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

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

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CN104267194B
CN104267194B CN201410489841.3A CN201410489841A CN104267194B CN 104267194 B CN104267194 B CN 104267194B CN 201410489841 A CN201410489841 A CN 201410489841A CN 104267194 B CN104267194 B CN 104267194B
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glp
peptide
antibody
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bsa
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CN104267194A (en
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范列英
刘颖冰
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Shanghai East Hospital
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification

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 bioengineering field, particularly relate to a peptide species and monoclonal, polyclonal antibody and kit.
Background technology:
The Chinese existing maturity-onset diabetes patient of statistical data display of 2010 reaches 9,240 ten thousand (incidences of disease 10%), and more alarming is adult's impaired glucose tolerance person (Impairedglucosetolerance, IGT) also having enormous quantity.How more effectively preventing diabetes, particularly diabetes B (type2diabetetsmellitus, T2DM) occurs, symptom management development effectively, reduces the generation of complication, has become one of focus that the whole nation pays close attention to jointly.
Alpha Cell of islet secretion hyperglycemic factor, β cells secrete insulin, both effects are contrary each other, in maintenance body blood sugar concentration, play most important effect.In alpha Cell of islet, the main expression product of hyperglycemic factor protogene is hyperglycemic factor, and in the L cell of intestinal mucosa, the product of hyperglycemic factor antigen gene expressed is sheared through prohormone converting Enzyme, wherein the section peptide chain of c-terminus is glucagon-like-peptide-1 (glucagon-likepeptide-1, GLP-1).GLP-1 has 2 kinds of biologically active forms, and the circulation being respectively GLP-1 (7-37) and GLP-1 (7-36) NH2, GLP-1 about 80% is active in GLP-1 (7-36).Biologically active GLP-1 is degraded into GLP-1 (9-37) and GLP-1 (9-36) NH2 of non-activity in vivo by dipeptidase (DPP-IV), neutral endopeptidase (NEP), half life period extremely short only 2min.
Research confirms, and GLP-1 promotes islet β cell insulin in concentration of glucose dependence mode, and reduces pancreas islet glucagon secretion thus reduce blood sugar.Normal person is after dining, and GLP-1 starts secretion, and then promotes insulin secretion, to reduce the fluctuation of postprandial blood sugar.But for T2DM patient, its " secretin's effect " is impaired, main manifestations is that after having meal, GLP-1 concentration elevation amplitude calibration ordinary person reduces to some extent, but it promotes that insulin secretion and hypoglycemic effect there is no obviously impaired.Zoopery and clinical research have proved that GLP-1 improves the blood sugar situation of T2DM animal model or patient significantly by number of mechanisms, wherein promote regeneration and the reparation of beta Cell of islet, the effect increasing beta Cell of islet quantity is particularly remarkable, and this is that the treatment of T2DM provides an extraordinary prospect.This concentration of glucose dependence hypoglycemic characteristic of GLP-1 is basis and the guarantee of its clinical practice security simultaneously, thus eliminates people may cause patient's severe hypoglycemia worry to existing Remedies for diabetes and scheme.In addition, animal and clinical studies show, some remedy measures, as Gastric bypass treatment non-obese patients with type Ⅰ DM, partial gastrectomy treatment of obesity and TCM Treatment of Diabetes etc., all by regulating circulation GLP-1 level to reach therapeutic purposes, instructing by GLP-1 level in peripheral blood before and after monitor therapy, adjusting therapeutic scheme simultaneously.
In recent years, multiple pharmacy corporation, as the Li Lai company of the U.S., Merck & Co., Inc., Bristol-Myers Squibb Co., Roche Groups of Switzerland etc. are all at development GLP-1 related drugs, what have is carrying out clinical testing, the clinical testing that completes had comes into the market, and shows GLP-1 one of conventional medicine that must become IGT, T2DM in the near future.
GLP-1 be applied to as new methods for the treatment of clinical before, must complete an important element task, that will be set up assay method fast and accurately exactly and judge whether really there is GLP-1 hyposecretion in patient body! Monitor GLP-1 content, for prerequisites that is all absolutely necessary such as the classification diagnosis of T2DM, personalized treatment and Different Individual dosages, the foundation of detection method is also study GLP-1 lacks diseases related pathogenesis and early stage Prevention Research experiment basis at GLP-1 such as IGT, T2DM, metabolic syndromes further in addition.There is not been reported quantitatively to detect at present the ELISA kit of total GLP-1 content in blood samples of patients at home.
