CN104267194B - Human glucagon-like peptide-1, antibody and kit thereof - Google Patents

Abstract

The invention discloses four kinds of glucagon-like-peptide-1s, disclose the monoclonal that can identify above-mentioned four peptide species, disclose the polyclonal antibody that can identify above-mentioned four peptide species, the invention also discloses a kind of detection serum, or the ELISA kit of blood plasma GLP-1 total content, include bioactive GLP-1 (7-37), GLP-1? (7-36)? NH2 peptide, the GLP-1 (9-37) of inactive and GLP-1? (9-36)? NH2 peptide, four kinds of peptide chain BSA fusions, the monoclonal antibody of above-mentioned four kinds of GLP-1 can be identified, polyclonal antibody and enzyme labeling polyclonal antibody.The present invention is that a kind of quick, stable, reliable diagnosis, treatment and examination of curative effect GLP-1 lack diseases related effective tool.

Description

Human glucagon-like peptide-1, antibody and kit thereof

Technical field:

The invention belongs to the field of bioengineering, and in particular relates to a polypeptide, monoclonal and polyclonal antibodies and kits thereof.

Background technique:

Statistics in 2010 show that there are 92.4 million adults with diabetes in China (incidence rate is 10%), and what is even more worrying is that there are still a huge number of adults with impaired glucose tolerance (Impaired glucose tolerance, IGT). How to prevent diabetes more effectively, especially the occurrence of type 2 diabetes mellitus (T2DM), effectively control the development of the disease, and reduce the occurrence of complications has become one of the focuses of the whole nation.

Pancreatic α-cells secrete glucagon and β-cells secrete insulin. The two functions are opposite to each other and play a vital role in maintaining the body's blood sugar concentration. In pancreatic α-cells, the main expression product of proglucagon gene is glucagon, while in L cells of intestinal mucosa, the product of proglucagon gene expression is cleaved by prohormone converting enzyme, in which carboxyl The segmented peptide chain at the end is glucagon-like peptide-1 (GLP-1). GLP-1 has two biologically active forms, namely GLP-1(7-37) and GLP-1(7-36)NH2, and about 80% of the circulating activity of GLP-1 comes from GLP-1(7-36). Biologically active GLP-1 is degraded in vivo by dipeptidase (DPP-IV), neutral endopeptidase (NEP) into inactive GLP-1(9-37) and GLP-1(9-36)NH2 , with a very short half-life of only 2 minutes.

Studies have confirmed that GLP-1 promotes insulin secretion from islet β cells in a glucose concentration-dependent manner, and reduces islet glucagon secretion to lower blood sugar. After a normal person eats a meal, GLP-1 starts to be secreted, which in turn promotes the secretion of insulin, so as to reduce the fluctuation of blood sugar after a meal. However, for patients with T2DM, their "incretin effect" is impaired, mainly manifested in that the increase of GLP-1 concentration after meals is less than that of normal people, but its effects of promoting insulin secretion and lowering blood sugar are not significantly affected. damage. Animal experiments and clinical studies have proved that GLP-1 can significantly improve the blood sugar of T2DM animal models or patients through various mechanisms, among which the role of promoting the regeneration and repair of islet β cells and increasing the number of islet β cells is particularly significant, which is T2DM. The treatment offers a very good outlook. At the same time, the glucose concentration-dependent hypoglycemic properties of GLP-1 are the basis and guarantee of its safety in clinical application, thus eliminating people's worries about severe hypoglycemia caused by existing diabetes treatment drugs and programs. In addition, animal and clinical studies have shown that some therapeutic measures, such as gastric bypass surgery for non-obese diabetes, partial gastrectomy for obesity, and traditional Chinese medicine for diabetes, can achieve therapeutic effects by regulating circulating GLP-1 levels. The purpose is to guide and adjust the treatment plan by monitoring the level of GLP-1 in peripheral blood before and after treatment.

In recent years, a number of pharmaceutical companies, such as Eli Lilly, Merck, Bristol-Myers Squibb in the United States, and Roche in Switzerland, are developing GLP-1-related drugs, some of which are in clinical trials, and some have completed Clinical trials have entered the market, indicating that GLP-1 will definitely become one of the commonly used therapeutic drugs for IGT and T2DM in the near future.

