CN102169118B - Method for detecting 8-oxo-deoxyguanosine (8-oxo-dG) - Google Patents

Abstract

The invention discloses a method for detecting 8-oxo-deoxyguanosine (8-oxo-dG). The method comprises the following steps of: adding a 8-oxo-dG containing object to be detected into a solid-phase carrier; adding a 8-oxo-dG resistant antibody and making 8-oxo-dG react with the 8-oxo-dG resistant antibody to form a specific combination; adding an anti-mouse IgG-HRP (Immunoglobulin G-Horseradish Peroxidase) polymer and making the anti-mouse IgG-HRP polymer react with the specific combination to form a ternary compound containing the 8-oxo-dG, the 8-oxo-dG resistant antibody and the anti-mouse IgG-HRP polymer; adding an ECL (Electrochemical Luminescence) chemical luminous substrate which emits light under the action of the ternary compound; and detecting the luminous intensity of the ECL chemical luminous substrate and calculating the content of the 8-oxo-dG according to the luminous intensity. The method disclosed by the invention has the advantages of high yield, low detection limit, low cross reaction, easiness for operation, short detection time, no need of complex pretreatment or the adoption of any expensive instrument and low detection cost.

Description

The quantitative detecting method of 8-oxo deoxyguanosine in serum or the urine

Technical field

The present invention relates to molecular biology and field of immunology, especially relate to the quantitative detecting method of 8-oxo deoxyguanosine in a kind of serum or the urine.

Background technology

Enzyme system in mitochondria, microsome, plasma membrane and the kytoplasm of body in cell and non-enzyme system can produce active oxygen radical in metabolic process.Simultaneously body also possibly produce active oxygen radical receiving under the conditions such as exogenous chemical substances and ionising radiation.Said active oxygen radical can form adduct such as deoxyguanosine with the base of DNA; Said deoxyguanosine adduct is under the effect of hydroxylase; Form the hydroxyl deoxyribonucleoside; Wherein 8-oxo deoxyguanosine (8-oxo-dG) is mainly a kind of in the hydroxyl deoxyribonucleoside hydroxyl, full name be 8-hydroxyl-2 ,-deoxyguanosine (8-oxo-dG), its chemical constitution is as shown in Figure 1.

8-oxo-dG can directly cause gene mutation and oncogene expression.Like reports such as Kuchino: 8-hydroxyl deoxy-guanine has the ability with any base pairing.The C of deoxy-guanine ₈After hydroxylation, the Disability that deoxy-guanine matches with the cytimidine specificity, the mispairing of base when causing dna replication dna, thus the form that several genes is suddenlyd change appears.Wood etc. once confirmed: in Escherichia coli, 8-oxo deoxyguanosine has the ability of inducing G → T displacement.Cheng etc. also report: 8-oxo deoxyguanosine can cause G → T and A → C displacement.8-oxo deoxyguanosine can mutagenesis, and the main cause of ras gene activation is point mutation.Kamiya etc. ^[4]Confirm: the proto-oncogene c-Ha-ras transfection NTS3T3 cell with the 12nd bit codon bases G of synthetic is replaced can make part cell generation vicious transformation.In the adduct of known about 20 kinds of DNA; 8-oxo-dG is easy to detect; And 8-oxo-dG is obviously relevant with gene mutation, and 8-oxo-dG has become the maximum DNA base oxidative damage of research and repaired product, becomes the most frequently used biological markers of researching DNA injury repair (Biomarker).

8-oxo-dG is a kind of extremely promising biological marker as endogenous or the extrinsic factor biomarker to the effect of DNA oxidative damage, the degree that can assess the vivo oxidation damage and repair through the detection of 8-oxo-dG.The mutual relationship of oxidative stress and dna damage, the relation of, environmental poisonous substance machine-processed to research DD, old and feeble mechanism, carcinogenesis and oxidative stress etc. are significant, also can be used for estimating the effect of antioxidant therapy DNA oxidative damage.The detection method of 8-oxo-dG mainly contains at present ³²Labelling method, immunofluorescence histochemistry detection method, enzyme linked immunological absorption (ELISA) method, high performance liquid chromatography-electrochemical method, gas chromatography mass spectrometry analytic approach and HPCE etc. behind the P.

