CN103076452B

CN103076452B - Method for testing adiponectin and application thereof

Info

Publication number: CN103076452B
Application number: CN201210588023.XA
Authority: CN
Inventors: 宁光; 黄珊; 王卫庆; 顾卫琼; 洪洁
Original assignee: SHANGHAI INST OF ENDOCRINE-METABOLIC DISEASE
Current assignee: SHANGHAI INST OF ENDOCRINE-METABOLIC DISEASE
Priority date: 2012-12-30
Filing date: 2012-12-30
Publication date: 2015-02-25
Anticipated expiration: 2032-12-30
Also published as: CN103076452A

Abstract

The invention discloses a method for testing adiponectin. The method comprises the following steps: preparing a buffering liquid standard product APN, adding and dissolving 10 milliliters of blood plasma specimen into 4,990 milliliters and diluting until the ratio is 1:500 to obtain a sample to be tested; adding 100 milliliters of sample to be tested or the standard product into each tube, adding I-APN solution and rabbit anti-APN antibody, mixing completely, incubating at room temperature for 24 hours and passing the night; adding rabbit serum and goat anti-rabbit IgG antibody the next day, mixing uniformly, incubating at the temperature of 4 DEG C for 20 minutes, centrifuging for 20 minutes, and absorbing and discarding the supernate; measuring the a~ ray quantity of each test tube; drawing a standard curve, namely drawing the standard curve on logarithmic coordinate paper by taking the APN concentration of the standard product as horizontal coordinate and B/B0 as the vertical coordinate; finding the corresponding APN concentration from the sample B/B0 curve; and calculating the APN concentration. The test method is simple in steps and accurate in measurement. By the test method, the clinical significance of diabetes serum adiponectin for forecasting the macroangiopathy at the early stage of diabetes and the severity degree thereof can be evaluated by analyzing the influence factors of the macroangiopathy of different types of impaired glucose regulation people.

Description

A kind of assay method of adiponectin concentration and application thereof

Technical field

The present invention relates to a kind of assay method and application thereof of adiponectin concentration.

Background technology

Adiponectin (APN) be a kind ofly to be synthesized by adipocyte, the cell factor of secretion, nineteen ninety-five is by the Late Cambrian in mouse 3T3 adipocyte such as Scherer.Encoding adiponectin gene is positioned chromosome 3q27 the mankind, and size is 17kb, comprises 3 extrons and 2 intrones.Genome-wide screening shows the susceptibility loci that this region exists diabetes B and Metabolic Syndrome.Adiponectin is the protein of the adipose tissue secretion that plays an important role in insulin sensitivity and inflammatory path at human body.Clinical research finds that adiponectin and obesity, diabetes, coronary heart disease, insulin resistance are closely related, has the characteristic of anti-endometrial hyperplasia after antiatherosclerosis formation, anti-inflammatory and injury of blood vessel.Current Cell culture invitro and tissue culture experiments confirm that adiponectin can directly act on atherosclerotic formation by following several mechanism: (1) adiponectin regulates and controls the inflammatory reaction of endothelial cell by activating CAMP-protein kinase A (cAMP-PKA) signal path.(2) suppress the function of macrophage, the growth of precursor macrophage can be suppressed, suppress endothelial cell to adhere to the expression of the factor, suppress macrophage to the picked-up of cholesteryl ester, thus suppress macrophage to the conversion of foam cells.(3) differentiation of endothelial cell, smooth muscle cell, propagation and migration [41-43] is suppressed.(4) adiponectin reduces insulin resistance by the triglyceride content in minimizing blood plasma free fatty acid and muscle and liver, improves glucose-lipid metabolism disorder.Adiponectin both can directly act on the forming process of coronary atherosclerosis as can be seen here, formed the relevant change of a series of hazards and the generation of remote effect vascular lesion and development again by causing with coronary sclerosis.

Human experimentation confirms, there is very strong correlativity between APN and body insulin sensitivity: RI degree is higher, and APN blood plasma level is lower; Conversely, APN level raises obviously can improve RI, APN improves insulin sensitivity by following mechanism: (1) adiponectin can increase 5-AMP activated protein kinase (AMPK) to acetyl-CoA carboxylase (ACC) phosphorylation, accelerate fatty acid oxidation, after making insulin receptor and acceptor, horizontal signal conduction is accelerated, reduce liver gluconeogenesis, promote glucose utilization, thus reach the object improving insulin resistance.(2) adiponectin plays certain antagonism to the B cell apoptosis that Adipocyte Factor and fatty acid cause, and promotes the utilization of glucose.(3) adiponectin can strengthen insulin sensitivity, and can improve pancreas B cell function, plays double protection to disease.(4) suppress the growth of macrophage, reduce its picked-up to lipid, thus reduce the conversion of macrophage to foam cells, in conjunction with injury of blood vessel place collagen accumulation under endangium, the termination procedure of inflammatory reaction can be participated in.

