Glucagon-like peptide-1 content detection pretreatment method

Abstract

The invention provides a pretreatment method for improving the content detection precision of glucagon-like peptide-1 (GLP-1). By adopting a specific enzyme inhibitor combination, the method can effectively prevent degradation of GLP-1 in a sample, so that a more reliable sample is provided for subsequent quantitative analysis. The method is particularly suitable for GLP-1 content detection in clinical diagnosis and research.

Description

Technical Field

[0001] The present invention provides a pretreatment method for improving the accuracy of glucagon-like peptide-1 (GLP-1) content detection. By adopting a specific enzyme inhibitor combination, the method can effectively prevent the degradation of GLP-1 in the sample, thereby providing a more reliable sample for subsequent quantitative analysis. This method is particularly suitable for GLP-1 content detection in clinical diagnosis and research. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is an important incretin that plays a key role in regulating postprandial blood glucose levels. However, since GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPP-4) in the body, its half-life in the blood is very short, about 2 minutes. Traditional detection methods often have difficulty accurately measuring the concentration of GLP-1 in samples, especially during storage and detection after collection, when GLP-1 is prone to further degradation. Therefore, it is of great significance to develop an effective pretreatment method to maintain the stability of GLP-1 in samples. Summary of the invention

[0003] 1. Technical issues

[0004] The present invention aims to solve the problem existing in the prior art, namely, how to effectively prevent the degradation of GLP-1 after sample collection to ensure the accuracy of subsequent test results.

[0005] 2. Solution

[0006] Specific stabilizers, such as DPP-4 inhibitor, mixed protease inhibitor cocktail (manufacturer: Merck, catalog number: 11697498001) or aprotinin, are added to the sample collection tube to prevent the degradation of GLP-1.

[0007] 3. Beneficial effects

[0008] The pretreatment method provided by the present invention significantly improves the stability of GLP-1 in the sample, thereby making subsequent detection more reliable and accurate, which is of great value for clinical diagnosis, drug development and diabetes research. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : The effect of each inhibitory system in Example 2 on preventing GLP-1 degradation. DETAILED DESCRIPTION

[0010] Example 1: 5x cocktail inhibitor can effectively inhibit the degradation of GLP-1 in the blood

[0011] The preliminary experimental results showed that a 5-fold diluted mixed protease inhibitor (hereinafter referred to as "cocktail inhibitor") showed a significant effect in inhibiting the degradation of GLP-1 in serum. Based on this finding, the present invention further designed an experimental scheme, that is, mixing a GLP-1 standard of known concentration with the above-mentioned cocktail inhibitor and a fresh mouse serum sample. Subsequently, the actual content of GLP-1 in the mixture was determined using an enzyme-linked immunosorbent assay kit (manufacturer: Merck, catalog number: E1036) to evaluate the specific inhibitory efficacy of the cocktail inhibitor on the degradation process of GLP-1.

[0012] Experimental procedures

[0013] 1) Take fresh blood from C57 mice and put it in an anticoagulant tube. After standing at room temperature for half an hour, centrifuge at 3000g for 15 minutes and keep the supernatant (serum) for later use.

[0014] 2) Prepare GLP-1 standard, and prepare experimental group and control group samples according to the grouping information shown in Table 1. Mix the experimental group samples and incubate them at 4°C for 30 minutes.

[0015] Table 1 Experimental group information

[0016]

[0017] 3) Take out the coated 96-well microplate in the GLP-1 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 GLP-1 in the sample to bind to the antibody at the bottom of the well.

[0018] 4) Discard the sample solution and wash 5 times with washing buffer.

[0019] 5) Add 100 μL of specific antibody labeled with horseradish peroxidase (HRP) to each well and incubate at 37°C for 1 hour.

[0020] 6) Discard the antibody solution and wash 5 times with washing buffer.

[0021] 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.

[0022] 8) When the color change reaches the expected level, add 100 μL of stop solution to each well to terminate the reaction.

[0023] 9) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0024] 10) Perform four-parameter fitting analysis on the OD values ​​of the standard control group samples measured at a wavelength of 450nm, and calculate the GLP-1 concentration of the experimental group samples according to the following formula:

[0025] Y=Bottom+(Top-Bottom)*X hillslope / (X hillslope +IC50 hillslope )

[0026] 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.

