Combined detection and application of biomarkers

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a combination and application of joint detection of biomarkers for Alzheimer's disease. The present invention provides an application of a biomarker group in detecting Alzheimer's disease, characterized in that the biomarker group includes: a) at least two of APOE4, GDF-15, UCH-L1 and PF4; or b) at least one of APOE4, GDF-15, UCH-L1 and PF4, as well as phosphorylated Tau protein and total Tau protein. The present invention also provides a kit for detecting Alzheimer's disease. The present invention contributes to a more effective detection and diagnosis of Alzheimer's disease, and provides a new path for the detection and prevention of AD.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the combination and application of joint detection of Alzheimer's disease biomarkers. Background Art

[0002] Alzheimer's disease (AD) is the most common age-related dementia and the most destructive neurodegenerative disease associated with aging, affecting approximately 50 million people worldwide. The main clinical manifestations of the disease are severe cognitive impairment, progressive memory loss, and retrograde and anterograde amnesia. Psychological and behavioral abnormalities are mainly manifested as irritability, euphoria, increased speech, emotional indifference, and erratic crying and laughing.

[0003] It is accompanied by severe histopathological changes, such as hippocampal degeneration or even loss. In 2018, the National Institute on Aging-Alzheimer's Association (NIA-AA) divided the development process of AD into the non-damage stage, the asymptomatic stage, the mild cognitive impairment stage, the early dementia stage, the middle dementia stage and the late dementia stage.

[0004] Mild cognitive impairment (MCI) is directly related to the onset of AD. For every unit increase in the cognitive function evaluation index, the risk of AD is reduced by 85% (RR = 0,15, 95% CI, 0.091-0.25) (Neurology,

[0005] 2019,59:198-205). According to the latest "Chinese Expert Consensus on Diagnosis and Treatment of Mild Cognitive Impairment Caused by Alzheimer's Disease 2024", the classification of MCI may have a suggestive significance for the prognosis of the disease. aMCI is likely to progress to typical AD, so monitoring MCI is an effective means to control the deterioration of AD.

[0006] At present, the main means of diagnosing Alzheimer's disease are: neuropsychological assessment scale, neuroimaging examination, cerebrospinal fluid and blood marker detection. Neuropsychological assessment scale has an indispensable diagnostic value in the quantitative analysis of cognitive ability and is an important part of the qualitative diagnosis system for cognitive impairment. The scale has the advantages of concise content and short testing time, but it also has the disadvantage of poor accuracy.

[0007] Neuroimaging tests, including amyloid protein PET and Tau protein PET, can trace the concentration and distribution of proteins in different brain regions, and have good sensitivity and specificity. However, the resources for neuroimaging tests are scarce, expensive, and not easily accessible. The concentration of biomarkers in cerebrospinal fluid can well reflect the situation in the brain, and the protein abundance is higher and the accuracy is better. However, as an invasive test, cerebrospinal fluid testing is difficult to sample and patient compliance is poor. Blood markers have the advantages of less trauma, strong repeatability, and wide acceptance, and are a good tool for early screening and auxiliary diagnosis of AD.

[0008] Although Roche has recently developed a series of cerebrospinal fluid test kits for diagnosing Alzheimer's disease

[0009] Although the test has been approved, cerebrospinal fluid is still required as the test sample, which is difficult to operate and has a very high sampling risk. At the same time, the subject compliance is poor. Blood samples are easier to obtain and are ideal test samples. At present, there are many studies on blood markers to assist in the differential diagnosis of Alzheimer's disease, but the diagnostic efficiency is poor. Summary of the invention

[0010] In a first aspect, the present invention provides an application of a biomarker panel in detecting AD, characterized in that the biomarker panel comprises:

[0011] a) at least two of APOE4, GDF-15, UCH-L1, and PF4; or

[0012] b) at least one of APOE4, GDF-15, UCH-L1 and PF4, and phosphorylated Tau protein and total Tau protein.

[0013] In some embodiments, the biomarker panel includes APOE4, GDF-15,

[0014] At least three of UCH-L1 and PF4.

[0015] In some embodiments, the biomarker panel includes APOE4, GDF-15,

[0016] At least two of UCH-L1 and PF4, as well as phosphorylated Tau protein and total Tau protein.

