CN104673755A - Anti-human macromolecule adiponectin monoclonal antibody and application thereof - Google Patents
Anti-human macromolecule adiponectin monoclonal antibody and application thereof Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain 8A8 with the preservation number of CGMCC No.9567, and an anti-human macromolecule adiponectin monoclonal antibody A22 produced by the hybridoma cell strain. The anti-human macromolecule adiponectin monoclonal antibody A22 can be used for quantitatively or qualitatively detecting human macromolecule adiponectin in a biological sample, wherein the method is an ELISA method. The invention also provides a kit for detecting macromolecule adiponectin. The monoclonal antibody is high in specificity and can only selectively identify adiponectin in a macromolecule form. The ELISA kit is high in repeatability, high in stability, and relatively high in specificity; the monoclonal antibody can be massively and repeatedly produced, and is advantaged in sustainable development. The kit for detecting the human macromolecule adiponectin does not need pretreatment separation of samples, and is convenient and quick, and convenient for testing a great batch of samples.
Description
Technical field
The invention belongs to immunology and biological technical field, relate to a kind of monoclonal antibody for specific recognition people polymer adiponectin and application thereof, more specifically, relate to and utilize the adiponectin enzyme-linked immuning adsorpting analysis of people's polymer (ELISA) test kit of this monoclonal antibody preparation and the clinical application of mass detection sample thereof.
Background technology
Adiponectin (adiponectin) is by apM1 genes encoding, the secreted protein of specifically expressing in adipocyte, monomer whose is made up of 244 amino-acid residues, and molecular weight is about 28KDa, structure belongs to collagen protein superfamily, similar with complement Clq to TNF.Adiponectin improves insulin sensitivity, anti-diabetic, atherosclerosis, anti-inflammatory because having and presses down the important protectiveness physiological function such as cancer and extensively concerned.Adiponectin is secreted in blood with homotrimer, the polymer of six aggressiveness that disulfide linkage relies on and 12-18 or higher polymerized form exists, wherein tripolymer 65kD is lower molecular weight (low molecular weight, LMW) adiponectin, six aggressiveness 150kD are middle-molecular-weihydroxyethyl (middle molecular weight, MMW) adiponectin, 12-18 and more high polymer form are high molecular (high molecular weight, HMW, 280-420kD or higher) adiponectin, in the majority with HMW form.The different polymerized form of research display adiponectin plays different physiological functions at different tissues by different receptor subtype, and macromolecular form may have more protective effect.Known adiponectin improves insulin sensitivity effect and depends on HMW form, and it is also mainly relevant with increase HMW adiponectin that motion loss of weight causes insulin sensitivity to improve; The effect that antidiabetic medicine tetrahydrothiazole diketone derivatives improves insulin resistant is closely related with increase HMW.Adiponectin also mainly relies on HMW form to cardiovascular provide protection, only has HMW could activate NF-KB transcription factor, and HMW minimizing is fallen ill more relevant to coronary heart diesease.It is the generation, the developer molecule basis that connect obesity and T2DM, metabolism syndrome and cardiovascular disorder that the adiponectin dyssecretosis that visceral fat accumulation causes, particularly HMW reduce.Increase the novel targets that HMW adiponectin also becomes metabolism syndrome association drug development.Therefore, specific detection polymer adiponectin has important value for disclosing the pathogenesis of above-mentioned relative disease, the early prediction of the screening of new drug target and relative disease, curative effect monitoring and Index for diagnosis.
But only will detect polymer adiponectin from containing the polymeric biological sample of various forms specifically, the immune analysis method based on conventional antibody faces the challenge.Main because HMW is that basic framework is polymerized to come by adiponectin monomer with tripolymer, although the adiponectin number of monomers of multi-form polymer polymerization is not etc., but all contain identical adiponectin monomer structure, also just containing epitope identical on monomer molecule, therefore the conventional antibody adopting the adiponectin monomer of procaryotic cell expression to prepare as immunogen, all more or less has cross reaction with various forms of polymer.