By to dissimilar IGR crowd macroangiopathy analysis of Influential Factors, evaluate the clinical meaning of diabetes Plasma adiponectin (APN) in prediction prediabetes macroangiopathy and the order of severity thereof.

Summary of the invention

The object of the invention is assay method and the application thereof in order to provide a kind of adiponectin, providing a kind of foundation to the situation of prediction prediabetes macroangiopathy.

Object of the present invention can be achieved through the following technical solutions.

An assay method for adiponectin concentration, the steps include:

(1) configure buffer standards product APN (concentration is respectively 200,100,50,25,12.5,6.25,3.125,1.56,0.78ng/ml, totally 9 pipes).

(2) plasma specimen 10ml add 4990ml dissolve damping fluid be diluted to 1:500, be sample to be tested.

(3) often pipe adds 100ml testing sample or standard items, then adds I-APN solution, the anti-APN antibody of rabbit, and fully mix, at room temperature (20 ~ 25 DEG C) hatch 24 hr overnight.

(4) add rabbit anteserum and goat anti-rabbit igg antibody (except control tube) next day, mixing, hatch 20 minutes for 4 DEG C, within centrifugal 20 minutes, (3000 × g) Aspirate supernatant discards.

(5) each test tube is measured quantity of X-rays X.

(6) Specification Curve of Increasing; On logarithmic paper with standard items APN concentration for horizontal ordinate, B/B0 (%) is ordinate drawing standard curve.

(7) B/B0 (%) checks in corresponding APN concentration (ng/ml) from curve per sample.

(8) APN concentration (ng/ml) calculates: gained APN concentration (ng/ml) value is multiplied by extension rate (500), is concentration of specimens.

The anti-APN antibody (13ml) of described 10 × times damping fluid (50ml) rabbit, I-APN label (13.5ml), standard items (restructuring APN sterling 0.75ml), quality-control product 1 and 2 (restructuring APN sterling 1ml), rabbit carrier (2ml), precipitation reagent (goat anti-rabbit immunoglobulin G (IgG) antibody, 3% polyglycol).Be purchased from the radioimmunoassay kits of Linco company of the U.S., CV < 6.21% in crowd, CV < 9.25% between crowd.

On the labelled antigen of measuring principle fixed concentration and a certain amount of antiserum incubated antibodies, the binding site of antigen is limited.If add unlabelled antigen, both are by the limited fixing site on competition binding antibody, and along with unlabelled antigen increases, the label be combined with antibody will reduce.Antibody conjugates is separated with free label, then counts wherein a part or two parts, the content of APN antigen in unknown sample can be checked in by the typical curve set up by standard items.

Method of testing of the present invention, step is simple, measure precisely, by dissimilar IGR crowd macroangiopathy analysis of Influential Factors, evaluate the clinical meaning of diabetes Plasma adiponectin (APN) in prediction prediabetes macroangiopathy and the order of severity thereof.

Accompanying drawing explanation

Fig. 1 is plasma A PN concentration comparison diagram between embodiment four groups;

Embodiment

Technical characterstic of the present invention is set forth further below in conjunction with accompanying drawing and specific embodiment:

Embodiment

2009-2011 collects Changning District central hospital, clinical doubtful coronary heart disease and be in hospital row coronary arteriography patient totally 210 example.The tolerance test of row oral glucose, is divided into according to WHO diagnostic criteria glycometabolism state in 1999: normal sugar metabolism group (NGT) 42 example (male sex 21 example, women 21 example, 60.3 ± 10.9 years old mean age); Fasting blood IGR group (IFG) 36 example (male sex 20 example, women 16 example, 62.8 ± 8.2 years old mean age); Impaired Glucose Tolerance Treated group (IGT) 92 example (male sex 54 example, women 38 example, 61.5 ± 9.9 years old mean age); Wherein IGT1 group 44 example, IGT2 group 48 example, IFG merges Impaired Glucose Tolerance Treated group (IFG+IGT) 40 example (male sex 25 example, women 15 example, 61.0 ± 9.5 years old mean age).All selected objects all get rid of liver, kidney, thyroid disease, and the acute metablize confusion, heart failure, infection, autoimmune disease or connective tissue disease, tumour and the past once accepted PTCA or CABG curer.Do not use the medicine affecting insulin secretion and insulin resistance.

