GIP content detection pretreatment method
By using blood collection vessels containing protease inhibitors and adding the DPP-4 inhibitor sitagliptin during blood collection, the problem of instability of GIP during in vitro detection was solved, significantly improving the stability and detection sensitivity of GIP, and providing more accurate data support.
Patent Information
- Application Number
- CN202411859030.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-04-22
AI Technical Summary
The prior art is difficult to accurately measure the GIP concentration in blood samples because GIP is rapidly degraded by DPP-4 in the body, resulting in a short half-life in the blood, and the samples are easily further degraded during storage and detection, resulting in errors and detection difficulties.
Blood collection vessels containing protease inhibitors and the addition of DPP-4 inhibitor sitagliptin to the blood sample were used to improve the stability of GIP.
By using blood collection vessels containing protease inhibitors and sitagliptin, the stability of GIP was significantly improved, and the effective protection rate of protease inhibitors was increased by more than 25%. Sitagliptin provided more than 10%, extending the sample storage time and detection sensitivity, and providing more accurate data support.
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Figure CN119860957A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technology, and in particular to a method for improving the stability of gastrointestinal hormones (such as glucose-dependent insulin peptide, i.e., GIP) in a blood sample, which is achieved by using a specific blood collection tube and a DPP-4 inhibitor. Background Art
[0002] GIP is a hormone secreted by intestinal L cells. It is involved in the regulation of postprandial blood glucose levels and is an important regulatory factor in insulin secretion. Accurately measuring the concentration of GIP in serum or plasma is crucial for studying metabolic diseases such as diabetes. However, since GIP is rapidly degraded by dipeptidyl peptidase IV (hereinafter referred to as "DPP-4") in the body after secretion, its half-life in the blood is about 7 minutes. Traditional detection methods often find it difficult to accurately measure the concentration of GIP in samples, especially during storage and detection after collection, GIP is prone to further degradation. Therefore, it is particularly necessary to develop a method that can effectively maintain the stability of GIP during in vitro detection. Summary of the invention
[0003] 1. Technical issues
[0004] The present invention aims to provide a method for improving the stability of GIP in vitro detection, so as to solve the detection problem caused by the instability of GIP in the prior art, ensure that the GIP in the sample can maintain its original state for a long time, reduce the error caused by degradation, and improve the test sensitivity.
[0005] 2. Technical solution
[0006] In order to solve the above technical problems, the present invention proposes two main technical solutions: ① using a blood collection tube containing a protease inhibitor. ② adding the DPP-4 inhibitor sitagliptin to the blood sample.
[0007] 3. Beneficial effects
[0008] The present invention significantly improves the stability of GIP (glucose-dependent insulin peptide) in blood samples by using blood collection tubes containing protease inhibitors and DPP-4 inhibitor sitagliptin. Among them, the effective protection rate of protease inhibitors on GIP is increased by more than 25%, and sitagliptin also provides more than 10% protection, effectively preventing the degradation of GIP, thereby enhancing the sample preservation time and detection sensitivity, providing more accurate data support for clinical diagnosis and scientific research, and promoting the exploration of new methods for the treatment of GIP-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 : BD in Example 1 TMThe effective protection rate of P800 blood collection tubes for GIP.
[0010] Figure 2 : The effective protection rate of DPP-4 against GIP in Example 2. DETAILED DESCRIPTION
[0011] Example 1: Evaluation of the effect of protease inhibitors
[0012] In order to explore the effect of protease inhibitors on GIP content detection, the present invention uses BD containing protease inhibitors. TM A comparative study was conducted using P800 blood collection tubes (manufacturer: BD, catalog number: 366420). Specifically, ordinary blood collection tubes were used as the control group, and the mouse GIP ELISA kit (manufacturer: Elabscience, catalog number: E-EL-M0539) was used to measure its content. TM P800 blood collection tubes were compared in parallel. The details are as follows:
[0013] Experimental procedures
[0014] 1) Take fresh blood from C57 mice, let it stand at 4°C for 1 hour, centrifuge it at 3000g for 15 minutes, and keep the supernatant (plasma) for later use.
[0015] 2) Prepare GIP standard and prepare samples in ordinary blood collection tubes and BD blood collection tubes according to the grouping information shown in Table 1. TM P800 blood collection tube, mix well and incubate at 4°C for 1 hour.
[0016] Table 1 Experimental group information
[0017]
[0018] 3) Take out the coated 96-well microplate from the GIP kit, add 100 μL of the prepared standard control group or experimental group sample to each well, and incubate at 37°C for 1.5 hours to allow the GIP in the sample to bind to the antibody at the bottom of the well.
[0019] 4) Discard the sample solution and wash 5 times with washing buffer.
[0020] 5) Add 100 μL of specific antibody labeled with horseradish peroxidase (HRP) to each well and incubate at 37°C for 1 hour.
[0021] 6) Discard the antibody solution and wash 5 times with washing buffer.
[0022] 7) Add 100 μL TMB (3,3',5,5'-tetramethylbenzidine) to each well and incubate at room temperature in the dark for 15-30 minutes to develop color.
[0023] 8) When the color change reaches the expected level, add 100 μL of stop solution to each well to terminate the reaction.
[0024] 9) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.