In order to overcome the interference of other molecular form, the immune analysis method of current detection HMW mostly needs sample to be carried out complicated pre-treatment, namely first by sample through column chromatography for separation, or first retain HMW with special protease cracking small molecules, total adiponectin immunoassay that re-using has the adiponectin antibody of cross reaction to set up with various forms polymer detects; Visible complex operation, inapplicable detection sample in enormous quantities.Therefore current development trend is that the adiponectin monoclonal antibody of preparation specificity for HMW form is for building immune analysis method direct-detection polymer adiponectin.Though have import reagent box to release at present, expensive, be difficult to carry out large batch of applying.In addition, except blood preparation, comprise the existence that the biological samples such as urine, saliva, synovial fluid, milk have adiponectin, whether also there is the clinical value detecting HMW and still lack application evidence.
Summary of the invention
Therefore, for addressing the deficiencies of the prior art, the object of this invention is to provide a kind of can specificity for the monoclonal antibody of people's polymer adiponectin, and a kind of easy and simple to handle, cost is low, can the ELISA kit of specific detection polymer fat connection.And by detection application large batch of in the biological specimen such as serum, blood plasma of the Diseases such as obesity, diabetes, obtaining can for the reference experience of clinical application.
On the one hand, the invention provides the hybridoma cell strain 8A8 (Classification And Nomenclature: human adiponectin monoclonal antibody hybridoma cell strain that a kind of preserving number is CGMCC No.9567, preservation date: on 08 18th, 2014, preserving number is CGMCC No.9567, preservation ground: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101).
On the other hand, the invention provides a kind of anti-human polymer adiponectin monoclonal antibody A22, the hybridoma cell strain 8A8 being CGMCC No.9567 by preserving number produces.
Anti-human polymer adiponectin monoclonal antibody of the present invention is prepared by following steps:
1) extract human blood polymer adiponectin and prepare immunogen;
2) the monoclonal antibody hybridoma cell strain of anti-human polymer adiponectin is prepared;
3) by the cloning of hybridoma and enlarged culturing;
4) a large amount of production, Purification and Characterization monoclonal antibody.
Again on the one hand, the invention provides and a kind ofly use the application of anti-human polymer adiponectin monoclonal antibody of the present invention in biological specimen quantitatively and/or in qualitative detection people polymer adiponectin, preferably, described people's polymer adiponectin is the form of 12-18 aggressiveness or more high molecular.Preferably, described biological specimen is the biological fluid sample containing adiponectin such as serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk and histocyte extracting solution or cells and supernatant etc.The method of described detection is semiquantitative native gel electrophoresis immunoblotting or quantitative ELISA method but is not limited to ELISA.
Preferably, described method is ELISA method, and the method comprises the following steps:
(1) solid carrier of anti-human polymer adiponectin monoclonal antibody of the present invention to testing sample and load is contacted;
(2) biotin labeled anti-human polymer adiponectin monoclonal antibody is added;
(3) add enzyme mark avidin, detect combined certification mark.
In the above-mentioned methods, testing sample can be the biological fluid sample containing adiponectin such as serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk and histocyte extracting solution or cells and supernatant etc.
Preferably, aforesaid method comprises the steps: that the polymer adiponectin solution first comprising concentration known with series substitutes testing sample repeating step (1) to (3), drawing standard curve; Again with testing sample repeating step (1) to (3), and draw the concentration of polymer adiponectin in testing sample according to typical curve.
Present invention also offers a kind of detection kit of people's polymer adiponectin, anti-human polymer adiponectin monoclonal antibody of the present invention is comprised as effective constituent in described test kit, preferably, described detection kit is ELISA kit, more preferably, ELISA in described ELISA kit adopts the sandwich non-competing binding pattern of same antibody, further preferably, introduces BA amplification system in described ELISA.
Preferably, described ELISA kit comprises:
For catching the anti-human polymer adiponectin monoclonal antibody in testing sample;
Biotin labeled anti-human polymer adiponectin monoclonal antibody;
Series comprises the solution of people's polymer adiponectin of concentration known;
Enzyme mark avidin;
Detect the instrument of enzyme mark avidin.
