Preferably, the marker combination is amyloid β 1-40 (Aβ1-40), amyloid β 1-42 (Aβ1-42) and tau protein (t-Tau).

Preferably, the marker combination is amyloid β-protein 1-42 (Aβ1-42), tau protein (t-Tau) and phosphorylated tau181 protein (pTau-181).

Preferably, the marker combination is amyloid β 1-40 (Aβ1-40), amyloid β 1-42 (Aβ1-42), tau protein (t-Tau) and phosphorylated tau181 protein (pTau-181).

The invention discloses a kit for detecting cognitive impairment, which comprises the marker combination.

Preferably, the kit further comprises quality control substances and/or standard substances.

Preferably, the test sample is selected from blood, serum or plasma.

The present invention discloses the use of the biomarker combination in preparing a cognitive impairment detection kit and/or reagent.

Preferably, the detection is selected from enzyme-linked immunosorbent assay (ELISA) detection, protein/peptide chip detection, immunoblotting detection, fluorescent immunoassay, chemiluminescent immunoassay or microfluidic immunoassay.

Preferably, the cognitive impairment includes mixed cortical and subcortical vascular dementia, senile dementia, Alzheimer's dementia and mild cognitive impairment.

The present invention discloses a protein marker combination including at least four or more of the six protein markers: amyloid β 1-40 (Aβ1-40), amyloid β 1-42 (Aβ1-42), tau protein (t-Tau), phosphorylated tau181 protein (pTau-181), phosphorylated tau217 protein (pTau-217), and phosphorylated tau231 protein (pTau-231).

The present invention discloses a blood protein marker combination as a biomarker, provides a detection reagent for detecting the corresponding protein marker, and provides the use of the protein marker or the detection reagent in preparing products for disease risk prediction, screening, prognosis evaluation, treatment effect monitoring or recurrence monitoring of cognitive impairment.

The invention discloses that the detection reagent may be a kit using enzyme-linked immunosorbent assay (ELISA), protein/peptide chip detection, immunoblotting, fluorescent immunoassay, chemiluminescent immunoassay or microfluidic immunoassay technology.

Preferably, the detection reagent used detects the biomarker through antigen-antibody specific reaction, such as ELISA reagent, fluorescent immunoassay reagent or chemiluminescent immunoassay reagent.

Preferably, nanozyme-enhanced chemiluminescent immunoassay reagent is used to detect each protein in the protein combination in the blood one by one to determine the concentration change of the detected protein marker in the blood.

The present invention discloses the selected protein markers and marker combinations as follows:

(1) Aβ1-42;

(2) Aβ1-42/Aβ1-40;

(3) pTau-181;

(4) tTau;

(5)Aβ1-42, Aβ1-42/Aβ1-40, tTau;

(6)Aβ1-42, pTau-181, tTau;

(7)Aβ1-42/Aβ1-40, pTau-181, tTau;

(8) Aβ1-42, Aβ1-42/Aβ1-40, pTau-181, tTau.

The protein marker combination of the present invention has high sensitivity and specificity as a diagnostic marker for cognitive impairment. The sensitivity of the combination of Aβ1-42, Aβ1-42/Aβ1-40, pTau-181, and tTau in the training cohort can reach 85.58%, the specificity can reach 91.11%, and the area under the ROC curve (AUC) can reach 0.913, which is significantly improved compared with the sensitivity, specificity, and AUC of a single protein marker.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of nanozyme-enhanced chemiluminescent immunoassay.

Figure 2. Differences in Aβ1-40 concentration levels in the blood of healthy people and patients with cognitive impairment.

Figure 3. Differences in Aβ1-42 concentration levels in the blood of healthy people and patients with cognitive impairment.

Figure 4. Differences in Aβ1-42/Aβ1-40 concentration levels in the blood of healthy people and patients with cognitive impairment.

Figure 5. Differences in pTau-181 concentration levels in the blood of healthy people and patients with cognitive impairment.

Figure 6. Differences in tTau concentration levels in the blood of healthy people and patients with cognitive impairment.

Figure 7. ROC curve analysis of Aβ1-40 subjects.

Figure 8. ROC curve analysis of Aβ1-42 subjects.

Figure 9. ROC curve analysis of Aβ1-42/Aβ1-40 subjects.