³²Labelling method behind the P, ³²Labelling technique is to grow up after the nineties in 20th century behind the P, have highly sensitive, amount of samples is few, advantages of wide application range.Its operating process comprises: the digestion of DNA, mark, chromatography, radioautograph.But the specificity of this method has much room for improvement, and causes radioactive contamination easily.

Enzyme linked immunological absorption (ELISA) method, the ELISA method of using monoclonal antibody has very high sensitivity and good repeatability.The development that is used for immune affinity column of the development of anti-8-oxo-dG polyclonal antibody and monoclonal antibody provides condition, can be used for analysis of biological samples, especially the oxidative damage product of DNA and RNA in the urine.This method method need not be carried out resolution process to DNA, need not use expensive instrument, and minute is short, and the sample preprocessor is simple, is a kind of method that using value is arranged very much.

High-efficient liquid phase chromatogram-electrochemical detector is analyzed (HPLC-ECD) method, is after enzyme hydrolysis sample with DNA separates through HPLC, to use ECD to measure.This method is the effective ways of a kind of quantitative measurement 8-oxo-dG of proposing such as Floyd.HPLC-ECD is used widely in detecting 8-oxo-dG; Has higher sensitivity; Its LDL is approximately 1/50 nucleosides, and required sample size is few, can not produce damaging and selectivity good to body; All can detect 8-oxo-dG in histocyte DNA and the urine, be widely used at present, comparatively ripe method.

Gas chromatography mass spectrometry analytic approach (GC-MS) is a kind of gas chromatograph and mass spectrometer to be united the use detection method, and the evaluation that can be the oxidation product in the dna damage provides reliable and clear and definite data, and it is limited to 1/50 nucleosides under detecting.Yet, can't be widely used in breadboard detection because mass spectrometer costs an arm and a leg.The DNA base must at first be carried out derivative reaction simultaneously, and this process causes the formation of accessory substance easily, thereby is easy to generate false-positive display result.

HPCE (HPCE) is to utilize 8-oxo-dG and other deoxyguanosines swimming direction and speed different and separating in electric field, uses retention time and carries out qualitatively, carries out quantitatively through external standard method.Its advantage is that sample size is little, separation efficiency is high, retention time is short, and is more rapider than the HPLC separation, uses UV-detector sensitivity lower, and the Applied Electrochemistry detecting device then improves sensitivity greatly.

In the above-mentioned multiple detection method; All have some defective, as utilize in the process that effect liquid phase chromatogram-electrochemical process detects, exist enzymolysis not exclusively, existence accessory substance, radioactive contamination, sense cycle be long; And it is high to detect cost, can only be confined to clinical practice very among a small circle.The ELISA immunological method is compared the HPLC method and is existed cross reaction to make the mensuration result higher.

Can know from existing above-mentioned detection method, 8-oxo-dG is separated with other interfering materials that detecting instrument is still waiting to improve to the sensitivity of the specificly-response of 8-oxo-dG.

Summary of the invention

Fundamental purpose of the present invention is to provide the quantitative detecting method of 8-oxo deoxyguanosine in a kind of blood or the urine, avoids the non-specific intersection of immune detection, simplifies and detects step.

The present invention proposes the quantitative detecting method of 8-oxo deoxyguanosine in a kind of serum or the urine, comprises step:

The determinand that will contain 8-oxo-dG joins in the solid phase carrier;

In solid phase carrier, add anti-8-oxo-dG antibody, make 8-oxo-dG and anti-8-oxo-dG antibody response form specificity junction mixture;

In solid phase carrier, add anti-mouse IgG-HRP polymer, make the reaction of anti-mouse IgG-HRP polymer and said specificity junction mixture form the ternary complex of 8-oxo-dG, anti-8-oxo-dG antibody and anti-mouse IgG-HRP polymer;

In solid phase carrier, add the ECL chemical luminous substrate, said ECL chemical luminous substrate is luminous under the effect of said ternary complex;

Obtain corresponding gray-scale value through chemiluminescence digital imagery analyser, according to the content of 8-oxo-dG in the gray-scale value calculating determinand, minimum detectability is 0.2734ng/ml, and the testing result deviation is less than 15%.

Preferably, the quantitative detecting method of 8-oxo deoxyguanosine in said blood or the urine, said solid phase carrier is the DE81 film.