Preferably, described test kit also comprises coating buffer, phosphate buffered saline buffer, blocking-up liquid, washings, antibody diluent, enzyme combination diluent, substrate application liquid and/or stop buffer.
Again on the other hand, the invention provides the application of a kind of people's polymer adiponectin detection kit in clinical sample detects, preferably, described clinical detection is mass detection, more preferably, the sample of described detection is biology liquid sample, is preferably selected from serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk and histocyte extracting solution or cells and supernatant.
On the other hand, the invention provides a kind of preparation method detecting the test kit of people's polymer adiponectin, comprise the following steps:
1) preparation experiment liquid;
2) HMW adiponectin standard substance are prepared;
3) enzyme plate insolubilized antibody and biotin labeled detection antibody is prepared.
Compared with prior art, beneficial effect of the present invention is:
Monoclonal antibody specificity of the present invention is strong, the adiponectin of a Selective recognition macromolecular form.ELISA kit of the present invention is reproducible, and stability is strong, and all adopts the monoclonal antibody of same of the present invention as the insolubilized antibody of key reagents and traget antibody, cost reduces, specificity is stronger, and this monoclonal antibody can duplication of production in enormous quantities, and Sustainable development is with the obvious advantage.The test kit of detection people polymer adiponectin of the present invention is without the need to pre-treatment separating sample, simple and efficient, is convenient to the mensuration of batch samples.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
The electrophorogram of Fig. 1 for using monoclonal antibody of the present invention to carry out the people's polymer adiponectin in non-denaturing polyacrylamide gel (native-PAGE) electrophoresis immune-blotting method biological specimen;
Wherein,
1a uses the people's polymer adiponectin in monoclonal antibody A22 of the present invention detection human serum;
1b is contrast, uses another monoclonal antibody C63 for total adiponectin to detect human serum;
1c uses the people's polymer adiponectin in monoclonal antibody A22 of the present invention detection saliva, synovial fluid and adipocyte culture supernatant.
Fig. 2 is the canonical plotting using monoclonal antibody ELISA method of the present invention to detect people's polymer adiponectin concentration;
Fig. 3 is that test kit of the present invention compares with the dependency that the HMW adiponectin ELISA kit of R & D company of the U.S. detects the result of human serum sample;
Fig. 4 is the graphic representation using the total adiponectin ELISA kit of ELISA kit of the present invention and phonix company of the U.S. to detect the content of adiponectin in human serum gel permeation chromatography elutriant collection tube;
Fig. 5 uses test kit of the present invention to detect HMW adiponectin in children and youth blood specimen and results contrast figure that is fat and metabolism syndrome relation;
Fig. 6 is the kaplan-merier analysis graph using the HMW adiponectin of test kit of the present invention detection patients with coronary heart disease and cardiocerebrovasculaevents events to be related.
Embodiment
Unless specifically stated otherwise, reagent used in following examples is analytical pure level reagent, and can be commercially available from regular channel.
the preparation of embodiment 1 anti-human polymer adiponectin monoclonal antibody
The preparation process of described anti-human polymer adiponectin monoclonal antibody hybridoma cell strain is:
1) preparation of polymer adiponectin:
The recombinant expressed adiponectin molecule of conventional gene is adopted to be difficult to obtain the only special monoclonal antibody for HMW adiponectin as immunogen.Because the level of adiponectin in human blood is up to mg/l level, the present invention collects healthy volunteer 100ml serum, therefrom extracts natural polymer adiponectin.
Concrete extracting method is: adopt sepharose 4B (the CNBr-Sepharose 4B that homemade mouse-anti human adiponectin monoclonal antibody C63 and CNBr activates, SIGMA company) coupling, 5mg antibody purification adds the CNBr-Sepharose 4B of 5mL, the Sepharose 4B of coupled antibody is loaded in chromatography column, balance with phosphate buffered saline buffer PBS, get 5ml serum upper prop at every turn, 4 DEG C of combinations are spent the night, non-binding constituents is washed away with PBS, with the citric acid-sodium citrate buffer solution elution human adiponectin of the pH3.0 containing 1mol/L NaCl, pH7.4 is neutralized to Tris-HCl, adding molecular weight cut-off again to is in the super filter tube of 100kDa, centrifugal ultrafiltration concentrates, be moved into separation and purification in gel permeation chromatography post again, collect the high molecular adiponectin elution peak of molecular weight >440kDa, row ultrafiltration and concentration more further, to obtain the human blood polymer adiponectin of high density, be used separately as immunogen Dispersal risk, or as Detection of antigen antibody, or be used as polymer adiponectin standard substance.
