Application of A20 protein detection reagent in preparation of detection kit

Abstract

The invention discloses application of a reagent for detecting A20 protein in preparation of a detection kit, and particularly relates to application of the reagent for detecting A20 protein in preparation of a kit for detecting lumbar vertebra bone mineral density of male patients with type 2 diabetes. The reagent for detecting the A20 protein is applied to preparation of a kit for diagnosing bone mass decline of male patients with type 2 diabetes mellitus. The combined biomarker provided by the invention can be used for preparing a diagnostic kit, has excellent sensitivity and specificity when being applied to diagnosis of bone mass decline of male patients suffering from type 2 diabetes mellitus, and is beneficial to early screening and recognition of high-risk groups suffering from osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to the application of a reagent for detecting A20 protein in the preparation of a detection kit. Background Art

[0002] The prevalence of diabetes in adults in my country is 11.2%, of which type 2 diabetes accounts for more than 90%. Diabetes can lead to a variety of complications, and there is increasing evidence that "diabetic bone disease" is also a complication of diabetes. In China, about 37.8% of patients with type 2 diabetes have osteoporosis. Osteoporosis is a metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure. With the aging of the population and changes in lifestyle, the prevalence of osteoporosis is on the rise. Fragility fractures are the most serious complication of osteoporosis, and the most common sites are the thoracic and lumbar spine, distal forearm and hip. The occurrence of fractures will lead to a significant increase in the disability and mortality rates of the elderly, bringing heavy medical and nursing burdens to families and society. Previous studies have shown that type 2 diabetes is an independent risk factor for brittle fractures at various sites, and bone density is an important influencing factor for the occurrence of brittle fractures. Therefore, exploring predictive markers of bone density in patients with type 2 diabetes has important clinical significance and application value. Summary of the invention

[0003] The object of the present invention is to address the deficiencies in the prior art and provide an application of a reagent for detecting A20 protein in the preparation of a detection kit.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] The first aspect is to provide the use of a reagent for detecting A20 protein in the preparation of a kit for detecting lumbar bone density in male patients with type 2 diabetes.

[0006] The second aspect is to provide the use of a reagent for detecting A20 protein in the preparation of a kit for diagnosing bone loss in male patients with type 2 diabetes.

[0007] Furthermore, the kit also includes a device for obtaining BMI and calf circumference, as well as a reagent for detecting triglycerides and a reagent for detecting low-density lipoprotein.

[0008] Among them, the device for obtaining BMI is a common height and weight measuring device, or a card is provided for the patient to fill in by himself; the device for obtaining the calf circumference is a common soft tape measure, etc.

[0009] Furthermore, the reagent for detecting A20 protein is an enzyme-linked immunosorbent assay reagent.

[0010] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0011] The combined biomarker provided by the present invention can be used to prepare a diagnostic kit, and when used to diagnose bone loss in male patients with type 2 diabetes, it has excellent sensitivity and specificity, which is helpful for early screening and identification of people at high risk of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Serum A20 levels in the normal bone mass group and the osteopenia group are shown.

[0013] Figure 2 Binary logistic analysis shows the association between A20 and osteopenia in men with type 2 diabetes.

[0014] Figure 3 Receiver operating characteristic (ROC) curve of A20 combined with BMI, triglycerides, low-density lipoprotein, and calf circumference as markers of bone loss in male patients with type 2 diabetes. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0016] (1) Research subjects: From January to December 2022, patients with type 2 diabetes were recruited from the inpatient department of the Department of Endocrinology, Tongji Hospital, Shanghai, and non-diabetic patients were recruited from the Ganquan Street Community Health Service Center, Shanghai, and the Yichuan Street Community Health Service Center, Shanghai.

[0017] Inclusion and exclusion criteria: A total of 183 non-diabetic patients and 189 type 2 diabetic patients were included. Patients with secondary osteoporosis, previous use of drugs that affect bone metabolism, malignant tumors, autoimmune diseases, and serum creatinine >100 μmol·L were excluded. -1 , ALT or AST > 80 U·L -1 , estimated glomerular filtration rate (eGFR) ≤ 90 ml·min -1 ·(1.73m 2 ) -1 .

[0018] (2) Collection of clinical data:

[0019] The subjects' baseline clinical data, including age, gender, smoking and drinking history, duration of type 2 diabetes, and medication history, were collected through a face-to-face questionnaire.

[0020] Short Physical Performance Battery (SPPB): The SPPB score is the sum of the scores of the following three tests: balance test, 4-meter gait test, and five sit-ups test. The total score of the SPPB is 12 points, and the higher the score, the stronger the physical function.

[0021] The human body parameters include waist circumference, hip circumference, thigh circumference, and calf circumference. All measurements were taken by a professional using a soft tape measure. The patient was standing, fasting, and breathing calmly. Each item was measured twice, and the average value was taken. The measurement results were expressed in cm. WHR = waist circumference / hip circumference.

[0022] All patients used an electronic handgrip dynamometer to test their grip strength, and the instrument was calibrated before testing. The grip strength of the left and right hands was measured three times, with an interval of 2 minutes between each measurement. The average grip strength of both hands was recorded and the results were expressed in kg.