Figure 10. ROC curve analysis of pTau-181 subjects.

Figure 11. ROC curve analysis of tTau-181 subjects.

Figure 12. ROC curve analysis of subjects in the Aβ1-42/Aβ1-40+tTau+pTau-181 combination.

Figure 13. ROC curve analysis of subjects in the combination of Aβ1-42+Aβ1-42/Aβ1-40+pTau-181.

Figure 14. ROC curve analysis of subjects in the Aβ1-42+Aβ1-42/Aβ1-40+tTau combination.

Figure 15. ROC curve analysis of subjects in the Aβ1-42+tTau+pTau181 combination.

Figure 16. ROC curve analysis of subjects in the combination of Aβ1-42+Aβ1-42/Aβ1-40+tTau+pTau181.

Figure 17. Comparison of ROC curves and AUCs of different protein marker combinations.

Figure 18. ROC curve analysis of subjects in the combination of Aβ1-42+Aβ1-42/Aβ1-40+pTau-181 in the validation set.

Figure 19. ROC curve analysis of subjects with the combination of Aβ1-42+Aβ1-42/Aβ1-40+tTau in the validation set.

Figure 20. ROC curve analysis of subjects with the combination of Aβ1-42+tTau+pTau181 in the validation set.

Figure 21. ROC curve analysis of subjects with the combination of Aβ1-42/Aβ1-40+tTau+pTau-181 in the validation set.

Figure 22. ROC curve analysis of subjects in the combination of Aβ1-42+Aβ1-42/Aβ1-40+tTau+pTau181 in the validation set.

DETAILED DESCRIPTION

The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

Example 1 Experimental samples for marker screening

The experimental samples are divided into training set samples and validation set samples.

The training set samples included: 194 clinical samples, including 90 healthy people and 104 patients with cognitive impairment. The Mini-Mental State Examination scores of patients with cognitive impairment were all <10 points, and the diagnosis was confirmed according to the AD diagnostic criteria recommended by the National Institute of Neurological Disorders and Language Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association. The characteristics of patients with cognitive impairment are shown in Table 1.

Table 1: Characteristics of patients with cognitive impairment in the training set samples

Sample size 104 age 55-94 years old Average age 78.6 years old male 43 cases female 61 cases Pathological subtype dementia 41 cases Mixed cortical and subcortical vascular dementia 13 cases Alzheimer's disease 27 cases Alzheimer's disease 17 cases Mild cognitive impairment 6 cases

The validation set samples included: 103 clinical samples, including 42 healthy people and 61 patients with cognitive impairment. The Mini-Mental State Examination scores of the patients with cognitive impairment were all <10 points, and the diagnosis was confirmed according to the AD diagnostic criteria recommended by the National Institute of Neurological Disorders and Language Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association. The characteristics of patients with cognitive impairment are shown in Table 2.

Table 2: Characteristics of patients with cognitive impairment in the validation set samples

Example 2 Establishment of Nanozyme Enhanced Chemiluminescent Immunoassay Method

1.1. Preparation of Nanozyme Enhanced Chemiluminescent Immunoassay Reagent

1.1.1. Preparation of streptavidin magnetic beads

Take 1 mL of streptavidin magnetic particles and dilute with 20 mM pH 7.4 PBS + 0.1% BSA + 0.0.1%

ProClin-300 was washed 3 times and diluted to 167 mL.

1.1.2. Preparation of Biotin-labeled Antibodies

Take 1.0 mg of the Aβ1-40 monoclonal antibody to be labeled, dilute it to 0.5 mg/mL with phosphate buffer, then add 27.3 μL of 2 mg/mL biotin ester, react at room temperature for 30 minutes, then use a 10kD ultrafiltration tube to centrifuge the labeled substance at 7500g centrifugal force for 10 minutes for purification, and take the remaining liquid after ultrafiltration to obtain the Aβ1-40 monoclonal antibody labeled with biotin.

According to the above steps and using the same method, biotin-labeled Aβ1-42 monoclonal antibody, biotin-labeled pTau181 monoclonal antibody, and biotin-labeled tTau monoclonal antibody were prepared respectively.