The quantitative detecting method of 8-oxo deoxyguanosine in blood provided by the invention or the urine; Existing relatively detection method; Its recovery is high, detectability is low, cross reaction is low, simple to operate; Lack (being no more than 3.5 hours) detection time, need not sample is carried out complicated processing in early stage, and need not to adopt expensive instrument, it is low to detect cost.

Description of drawings

Fig. 1 be 8-hydroxyl-2 ,-chemical structural drawing of deoxyguanosine;

Fig. 2 is a 8-oxo-dG content detection principle schematic of the present invention;

Fig. 3 be the present invention according to master sample concentration and corresponding gray thereof, the canonical plotting that match forms.

Embodiment

Through embodiment the present invention is further explained below.

Referring to Fig. 2, the principle that the present invention detects 8-oxo-dG is following: 8-oxo-dG and anti-8-oxo-dG antibody response are formed specificity junction mixture; Add anti-mouse-HRP polymer to specificity junction mixture, make the reaction of anti-mouse-HRP polymer and said specificity junction mixture form the ternary complex of 8-oxo-dG, anti-8-oxo-dG antibody and anti-mouse-HRP polymer; In ternary complex, add the ECL chemical luminous substrate then, said ECL chemical luminous substrate is luminous under the effect of said ternary complex; Detect the luminous intensity of ECL chemical luminous substrate then, calculate the content of 8-oxo-dG in the determinand according to luminous intensity.

The detection of embodiment 1:8-oxo-dG content

The present invention adopts above-mentioned detection method to detect the content of 8-oxo-dG in the determinand, and the typical curve of making according to the standard items measurement result calculates the content of 8-oxo-dG in the determinand.The production standard curve need be used 8-oxo-dG master sample concentration and comprise: 20ng/ml, 10ng/ml, 4ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml be the titer of totally 6 variable concentrations.The master sample of this 6 variant concentration is through PBS damping fluid (pH 7.4 for sodium dihydrogen phosphate 0.01M/L, NaCl 0.15M/L) the 8-oxo-dG standard items to be diluted to.

8-oxo-dG content in production standard curve, the detection determinand, and it is following to analyze specific detailed process:

S1, the template that will include the DE81 film lie on the clean filter paper; With concentration is that the titer of 0ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml, 20ng/ml is numbered in A1, A2, A3, A4, A5, A6, the A7 hole every hole point sample 3 μ l successively in the point sample DE81 film.The sample point sample to be checked that with concentration is 15ng/ml is numbered in A8, the A9 hole each hole point sample 3 μ l in the DE81 film; Concentration be the sample point sample to be checked of 1.5ng/ml in A10 and A11 hole, each hole point sample 3 μ l.With concentration is that the dG of 10ng/ml disturbs the sample point sample to being numbered in A12 and the A13 hole each hole point sample 3 μ l.With PBS damping fluid point sample to being numbered in A14 to the A23 hole point sample 3 μ l in each hole.The master sample point sample of 4ng/ml is numbered in the A24 hole point sample 3 μ l.

S2, said template placed under the room temperature DE81 film was parched to natural perk in 30～60 minutes.

S3, open template the DE81 film is put into reaction box, wash film 2 times with the deionized water jolting, each 1 minute; Outwell the deionized water after the washing; (1%BSA dilutes with PBS, and BSA requires to use fragment 5 to add confining liquid then; Purity is more than 95%), reaction box placed in 37 ℃ the constant temperature oven jolting sealing 20 minutes.

S4, outwell confining liquid, add anti-8-oxo-dG antibody, and reaction box placed in 37 ℃ the constant temperature oven jolting to hatch 60 minutes through the PBS dilution.

S5, outwell unnecessary anti-8-oxo-dG antibody liquid, and (PBS, 0.1%Tween-20) the jolting washing is 3 times, washs 5 minutes at every turn with cleansing solution.

S6, add anti-mouse IgG-HRP polymkeric substance, and reaction box placed in 37 ℃ the constant temperature oven jolting to hatch 10 minutes through the PBS dilution.

S7, (PBS, 0.1%Tween-20) jolting washing is 3 times, washs 5 minutes at every turn with cleansing solution.Add ECL substrate solution liquid, the DE81 film in the reaction box is crossed film and is soaked, and picks up counting, accurately react 1 minute after, with thieving paper the DE81 film is blotted, then the DE81 film is put into the press mold transparent film, extract rest solution.