Summary of the invention:
The object of the present invention is to provide a kind of human glucagon-like-peptide-1, antibody and kit thereof, described this human glucagon-like-peptide-1, antibody and kit thereof will solve in prior art the technical matters of the ELISA kit not having total GLP-1 content in suitable detection blood samples of patients.
A kind of humanized GLP-1 peptide chain, amino acid sequence is as follows:
GLP-1(7-37):HAEGTFTSDVSSTLEGQAAKEFIAWLVKGRG
A kind of humanized GLP-1 peptide chain, amino acid sequence is as follows:
GLP-1(7-36):HAEGTFTSDVSSTLEGQAAKEFIAWLVKGR
A kind of humanized GLP-1 peptide chain, amino acid sequence is as follows:
GLP-1(9-37):EGTFTSDVSSTLEGQAAKEFIAWLVKGRG
A kind of humanized GLP-1 peptide chain, amino acid sequence is as follows:
GLP-1(9-36):EGTFTSDVSSTLEGQAAKEFIAWLVKGR
Present invention also offers a kind of monoclonal antibody, it is characterized in that: the polypeptide described in claim 1,2,3 or 4 can be identified.
Present invention also offers a kind of polyclonal antibody, it is characterized in that: the polypeptide described in claim 1,2,3 or 4 can be identified, and marked by HRP.
Further, described monoclonal antibody is by preserving number secreted by the hybridoma cell strain of CCTCCNO:C2014108.
Present invention also offers a kind of hybridoma cell strain, its preserving number is CCTCCNO:C2014108.
The invention provides a kind of kit, containing the polypeptide described in Claims 1 to 4 or monoclonal antibody according to claim 5 or polyclonal antibody according to claim 6.
Further, described kit is for detecting the GLP-1 total content of biologically active in serum or blood plasma and inactivation.
Present invention also offers a kind of method adopting the method for ELISA to measure GLP-1 total content in blood or blood plasma, comprise the steps:
1) step of a preparation feedback plate, in the step of described preparation feedback plate, preserving number is adopted to be that the monoclonal antibody of CCTCCNO:C2014108 is as coated antibody, be buffered liquid with bag and do 1:1000 dilution, be added in microwell plate, every hole 100ul, puts in 2 ~ 5 DEG C of refrigerators, takes out after 20 ~ 50 hours, with distilled water flushing 2 ~ 6 times, pat dry, in microwell plate, add 0.5%BSA-PBS, every hole 100ul, to put in 30 ~ 40 DEG C of water-baths 1 ~ 3 hour, take out, with distilled water flushing 2 ~ 6 times, pat dry;
2) step of a configuration standard liquid, first configure damping fluid, described damping fluid is 0.5%BSA-0.05%Tween20-PBS, then gets antigenic synthetic peptide GLP-1 (9-36) NH2, join with damping fluid, be mixed with the solution freezen protective of 74.5uM;
3) get above-mentioned GLP-1 (9-36) fusion (74.5umol/L) confining liquid to close, every hole 100ul, to put in 30 ~ 40 DEG C of water-baths 1 ~ 3 hour, take out, with distilled water flushing 2 ~ 6 times, pat dry;
4) add HRP-and mark the anti-GLP-1 antibody of rabbit, do 1:1500 dilution with pH7.2,0.5%BSA-0.05%Tween20-0.15molPBS, be added in microwell plate, every hole 100ul, put in 2 ~ 5 DEG C of refrigerators, take out after 20 ~ 50 hours, with distilled water flushing 2 ~ 6 times, pat dry;
5) add o-phenylene diamine substrate colour developing, room temperature 15 minutes, by 2mol sulfuric acid color development stopping;
6) use predominant wavelength 492nm, pay wavelength 640nm colorimetric;
7) each 96 hole ELISA Plate all establish 0 ~ 2980nmol/L typical curve and blank well, are horizontal ordinate with absorbance, be that ordinate draws typical curve, find the concentration of this sample GLP-1 with the absorbance of each sample to be tested on typical curve with concentration.