[0025] 10) Perform four-parameter fitting analysis on the OD values of the standard control group samples measured at a wavelength of 450 nm, and calculate the GIP concentration of the experimental group samples according to the following formula:
[0026] Y=Bottom+(Top-Bottom)*X hillslope / (X hillslope +IC50 hillslope )
[0027] Among them, Y is the drug concentration or standard concentration; X is the absorbance value read by the instrument; Bottom is the lower asymptote of the curve; Top is the upper asymptote of the curve; HillSlope is the slope factor, which controls the steepness of the curve; IC50 is the half-maximal inhibition concentration, the inflection point of the curve.
[0028] Experimental Results
[0029] According to the fitting equation Y = 1926 + (199102444-1926) * X 3.098 / (X 3.098 +20.38 3.098 ) The GIP concentration of each well is calculated as shown in the following table:
[0030] Table 2 GIP concentration test results and effective protection rate of each group
[0031]
[0032] *Effective protection rate (%) = (calculated concentration - standard concentration 0 calculated concentration) / standard concentration * 100%
[0033] The experimental results show that (see Table 2, Figure 1 ), BD TM The effective protection rate of protease inhibitors in P800 blood collection tubes against GIP at all concentrations is above 60%. TM The effective protection rate of GIP in samples by P800 blood collection tube increased by more than 25%.
[0034] Example 2: Evaluation of the efficacy of DPP-4 inhibitors
[0035] In order to further explore the protective effect of DPP-4 inhibitors on GIP in blood samples, the present invention uses sitagliptin to conduct research. By adding 500 μM sitagliptin to GIP standards of different concentrations, mixing with fresh mouse plasma and incubating, the actual content of GIP in the mixture was determined using a mouse GIP enzyme-linked immunosorbent assay kit (manufacturer: Elabscience, catalog number: E-EL-M0539), so as to evaluate the specific inhibitory efficacy of the DPP-4 inhibitor on the GIP degradation process.
[0036] Experimental procedures
[0037] 1) Take fresh blood from C57 mice, let it stand at 4°C for 1 hour, centrifuge it at 3000g for 15 minutes, and keep the supernatant (plasma) for later use.
[0038] 2) Prepare GIP standard and samples according to the grouping information shown in Table 3 below, mix well and incubate at 4°C for 1 hour.
[0039] Table 3 Experimental group information
[0040]
[0041] 3) Take out the coated 96-well microplate from the GIP kit, add 100 μL of the prepared standard control group or experimental group sample to each well, and incubate at 37°C for 1.5 hours to allow the GIP in the sample to bind to the antibody at the bottom of the well.
[0042] 4) Discard the sample solution and wash 5 times with washing buffer.
[0043] 5) Add 100 μL of specific antibody labeled with horseradish peroxidase (HRP) to each well and incubate at 37°C for 1 hour.
[0044] 6) Discard the antibody solution and wash 5 times with washing buffer.
[0045] 7) Add 100 μL TMB (3,3',5,5'-tetramethylbenzidine) to each well and incubate at room temperature in the dark for 15-30 minutes to develop color.
[0046] 8) When the color change reaches the expected level, add 100 μL of stop solution to each well to terminate the reaction.
[0047] 9) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.
[0048] 10) Perform four-parameter fitting analysis on the OD values of the standard control group samples measured at a wavelength of 450 nm, and calculate the GIP concentration of the experimental group samples according to the following formula:
[0049] Y=Bottom+(Top-Bottom)*X hillslope / (X hillslope +IC50 hillslope )
[0050] Among them, Y is the drug concentration or standard concentration; X is the absorbance value read by the instrument; Bottom is the lower asymptote of the curve; Top is the upper asymptote of the curve; HillSlope is the slope factor, which controls the steepness of the curve; IC50 is the half-maximal inhibition concentration, the inflection point of the curve.
[0051] Experimental Results
[0052] According to the fitting equation Y = 1854 + (166674148-1854) * X 3.397 / (X 3.397 +19.52 3.397 ) The GIP concentration of each well is calculated as shown in the following table:
[0053] Table 4 GIP concentration test results and effective protection rate of each group
[0054]
[0055] *Effective protection rate (%) = (calculated concentration - standard concentration 0 calculated concentration) / standard concentration * 100%
[0056] The experimental results show that (see Table 4, Figure 2 ), compared with the DPBS group, the effective protection rate of sitagliptin on GIP in the samples was only increased by more than 10%.
Claims
1. A method for improving the stability of GIP in a blood sample, characterized in that: The sample is collected using a blood collection tube containing a protease inhibitor, which can increase the effective protection rate of GIP to more than 60%, which is at least 25% higher than that of a sample without a protease inhibitor.
2. A method for improving the stability of GIP in a blood sample, characterized in that: The DPP-4 inhibitor sitagliptin is added to the blood sample at a concentration of ≥500 μM. The sitagliptin can increase the effective protection rate of GIP by at least 10% compared with the control group (such as the DPBS treatment group).
3. The method according to claim 1, wherein the protease inhibitor can effectively prevent GIP degradation, ensure that the sample remains stable during long-term storage, and improve the sensitivity of GIP content detection.
4. The method according to claim 1, further comprising comparing the samples using protease inhibitors in parallel with the samples not using protease inhibitors to evaluate the GIP protective effect.
5. The method according to claim 2, wherein the DPP-4 inhibitor sitagliptin is mixed with different concentrations of GIP standards and then incubated with fresh mouse plasma, and then the GIP content is measured.
6. The method according to claim 2, further comprising comparing the sitagliptin-treated samples with the untreated DPBS group samples to evaluate the effect of the DPP-4 inhibitor on GIP protection.