CN105021548A - Arsenic-fibrinogen chelate as well as preparation method and application thereof - Google Patents
Arsenic-fibrinogen chelate as well as preparation method and application thereof Download PDFInfo
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- 239000013522 chelant Substances 0.000 title claims abstract description 123
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- 238000001514 detection method Methods 0.000 claims abstract description 18
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Abstract
The invention provides an arsenic-fibrinogen chelate and a preparation method and application thereof, the arsenic-fibrinogen chelate is formed by chelating arsenic ions and fibrinogen through sulfydryl or/and cysteine residues, and can be used for preparing a reagent for detecting the arsenic-fibrinogen chelate of a human body. The invention proves that arsenic ions can directly act on fibrinogen for the first time. The invention establishes a qualitative and quantitative detection method of arsenic-fibrinogen chelate to detect the fibrinogen content in the crowd in one region, thereby indirectly reflecting the arsenic pollution degree in one region and the influence on the health of the crowd. The quantitative detection method of the arsenic-fibrinogen chelate, which is established by the invention, has high accuracy, repeatability and good repeatability.
Description
Technical field
The present invention relates to the immunology detection of arsenic ion, be specifically related to a kind of arsenic-fibrinogen chelate and its preparation method and application.
Background technology
Its physiological function of fibrinogen (fibrinogen, Fg) mainly participates in coagulation process directly as factor I.In blood coagulation common pathway, the first cracking fibrinogen of fibrin ferment two A α chain aminoterminal Arg16-Gly17 discharge a pair fibrinogen peptide A, form fibrinogen monomer I; A pair fibrinogen peptide B is discharged at cracking fibrinogen two B β chain aminoterminal Arg14-Gly15, form fibrinogen monomer II, the polymerization position of exposed fibers proteinogen monomer, by Non-covalent binding, defines the unstable former monomer of soluble fibrin.At factor XIII and the Ca of activation
2+effect under, fibrinogen monomer is cross-linked mutually, generates stable soluble fibrin former, and holds wherein by the visible component of blood, forms firmly thrombus.
Except participation blood coagulation, fibrinogen also has other several functions, and as being combined with platelet membrane glycoprotein Ⅱb/III a, mediate platelet aggregation reacts, and participates in atherosclerotic and tumour hematogenous metastasis etc.; Fibrinogen level also affects blood viscosity, it is found that plasma fibrinogen level rising is the important risk factor of cardiovascular and cerebrovascular, thrombotic diseases especially in recent years.Plasma fibrinogen is also acute phase protein, much stress under situation, as infection, severe trauma etc. also can raise in the short time.
Arsenic is that one common are noxious metals and environmental contaminants, general population's contact or take in the main path of arsenic and have: suck air that arsenic pollutes, absorptions arsenic container or pack the beverage of splendid attire or food, absorption contains arsenic medicine etc.Although the infringement of arsenic to body is fairly obvious, its mechanism causing body damage also waits research.There are positive trivalent and positive pentavalent two kinds of modal oxidation state in arsenic, a series of changes such as inorganic arsenic can be oxidized in nature and organic metabolic process, reduce, methylate, glycosylation, forms the arsenic of different shape.Inorganic arsenic is considered to the very strong form of toxicity, is listed in carcinogen, wherein arsenite (iAs
iII) toxicity be better than arsenate (iAs
v); The arsenic Difference In Toxicity of different shape is very large, monomethyl arsenious acid (monomethylarsonous acid, MMA
iII) and dimethyl arsenious acid (dimethylarsinous acid, DMA
iII) all there is severe toxicity, and its pentavalent form (MMA
v, DMA
v) then toxicity is not strong.Research is thought, arsenious toxicity is by producing with protein sulfhydryl effect.Arsenic and protein combination can affect the composition of protein, structure and function, affect this protein and are combined with other functional proteins, therefore study arsenic and protein bound to understanding the toxicity of arsenic and significant to the damage mechanisms of body.