Monoclonal provided by the invention, polyclonal antibody, the immunologic detection method that can be used for setting up other measures humanized GLP-1 total content, as collaurum detect fast, immunoturbidimetry etc.
A kind of hybridoma cell strain of the present invention, its Classification And Nomenclature is: hybridoma cell strain 2D11-2, this cell line is preserved in China typical culture collection center (CCTCC), the address of China typical culture collection center is: Wuhan City, Hubei Province Wuhan University (Luo Jia Shan, wuchang, wuhan), preservation date is on July 1st, 2014, and preserving number is CCTCCNO:C2014108.
ELISA kit provided by the invention, measuring the GLP-1 range of linearity in serum is 1490nmol/L ~ 23.5nmol/L, and sensitivity for analysis is 23.5nmol/L.Detect through concept clinical and analyze, the total GLP-1 term of reference of health adult's Diagnostic Value of Fasting Serum is 79.5 ± 39.7nmol/L.The total GLP-1 content of IGT and T2DM patient Diagnostic Value of Fasting Serum is higher than normal glucose tolerance group, and significantly raise the 1 hour after the meal total GLP-1 level of normal glucose tolerance group serum, 2h returns to fasting level.But in IGT and T2DM patients serum, 1hGLP-1 does not rise and significantly reduces on the contrary after the meal, and 2h is close to fasting level.The invention of concept clinical testing result proved provide not only the hematological indices of the early diagnosiss such as auxiliary IGT and T2DM, and is that the clinical practice of GLP-1 class biopreparate provides necessary monitoring index.Also for the other therapies of some treatment diabetes, obesity provides examination of curative effect index.In addition except IGT and T2DM, studies have reported that recently metabolic syndrome, coronary heart disease, Alzheimer disease (Alzheimer ' sdisease, AD) all relevant with GLP-1 diacrisis, various GLP-1 polypeptide provided by the invention and antibody thereof, ELISA kit all play an important role in relevant disease pathogenesis, study on prevention.
This " the secretin's effect " that the present invention be directed to diabetes B (T2DM) patient is impaired, and Prof. Du Yucang GLP-1 preparation can promote beta Cell of islet to regenerate and repair, effectively improves the blood sugar situation of T2DM animal model or patient, sets up GLP-1 total content ELISA detection method in blood.Testing result finds IGT, T2DM patient, and GLP-1 aggregate level calibration ordinary person is higher on an empty stomach, but GLP-1 secretion is after the meal significantly not enough.The foundation of GLP-1 total content ELISA immue quantitative detection reagent box plays a significant role in the diseases related early diagnosis of the GLP-1 diacrisises such as IGT, T2DM, treatment and curative effect monitoring field.
Four kinds of humanized GLP-1 peptide chains provided by the invention and monoclonal, polyclonal antibody and Quantikine ELISA kits, for clinically to provide fast, stable, reliably GLP-1 lack the effective tool of diseases related diagnosis, treatment and examination of curative effect.
Accompanying drawing illustrates:
Fig. 1 is GLP-1 total content ELISA examination criteria curve.
Fig. 2 is the total GLP-1 content distribution of healthy people's serum.
Embodiment:
Embodiment 1 designs and synthesizes humanized's biologically active and non-activity GLP-1 polypeptide
According to people's biologically active that NCBI official website issues, non-activity GLP-1 amino acid sequence, carry out artificial bio-membrane's synthesis, purifying (Fig. 1 synthesizes the amino acid sequence of peptide chain).
Embodiment 2GLP-1 polypeptide and monoclonal thereof and Anti-TNF-α body characteristics
7-37,7-36,9-37,9-36 polypeptide of preparation biosynthesizing GLP-1 and BSA fusion.Adopt GLP-1 (9-36) NH2 fusion, prepare polyclonal antibody with booster immunization method immunizing rabbit, and mark horseradish peroxidase.Adopt hybridoma cell fusion technology, preparation GLP-1 (9-36) NH2 monoclonal antibody.