Before GLP-1 is used as a new treatment method in clinic, an important basic work must be completed, that is, to establish a rapid and accurate determination method to determine whether there is a real deficiency of GLP-1 secretion in the patient's body! Accurate monitoring of GLP-1 content changes is an essential prerequisite for T2DM type diagnosis, personalized treatment, and different individual drug dosages. In addition, the establishment of detection methods is also a prerequisite for further research on the role of GLP-1 in IGT, T2DM, and metabolism. Syndrome and other GLP-1 deficiency-related diseases pathogenesis and experimental basis for early prevention research. At present, the ELISA kit for the quantitative detection of the total GLP-1 content in the blood of patients has not been reported in China.

Invention content:

The object of the present invention is to provide a kind of human glucagon-like peptide-1, antibody and its kit, and the described human glucagon-like peptide-1, antibody and its kit will solve the problems in the prior art. There is a technical problem with the absence of a suitable ELISA kit for the detection of total GLP-1 levels in the patient's blood.

A human GLP-1 peptide chain, the amino acid sequence is as follows:

GLP-1(7-37):HAEGTFTSDVSSTLEGQAAKEFIAWLVKGRG

A human GLP-1 peptide chain, the amino acid sequence is as follows:

GLP-1(7-36):HAEGTFTSDVSSTLEGQAAKEFIAWLVKGR

A human GLP-1 peptide chain, the amino acid sequence is as follows:

GLP-1(9-37):EGTFTSDVSSTLEGQAAKEFIAWLVKGRG

A human GLP-1 peptide chain, the amino acid sequence is as follows:

GLP-1(9-36):EGTFTSDVSSTLEGQAAKEFIAWLVKGR

The present invention also provides a monoclonal antibody, which is characterized in that it can recognize the polypeptide described in claim 1, 2, 3 or 4.

The present invention also provides a polyclonal antibody, which is characterized in that it can recognize the polypeptide described in claim 1, 2, 3, or 4 and is labeled with HRP.

Further, the monoclonal antibody is secreted by the hybridoma cell line with the preservation number CCTCCNO: C2014108.

The present invention also provides a hybridoma cell strain, the preservation number of which is CCTCCNO: C2014108.

The present invention provides a kit containing the polypeptide according to claims 1-4, or the monoclonal antibody according to claim 5, or the polyclonal antibody according to claim 6.

Further, the kit is used to detect the total content of biological activity and inactivated GLP-1 in serum or plasma.

The present invention also provides a method for measuring the total content of GLP-1 in blood or plasma by ELISA, comprising the steps of:

1) A step of preparing the reaction plate. In the step of preparing the reaction plate, the monoclonal antibody with the preservation number CCTCCNO: C2014108 is used as the coating antibody, diluted 1:1000 with the coating buffer, and added to the microplate In the well plate, 100ul per well, put in the refrigerator at 2-5°C, take it out after 20-50 hours, rinse with distilled water 2-6 times, pat dry, add 0.5% BSA-PBS in the micro-well plate, 100ul per well, Place in a water bath at 30-40°C for 1-3 hours, take it out, rinse with distilled water 2-6 times, and pat dry;

2) A step of configuring the standard solution, first configure the buffer, the buffer is 0.5% BSA-0.05% Tween20-PBS, then take the synthetic peptide antigen GLP-1(9-36)NH2, and prepare it with the buffer, Formulated into a 74.5uM solution for cryopreservation;

3) Take the above-mentioned GLP-1(9-36) fusion protein (74.5umol/L) and seal it with blocking solution, 100ul per well, put it in a water bath at 30-40°C for 1-3 hours, take it out, and rinse it with distilled water for 2-6 times , pat dry;

4) Add HRP-labeled rabbit anti-GLP-1 antibody, make a 1:1500 dilution with pH7.2, 0.5% BSA-0.05% Tween20-0.15molPBS, add to a microwell plate, 100ul per well, place at 2-5°C In the refrigerator, take it out after 20-50 hours, rinse with distilled water 2-6 times, and pat dry;

5) add o-phenylenediamine substrate for color development, room temperature for 15 minutes, stop color development with 2mol sulfuric acid;

6) Use the main wavelength of 492nm and the secondary wavelength of 640nm for colorimetry;

7) Each 96-well ELISA plate is equipped with a standard curve of 0-2980nmol/L and a blank well. Draw the standard curve with the absorbance as the abscissa and the concentration as the ordinate, and draw the absorbance value of each sample to be tested on the standard curve. Find out the concentration of GLP-1 in this sample.