2) preparation of mouse hybridoma cell strain
Choose 6-8 Balb/C female mice in age in week (purchased from Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences), with the dosage of the polymer adiponectin extracted in blood according to 1-5ug, booster immunization mouse before initial immunity, repeatedly booster immunization and fusion.Get mouse boosting cell and mouse sp2/0 myeloma cell (basis institute of Chinese Academy of Medical Sciences cell centre), the polyethylene glycol method of ordinary method is adopted to be fused into hybridoma, inoculate 24 well culture plates and cultivate screening with HAT selectivity, indirect ELISA detection specificity antibody-secreting.
The operating process of described indirect ELISA is (1) people HMW adiponectin coated elisa plate; (2) add mouse serum/cells and supernatant 100ul/ hole, room temperature reaction 1 hour, washes plate; (3) add horseradish enzyme-sheep anti-mouse igg (sigma company) 100ul/ hole, room temperature 1 hour, washes plate; (4) 3,3', 5,5' tetramethyl benzidine (TMB) substrate color reaction 10 minutes; (5) after termination reaction, microplate reader reads absorbancy (OD) value at wavelength 450nm/620nm.
By the positive hybridoma enlarged culturing detected, adopt limiting dilution assay mono-clonal repeatedly, obtain the hybridoma cell strain of the monoclonal antibody A22 of the anti-human polymer adiponectin of stably excreting thus.
Contriver is by this hybridoma cell strain (called after 8A8, Classification And Nomenclature: human adiponectin monoclonal antibody hybridoma cell strain, preservation date: on 08 18th, 2014, preserving number is CGMCC No.9567, preservation ground: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101), for the production of monoclonal antibody.
3) production in enormous quantities of monoclonal antibody, Purification and Characterization: adopt mice celiac inoculation, namely adopt Balb/c mouse in abdominal injection 0.5ml white oil, abdominal injection 1 × 10 again after a week
6individual hybridoma, collects ascites, adopts indirect ELISA to measure adiponectin antibody titers in ascites, and result display titre is up to 5 × 10
4, obtain the ascites containing a large amount of monoclonal antibody thus.
For obtaining high purity monoclonal antibody, adopt affinity chromatography method monoclonal antibody purification ascites.Concrete grammar is: adopt the Agarose microbead Protein A sepharose CL-4B of sigma company to prepare affinity column, ascites is with upper prop after the dilution of Tris-Hcl (PH9.0) damping fluid, 4 DEG C of combinations are spent the night, with the non-associated proteins of 0.1M Tris-Hcl buffer solution elution, use the citrate phosphate buffer wash-out specific antibody of the 0.1M of PH3.0 again, in and pH value after frozen.Obtain the anti-human polymer adiponectin monoclonal antibody of large-batch high-purity thus.
embodiment 2 monoclonal antibody of the present invention adopts immunoblotting to evaluate binding characteristic
Human serum, saliva, synovial fluid and people's adipocyte culture supernatant are comprised with non-denaturing polyacrylamide gel (native-PAGE) electrophoretic separation to the biological liquid sample containing different polymerized form adiponectin, polymer adiponectin monoclonal antibody A22 of the present invention is adopted to carry out Western blot, evaluate and the binding ability of various polymerized form adiponectin, and with identify that with another kind the monoclonal antibody of total adiponectin is made comparisons.Above-mentioned biological specimen should containing comprising the LMW tripolymer (about 65kDa) of adiponectin, MMW six aggressiveness (150kDa) and HMW 12-18 aggressiveness or higher polymer (280-420kDa, 660kDa)
Result is as Fig. 1 a, 1c display, and apply anti-human HMW adiponectin monoclonal antibody of the present invention, immunoblotting assay only identifies the polymer adiponectin of more than 440KD in the above-mentioned biological samples such as serum; Compare with monoclonal antibody C63, monoclonal antibody A22 is not in conjunction with MMW and the LMW adiponectin in serum.