The 7-37 of the anti-GLP-1 of rabbit (9-36) NH2 fusion polyclonal antibody and GLP-1 identified by table 1, 7-36, 9-37, the reactivity (reaction titre) of 9-36 polypeptide BSA fusion antigen, antiserum reacts with polypeptide-BSA antigen protein after three times of serial dilutions, and after two anti-HRP signal iodines, 96 hole elisa plates demonstrate the absorbance (OD) in difference and gradient curve, to confirm that the anti-GLP-1 of prepared rabbit (9-36) NH2 polyclonal antibody can identify GLP-1 (7-37), GLP-1 (7-36), GLP-1 (9-37), GLP-1 (9-36) antigenic peptides, and have higher tiring.
Be monoclonal antibody prepared by antigen with GLP-1 (9-36) NH2, then adopt GLP-1 (7-37), GLP-1 (7-36), GLP-1 (9-37) antigenic peptides to screen respectively simultaneously.Table 2 shows the monoclonal antibody (2D11-2) screened, with each GLP-1 antigenic peptides fusion response curve, show that screened mouse-anti GLP-1 (9-36) NH2 fusion monoclonal antibody identifies other GLP-1 polypeptide BSA fusion equally, tire close.After this hybridoma cell strain (2D11-2) being carried out clone's expansion cultivation, deliver China typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province, its preserving number is CCTCCNO:C2014108;
In analysis GLP-1 polyclonal antibody and antigen-reactive characteristic test, determine that four antigenic peptides fusions of GLP-1 are all with 250nmol/L concentration by chessboard method preliminary screening, 100ul/ hole bag is by elisa plate, ambient temperature overnight, after reaction plate is closed, different dilutability antiserum 100ul/well is added in reacting hole, room temperature was washed three times after 1 hour, add 100ul/well dilutes HRP coupling goat anti-rabbit igg with 1:2000 again, room temperature washs four times after 1 hour again, add HRP substrate solution (TMB and hydrogen peroxide) and develop the color 10 minutes, read OD492/640nm value.GLP-1 monoclonal antibody screening technique is the same, and the colour developing antibody of enzyme labeling changes the sheep anti-mouse igg of HRP coupling into.
The anti-GLP-1 polyclonal antibody of table 1. rabbit and GLP-1 antigenic peptides response characteristic
Table 2.GLP-1 monoclonal antibody (2D11-2) and GLP-1 antigenic peptides response characteristic
From table 1,2 interpretation of result, prepared GLP-1 (9-36) polyclonal antibody and the monoclonal antibody screened all can identify four antigenic peptides of GLP-1, and the epitope that instruction book clone, polyclonal antibody identify is epitope total in GLP-1 (9-36), (9-37), (7-36), (7-37) peptide chain molecule.
The total GLP-1 quantitative ELISA of embodiment 3 blood plasma is set up
One, reaction plate preparation
1, coated elisa plate: preserving number be the monoclonal antibody (2D11-2) of CCTCCNO:C2014108 as coated antibody, with bag be buffered liquid (CB) do 1:1000 dilution, be added in microwell plate, every hole 100ul.Put 4 DEG C, 48 hours.Take out, with distilled water flushing 5 times, pat dry.
2, close: in microwell plate, add 0.5%BSA-PBS, every hole 100ul.Put 37 DEG C, 2 hours.Take out, with distilled water flushing 5 times, pat dry.
Two, titer preparation:
1, damping fluid: 0.5%BSA-0.05%Tween20-PBS.
2, get antigenic synthetic peptide GLP-1 (9-36) NH2, be mixed with 74.5uM freezen protective with damping fluid.
Three, experimental procedure:
1, standard is added: get above-mentioned GLP-1 (9-36) fusion (74.5umol/L) confining liquid and (make doubling dilution, every hole 100ul, 37 DEG C, 2 hours.Wash 5 times with distilled water, pat dry.
2, add HRP-and mark the anti-GLP-1 antibody of rabbit, do 1:1500 dilution with pH7.2,0.5%BSA-0.05%Tween20-0.15molPBS.Be added in microwell plate, every hole 100ul.Put 4 DEG C, 48 hours.Take out, with distilled water flushing 5 times, pat dry.
3, add o-phenylene diamine substrate colour developing, room temperature 15 minutes, by 2mol sulfuric acid color development stopping.
4, use predominant wavelength 492nm, pay wavelength 640nm colorimetric.
Four, experimental result:
Table 3. continuous 5 days titer testing results (absorbance)
Experimental result shows, sensing range is 1490nmol-23.5nmol/L, and sensitivity for analysis is 23.5nmol/L.