The monoclonal and polyclonal antibodies provided by the present invention can be used to establish other immunoassay methods to determine the total content of human GLP-1, such as colloidal gold rapid detection, immunoturbidimetric method, and the like.

A kind of hybridoma cell strain of the present invention, its classification name is: hybridoma cell strain 2D11-2, and this cell strain is preserved in China Type Culture Collection Center (CCTCC), and the address of China Type Culture Collection Center is: Hubei Wuhan University (Luojia Mountain, Wuchang, Wuhan City), Wuhan City, Province, the preservation date is July 1, 2014, and the preservation number is CCTCCNO: C2014108.

The ELISA kit provided by the invention has a linear range of 1490 nmol/L to 23.5 nmol/L for measuring GLP-1 in serum, and an analysis sensitivity of 23.5 nmol/L. After preliminary clinical detection and analysis, the reference range of fasting serum total GLP-1 in healthy adults is 79.5±39.7nmol/L. The fasting serum total GLP-1 content of IGT and T2DM patients was higher than that of the normal glucose tolerance group, and the serum total GLP-1 level of the normal glucose tolerance group was significantly increased 1 hour after the meal, and returned to the fasting level at 2 hours. However, in the serum of IGT and T2DM patients, GLP-1 did not increase at 1h after a meal but decreased significantly, and was close to the fasting level at 2h. Preliminary clinical test results prove that the present invention not only provides hematological indicators for early diagnosis of IGT and T2DM, but also provides necessary monitoring indicators for the clinical application of GLP-1 biological preparations. It also provides efficacy detection indicators for some other treatments for diabetes and obesity. In addition, in addition to IGT and T2DM, recent studies have reported that metabolic syndrome, coronary heart disease, and Alzheimer's disease (Alzheimer's disease, AD) are all related to abnormal secretion of GLP-1. The various GLP-1 polypeptides and their Antibodies and ELISA kits all play an important role in the research on the pathogenesis and prevention of related diseases.

The present invention is aimed at the impairment of the "incretin effect" in patients with type 2 diabetes (T2DM), and the artificially synthesized GLP-1 preparation can promote the regeneration and repair of pancreatic islet β cells, and effectively improve the blood sugar status of T2DM animal models or patients , to establish an ELISA detection method for the total content of GLP-1 in blood. The test results found that the overall level of fasting GLP-1 in IGT and T2DM patients was higher than that of normal people, but the postprandial GLP-1 secretion was significantly insufficient. The establishment of the ELISA quantitative detection kit for the total content of GLP-1 will play an important role in the early diagnosis, treatment and curative effect monitoring of diseases related to abnormal GLP-1 secretion such as IGT and T2DM.

The four kinds of human GLP-1 peptide chains and their monoclonal and polyclonal antibodies and quantitative ELISA kits provided by the present invention provide rapid, stable and reliable diagnosis, treatment and curative effect of GLP-1 deficiency-related diseases for clinic Effective tool for detection.

Description of drawings:

Figure 1 is the standard curve for the detection of the total content of GLP-1 by ELISA.

Figure 2 is the distribution of serum total GLP-1 content in normal physical examination subjects.

detailed description:

Example 1 Design and Synthesis of Human Bioactive and Inactive GLP-1 Polypeptides

According to the amino acid sequence of human biological activity and inactivity GLP-1 released on the official website of NCBI, the artificial biosynthesis and purification were carried out (Figure 1 amino acid sequence of the synthetic peptide chain).

Embodiment 2GLP-1 polypeptide and monoclonal and polyclonal antibody characteristics thereof

Preparation of 7-37, 7-36, 9-37, 9-36 polypeptide and BSA fusion protein of biosynthetic GLP-1. Using GLP-1(9-36) NH2 fusion protein, rabbits were immunized by booster immunization method to prepare polyclonal antibody and labeled with horseradish peroxidase. The GLP-1(9-36)NH2 monoclonal antibody was prepared by hybridoma cell fusion technology.