[0017] In some embodiments, the biomarker panel includes APOE4, GDF-15,

[0018] At least three of UCH-L1 and PF4, as well as phosphorylated Tau and total Tau.

[0019] Furthermore, the ratio of the phosphorylated Tau protein content to the total Tau protein content is used as an evaluation index, rather than the phosphorylated Tau protein or total Tau protein content alone.

[0020] In some embodiments, the biomarker panel further includes NFL and / or GFAP.

[0021] In some embodiments, the biomarker panel includes GDF-15, PF4, and one or more of APOE4, UCH-L1, and phosphorylated Tau protein and total Tau protein.

[0022] In some embodiments, the biomarker panel includes APOE4, GDF-15,

[0023] PF4.

[0024] In some embodiments, the biomarker panel includes APOE4, UCH-L1,

[0025] GDF-15, PF4.

[0026] In some embodiments, the biomarker panel includes APOE4, UCH-L1,

[0027] GDF-15, PF4, and phosphorylated Tau protein and total Tau protein.

[0028] In some embodiments, the biomarker panel includes APOE4, UCH-L1,

[0029] GDF-15, PF4, NFL and phosphorylated Tau protein and total Tau protein.

[0030] In some embodiments, the biomarker panel includes APOE4, UCH-L1, GDF-15, PF4, GFAP, and phosphorylated Tau protein and total Tau protein.

[0031] In some embodiments, the phosphorylated Tau protein is any one of p-tau181, p-tau217, p-tau231 and p-tau205.

[0032] In some embodiments, the phosphorylated Tau protein is p-tau181 and / or p-tau217.

[0033] In some embodiments, the biomarker panel is used to test a blood sample.

[0034] In a second aspect, the present invention provides a kit for AD, characterized in that the kit comprises a detection reagent; the detection reagent comprises chemical magnetic beads and a chemiluminescent substrate, and the biomarker group comprises:

[0035] a) at least two of APOE4, GDF-15, UCH-L1, and PF4; or

[0036] b) at least one of APOE4, GDF-15, UCH-L1 and PF4, and phosphorylated Tau protein and total Tau protein.

[0037] In some embodiments, the phosphorylated Tau protein is any one of p-tau181, p-tau217, p-tau231 and p-tau205.

[0038] It is understandable that in the present invention, phosphorylated Tau protein and total Tau protein are detected as biomarkers. When used to evaluate the correlation between relevant markers and related diseases, the content of phosphorylated Tau protein and total Tau protein is not used as an indicator, but the ratio of phosphorylated Tau protein content to total Tau protein content is used as an evaluation indicator to avoid the influence of individual differences.

[0039] In the present invention, "Alzheimer's disease" or "AD" includes symptoms or stages in the development of AD, such as the MCI stage.

[0040] Compared with the prior art, the beneficial technical effects of the present invention include at least the following aspects:

[0041] The combined use of the biomarkers provided by the present invention can greatly improve the specificity and sensitivity of AD, contribute to more effective detection and diagnosis of Alzheimer's disease, provide a new path for the detection and prevention of AD, and is particularly suitable for the early diagnosis of AD.

[0042] The present invention uses the ratio of phosphorylated Tau protein to total Tau protein instead of measuring phosphorylated Tau protein alone, thereby avoiding the problem of inconsistent total Tau protein content due to factors such as age, personal health status and individual differences, and thus avoiding clinical judgments that are prone to errors.

[0043] The existing means of detecting APOE4 risk genes are mainly through molecular detection, while other proteins are detected by immunoassay. If APOE4 and other markers are to be detected simultaneously, samples need to be processed separately and then performed using different instruments. The present invention provides a magnetic microparticle chemiluminescence joint detection kit for auxiliary diagnosis of AD, which reduces the trouble of sample processing and testing and increases comparability.

[0044] The sample used in the present invention is peripheral blood, which replaces the invasive cerebrospinal fluid.