Embodiment 4GLP-1ELISA reagent clinical principium is applied
One, research object
Collect physical examination of healthy population limosis vein blood 108 example, specify that fasting blood-glucose is normal, hepatic and renal function normal, blood lipids index is all normal, non-diabetic and other cardiovascular disease history, the wherein male sex 52 example, 25 ~ 76 years old age, women 56 example, 25 ~ 75 years old age.
Collect IGT, T2DM patient 120 example of new diagnosis, collection carbohydrate tolerance test normal person 50 example, above-mentioned object at least comprises on an empty stomach, rear 1h and 2h tri-the time point samples of clothes sugar, accumulative blood sample 510.All patients all do not accept GLP-1 or its analog drug therapy.
Adopt common tube or short solidifying pipe to collect blood sample, collect serum-20 DEG C frozen, detect for the total GLP-1 of serum.Other index blood sample is conventionally collected, is detected.
Two, the total GLP-1 assay of serum
Above-mentioned all serum samples are concentrated according to operation steps shown in embodiment 3 and are tested, and each 96 hole ELISA Plate all establish 0 ~ 2980nmol/L typical curve and blank well.
Three, the routine biochemistry such as blood sugar, glycosylated hemoglobin project detects
Adopt German Roche Diagnistics Products Co., Ltd automatic clinical chemistry analyzer and matched reagent thereof, according to the above-mentioned sample blood sugar of clinical examination conventional sense, insulin (In), cholesterol, triglyceride, low-density lipoprotein (LDL), high-density lipoprotein (HDL) (HDL), C reactive protein (CRP), apply Japanese TOSOHG8 type glycolated hemoglobin analysis and matched reagent detection glycosylated hemoglobin (HbA1c).
Four, result
1. the total GLP-1 content of normal person's Diagnostic Value of Fasting Serum
According to embodiment 3 method centralized detecting, secure good health adult total GLP-1 content 79.5 ± 39.7nmol/L on an empty stomach, Fig. 2 shows the distribution situation of the total GLP-1 content of 108 routine normal person's Diagnostic Value of Fasting Serum, therefrom can find that the fasting baseline difference of GLP-1 in glycometabolism normal population is comparatively large, content >100nmol/L accounts for 13.89%.
2.IGT, T2DM Clinical Application Analysis
All clinical sample testing results are in table 4.Analyze the result detected, the total GLP-1 content of T2DM patient's Diagnostic Value of Fasting Serum is higher than normal group, and IGT group is higher but not yet reach significant difference.Three group objects detect the total GLP-1 content of serum while accepting sugar tolerance test, find that NGT group is significantly increased to 2h at the total GLP-1 content of 1h serum after the meal and falls after rise gradually, and IGT group and T2DM group all present 1h after the meal does not rise anti-trend of falling, remarkable to 2h serum total GLP-1 rise after the meal, there is GLP-1 hyposecretion in prompting IGT group and T2DM group.
In addition, observe each row research object and carrying out the total GLP-1 content of serum in sugar tolerance test, find 50 routine NGT empty stomaches, 1h, 2hGLP-1 variation tendency is consistent after the meal.Having in 52 routine IGT patients in 5 examples, 68 routine T2DM has 3 routine GLP-1 variation tendencies consistent with NGT, occurred that GLP-1 level significantly raised at 1 hour after the meal, 2 as a child fell after rise, showing that not all IGT, T2DM patient exists GLP-1 hyposecretion after the meal, may there is other reason in the abnormal carbohydrate metabolism of patient.
Clinical biochemistry indications situation under the routine research object fasted conditions of table 4.170
NGT: normal glucose tolerance group; IGT: impaired glucose tolerance group; T2DM:2 patients with type Ⅰ DM group; FBG: fasting blood-glucose; In: insulin; TG: triglyceride; TC: cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein (HDL); HbA1c: glycosylated hemoglobin; CRP:C reactive protein.