In addition, result also illustrates that anti-human HMW adiponectin monoclonal antibody of the present invention can be used for the sxemiquantitative immune-blotting method of HMW adiponectin in the multiple biological specimens such as blood plasma, saliva, synovial fluid.
the preparation of embodiment 3 anti-human HMW adiponectin monoclonal antibody ELISA immue quantitative detection reagent box
People HMW adiponectin detection by quantitative ELISA kit composition mainly comprises: wrap and comprised by the enzyme plate of A22 monoclonal antibody, biotin labeled detection antibody (A22), HMW adiponectin standard substance, enzyme mark avidin and various experiment liquid: (1) coating buffer: 0.1M NaHCO3-Na
2cO3 (PH9.6-9.8) damping fluid, (2) phosphate buffered saline buffer (PBS): containing the 0.01M Na of 0.15mol/L NaCL
2hPO4-NaH
2pO4 (PH7.2) damping fluid, (3) liquid (1%BSA-PBST) is blocked: 1%BSA-0.01M PBS-TWEEN-20, (4) washings (PBS-T), (5) antibody diluent: get 1L 0.01M PBS and add BSA 10g, ox gamma Globulin 1g, Tween-200.5ml, (6) enzyme combination diluent: get 1L 0.01M PBS and add BSA 2g, Thiomersalate 0.1g, TWEEN-20 0.5ml, (7) substrate application liquid: TMB-HCL1 sheet (1mg tablet) adds 10ml Sodium peroxoborate solution.(8) stop buffer: 1M H
2sO
4.
Step prepared by standard substance is: collect normal human serum 100ml, supercentrifuge 22000g, 4 DEG C of centrifugal 20min.Get supernatant and detect its people HMW adiponectin content with the HMW adiponectin ELISA kit (DHWAD0) of R & D company of the U.S., get average as people HMW adiponectin standard substance mother liquid concentration.Add sanitas Thiomersalate to 0.01% concentration, and filtration sterilization packing is frozen in-80 DEG C, using as reference standard.Standard substance and each concentration point of quality control product is mixed with 1%BSA-PBS dilution during use.
The preparation of biotin labeled detection antibody: monoclonal antibody purification A22 of the present invention is diluted to 1mg/ml with 0.1M carbonate buffer solution, adopt N-hydroxy-succinamide vitamin H (BNHS) mark of SIGMA company, it is 5 ~ 10:1 that BNHS and antibody molecule mark the ratio added, and can obtain best background and signal to noise ratio.Traget antibody working concentration 0.25-0.5 μ g/ml.
People HMW adiponectin detection by quantitative ELISA kit working method is:
(1) enzyme plate prepares: the anti-human HMW adiponectin monoclonal antibody A22 of affinitive layer purification, 5-10 μ g/ml coated elisa plate capillary strip is diluted to carbonate buffer solution, 4 DEG C spend the night after add block fluid-tight close 1h, with PBST washing after deposit for subsequent use in 4 DEG C.
(2) sample process: with doing 1:1000 dilution containing sample diluent before serum/slurry sample measures.Other sample such as synovial fluid need be made about 1:100 and dilute; Milk need detect after removing lipid through ultrasonication, ultracentrifugation; Saliva and cells and supernatant 3000rpm centrifugal after get supernatant direct-detection.
(3) double-antibody sandwich: people HMW adiponectin standard substance or sample to be tested and each 50 μ l of biotinylated detection antibody are together added and wraps by hole, shaken at room temperature reaction 12h.
(4) integrated enzyme reaction: the horseradish peroxidase 100 μ l adding streptavidin mark after washing 4 times reacts 0.5h.
(5) color reaction: add substrate solution (containing 0.03% Sodium peroxoborate and 0.01% hydrochloric acid TMB) 100 μ l after washing 4 times again, vibration colour developing 10 minutes.
