Double-antibody sandwich ELISA detection kit for rat PYY and application

Abstract

The invention discloses a double-antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) kit for rat PYY (Protein Yeast Yeast) and The kit comprises an antibody-coated elisa plate, a biotin-labeled detection antibody, an HRP-labeled streptavidin, a PYY standard substance, a sample diluent, a scrubbing solution, a substrate developing solution and a stop solution. The detection method comprises the following steps: adding a PYY standard substance and a to-be-detected sample into the antibody-coated elisa plate, sequentially adding the biotin-labeled detection antibody and the HRP-labeled streptavidin, adding a substrate for color development, detecting an OD value, and calculating the concentration of PYY in the to-be-detected sample through a standard curve. According to the present invention, the monoclonal antibody is adopted as the coating antibody, the polyclonal antibody is adopted as the detection antibody, and the biotin-streptavidin amplification system is adopted, such that the specificity is strong, the sensitivity is high, the operation is simple and convenient, and the effective method is provided for the preclinical research of the weight loss and metabolism patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a double antibody sandwich ELISA detection kit for rat PYY and its application. Background Art

[0002] Obesity is a multi-system disease state with multiple associated complications such as musculoskeletal problems, obstructive sleep apnea, cardiovascular disease, infertility and cancer (endometrial, breast, ovarian, prostate, liver, gallbladder, kidney and colon) as well as severe metabolic sequelae, most importantly type 2 diabetes, due to its high morbidity and mortality through vascular complications such as ischemic heart disease, stroke and peripheral vascular disease. How to more effectively prevent obesity and diabetes, effectively control the progression of the disease and reduce the occurrence of complications has become one of the focuses of common concern of the whole nation.

[0003] The intestine is an important endocrine organ that can secrete a variety of metabolism-related hormones, regulate the body's glucose and lipid metabolism, and affect the occurrence of obesity and diabetes. Compared with normal individuals, the intestinal hormone levels of obese and T2DM patients change, leading to metabolic disorders, which may be one of the causes of obesity and the development of T2DM. The intestinal hormones closely related to energy metabolism are mainly: glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), cholecystokinin (CCK), peptide tyrosine tyrosine (PYY) and ghrelin. PYY is a gastrointestinal hormone composed of 36 amino acids, which is secreted from L cells together with GLP-1 and oxytocin. PYY (3-36) peptide is derived from full-length PYY (1-36) through processing by the enzyme DPP-IV and binds to neuropeptide Y (NPY) Y2 and Y5 receptors. PYY(3-36) exhibits anorectic effects via the Y2 receptor in the arcuate nucleus; infusion of PYY(3-36) in human volunteers reduced food intake by 33% over 24 hours.

[0004] Studies using a combination of PYY(3-36) and GLP-1 have demonstrated an additive effect in reducing food intake in humans. Functional MRI imaging studies have shown that when subjects are shown pictures of food, brain regions associated with appetite and interest in food are activated. Administration of PYY(3-36) and GLP-1 alone reduced activation in these regions. Co-infusion of PYY(3-36) and GLP-1 resulted in a synergistic effect with near inhibition of these regions, which correlated with appetite suppression as assessed by food intake studies. Co-administration of PYY(3-36) and oxytocin also had an additive effect in reducing food intake in humans compared to each peptide alone. PYY analogs may in the future be used in combination with GLP-1 or other gut hormones to enhance weight loss.

[0005] With the increase of obese people in my country, the development of related drugs is also increasing. The detection of GLP-1 and PYY in preclinical rodents has become an effective marker for the development of metabolic diseases. Obtaining a highly sensitive PYY ELISA detection kit is of great significance for the preclinical diagnosis and scientific research of weight loss and type 2 diabetes. Summary of the invention

[0006] The present invention provides a rat PYY double antibody sandwich ELISA detection kit; Another object of the present invention is to provide a method for using the PYY double antibody sandwich ELISA detection kit; Another object of the present invention is to provide a PYY double antibody sandwich ELISA detection kit for use in detecting the PYY content in rat plasma.

[0007] The above-mentioned purpose of the present invention is achieved by the following technical means: Provided is a double antibody sandwich ELISA detection kit for rat PYY, which comprises an enzyme-labeled plate pre-coated with a monoclonal antibody, a biotin-labeled detection antibody, horseradish peroxidase-labeled streptavidin (HRP-Streptavidin), a PYY standard, a sample diluent, a washing solution, a TMB substrate colorimetric solution and a termination reaction solution.

[0008] The 96-well ELISA plate coated with the monoclonal antibody is a rat PYY monoclonal antibody, which is purchased commercially; The biotin-labeled detection antibody is a rat PYY polyclonal antibody, which is purchased commercially; The PYY standard was obtained from Novus Company; The TMB substrate color developing solution was purchased commercially; The sample diluent is purchased commercially; The termination reaction solution was 2M H 2 SO 4 .