Current known arsenic can suppress more than 200 kind of enzyme, no matter be that trivalent arsenic or organo-arsenic (comprise oxidation arsenobenzene, phenylarsine (PAO)) there is the effect of inhibitory enzyme, these enzymes comprise glutathione reductase, glutathione transferase, glutathione peroxidase, sulphur hydrogen reduction compound enzyme, DNA ligase, Arg-tRNA protein transferase, oxidized form diglutathione spermidine reductase, I κ B activates β enzyme (IKK β), pyruvic acid activates Galectins 1 enzyme, tyrosine phosphatase proteinase, JNK phosphatase, Wip1 phosphatase, E3 connects c-CBL and SIAH1.Research is thought, arsenic mainly by being incorporated on sulfydryl or cysteine residues in protein or peptide chain combines, plays a role.
There is large metering method both at home and abroad carries out the detection of blood arsenic at present, comprise ELISA, atomic absorption spectrum (Atomic AbsorptionSpectroscopy, AAS) quantitative assay, icp ms (inductively coupled plasma massspectrometry, ICP-MS) quantitative assay etc.But these methods can only detect non-specific blood arsenic, and majority can only point out index as Disease activity, and lack disease specific value.
Summary of the invention
For the with serious pollution problem of arsenic, the object of the present invention is to provide a kind of arsenic-fibrinogen chelate and preparation method thereof, and set up the qualitative and quantitative analysis method of arsenic-fibrinogen chelate, quantitatively to detect the application of arsenic-fibrinogen chelate in the regional arsenic pollution level of evaluation one.By quantitatively detecting arsenic-fibrinogen chelate in a regional crowd, indirectly can reflect the situation that this regional crowd pollutes by arsenic, thus indirectly reflecting this regional arsenic pollution level.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of arsenic-fibrinogen chelate, this arsenic-fibrinogen chelate be arsenic ion and fibrinogen by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned arsenic-fibrinogen chelate, i.e. external synthetic method, comprises the following steps:
The synthesis of A) arsenic-fibrinogen huge legendary turtle compound: add arsenic ion and carry out chelatropic reaction purifying in the fibrinogen that comes from human body or the fibrinogen according to biological method restructuring, obtain reaction solution;
B) arsenic-fibrinogen chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted fibrinogen, specific antibody and arsenic ion in reaction solution, obtain arsenic-fibrinogen chelate.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibrinogen chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
Described can with the filler of fibrinogen specific binding be adsorbed with can with the silica gel of fibrinogen specific binding material or resin;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
Described can with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-fibrinogen chelate.
As preferably, the preparation method of above-mentioned arsenic-fibrinogen chelate, also comprises the following authentication step to arsenic-fibrinogen chelate, specifically comprises the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-fibrinogen chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: the protein band finding out metal on glue bed, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing arsenic and detection arsenic.
The present invention also provides the application of a kind of arsenic described above-fibrinogen chelate in preparation human body in the reagent of arsenic-fibrinogen chelate or kit.
The present invention also provides a kind of and at least comprises the kit of arsenic described above-fibrinogen chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching fibrinogenic material or catching arsenic.
The present invention also provides a kind of method of quantitative detection arsenic-fibrinogen chelate, using the above-mentioned arsenic-fibrinogen chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-fibrinogen chelate and enzyme linked immunological combined techniques, purification arsenic-fibrinogen chelate and atomic absorption spectrum combined techniques, purification arsenic-fibrinogen chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Compared to existing technology, beneficial effect of the present invention is:
1. the present invention's external synthesis first or extract arsenic-fibrinogen chelate from whole blood;
2. the present invention proposes arsenic-fibrinogen chelate first and can be used for preparing application in the reagent or kit detecting arsenic-fibrinogen chelate in blood sample;
3. present invention achieves the specific recognition of arsenic-fibrinogen chelate and quantitatively detect, so that the content of arsenic-fibrinogen chelate in quantitative detection crowd, to evaluate the application of a regional arsenic pollution level, the arsenic polluted water for industrial area is flat provides indirect indexes.The accuracy of arsenic-fibrinogen chelate quantitative detecting method that the present invention sets up is high, reproducible.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-fibrinogen chelate;
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of arsenic of the present invention-fibrinogen chelate.