(6) termination reaction: with 1mol/L sulfuric acid termination reaction.
(7) matched curve with read value: in microplate reader, read the OD value that wavelength is 450nm/620nm, adopt 4 parameter methods to fit typical curve, directly draw typical curve and concentration by the program finished.
the evaluation of methodology of embodiment 4 people HMW adiponectin detection by quantitative ELISA kit and comparing
The indexs such as system examination response curve, sensitivity, specificity, precision, accuracy, and compare with commercial kit.
1, typical curve: sensitivity and sensing range
This kit standard concentration point is 0,0.5,1.25,2.5,5,10,20,40ng/ml, as X-axis, make Y-axis with absorbancy OD (450-620nm) value, see Fig. 2, degree of fitting R with the typical curve of four parametric line mode matchings
2=1.Add sensitivity that 3SD mode calculates to 0.1ng/ml with 0 standard, the working range 0.5 ~ 40ng/ml of curve, blood specimen dilutes 1000 times and can detect.
2, specificity
1) the animal derived cross reaction of different genera: get ox, sheep, mouse, rat and rabbit anteserum, press 1:1 respectively, 1:10,1:100 doubly dilute, measures adiponectin content with ELISA, is showed no cross reaction.
2) cross reaction is had no with the adiponectin monomer of 100ng/ml structure associated protein people phylaxin, TNF α, C1Q and escherichia coli expression.
3) with the polymer adiponectin of different molecular weight: be separated through Ultrogel ACA34 (1 × 50cm) chromatography column with Healthy People pooled serum 4 DEG C, with 0.1%BSA-PBS wash-out, 0.5ml/ pipe is collected, test kit of the present invention and U.S. Phoenix adiponectin ELISA kit (lot number: EK-ADI-01) is used to measure the content of adiponectin in each collection tube, the result that each pipe concentration dilution 10 times measures as shown in Figure 3, Phoenix adiponectin ELISA at least detects 3 adiponectin elution peaks, confirm through non-denaturing polyacrylamide gel (native-PAGE) electrophoresis immunoblotting, be followed successively by the HMW adiponectin of 280 ~ 660KD, the MMW molecule of 150KD and 65KD LMW, and this test kit only detects the elution peak of high molecular position, illustrate that Phoenix ELISA detects total adiponectin, test kit specific assay polymer adiponectin of the present invention.
4) precision and accuracy: normal people's pooled serum, criticize after diluting 1000 times with standard dilution interior and batch between respectively replication 10 times, measured value average is 5.1 ± 0.35 μ g/ml, and variation within batch coefficient cv is 2.8%; Batch between replication CV be 7.1%.Accuracy with rate of recovery evaluation, with namely get normal people's pooled serum dilute 1000 times after 1ml add respectively 3ng, 10ng, 30ng the present invention extract human serum HMW adiponectin, the calculating rate of recovery is 92.2%-107.5%.Illustrate that test kit of the present invention accurately, reliably.
5) methodology comparison: Diagnostic Value of Fasting Serum sample totally 40 person-portions of getting normal people and diabetic subject, apply test kit of the present invention and adiponectin detects simultaneously in U.S. R & D HMW adiponectin ELISA kit (catalog number (Cat.No.): DHWAD0), both measurement results are compared through linear regression analysis, result as shown in Figure 4, correlation coefficient r=0.94, both dependencys are good.
Compare with commercial kit as seen, test kit detected result of the present invention is consistent, but self-control monoclonal antibody can be mass-produced, and cost is low, and price advantage is very outstanding.
embodiment 5 health adult and diabetes B patients serum HMW adiponectin detect
Include in altogether from community's screening for diabetes object 320 example, empty stomach 10-12 hour overnight, m seq is row 2h-75g OGTT all.By ADA standard diagnostics diabetes B patient 45 example, contrast 220 example of pre-diabetes (IGT) 55 example and normal glucose tolerance (NGT), test kit of the present invention is adopted to detect HMW adiponectin in blood, result is as shown in Table 1: HMW adiponectin significantly reduces (p<0.01) along with increasing the weight of of impaired glucose tolerance degree, compare with normal control, pre-diabetes reduces by 30%, and diabetes B person reduces by 55%.Correlation analysis display serum HMW adiponectin and BMI, empty stomach and postprandial blood sugar are all in negative correlation, and correlation coefficient r is respectively :-0.44 ,-0.23 ,-0.35.