[0045] This greatly reduces the patient's pain and the difficulty of sampling, while also providing excellent diagnostic performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art,

[0047] The following is a brief introduction to the drawings required for use in the embodiments or prior art descriptions. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0048] Figure 1 ROC curve for the marker combination in diagnosing Alzheimer's disease;

[0049] Figure 2 ROC curve for the marker combination in diagnosing mild cognitive impairment. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0051] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0052] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0053] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0054] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0055] In this specification, some embodiments may be disclosed in a format of being in a certain range. It should be understood that such descriptions of "being in a certain range" are merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2,

[0056] 3, 4, 5 and 6. The above rules apply regardless of the breadth of the range.

[0057] Embodiment 1

[0058] Subjects:

[0059] According to the diagnosis of the hospital, this embodiment selected elderly people over 60 years old with similar age, similar gender ratio and similar education level, and passed the Mini Mental State Examination (MMSE) test. Finally, 32 patients in the AD group, 30 patients in the MCI group, and 35 patients in the control group were screened, and the specific information is shown in Table 1. The blood samples of the above people were properly collected according to known procedures.

[0060] Table 1

[0061] Grouping Number of cases age Gender (female / male) Years of education MMSE AD Group 32 73.81±4.60 15 / 17 5.88±1.66 18.06±2.40 MCI Group 30 75.77±3.40 14 / 16 6.03±1.10 23.63±1.47 Control group 35 72.54±4.29 19 / 16 6.06±1.28 28.14±1.12

[0062] Detection of markers:

[0063] The present invention detects the concentration of each marker in the blood sample of the subject, including:

[0064] New markers include platelet factor 4 (PF4), growth differentiation factor-15 (GDF-15), ubiquitin carboxyl-terminal hydrolase-1 (UCH-L1), and the risk gene APOE4; plus one or more biomarkers that have been confirmed by a large number of previous studies for the diagnosis of Alzheimer's disease, including p-Tau181, p-Tau217, and p-Tau231, which characterize abnormal hyperphosphorylation of Tau protein in Aβ pathology, p-Tau205, which characterizes Tau pathology, GFAP, which characterizes astrocyte activation, and NFL, which characterizes neurodegeneration.

[0065] The concentration of the above markers in the measured sample is brought into one of the algorithm models such as logistic regression or support vector machine (SVM) to calculate the probability of suffering from Alzheimer's disease.

[0066] For Logistic regression, the regression equation is as follows:

[0067]

[0068] Wherein C is the natural constant obtained by regression analysis, α is the coefficient of each marker obtained by regression analysis, i is the concentration of each marker, and n is the number of markers greater than or equal to 2.

[0069] After calculating Logit(P) through the regression equation, substitute it into the following formula to calculate the P value:

[0070]

[0071] After obtaining the P value, perform ROC curve analysis based on the P value to obtain the Cut off

[0072] The cognitive impairment of the sample to be tested can be determined based on the Cut off value.

[0073] Support vector machine (SVM) is a binary classification model. Its purpose is to find a hyperplane to segment samples. The principle of segmentation is to maximize the interval, which is ultimately converted into a convex quadratic programming problem to solve.

[0074] Its basic model is a linear classifier with the largest interval defined in the feature space, which is different from the perceptron. It has many unique advantages in solving small sample, nonlinear and high-dimensional pattern recognition.

[0075] In this embodiment, the concentration of the above markers is detected by magnetic microparticle chemiluminescence immunoassay. The above samples are added to the components of each kit, and the instrument automatically adds samples, incubates, washes and detects, and reads the concentration results of each marker. The above concentration results are transformed into natural logarithms, and Logistic regression analysis (using R language programming or Medcalc software) is performed to obtain the regression equation, and ROC analysis is performed to obtain the corresponding AUC value and the values ​​of specificity and sensitivity.

[0076] Sensitivity (also known as true positive rate, sensitivity, and sensitivity) refers to the proportion of samples that are actually positive that are judged to be positive (the ability to correctly judge cases that are actually sick as sick, that is, the probability that the patient is judged to be positive). The higher the sensitivity, the lower the missed diagnosis rate. Specificity (also known as true negative rate) refers to the proportion of samples that are actually negative that are judged to be negative (the ability to correctly judge cases that are actually not sick, that is, the proportion of negative test results). The higher the specificity, the lower the misdiagnosis rate.