The total GLP-1 mutation analysis of table 5.IGT, T2DM patient's post-prandial serum
* IGT group or T2DM group compare with NGT group (* p<0.05, * * p<0.01); Δ after the meal 1h, 2h with compare on an empty stomach ( Δp<0.05, Δ Δp<0.01).Under fasted conditions, T2DM group GLP-1 level is significantly higher than NGT group (p=0.006), but with IGT group without significant difference.In each group, GLP-1 level compares after the meal, and NGT group after the meal 1hGLP-1 level significantly rises, and falls after rise to fasting level after 2h, and IGT group after the meal 1h reduces (p=0.000), and 2h is still lower than fasting level (p=0.003) after the meal.T2DM group after the meal 1hGLP-1 level significantly reduces (p=0.011), after the meal 2hGLP-1 level, numerically still low, but with fasting level without significant difference (p=0.056).
Conclusion: the total GLP-1ELISA detection method of the serum (blood plasma) adopting the present invention to set up, first setting up normal person's Diagnostic Value of Fasting Serum GLP-1 term of reference is 79.5 ± 39.7nmol/L, and finds that between normal individual, the fasting baseline of GLP-1 differs greatly.Analyze total GLP-1 content in IGT, T2DM patient empty stomach, after the meal Different periods blood, find that in 120 routine IGT, T2DM patients, 112 routine patients exist GLP-1 hyposecretion after the meal.Significance of the present invention is the total GLP-1 quantitative ELISA detection method of disclosed serum (blood plasma), total GLP-1 content in dynamic Quantitative Monitoring blood, lacks the pathogenesis of diseases related (metabolic syndrome, coronary heart disease, senile dementia etc.) for the classification diagnosis of T2DM, personalized treatment and other GLP-1 and early stage Prevention Research has established experiment basis.In addition, detect total GLP-1 content in blood, coordinate biologically active GLP-1 to detect, can fully understand the situation of GLP-1 secretion, inactivation in subject, be GLP-1 relevant rudimentary and the requisite instrument of clinical research.

Claims (4)

1. a monoclonal antibody, is characterized in that: be by preserving number secreted by the hybridoma cell strain of CCTCCNO:C2014108.
2. a hybridoma cell strain, its preserving number is CCTCCNO:C2014108.
3. a kit, is characterized in that: containing monoclonal antibody according to claim 1.
4. adopt the method for ELISA to measure a method for GLP-1 total content in blood or blood plasma, it is characterized in that comprising the steps:
1) step of a preparation feedback plate, in the step of described preparation feedback plate, adopts preserving number to be that the monoclonal antibody of CCTCCNO:C2014108 is as coated antibody, be buffered liquid with bag and do 1:1000 dilution, be added in microwell plate, every hole 100ul, put in 2 ~ 5 DEG C of refrigerators, take out after 20 ~ 50 hours, with distilled water flushing 2 ~ 6 times, pat dry, in microwell plate, add 0.5%BSA-PBS, every hole 100ul, to put in 30 ~ 40 DEG C of water-baths 1 ~ 3 hour, take out, with distilled water flushing 2 ~ 6 times, pat dry;
2) step of a configuration standard liquid, first configures damping fluid, and described damping fluid is 0.5%BSA-0.05%Tween20-PBS, then gets antigenic synthetic peptide GLP-1 (9-36) NH 2, join with damping fluid, be mixed with the solution freezen protective of 74.5uM;
3) get the solution of the above-mentioned 74.5uM prepared, with confining liquid dilution, every hole 100ul, to put in 30 ~ 40 DEG C of water-baths 1 ~ 3 hour, takes out, and with distilled water flushing 2 ~ 6 times, pats dry;
4) add HRP-and mark rabbit anti-GLP-1 antibody, do 1:1500 dilution with pH7.2,0.5%BSA-0.05%Tween20-0.15molPBS, be added in microwell plate, every hole 100ul, put in 2 ~ 5 DEG C of refrigerators, take out after 20 ~ 50 hours, with distilled water flushing 2 ~ 6 times, pat dry;
5) add o-phenylene diamine substrate colour developing, room temperature 15 minutes, by 2mol sulfuric acid color development stopping;
6) predominant wavelength 492nm, commplementary wave length 640nm colorimetric is used;
7) each 96 hole ELISA Plate all establish 0 ~ 2980nmol/L typical curve and blank well, are horizontal ordinate with absorbance, be that ordinate draws typical curve, find the concentration of this sample GLP-1 with the absorbance of each sample to be tested on typical curve with concentration.
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