Result illustrates, the generation of the HMW adiponectin that test kit of the present invention detects and diabetes B develops closely related, can be applied to the relevant clinical research of diabetes B.
Table 1 different sugar tolerance crowd serum HMW adiponectin
* compare with normal control, P<0.05
the foundation of embodiment 6 children and youth serum HMW adiponectin normal reference value and clinical epidemiology applied research thereof
Choose from Beijing area metabolism syndrome research queue 3202 6-18 year children and teenager's (comprising 2170 metabolism syndrome high risk childs, teenager and 1032 normal healthy controls) carry out the evaluation of scale of construction index (height, body weight, waistline, blood pressure), fasting plasma glucose, lipid determination and Paederus fuscipes Curtis degree, HMW adiponectin ELISA of the present invention is adopted to measure the concentration of Diagnostic Value of Fasting Serum polymer adiponectin, set up children and youth normal reference value, analyze the relation of polymer adiponectin and children obesity, metabolism syndrome.
As a result 1) polymer adiponectin term of reference is set up: select healthy children 1032 (man 430) without any metabolism syndrome component as the reference crowd setting up normal value according to IDF metabolism syndrome standard sieve in 2009, the polymer adiponectin recorded is by sex hundredths distribution statistics, average 4.9 ± the 3.0ug/ml of result boy, 5-95 hundredths is 1.5-8.6ug/ml; Girl 5.6 ± 3.5ug/ml, 5-95 hundredths 2.0-10.1ug/ml, gender difference significantly (P<0.01).2) the serum HMW adiponectin recorded is shown in Fig. 5 with relation that is fat and metabolism syndrome, to compare with healthy population as seen, the average adiponectin of Obesity group reduces by 21% (p<0.001), and the fat metabolism syndrome group that merges reduces by 36%.
Result illustrates, test kit of the present invention establishes fairly perfect normal reference value based on Samples detection in enormous quantities, and Preliminary Applications proves that HMW adiponectin decline degree is closely related with the increase of fat and fat cardiovascular risk; HMW adiponectin can be further used as the biomarker distinguishing fat hypotype, namely the obesity of health type obesity and high risk is distinguished by detecting HMW adiponectin, to save public health resources, intervening measure is more targetedly implemented, for clinical and the further high volume applications of epidemiology provide reference frame to high risk obese patient.
embodiment 7 patients with coronary heart disease serum adiponectin levels detects
Collect through coronary angiography inspection clarify a diagnosis coronary heart disease patient totally 449 example, the wherein male sex 305, 66 ± 11 years old mean age, carry out the Clinical Follow-up in 31 ± August by a definite date, a situation arises to observe main heart and brain adverse events (MACCE), specifically comprise cardiac death, non-cardiac death, target vessel revascularization, the acute coronary syndrome occurred between follow-up period, cerebral apoplexy or transient cerebral ischemia, heart function deterioration etc., this test kit is adopted to detect baseline Diagnostic Value of Fasting Serum adiponectin, inquire into the relation between itself and prognosis of patients with coronary artery disease (MACCE occurs).There is the patients with coronary heart disease of MACC event in result display, baseline HMW adiponectin (average 2.1 μ g/ml) is starkly lower than and event person (2.6ug/ml does not occur, p<0.05), Kaplan-meier survival curve is adopted to evaluate the prognostic value of HMW adiponectin, as shown in Figure 6, there is MACCE event and be significantly higher than HMW adiponectin high group (p<0.05) in the patients with coronary heart disease group that HMW adiponectin is low to result.COX regression analysis shows, the risk that MACCE event occurs low-level HMW patient be high-level group at the male sex 1.656 times of (95%CI 1.074,2.554), women 2.802 (1.425-5.512), women HMW reduces higher than male sex occurrence risk.Point out low-level HMW adiponectin and prognosis of patients with coronary artery disease bad closely related.