Table 1 identifies the reactivity of rabbit anti-GLP-1 (9-36) NH2 fusion protein polyclonal antibody and 7-37, 7-36, 9-37, 9-36 polypeptide BSA fusion protein antigen of GLP-1 (reaction drop degree), the antiserum reacted with the polypeptide-BSA antigen protein after three-fold serial dilution, and after the signal amplification reaction of the secondary antibody-HRP, it showed different absorbance (OD) and gradient curves on the 96-well ELISA plate. It is confirmed that the prepared rabbit anti-GLP-1(9-36)NH2 polyclonal antibody can recognize GLP-1(7-37), GLP-1(7-36), GLP-1(9-37), GLP-1 (9-36) antigenic peptide, and has higher potency.

Monoclonal antibody prepared with GLP-1(9-36)NH2 as antigen, and GLP-1(7-37), GLP-1(7-36), GLP-1(9-37) antigen peptide filter. Table 2 shows the reaction curves of the screened monoclonal antibody (2D11-2) with each GLP-1 antigen peptide fusion protein, indicating that the screened mouse anti-GLP-1(9-36) NH2 fusion protein monoclonal antibody is also Recognizes other GLP-1 polypeptide BSA fusion proteins with similar potency. After the hybridoma cell line (2D11-2) was subjected to cloning and expansion culture, it was sent to the China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, Hubei Province for preservation, and its preservation number is CCTCC NO: C2014108;

In the experiment of analyzing the reaction characteristics of GLP-1 polyclonal antibody and antigen, the four antigenic peptide fusion proteins of GLP-1 were preliminarily screened by the checkerboard method to coat the ELISA plate with a concentration of 250nmol/L and 100ul/well, and react overnight at room temperature. After the plate is blocked, add 100ul/well of different dilutions of antiserum into the reaction well, wash three times after 1 hour at room temperature, add 100ul/well to dilute HRP-coupled goat anti-rabbit IgG at 1:2000, and wash again after 1 hour at room temperature Four times, add HRP substrate solution (TMB and hydrogen peroxide) and develop color for 10 minutes, read OD492/640nm value. The GLP-1 monoclonal antibody screening method was the same as above, and the enzyme-labeled chromogenic antibody was replaced with HRP-coupled goat anti-mouse IgG.

Table 1. Reaction characteristics of rabbit anti-GLP-1 polyclonal antibody and GLP-1 antigen peptide

Table 2. Reaction characteristics of GLP-1 monoclonal antibody (2D11-2) and GLP-1 antigen peptide

From the analysis of the results in Tables 1 and 2, the prepared GLP-1 (9-36) polyclonal antibody and the screened monoclonal antibody can all recognize four antigenic peptides of GLP-1, indicating that monoclonal and polyclonal antibodies The identified antigenic epitope is the common antigenic epitope in GLP-1 (9-36), (9-37), (7-36), (7-37) peptide chain molecules.

Example 3 Plasma Total GLP-1 Quantitative ELISA Establishment

1. Reaction plate preparation

1. Coated microtiter plate: The monoclonal antibody (2D11-2) with the preservation number CCTCCNO: C2014108 is used as the coating antibody, diluted 1:1000 with coating buffer (CB), and added to the microwell plate, every Hole 100ul. Place at 4°C for 48 hours. Remove, rinse 5 times with distilled water, and pat dry.

2. Sealing: Add 0.5% BSA-PBS to the microwell plate, 100ul per well. Set at 37°C for 2 hours. Remove, rinse 5 times with distilled water, and pat dry.

2. Preparation of standard solution:

1. Buffer: 0.5% BSA-0.05% Tween20-PBS.

2. Take the synthetic peptide antigen GLP-1(9-36)NH2 and prepare it with buffer solution to 74.5uM for cryopreservation.

3. Experimental steps:

1. Add standard: Take the above-mentioned GLP-1(9-36) fusion protein (74.5umol/L) and use blocking solution (for doubling dilution, 100ul per well, 37°C, 2 hours. Wash 5 times with distilled water and pat dry .

2. Add HRP-labeled rabbit anti-GLP-1 antibody, and make a 1:1500 dilution with pH7.2, 0.5% BSA-0.05% Tween20-0.15molPBS. Added to the microwell plate, 100ul per well. Place at 4°C for 48 hours. Remove, rinse 5 times with distilled water, and pat dry.

3. Add o-phenylenediamine substrate for color development, room temperature for 15 minutes, stop color development with 2mol sulfuric acid.