[0027] Experimental Results

[0028] According to the fitting equation Y = 10.28 + (3703756-10.28) * X 1.548 / (X 1.548 +408.3 1.548 ) The GLP-1 concentration of each well is calculated as shown in the following table:

[0029] Table 2 GLP-1 concentration fitting calculation results and inhibitory effects of the experimental group

[0030]

[0031] Note:

[0032] * The OD value data of each well in the table is the result after zeroing the zero well;

[0033] #: Inhibition rate (%) = calculated concentration of experimental group / calculated concentration of GLP-1 standard control group * 100%

[0034] The experimental results showed that a 5-fold diluted cocktail inhibitor could effectively inhibit the degradation of GLP-1, and the inhibition rate of each concentration was over 60%.

[0035] Example 2: Evaluation of the inhibitory effects of aprotinin and DPP4-I

[0036] To further explore the anti-degradation effect of GLP-1, the present invention added different concentrations of aprotinin and dipeptidyl peptidase-4 inhibitor (DPP4-I) to a system containing a 5-fold diluted cocktail inhibitor. By systematically evaluating the effects of these ingredients on GLP-1 degradation, the aim is to optimize the inhibitor combination to achieve the best protective effect.

[0037] Experimental procedures

[0038] 1) Prepare the experimental group inhibition system samples as shown in the following table (GLP-1 standard is not added in this step):

[0039] Table 3 Inhibitors and their final concentrations in the experimental groups

[0040]

[0041] 2) Take fresh blood from C57 mice and put it in an anticoagulant tube. Mix it evenly with the inhibitor system prepared in step 1 without standard substance in a ratio of 1:1. After standing at room temperature for half an hour, centrifuge at 3000g for 15 minutes and keep the supernatant.

[0042] 3) Add the GLP-1 standard of the corresponding concentration in Table 3 to the serum obtained in step 2, mix well and incubate at 4°C for 30 minutes.

[0043] 4) Take out the coated 96-well microplate in the GLP-1 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 GLP-1 in the sample to bind to the antibody at the bottom of the well.

[0044] 5) Discard the sample solution and wash 5 times with washing buffer.

[0045] 6) Add 100 μL of specific antibody labeled with horseradish peroxidase (HRP) to each well and incubate at 37°C for 1 hour.

[0046] 7) Discard the antibody solution and wash 5 times with washing buffer.

[0047] 8) Add 100 μL TMB (3,3',5,5'-tetramethylbenzidine) to each well and incubate at room temperature in the dark for 15-30 minutes.

[0048] 9) When the color change reaches the expected level, add 100 μL of stop solution to each well to terminate the reaction.

[0049] 10) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0050] 11) Perform four-parameter fitting analysis on the OD values ​​of the standard control group samples measured at a wavelength of 450nm, and calculate the GLP-1 concentration of the experimental group samples according to the following formula:

[0051] Y=Bottom+(Top-Bottom)*X hillslope / (X hillslope +IC50 hillslope )

[0052] 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.

[0053] Experimental Results

[0054] According to the fitting equation Y = 1.78 + (2890389-1.78) * X 1.245 / (X 1.245 +1267 1.245 ) The GLP-1 concentration of each well is calculated as shown in the following table:

[0055] Table 4 GLP-1 concentration fitting calculation results and inhibitory effects of the experimental group

[0056]

[0057] Note: * The OD value data of each hole in the table is the result after zero hole zeroing;

[0058] #The calculated concentrations were all higher than the concentration of the added GLP-1 standard bottle, which suggests that the inhibitor system may have simultaneously inhibited the degradation of GLP-1 in serum.

[0059] The experimental results showed that in a system containing a 5-fold diluted cocktail inhibitor, 1μM aprotinin and 5μM DPP4-I, the combination exhibited the best effect in preventing GLP-1 degradation.

Claims

1. A pretreatment method for improving the detection accuracy of glucagon-like peptide-1 (GLP-1) content, characterized in that: The method comprises the following steps: Add a specific inhibitor combination to the sample collection tube to prevent the degradation of GLP-1 in the sample; The stabilizer combination includes a dipeptidyl peptidase IV inhibitor (DPP-4 inhibitor), a cocktail inhibitor and aprotinin.

2. The method according to claim 1, characterized in that The concentration of the DPP-4 inhibitor is ≥5 μM.

3. The method according to claim 1 or 2, characterized in that: The cocktail inhibitor concentration is a 5-fold or less dilution concentration.

4. The method according to any one of claims 1 to 3, characterized in that: The concentration of aprotinin is ≥1 μM.

5. The method according to any one of claims 1 to 4, characterized in that: The inhibitor combination in the sample collection tube can significantly improve the stability of GLP-1 in the sample, thereby ensuring the accuracy of subsequent test results.

6. The method according to any one of claims 1 to 5, characterized in that: The method is particularly suitable for GLP-1 content detection in clinical diagnosis, drug development and diabetes research.

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