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Use of polypeptide combination markers in diabetes diagnosis and its kit and method

A diagnostic kit and marker technology, applied in disease diagnosis, chemical instruments and methods, biological testing, etc., can solve the problems of low specificity, lack of sensitivity of markers, etc., and achieve high diagnostic specificity, high sensitivity, and high development The effect of the applied value

Active Publication Date: 2022-05-31
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of lack of markers for early warning and diagnosis of diabetes, low sensitivity and low specificity, and to provide a combination of polypeptide markers by using non-targeted peptidomics to discover differential polypeptides and targeted peptidomics verification. Its application and kit in early warning / diagnosis of diabetes, and also provides a method for the quantitative analysis and detection of the above-mentioned combined polypeptide markers

Method used

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  • Use of polypeptide combination markers in diabetes diagnosis and its kit and method
  • Use of polypeptide combination markers in diabetes diagnosis and its kit and method
  • Use of polypeptide combination markers in diabetes diagnosis and its kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Screening of differential polypeptides by standard-free quantitative method

[0045] Clinical samples: 23 human serum samples (including 7 healthy people, 7 prediabetic patients, and 9 type 2 diabetes patients) were from Shanghai Sixth People's Hospital. Patients with prediabetes and type 2 diabetes were diagnosed or excluded by experts, and the three groups were matched for age, body mass index (BMI), blood sugar and other indicators. Blood samples were approved by the local hospital ethics committee, and all subjects signed informed consent. Blood samples were stored in a -80°C refrigerator after collection.

[0046] Extraction of serum endogenous polypeptides: Ultrafiltration centrifugation was used. Specific operation process: Take the blood sample from the -80°C refrigerator and thaw it in an ice bath, vortex to mix, and centrifuge at 20,000g for 5min to discard the lipid. Take 25 μL of each serum sample, add 125 μL of deionized water and boil for 5 m...

Embodiment 2

[0054] Example 2: Dimethyl-labeled polypeptides and targeted MRM verification

[0055] Clinical samples: 18 human serum samples (including 9 serum samples from healthy individuals and 9 serum samples from patients with type 2 diabetes) were obtained from Shanghai Sixth People's Hospital. Type 2 diabetes patients were diagnosed or excluded by experts, and the three groups of subjects were matched for age, body mass index (BMI), blood sugar and other indicators. There were no complications such as hypertension and hyperglycemia in the diabetic patients. Blood samples were approved by the local hospital ethics committee, and all subjects signed informed consent. Blood samples were stored in a -80°C refrigerator after collection.

[0056] Extraction of Serum Endogenous Polypeptides: Reference Example 1.

[0057] Synthesis of peptides: According to the information in Table 3 above, the structure of the synthetic target peptides was reconfirmed by comparing their MS spectra.

[...

Embodiment 3

[0079] Example 3: Determination of Early Warning / Diagnostic Markers

[0080] From the above quantitative results, it can be seen that the contents of the polypeptides GVLSSRQLGLPGPPDVPDHAAYHPF(245), GVLSSRQLGLPGPPDVPDHAA(244), GLPGPPDVPDHAAYHPF(251), DIQMTQSPSSVSASVGD(254), and GSEMVVAGKLQ(261) changed significantly in the healthy and diabetic patients.

[0081] Using SPSS statistical software, through binary logistic regression analysis, five peptides were regressed as combined marker variables, and ROC (receiver operating characteristic) curve was used to evaluate the sensitivity and specificity of combined markers. Candidate markers were identified with AUC greater than 0.8.

[0082] The five peptides are regressed as the combined marker variable X, and the binary logistic regression equation is:

[0083] X=16.383+9.702×C(245)+27.806×C(244)-45.662×C(251)-17.920×C(254)-37.420×C(261)

[0084] Prob(diabetes)=1 / [1+e -X ]

[0085] C represents the concentration of the endoge...

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Abstract

The invention relates to the field of medical biological detection, in particular to the new application of polypeptide combination markers in the preparation of diabetes diagnostic reagents or kits. The present invention provides a method and kit for quantitatively detecting endogenous polypeptides in serum by using liquid chromatography-mass spectrometry multiple reaction monitoring (multiple reaction monitoring, MRM). Through the reagents and methods of the present invention, it is possible to quantitatively determine the endogenous polypeptides in human serum The content of the five endogenous polypeptide markers, and then calculate the combined marker variable and the determined cut-off value based on the binary logistic regression equation, for the early warning and / or diagnosis of diabetes. The kit and detection method of the invention have good stability and high specificity, and have the potential of clinical promotion.

Description

technical field [0001] The present invention relates to medical biological detection technology and the fields of diagnosis, prevention and treatment of metabolic diseases, in particular, to the preparation of reagents for early warning and / or diagnosis of diabetes based on a method for quantitative determination of endogenous polypeptides in human plasma and a combination of polypeptide markers Box and method. Background technique [0002] According to the International Diabetes Federation data in 2017, there are 415 million people with diabetes worldwide, of which type 2 diabetes (T2DM) accounts for more than 95%. The number of people with diabetes worldwide is expected to increase to 552 million by 2030. Diabetes is the third major disease that endangers human health after cardiovascular disease and tumor. At present, although the diagnostic criteria for type 2 diabetes have been clarified, and there are gold standards such as oral glucose tolerance test (OGTT), due to ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C07K14/47C07K7/08C07K7/06
CPCG01N33/6848G01N33/6893C07K14/47C07K7/08C07K7/06G01N2800/042G01N2800/50
Inventor 吴仁安王莉牛欢周孝禹
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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