[0023] (3) Testing of blood biochemical indicators:

[0024] Blood sample collection: Fasting venous blood was collected by nurses between 8:00 and 10:00 a.m. and immediately placed on ice. The samples were centrifuged at 4°C, 3000 rpm / min for 5 minutes to separate the serum. The samples were stored at -80°C.

[0025] Hemoglobin was quantitatively detected by cyanomethemoglobin colorimetry. Electrolytes, lipid indexes, liver function, and serum creatinine were measured by fully automatic serum chemistry analyzer (Beckman, AU5800). HbA1c was measured by whole blood high performance liquid chromatography. Fasting insulin level was measured by electrochemiluminescence (Roche, COB8000). Fasting blood glucose was measured by oxidase method; serum OC, CTX, and PINP were measured by electrochemiluminescence (Roche, Cobas601); and serum TRACP-5b level was measured by enzyme-linked immunosorbent assay.

[0026] (4) Bone density measurement: Dual-energy X-ray absorptiometry (DXA; HOLOGIC Discovery; coefficient of variation <1%) was used to detect bone density at specific sites (lumbar vertebrae 1-4, femoral neck, and total hip joint). To minimize errors, each operation was performed by the same technician. The unit of bone density is grams per square centimeter (g / cm 2 ). A BMD T-score < -1.0 at the hip and / or lumbar spine is considered osteopenia.

[0027] (5) Enzyme linked immunosorbent assay (ELISA) was used to measure the concentration of serum A20 protein:

[0028] The enzyme-linked immunosorbent assay (ELISA) kit (CUSABIO, Shanghai, China) was used. The sensitivity was 5.8 pg / mL. The intra-group and inter-group differences were <10%.

[0029] Operation steps: ① Move all reagents to room temperature (18-25℃) and equilibrate for at least 30 minutes, prepare reagents and set aside. ② Sample addition: Set up standard wells and sample wells to be tested. Add 100μL of standard or sample to be tested to each well, shake gently to mix, cover with plate sticker, and incubate at 37℃ for 2 hours. ③ Discard the liquid, spin dry, and do not wash. ④ Add 100μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1 hour. ⑤ Discard the liquid in the well, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200μL / well, spin dry. ⑥ Add 100μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1 hour. ⑦ Discard the liquid in the well, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200μL / well, spin dry. ⑧Add 90μl of substrate solution to each well in sequence, and color at 37℃ in the dark for 15-30 minutes. ⑨Add 50μl of stop solution to each well in sequence to terminate the reaction. ⑩Measure the optical density (OD value) of each well in sequence at a wavelength of 450nm within 5 minutes after the reaction is terminated using an ELISA reader.

[0030] (6) SPSS 20.0 software was used to perform statistical analysis on the data. The Shapiro-Wilk method was used to test the normality of the data. Continuous variables were described as Mean±SD if they were normally distributed, and as medians if they were skewed (p25-p75). Categorical parameters were described as percentages and numbers. For normally distributed variables, the t-test was used to compare the differences between different groups. Otherwise, the Mann-Whitney U test was used for analysis. The chi-square test was used to analyze the differences in categorical variables. The association between A20 and lumbar spine bone mineral density was analyzed using multivariate linear regression. The association between A20 and bone loss was analyzed using binary logistic regression. P<0.05 was considered statistically significant.

[0031] The results show:

[0032] refer to Figure 1 In male patients with type 2 diabetes, the serum A20 level in the osteopenia group was lower than that in the normal bone mass group. In male non-diabetic patients, there was no difference in serum A20 level between the osteopenia group and the normal bone mass group. In postmenopausal women without diabetes and type 2 diabetes, there was no difference in serum A20 level between the osteopenia group and the normal bone mass group.

[0033] A20 was transformed into natural logarithm for analysis. Pearson analysis showed that A20 was positively correlated with lumbar spine BMD in male patients with type 2 diabetes, but not with femoral neck and total hip BMD. Univariate regression results showed that A20, insulin use, and duration of diabetes were positively correlated with lumbar spine BMD (see Table 1).

[0034] Table 1

[0035]

[0036]

[0037] The results of multiple linear regression showed that after further correction for fasting blood glucose, insulin use, and duration of diabetes, A20 was still positively correlated with lumbar spine bone mineral density (Table 2).

[0038] Table 2

[0039]

[0040] refer to Figure 2 The results of binary logistic regression showed that after further correction of body mass index (BMI), triglycerides, low-density lipoprotein, and calf circumference in male patients with type 2 diabetes, the risk of osteopenia decreased by 58% for every 1 unit increase in A20.

[0041] refer to Figure 3 , ROC analysis results showed that A20 combined with BMI, triglycerides, low-density lipoprotein, and calf circumference had a good value in diagnosing osteopenia (male patients with type 2 diabetes), with an area under the curve of 0.800 (95% CI: 0.675-0.925).

[0042] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of the reagent for detecting A20 protein in the preparation of a kit for detecting lumbar bone density in male patients with type 2 diabetes.

2. Application of the reagent for detecting A20 protein in the preparation of a kit for diagnosing bone loss in male patients with type 2 diabetes.

3. The use according to claim 1 or 2, characterized in that: The kit also includes a device for obtaining BMI and calf circumference, a reagent for detecting triglycerides, and a reagent for detecting low-density lipoprotein.

4. The use according to claim 1 or 2, characterized in that: The reagent for detecting A20 protein is an enzyme-linked immunosorbent assay reagent.

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