Diagnosis and criteria for classification:

Adopt the World Health Organization (WHO) (WHO) classification diagnosis standard in 1999: fasting blood IGR (IFG),

FPG6.1 ~ <7.0mmol/L, 2hPG<7.8mmol/L after clothes sugar; Impaired Glucose Tolerance Treated (IGT), 2hPG<11.1mmol/L after FPG<6.1mmol/L, 7.8mmol/L≤clothes sugar; IFG merges Impaired Glucose Tolerance Treated (IFG+IGT), 2hPG<11.1mmol/L after FPG6.1 ~ <7.0mmol/L, 7.8mmol/L≤clothes sugar.To IGT crowd with 2hPG 10.0mmol/L for the further layering of node, IGT1 group: 7.8mmol/L≤take sugared 2hPG<10.0mmol/L; 2hPG<11.1mmol/L after IGT2 group: 10.0mmol/L≤clothes sugar.Every routine experimenter on an empty stomach after 8-10 hour in morning next day 8AM do 75g dextrose tolerance test, 75g glucose is dissolved in 250-300 ml water, drink in five minutes, fasting blood-glucose (fasting blood glucose is surveyed respectively at empty stomach, clothes sugar latter 2 hours venous blood samples, FBG), 2h blood sugar (2hpost-challenge glucose after clothes sugar, 2hPG), adopt determination of glucose oxidase, U.S.'s Beckman full automatic biochemical apparatus completes.Detect triglyceride (TG), cholesterol (TC), highdensity lipoprotein-cholesterol (HDL-c) LDL-C (LDL-c) on an empty stomach simultaneously, and survey the indexs such as FPI (fastinginsulin, FINS), adiponectin, c reactive protein.Every routine experimenter all surveys body weight (kg), height (m), blood pressure (mmHg)

Calculate body mass index BMI (kg/m2)=body weight/height 2 of every patient.Evaluate insulin resistance index and calculate insulin resistance index HOMA-IR=(FINS × FBG)/22.5.

All those selected at the above-mentioned limosis vein blood sample of extraction simultaneously, leave and take 2 milliliters of venous blood, with after 2%EDTA anti-freezing centrifugal 15 minutes, rotating speed 3000r/min, left and took supernatant blood plasma 0.5 milliliter, and place-80 DEG C of Refrigerator stores, the holding time is less than 3 months.

Assay method:

(5) each test tube is measured quantity of X-rays X.

The comparison of each group of plasma A PN concentration:

Impaired glucose tolerance and IFG merge the plasma A PN concentration of impaired glucose tolerance group significantly lower than IFG group, and difference has statistical significance (P value is respectively 0.013 and 0.002); Also remarkable have statistical significance (P value is respectively 0.004 and 0.000) lower than normal glucose tolerance group difference; And there is no significant difference (P=0.419) between impaired glucose tolerance group and IFG merging impaired glucose tolerance group; Compared with IFG group, though the APN concentration of normal glucose tolerance group is higher, no significant difference (P=0.779).In table 1 and Fig. 1.

Between four groups, table 1, plasma A PN concentration compares

	NGT group	IFG group	IGT group	IFG+IGT group

Number of cases	42	36	92	40
					APN(ng·ml ^-1)	12.15±3.85	11.92±2.94	9.95±3.58*#	9.33±3.57**#

Note: compare with NGT, * P<0.05, * * P<0.01; Compared with IFG group, #P<0.05, ##P<0.01

Claims

1. an assay method for adiponectin concentration, is characterized in that: the steps include:

(1) buffer standards product APN is configured;

(2) plasma specimen 10ml add 4990ml dissolve damping fluid be diluted to 1:500, be sample to be tested;

(3) often pipe adds 100ml testing sample or standard items, then adds I-APN solution, the anti-APN antibody of rabbit, fully mixes, hatches 24 hr overnight at 20 ~ 25 DEG C of temperature;

(4) rabbit anteserum and goat anti-rabbit igg antibody is added next day, except control tube, mixing, hatch 20 minutes for 4 DEG C, within centrifugal 20 minutes, Aspirate supernatant discards;

(5) each test tube is measured quantity of X-rays X;

(6) Specification Curve of Increasing; On logarithmic paper with standard items APN concentration for horizontal ordinate, B/B0 (%) is ordinate drawing standard curve;

(7) B/B0 checks in corresponding APN concentration from curve per sample;

(8) APN concentration ng/ml calculates: gained APN concentration ng/ml value is multiplied by extension rate, is concentration of specimens.

2. the assay method of a kind of adiponectin concentration according to claim 1, is characterized in that: step 1) in, the concentration of buffer standards product APN is respectively 200,100,50,25,12.5,6.25,3.125,1.56,0.78ng/ml, totally 9 pipes.