[0077] The receiver operating characteristic curve (ROC) is a comprehensive indicator that reflects the sensitivity and specificity of continuous variables and is used for binary classification. The area under the ROC curve (AUC) is used to indicate the accuracy of prediction. The higher the AUC value, the larger the area under the curve, indicating a higher prediction accuracy.

[0078] This embodiment is aimed at the specific application of Logistic regression analysis, and the following examples are given:

[0079] The combined regression equation for the joint detection of four markers APOE4+UCH-L1+GDF-15+PF4 to distinguish the AD group from the control group:

[0080] Logit(P)=-26.64+3.64*APOE4+1.46*Ln(GDF-15)-1.66*Ln

[0081] (PF4)+3.97*Ln(UCH-L1);

[0082] The obtained AUC = 0.945, with a specificity of 91.43% and a sensitivity of 93.75%,

[0083] The results were significantly better than those of either biomarker alone or the combination of the two biomarkers.

[0084] Embodiment 2

[0085] This example examines the performance of the following substances as biomarkers for diagnosing Alzheimer's disease or mild cognitive impairment. The substances examined in this example include: ①

[0086] APOE4 (Apolipoprotein E4), ②UCH-L1 (ubiquitin carboxyl-terminal hydrolase-1),

[0087] ③GDF-15 (growth differentiation factor-15), ④PF4 (platelet factor 4), ⑤

[0088] p-tau181 / t-tau、⑥p-tau217 / t-tau、⑦p-tau231 / t-tau、⑧

[0089] p-tau205 / t-tau, ⑨NFL (neurofilament light chain), ⑩GFAP (glial fibrillary acidic protein).

[0090] Table 2

[0091]

[0092]

[0093] The results in Table 2 show that the AUCs of GDF-15 and PF4 can reach a level comparable to that of the reported Alzheimer's disease markers APOE4, UCH-L1, NFL, and GFAP, and therefore can be used as AD markers.

[0094] The AUCs of the six markers, NFL and GFAP, were not high when tested individually, ranging from 0.7 to 0.8. The effectiveness of phosphorylated Tau and total Tau as AD markers has been widely verified. The ratio of phosphorylated Tau to total Tau can avoid individual differences. The AUC of the ratio of phosphorylated Tau to total Tau as an evaluation index is between 0.8 and 0.9. From the perspective of the indicators of a single marker or evaluation factor, there is still a lot of room for improvement.

[0095] Embodiment 3

[0096] This example further investigated the AUC, specificity and sensitivity of the pairwise combinations of the markers. The results are shown in Table 3.

[0097] Table 3

[0098]

[0099]

[0100] According to common understanding in the art, compared with a single marker, a combination of two or more markers often improves its diagnostic performance, which is specifically reflected in the improvement of AUC.

[0101] The results in Table 3 show that not all combinations of two markers can improve the AUC. For example, when NFL or GFAP is combined with APOE4, the corresponding AUC is not much different from that before the combination (APOE4 alone), and may even reduce the sensitivity of APOE4. The combination of NFL and GDF-15 also proves that the corresponding AUC is not much different from that before the combination (GDF-15 alone), and may even reduce the sensitivity of GDF-15.

[0102] In general, compared with the pre-combination (PF4, APOE4, UCH-L1, GDF-15 alone), the addition of the p-tau / t-tau ratio increased the AUC to above 0.8, with excellent diagnostic performance. Similarly, compared with the pre-combination (PF4, APOE4, UCH-L1, GDF-15 alone), the pairwise combination of PF4, APOE4, UCH-L1, and GDF-15 also increased the AUC to above 0.8, with excellent diagnostic performance.

[0103] It is worth noting that compared with before the combination (APOE4, UCH-L1, GDF-15 alone), the addition of PF4 can increase the specificity from approximately 80% to more than 90%, among which the specificity of the combination of PF4 and UCH-L1 can reach 94.29%, and the specificity of the combination of PF4 and APOE4 can reach 100%.

[0104] Embodiment 4

[0105] From the experimental results of Example 3, although APOE4, UCH-L1, GDF-15,

[0106] The AUCs of the two-by-two combinations of the four biomarkers PF4 or the combination with the p-tau / t-tau ratio can all be increased to above 0.8. However, this example found that it was the specificity that was substantially increased, and the sensitivity was not significantly improved.