1.1.3. Preparation of Antibodies for Labeling Nanozymes

Take 0.1 mg of the Aβ1-40 monoclonal antibody to be labeled, dilute it to 0.5 mg/mL with 50 mM MES buffer at pH 5.0, then add 1 mL of 10 mg/mL nanozyme, react at room temperature for 2 hours, then add 20 μL of 1 M Tris solution, react at room temperature for 10 minutes, then centrifuge the marker at 10000 g for 15 minutes, remove the supernatant and resuspend the collected material with 50 mM MES buffer at pH 5.0 to obtain the Aβ1-40 monoclonal antibody labeled with nanozyme.

According to the above steps, the same method was used to prepare Aβ1-42 monoclonal antibody labeled with nanozyme, pTau181 monoclonal antibody labeled with nanozyme, and tTau monoclonal antibody labeled with nanozyme.

The detection principle of nanozyme enhanced chemiluminescence immunoassay is shown in Figure 1.

1.2. Clinical sample testing

The test was performed using a fully automatic chemiluminescence immunoassay analyzer. The instrument sequentially added 50 μL of the sample to be tested, 50 μL of Aβ1-40 monoclonal antibody labeled with biotin, and 50 μL of Aβ1-40 monoclonal antibody labeled with nanozyme into the reaction cup, and then incubated at 37°C for 30 minutes. The Aβ1-40 antigen in the sample reacted with the antibody to form a sandwich complex of "capture antibody-antigen-detection antibody". Then 50 μL of streptavidin magnetic beads were added. The biotin on the antibody in the sandwich complex specifically bound to the streptavidin on the magnetic beads, thereby binding the sandwich complex to the magnetic beads. After washing to remove other unbound substances, substrate solution A (hydrogen peroxide) and substrate solution B (sodium hydroxide) were added. The nanozyme labeled on the antibody catalyzed the luminescence in the substrate to generate photons. The photons were collected by the detection device and converted into light signals. The content of Aβ1-40 in the sample was positively correlated with the intensity of the luminescence signal. The concentration of Aβ1-40 in the sample could be quantitatively calculated based on the calibration curve.

According to the above detection method, the prepared Aβ1-42, pTau181, and tTau nanozyme enhanced chemiluminescence immunoassay reagents were used to detect the concentrations of Aβ1-42, pTau181, and tTau in the samples respectively.

Negative/positive judgment of protein combination:

For each protein marker, the test result was compared with the cutoff value, and a positive reaction was defined as a value ≥ the cutoff value; a negative reaction was defined as a value < the cutoff value.

Since the positive rate of a single protein marker is low, in order to increase the positive rate of protein marker detection, the results of multiple protein markers are combined when analyzing the results to determine the prediction effect. The rule is: when multiple protein markers are detected in a sample, as long as one or more of the protein markers show positive results, the protein marker combination result is judged to be positive; if all protein markers are negative, the result is judged to be negative.

Sensitivity and specificity judgment

Sensitivity: The proportion of cases with positive results of protein marker combination test among all cases diagnosed with the disease by gold standard.

Specificity: The proportion of subjects with negative results for the protein marker combination test among all subjects diagnosed as disease-free by the gold standard.

Example 3 Determination of marker concentration in training set samples and establishment of ROC curve

The nanozyme-enhanced chemiluminescence immunoassay method established in Example 2 was used to measure the concentrations of the four markers Aβ1-40, Aβ1-42, pTau181, and tTau in 194 clinical samples of the ear in the training set, and the concentration difference level diagrams of different markers in the blood of healthy people and patients with cognitive impairment (Figures 2-6) and ROC curves of different marker combinations (Figures 7-17) were established. The measurement results are as follows.

When the specificity is set to no less than 90%, the sensitivity and specificity results of a single protein marker in a blood sample as a diagnostic marker for cognitive impairment are shown in Table 3 below.

Table 3: Sensitivity and specificity of individual protein markers in the training set

Serial number Markers Sensitivity Specificity AUC 1 Aβ42 56.73% 90.00% 0.815 2 Aβ1-42/Aβ1-40 66.35% 90.00% 0.728 3 pTau181 61.54% 90.00% 0.792 4 tTau 68.37% 90.00% 0.826

Different protein combinations were selected from the four protein markers. The results showed that as the number of markers increased, the detection sensitivity gradually increased. When the four protein markers were used in combination, the sensitivity could reach 85.58% and the specificity could reach 91.11%. Compared with a single protein marker, the combination of multiple protein markers could significantly improve the sensitivity. The detection results are shown in Table 4.