4. Use the main wavelength of 492nm and the secondary wavelength of 640nm for colorimetry.

4. Experimental results:

Table 3. Standard solution detection results (absorbance) for 5 consecutive days

The experimental results show that the detection range is 1490nmol-23.5nmol/L, and the analytical sensitivity is 23.5nmol/L.

Example 4 GLP-1ELISA Reagent Clinical Preliminary Application

1. Research object

Collected 108 cases of fasting venous blood from healthy subjects, and it was confirmed that the fasting blood sugar, liver and kidney function were normal, and blood lipid indicators were normal, without diabetes and other cardiovascular disease history, including 52 male cases, aged 25-76 years, and 56 female cases, aged 25 ~75 years old.

Collect 120 newly diagnosed patients with IGT and T2DM, and collect 50 cases of normal glucose tolerance test. The above-mentioned subjects include at least three time points of fasting, 1h and 2h after taking glucose, and a total of 510 blood samples. None of the patients received GLP-1 or its analogue drug treatment.

Blood samples were collected in ordinary tubes or coagulation-promoting tubes, and the collected serum was frozen at -20°C for detection of serum total GLP-1. Other indicators Blood samples were collected and tested according to conventional methods.

2. Determination of serum total GLP-1 content

All the above-mentioned serum samples were tested intensively according to the operation steps shown in Example 3, and each 96-well microtiter plate was equipped with a 0-2980 nmol/L standard curve and blank wells.

3. Detection of routine biochemical items such as blood sugar and glycosylated hemoglobin

Using the automatic biochemical analyzer and its supporting reagents from Roche Diagnostic Products Company of Germany, the blood glucose, insulin (In), cholesterol, triglyceride, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) of the above samples were detected according to the clinical test routine. ), C-reactive protein (CRP), and glycosylated hemoglobin (HbA1c) were detected using a Japanese TOSOH G8 glycosylated hemoglobin analyzer and supporting reagents.

4. Results

1. Normal fasting serum total GLP-1 content

According to the concentrated detection method of Example 3, the fasting total GLP-1 content of healthy adults was obtained as 79.5 ± 39.7nmol/L. Figure 2 shows the distribution of 108 normal people's fasting serum total GLP-1 content, from which it can be found that people with normal glucose metabolism The fasting basal value of GLP-1 was quite different, and the content>100nmol/L accounted for 13.89%.

2. Clinical application analysis of IGT and T2DM

The test results of all clinical samples are shown in Table 4. Analysis and detection results showed that the fasting serum total GLP-1 content of T2DM patients was higher than that of the normal group, and that of the IGT group was higher, but the statistical difference was not yet reached. The total serum GLP-1 content of the three groups was tested while receiving the glucose tolerance test. It was found that the total serum GLP-1 content of the NGT group increased significantly at 1 h after the meal and gradually fell back at 2 h, while the IGT group and the T2DM group showed no difference at 1 h after the meal. There was a trend of rising instead of falling, and the recovery of total serum GLP-1 was not significant until 2 hours after the meal, suggesting that there was a defect in GLP-1 secretion in the IGT group and the T2DM group.

In addition, observing the change of serum total GLP-1 content in each column of research subjects during the glucose tolerance test, it was found that 50 cases of NGT fasting, postprandial 1h, 2hGLP-1 change trend is consistent. The change trend of GLP-1 in 5 of 52 IGT patients and 3 of 68 T2DM patients was consistent with NGT, and the GLP-1 level increased significantly 1 hour after a meal, and then fell back after 2 hours, indicating that not all IGT, T2DM patients All patients have insufficient secretion of GLP-1 after meals, and there may be other reasons for abnormal glucose metabolism in patients.

Table 4. Clinical and biochemical indicators of 170 research subjects in the fasting state

NGT: normal glucose tolerance group; IGT: abnormal glucose tolerance group; T2DM: type 2 diabetes group; FBG: fasting blood glucose; In: insulin; TG: triglyceride; TC: cholesterol; LDL: low-density lipoprotein; HDL: high Density lipoprotein; HbA1c: glycosylated hemoglobin; CRP: C-reactive protein.