Result illustrates, the HMW adiponectin that test kit of the present invention detects can be used as the important indicator of prognosis of patients with coronary artery disease assessment.
Claims (10)
1. a preserving number is the hybridoma cell strain 8A8 of CGMCC No.9567.
2. an anti-human polymer adiponectin monoclonal antibody A22, the hybridoma cell strain 8A8 being CGMCC No.9567 by preserving number produces.
3. prepare a method for anti-human polymer adiponectin monoclonal antibody as claimed in claim 2, said method comprising the steps of:
1) extract human blood polymer adiponectin and prepare immunogen;
2) the monoclonal antibody hybridoma cell strain of anti-human polymer adiponectin is prepared;
3) by the cloning of hybridoma and enlarged culturing;
4) a large amount of production, Purification and Characterization monoclonal antibody.
4. anti-human polymer adiponectin monoclonal antibody as claimed in claim 2 in biological specimen quantitatively and/or the application of qualitative detection people polymer adiponectin;
Preferably, described people's polymer adiponectin is the form of 12-18 aggressiveness or more high molecular;
Preferably, described biological specimen is biology liquid sample, is preferably selected from serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk, histocyte extracting solution or cells and supernatant;
Described detection uses semiquantitative native gel electrophoresis immunoblotting or quantitative ELISA method.
5. method according to claim 4, wherein, described method is ELISA method, and the method comprises the following steps:
(1) solid carrier of anti-human polymer adiponectin monoclonal antibody of the present invention to testing sample and load is contacted;
(2) biotin labeled anti-human polymer adiponectin monoclonal antibody is added;
(3) add enzyme mark avidin, detect combined vitamin H;
Preferably, described testing sample is biology liquid sample, is preferably selected from serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk, histocyte extracting solution or cells and supernatant.
6. method according to claim 5, wherein, also comprises the steps: that the polymer adiponectin solution first comprising concentration known with series substitutes testing sample repeating step (1) to (3), drawing standard curve; Again with testing sample repeating step (1) to (3), and draw the concentration of polymer adiponectin in testing sample according to typical curve.
7. detect a test kit for people's polymer adiponectin, wherein, in described test kit, comprise anti-human polymer adiponectin monoclonal antibody as claimed in claim 2;
Preferably, described detection kit is ELISA kit;
More preferably, the ELISA in described ELISA kit adopts the sandwich non-competing binding pattern of same antibody;
Further preferably, BA amplification system is introduced in described ELISA.
8. test kit according to claim 7, wherein, described ELISA kit comprises:
For catching the anti-human polymer adiponectin monoclonal antibody in testing sample;
Biotin labeled anti-human polymer adiponectin monoclonal antibody;
A series of solution comprising people's polymer adiponectin of concentration known;
Enzyme mark avidin;
Detect the instrument of enzyme mark avidin.
9. test kit according to claim 8, wherein, described test kit also comprises coating buffer, phosphate buffered saline buffer, blocking-up liquid, washings, antibody diluent, enzyme combination diluent, substrate application liquid and/or stop buffer.
10. the application of test kit in clinical sample detects as claimed in claim 8 or 9, described test kit is used for the people's polymer adiponectin quantitatively and/or in qualitative detection biological specimen; Preferably, described clinical sample is detected as mass detection; More preferably, described testing sample is biology liquid sample, is selected from serum/slurry, saliva, cerebrospinal fluid, synovial fluid, milk, histocyte extracting solution or cells and supernatant.
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| CN111505317A (en) * | 2020-04-21 | 2020-08-07 | 江西乐成生物医疗有限公司 | Adiponectin determination reagent quality control product and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111505317A (en) * | 2020-04-21 | 2020-08-07 | 江西乐成生物医疗有限公司 | Adiponectin determination reagent quality control product and preparation method thereof |
| CN111505317B (en) * | 2020-04-21 | 2023-09-29 | 江西乐成生物医疗有限公司 | Quality control product of adiponectin determination reagent and preparation method thereof |
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