Table 4: Sensitivity and specificity of different protein marker combinations as diagnostic markers for cognitive impairment

Judging from the test results, the combined sensitivity of Aβ1-42, Aβ1-42/Aβ1-40, pTau-181, and tTau in the training set can reach 85.58%, the specificity can reach 91.11%, and the area under the ROC curve (AUC) can reach 0.913, which are significantly improved compared with single protein markers in terms of sensitivity, specificity, and AUC.

Among them, the sensitivity of the combination of four protein markers, Aβ42+Aβ42/Aβ40+pTau181+tTau, in different pathological subtypes of cognitive impairment is higher than that of the combination of four protein markers as biomarkers for detecting different cognitive impairments. The sensitivity is all above 80%. The test results are shown in Table 5.

Table 5: Sensitivity of the four protein markers in different pathological subtypes of cognitive impairment in the training set

Pathological subtype Sensitivity dementia 80.49% Mixed cortical and subcortical vascular dementia 84.62% Alzheimer's disease 92.59% Alzheimer's disease dementia 82.35% Mild cognitive impairment 100.00%

Example 4. ROC curve determination of different marker combinations in validation set samples

The nanozyme-enhanced chemiluminescent immunoassay method established in Example 2 was used to measure the concentrations of the four markers Aβ1-40, Aβ1-42, pTau181, and tTau in 103 clinical samples in the validation set, and ROC curve analysis was performed for different combinations of three or four markers. The results are as follows.

When the specificity is set to no less than 90%, the sensitivity of a single protein marker in a blood sample as a diagnostic marker for cognitive impairment is shown in Table 6 below.

Table 6: Sensitivity and specificity of individual protein markers in the validation set

The sensitivity and specificity test results of the combination of three or four markers obtained in Example 3 as diagnostic markers for cognitive impairment are shown in Table 7 and Figures 18 to 22.

Table 7: Sensitivity and specificity of different protein marker combinations as diagnostic markers for cognitive impairment in the validation set

Serial number Markers Sensitivity Specificity AUC 1 Aβ1-42+Aβ1-42/Aβ1-40+pTau181 81.97% 92.86% 0.874 2 Aβ1-42+Aβ1-42/Aβ1-40+tTau 85.25% 92.86% 0.891 3 Aβ1-42+tTau+pTau181 85.25% 92.86% 0.891 4 Aβ1-42/Aβ1-40+tTau+pTau181 86.89% 92.86% 0.899 5 Aβ1-42+Aβ1-42/Aβ1-40+pTau181+tTau 90.16% 92.86% 0.915

Judging from the test results, using Aβ1-42 in the training set, the combined sensitivity of Aβ1-42/Aβ1-40, pTau-181, and tTau can reach 90.16%, the specificity can reach 92.86%, and the area under the ROC curve (AUC) can reach 0.915, which are basically consistent with the results of the training set.

Among them, the sensitivity of the combination of four protein markers, Aβ42+Aβ1-42/Aβ1-40+pTau181+tTau, in different pathological subtypes of cognitive impairment is higher than that of the combination of four protein markers, which is used as a biomarker for detecting different cognitive impairments, and its sensitivity is also above 80%. The test results are shown in Table 8.

Table 8: Sensitivity of the four protein markers in different pathological subtypes of cognitive impairment in the validation set

Pathological subtype Sensitivity dementia 85.71% Mixed cortical and subcortical vascular dementia 83.33% Alzheimer's disease 92.86% Alzheimer's disease dementia 100.00%

The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims (3)

1. Use of a biomarker combination in the preparation of a cognitive impairment detection kit and/or reagent, characterized in that the biomarker consists of β-amyloid 1-42/β-amyloid 1-40, β-amyloid 1-42, t-Tau protein and phosphorylated tau181 protein, and the cognitive impairment is mixed cortical and subcortical vascular dementia. 2. The use according to claim 1, characterized in that the detection is selected from enzyme-linked immunosorbent assay (ELISA) detection, protein/peptide chip detection, immunoblotting detection, fluorescent immunoassay, chemiluminescent immunoassay or microfluidic immunoassay. 3. The method according to any one of claims 1 to 2, wherein the test sample is selected from blood, serum or plasma.