S8, after 6 minutes the DE81 film is put into that chemiluminescence digital imagery analyser is taken and analyze, obtain corresponding gray-scale value.

Be numbered the gray-scale value of counter sample in A1 to the A24 hole.ECL reagent reaches peak value from the reaction beginning after 6 minutes, and it is more accurate that take the gray-scale value that obtains this moment.

Wherein, the master sample corresponding gray of 7 variable concentrations such as following table 1.

Table 1

The hole numbering

A7

A6

A5

A4

A3

A2

A1

Concentration

20ng/ml

10ng/ml

4ng/ml

2ng/ml

1ng/ml

0.5ng/ml

0ng/ml

Gray-scale value

139158.6

77043.3

29609

15821.1

7598.3

3785.6

0

According to the variable concentrations value and the corresponding gray of above-mentioned 7 master samples, with the match of straight line mathematical model, obtain typical curve, its coefficient R like Fig. 3 ²Value is 0.9973.

Precision in the computational analysis:

With replication precision sample in once testing, calculate the mean value of gray scale measured value

And standard deviation (S), precision in then analyzing $(\mathrm{CV}\%)=S/\stackrel{\‾}{x}\×100\%.$

Wherein, the concentration of specimens in the present embodiment is 4ng/ml, and this concentration samples surveys twice, and it corresponds to A5 and A24 on the DE81 film, and corresponding gray is respectively: 25984,28719, thus calculate: average gray X ₁=be 27351.5; Standard deviation S1=2461.6; Precision in analyzing $(\mathrm{CV}\%)=S/\stackrel{\‾}{x}\×100$ $\%==2461.6/27351.7\×100\%=8.99\%.$

Precision between analysis:

Replication precision sample (n>=10 in continuous three different experiments; Concentration of specimens is 4ng/ml); Calculate the mean value

and the standard deviation (S) of gray scale measured value, precision between analysis

Repeat above-mentioned steps S2-S8 with two other DE81 film, detectable concentration is the gray-scale value of 4ng/ml master sample.

Wherein, the concentration that detects for three times is that the master sample corresponding gray of 4ng/ml is respectively: 27750,27370; 28669,30533; 26898,26397, thus calculate: average gray X ₂=be 28269.4; Standard deviation S ₂=3742.5; Precision between then analyzing $(\mathrm{CV}\%)=S/\stackrel{\‾}{x}\×100\%=3742.5/28269.4\×100\%$ $=13.24\%.$

Detectability

The gray scale measured value of 10 PBS damping fluids of replicate determination; It corresponds to A14 to A23 on the DE81 film; According to the concentration value of each parallel samples of typical curve calculating, try to achieve sample mean and standard deviation (S); Then

Wherein, The gray scale measured value of above-mentioned 10PBS damping fluid is respectively 1788,1821,1220,970,1208,1630,1097,1927,882,2122; Calculate in the 10PBS damping fluid according to typical curve that 8-oxo-dG content is respectively 0.21,0.21,0.14,0.11,0.14,0.18,0.12,0.22,0.10,0.25ng/ml; Therefore calculate: the mean concentration of 10 PBS samples is 0.170738ng/ml; Standard deviation S is 0.05133,

The recovery

With PBS the 8-oxo-dG standard items are diluted to 15ng/ml and two concentration of 1.5ng/ml as recovery test sample book, the diplopore parallel testing calculates test sample book concentration A according to its gray-scale value and typical curve; The recovery ₁=A/15 * 100%; The recovery ₂=A/1.5 * 100%.

Wherein, concentration is that the 15ng/ml master sample corresponds to A8 and A9 on the DE81 film, and its corresponding gray is 51386,48681, calculates concentration according to typical curve and is respectively 13.3ng/ml, 12.6ng/ml; Concentration is that the 1.5ng/ml master sample corresponds to A10 and A11 on the DE81 film, and its corresponding gray is 51388,5668, calculates concentration according to typical curve and is respectively 1.26ng/ml, 1.39ng/ml.

For concentration is the recovery of 15ng/ml master sample ₁=A/15 * 100%=(13.3+12.6)/15 * 100%=86.3%.

For concentration is the recovery of 1.5ng/ml master sample ₁=A/1.5 * 100%=(1.26+1.39)/1.5 * 100%=88.3%.

Can know that from above-mentioned 15ng/ml and 1.5ng/ml master sample testing result its deviation is less than 15%.