[0009] The invention also provides a method for determining the PYY content in rat serum or plasma using ELISA, comprising the following steps: 100 μl of standard or test sample of different concentrations was added to the ELISA plate pre-coated with PYY monoclonal antibody, incubated at 37°C for 1.5 h, washed, added with biotin-labeled PYY polyclonal antibody, incubated at 37°C for 1 h, washed, added with HRP-labeled streptavidin, incubated at 37°C for 0.5 h, washed, added with TMB substrate colorimetric solution, incubated at 37°C in the dark for 15 min, and added with 2MH 2 SO 4After the stop solution, the OD value of each well was measured at a wavelength of 450 nm using an ELISA reader. A PYY standard curve was drawn and the concentration of PYY in the sample to be tested was calculated using the standard curve.

[0010] The ELISA kit provided by the present invention has a linear range of 15.63 pg / mL to 500 pg / mL for determining PYY in rat serum or plasma, and an analytical sensitivity of 9.38 pg / mL.

[0011] The quantitative ELISA kit provided by the present invention provides an effective tool for rapid, stable and reliable PYY detection in the preclinical stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the standard curve for detecting rat PYY standard. DETAILED DESCRIPTION

[0013] The present invention is further described below in conjunction with experimental examples and specific implementation methods. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0014] (1) Add the diluted PYY standard of different concentrations and the sample to be tested to the ELISA plate coated with PYY monoclonal antibody, 100 μl per well. Set up a negative control well at the same time, add an equal volume of sample dilution, cover with a sealing film, incubate at 37°C for 1.5 h, wash three times, and pat dry; (2) Add biotin-labeled PYY polyclonal antibody, 100 μl per well, cover with sealing film, incubate at 37°C for 1 h, wash three times, and pat dry; (3) Add HRP-labeled streptavidin, 100 μl per well, cover with sealing film, incubate at 37°C for 0.5 h, wash 5 times, and pat dry; (4) Add TMB substrate colorimetric solution, 100 μl per well, incubate at 37°C in the dark for 15 min, add 2 M H 2 SO 4 The reaction was terminated with stop solution, 100 μl per well; (5) Read the OD value of each well at 450 nm using an enzyme reader and draw a standard curve based on the obtained OD values. Figure 1 shown.

[0015] Blood samples were collected using coagulation tubes, and rat plasma was collected and frozen at -80°C for plasma PYY detection.

[0016] All the above plasma samples were centrally tested according to the operation steps shown in Example 1. The rat plasma samples were added to the antibody-coated ELISA plate, 100 μl per well, and positive and negative controls were set at the same time. The samples were incubated at 37°C for 1.5 h, and the biotin-labeled detection antibody was added after washing, and the samples were incubated at 37°C for 1 h. The HRP-labeled streptavidin was added after washing, and the samples were incubated at 37°C for 0.5 h. The TMB substrate colorimetric solution was added after washing, and the samples were incubated at 37°C in the dark for 15 min. After the reaction was terminated, the OD value at 450 nm was detected by an ELISA instrument. The content of PYY in rat plasma was calculated to be 58.62 pg / mL according to the standard curve.

Claims

1. A double antibody sandwich ELISA detection kit for rat PYY, characterized in that: The composition includes an ELISA plate coated with an antibody, a biotin-labeled detection antibody, a horseradish peroxidase-labeled streptavidin (HRP-Streptavidin), a PYY standard, a sample diluent, a concentrated washing solution, a TMB substrate color development solution and a 2M H2SO4 reaction termination solution.

2. The ELISA kit according to claim 1, characterized in that The antibody-coated ELISA plate, wherein the coating antibody is a rat PYY-specific antibody.

3. The ELISA kit according to claim 1, characterized in that The biotin-labeled detection antibody is a biotinylated detection antibody specific to rat PYY.

4. Use of the PYY double antibody sandwich ELISA kit according to claims 1-3 in in vitro detection of rat PYY, characterized in that: The following steps are involved: (1) Add the diluted sample to be tested to the ELISA plate coated with PYY monoclonal antibody, 100 μl per well, set up positive control and negative control wells, incubate at 37°C for 1.5 h, discard the solution, and pat dry; (2) Add biotin-labeled detection antibody, 100 μl per well, incubate at 37°C for 1 h, wash three times, and pat dry; (3) Add HRP-labeled streptavidin, 100 μl per well, incubate at 37°C for 0.5 h, wash five times, and pat dry; (4) Add TMB substrate colorimetric solution, 100 μl per well, incubate at 37°C in the dark for 15 min, add 2 M H2SO4 stop solution, 100 μl per well, to terminate the reaction; (5) Read the OD value of each well at 450 nm using an ELISA reader.

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