[0107] This example further investigated the combination of the above biomarkers, and the results are shown in Table 4.

[0108] Table 4

[0109]

[0110] In general, compared with the pairwise combination of the above biomarkers, the three-item combination of the above biomarkers has better diagnostic performance, with an AUC of about 0.9.

[0111] This example found that the combination of GDF-15 and PF4 with APOE, and the combination of GDF-15 and PF4 all had low sensitivity. Unexpectedly, the simultaneous addition of GDF-15 and PF4 can improve the diagnostic performance of other biomarkers (such as APOE, p-tau205 / t-tau) in terms of both specificity and sensitivity, that is, while the AUC is above 0.9, the specificity and sensitivity are maintained at a high level (such as 90%) and the difference is not large. Specifically, the AUC of the APOE4+GDF-15+PF4 combination reached 0.907, and the AUC of the APOE4+UCH-L1+GDF-15+PF4 combination reached 0.954. From Figure 1 and Figure 2 It can be seen from the results that the simultaneous addition of GDF-15 and PF4 significantly improves the AUC.

[0112] In addition, the data in Table 4 also prove that the addition of GDF-15 or PF4 alone,

[0113] Although it can also improve AUC to a certain extent, compared with other combinations, its effect on improving AUC is not very obvious, which is particularly reflected in the combinations of APOE4+GDF-15+NFL and APOE4+PF4+GFAP.

[0114] The above results of the present invention all show that NFL and GFAP are not suitable for combination with other biomarkers to diagnose Alzheimer's disease or mild cognitive impairment. Surprisingly, the simultaneous addition of GDF-15 and PF4 can enable GFAP and NFL to be used for the diagnosis of Alzheimer's disease or mild cognitive impairment, and can improve the AUC in terms of specificity and sensitivity. The corresponding AUC can even reach more than 95% (for example, 100%). Specifically, the AUC for diagnosing Alzheimer's disease population reached 0.963, the specificity was 100.00%, and the sensitivity was 93.75%; the AUC for diagnosing mild cognitive impairment population was 0.963, the specificity was 100.00%, and the sensitivity was 93.75%; the AUC for diagnosing mild cognitive impairment population was 0.963, the specificity was 100.00%, and the sensitivity was 93.75%.

[0115] The specificity was 100.00% and the sensitivity was 90.00%.

[0116] In summary, the combination of markers provided by the present invention can significantly improve the diagnostic efficacy, and has consistent performance in diagnosing Alzheimer's disease population and mild cognitive impairment population.

[0117] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Use of a biomarker group in preparing a kit for detecting Alzheimer's disease, characterized in that: The biomarker group includes: APOE4, GDF-15, UCH-L1 and PF4; the biomarker group is used to detect blood samples.

2. The use according to claim 1, characterized in that The biomarker panel also includes phosphorylated Tau protein and total Tau protein.

3. The use according to claim 2, characterized in that The ratio of phosphorylated Tau protein content to total Tau protein content was used as the evaluation index.

4. The use according to claim 1, characterized in that The biomarker panel also includes NFL and / or GFAP.

5. The use according to claim 2, characterized in that The phosphorylated Tau protein is any one of p-tau181, p-tau217, p-tau231 and p-tau205.

6. The use according to claim 2, characterized in that The phosphorylated Tau protein is p-tau181 and / or p-tau217.

7. A kit for detecting Alzheimer's disease, characterized in that: The kit includes a detection reagent for a biomarker group, wherein the detection reagent contains chemical magnetic beads and a chemiluminescent substrate. The biomarker group includes: APOE4, GDF-15, UCH-L1 and PF4. The biomarker group is used to detect a blood sample.

8. The kit according to claim 7, characterized in that The biomarker panel also includes phosphorylated Tau protein and total Tau protein.

9. The kit according to claim 7, characterized in that The biomarker panel also includes NFL and / or GFAP.

10. The kit according to claim 8, characterized in that The phosphorylated Tau protein is any one of p-tau181, p-tau217, p-tau231 and p-tau205.

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