Table 5. Analysis of changes in serum total GLP-1 after meals in patients with IGT and T2DM

*Comparison between IGT group or T2DM group and NGT group (*p<0.05, **p<0.01); ^{ΔComparison between 1h and 2h postprandial and fasting (Δp<0.05, ΔΔp <} 0.01). In the fasting state, the level of GLP-1 in the T2DM group was significantly higher than that in the NGT group (p=0.006), but there was no significant difference from the IGT group. Comparing the postprandial GLP-1 levels in each group, the postprandial 1h GLP-1 level in the NGT group increased significantly, and fell back to the fasting level after 2h, while the IGT group decreased 1h postprandially (p=0.000), and was still lower than the fasting level at 2h postprandial level (p=0.003). The postprandial 1hGLP-1 level in the T2DM group was significantly lower (p=0.011), and the postprandial 2hGLP-1 level was still low numerically, but had no significant difference from the fasting level (p=0.056).

Conclusion: adopt the serum (plasma) total GLP-1 ELISA detection method established by the present invention, first establish normal people fasting serum GLP-1 reference range is 79.5 ± 39.7nmol/L, and find the fasting basic value difference of GLP-1 among normal individuals larger. Analyzing the total GLP-1 content in the blood of IGT and T2DM patients at different time periods after fasting and after meals, it was found that 112 of 120 IGT and T2DM patients had postprandial GLP-1 secretion defects. The significance of the present invention lies in that the disclosed serum (plasma) total GLP-1 quantitative ELISA detection method can dynamically and quantitatively monitor changes in total GLP-1 content in blood, and is useful for typing diagnosis of T2DM, personalized treatment and other GLP-1 The lack of research on the pathogenesis and early prevention of related diseases (metabolic syndrome, coronary heart disease, senile dementia, etc.) has laid an experimental foundation. In addition, the detection of total GLP-1 content in the blood, combined with the detection of bioactive GLP-1, can fully understand the secretion and inactivation of GLP-1 in the subject, and is an indispensable tool for basic and clinical research on GLP-1 .

Claims (4)

1. A monoclonal antibody, characterized in that: it is secreted by a hybridoma cell line whose preservation number is CCTCCNO: C2014108. 2. A hybridoma cell line, the preservation number of which is CCTCCNO: C2014108. 3. A kit, characterized in that: it contains the monoclonal antibody according to claim 1. 4. A method adopting ELISA to measure the total content of GLP-1 in blood or plasma, is characterized in that comprising the steps: 1) A step of preparing the reaction plate. In the step of preparing the reaction plate, the monoclonal antibody with the preservation number CCTCCNO: C2014108 is used as the coating antibody, diluted 1:1000 with the coating buffer, and added to the microplate In the well plate, 100ul per well, put it in the refrigerator at 2~5℃, take it out after 20~50 hours, rinse with distilled water 2~6 times, pat dry, add 0.5%BSA-PBS in the microwell plate, 100ul per well, Place in a water bath at 30-40°C for 1-3 hours, take it out, rinse with distilled water 2-6 times, and pat dry; 2) A step of preparing the standard solution, first prepare the buffer solution, the buffer solution is 0.5%BSA-0.05%Tween20-PBS, then take the synthetic peptide antigen GLP-1(9-36)NH ₂ and prepare it with the buffer solution , formulated into a 74.5uM solution for cryopreservation; 3) Take the above-prepared 74.5uM solution, dilute it with blocking solution, 100ul per well, put it in a water bath at 30-40°C for 1-3 hours, take it out, rinse it with distilled water 2-6 times, and pat dry; 4) Add HRP-labeled rabbit anti-GLP-1 antibody, make a 1:1500 dilution with pH7.2, 0.5%BSA-0.05%Tween20-0.15molPBS, add to the microwell plate, 100ul per well, place at 2~5℃ In the refrigerator, take it out after 20-50 hours, rinse with distilled water 2-6 times, and pat dry; 5) Add o-phenylenediamine substrate for color development, room temperature for 15 minutes, stop color development with 2mol sulfuric acid; 6) Use the main wavelength of 492nm and the secondary wavelength of 640nm for colorimetry; 7) Each 96-well ELISA plate is equipped with a standard curve of 0-2980nmol/L and a blank well. Draw the standard curve with the absorbance as the abscissa and the concentration as the ordinate, and use the absorbance value of each sample to be tested on the standard curve Find out the concentration of GLP-1 in this sample.