Specificity

Select for use deoxyguanosine (dG) as the interference test test sample book, dG is diluted to 10ng/ml, the diplopore parallel testing calculates test sample book concentration B according to its gray-scale value and typical curve.

In this instance; The dG sample corresponds to A12 and A13 on the DE81 film, its corresponding gray is respectively 1264,734, calculates corresponding concentration according to typical curve and is respectively 0.31ng/ml and 0.18ng/ml; This shows that its cross reaction is low, do not influence the normal detection of 8-oxo-dG content.

Can find out from the foregoing description, the method that 8-oxo-dG of the present invention detects, the recovery is high, detectability is low; Cross reaction is low; It is simple to operate simultaneously, detection time short (being no more than 3.5 hours), need not that sample is carried out complicated early stage and handles; And need not to adopt expensive instrument, it is low to detect cost.Therefore 8-oxo-dG detection method of the present invention can be extensively utilized, the content of 8-oxo-dG in human serum or the urine can be detected.

The above DE81 film is available from Britain WHATMAN, and its product article No. is 3658-917.This DE81 film has following performance: thin (0.20mm), flow velocity are that 95mm/30min, ion-exchange intensity are that 18.0 μ eq/cm2, material therefor are DEAE-cellulose (DEAE) paper, and it has the weak base anion exchanger of diethyl chloroethyl functional group.

Said 8-oxo-dG standard items: for buy obtaining from SIGMA, its purity grade is>=98%LDC, and full name is 8-Oxo-7, and 8-dihydro-2 '-deoxyguanosine, english abbreviation are 8-Oxo-dG, its molecular formula: C ₁₀H ₁₃N ₅O ₅Molecular weight: 283.24; Article No.: 88847-89-6.

Said anti-mouse IgG-HRP polymkeric substance: be anti-mouse glucosan skeleton poly IgG-HRP; This polymkeric substance uses dextran chain to be skeleton, on dextran chain, forms an antibody-enzyme chain to a plurality of (50～100) anti-mouse IgG and HRP enzyme molecular labeling; Can detection signal be amplified; Shorten incubation time simultaneously, this anti-mouse IgG-HRP polymkeric substance steps neoformation technology company limited by Foochow and produces preparation, and article No. is kit-5903.

Said ECL luminous substrate: principal ingredient is luminol and derivant thereof, and luminol and derivant thereof are under peroxidase such as HRP catalytic action, with superoxide such as H ₂O ₂React luminous.This ECL luminous substrate is by the PIERCE manufacturer production.

Said chemiluminescence digital imagery analyser: be used to take the light signal that Western blotting sends, can the analytical standard article etc. the spot gray-scale value, generate typical curve and calculate sample value according to typical curve.This chemiluminescence digital imagery analyser is produced by SSTK Bio-Tech. (Shenzhen) Co., Ltd., and its corresponding medicine equipment registration certificate number is No. the 2400742nd, Guangdong food medicine prison tool (standard) word 2009.

Said anti-8-oxo-dG antibody full name is a mouse-anti people 8-oxo-dG monoclonal antibody, is produced by U.S. abcam company, and its product article No. is ab48508.The antibody type is IgG ₁, the about 150KD of molecular weight.

Claims (2)

1. the quantitative detecting method of 8-oxo deoxyguanosine in serum or the urine comprises step:

The determinand that will contain 8-oxo-dG joins in the solid phase carrier;

In solid phase carrier, add anti-8-oxo-dG antibody, make 8-oxo-dG and anti-8-oxo-dG antibody response form specificity junction mixture;

In solid phase carrier, add anti-mouse IgG-HRP polymer, make the reaction of anti-mouse IgG-HRP polymer and said specificity junction mixture form the ternary complex of 8-oxo-dG, anti-8-oxo-dG antibody and anti-mouse IgG-HRP polymer;

In solid phase carrier, add the ECL chemical luminous substrate, said ECL chemical luminous substrate is luminous under the effect of said ternary complex;

Obtain corresponding gray-scale value through chemiluminescence digital imagery analyser, according to the content of 8-oxo-dG in the gray-scale value calculating determinand, minimum detectability is 0.2734ng/ml, and the testing result deviation is less than 15%.

2. the quantitative detecting method of 8-oxo deoxyguanosine is characterized in that in blood according to claim 1 or the urine, and said solid phase carrier is the DE81 film.

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