Embodiment
Below in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention; Agents useful for same, material, as non-specified otherwise, are all considered as to buy from commercial mode:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Catching fibrinogenic material is antifibrin original antibody, by commercially available acquisition, as the human fibrin original antibody anti-human Fibrinogen that article No. is " NovusA-0577 ", in following embodiment, described material with fibrinogen specific binding, antifibrin original antibody, anti-Fg antibody are and catch fibrinogenic material;
Enzyme labelled antibody is the one in the antibody containing the enzyme labeling such as horseradish peroxidase, alkaline phosphatase;
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example: the Na getting 1.5g
2cO
3with the NaHCO of 2.93g
3dissolving adds ddH
2o is settled to 1000mL;
Lavation buffer solution is the 0.15M PBS solution of pH7.4, compound method example: the KH getting 0.2g
2pO
4, 2.90g Na
2hPO
412H
2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH
2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin(BSA), adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2M H
2sO
4, compound method example: the ddH getting 178.3mL
2o, enriching H
2sO
4be settled to 200mL;
Substrate is methyl biphenyl amine (TMB) solution, compound method example: get the methyl biphenyl amine ethanolic solution that 0.5mL concentration is 2g/L, add substrate dilution and be diluted to 10mL;
Substrate buffer solution pH is 5.0, wherein Na
2hPO
4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, compound method example: get 1.42gNa
2hPO
4, 0.96g citric acid, then add ddH
2o to 50mL, to obtain final product;
The compound method example of eluent: papain pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/mL, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min, obtain eluent;
Sample-loading buffer can be formulated by the component of following ratio: Tris-HCl:1% bromophenol blue: ddH
2o: glycocoll=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
The compound method of electrophoretic buffer is as follows: get 3.0g Tris, 14.4g glycocoll, be dissolved in 800mLddH
2in O, after adjusting pH to 8.3, be settled to 1L;
The material of the catching arsenic mouse-anti As mAb that to be article No. be " Guangzhou Ran Ke company RK15728 ", being with the material of arsenic specific binding, anti-As antibody, two anti-, anti-arsenic antibody described in following embodiment catches to obtain the material of arsenic;
The invention provides a kind of arsenic-fibrinogen chelate, arsenic ion and fibrinogen by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of arsenic-fibrinogen chelate, comprises the following steps:
The synthesis of A) arsenic-fibrinogen huge legendary turtle compound: add arsenic ion and carry out chelatropic reaction purifying in the fibrinogen that comes from human body or the fibrinogen according to biological method restructuring, obtain reaction solution;
B) arsenic-fibrinogen chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted fibrinogen, specific antibody and arsenic ion in reaction solution, obtain arsenic-fibrinogen chelate, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibrinogen chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
Described can with the filler of fibrinogen specific binding be adsorbed with can with the silica gel of fibrinogen specific binding material or resin;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
Described can with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-fibrinogen chelate.
C): to the qualification of arsenic-fibrinogen chelate, specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-fibrinogen chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: the protein band finding out metal on glue bed, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing arsenic and detection arsenic.
The present invention also provides a kind of and at least comprises the kit of arsenic described above-fibrinogen chelate as standard items.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprising: containing can be used for catching the coating buffer of fibrinogenic material, confining liquid, lavation buffer solution, the material of arsenic can being caught as two anti-, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative controls etc.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprising: containing the coating buffer, confining liquid, lavation buffer solution, eluent, positive control, negative control etc. that can be used for catching fibrinogenic material.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprising: containing the coating buffer, confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, negative control etc. that can be used for catching fibrinogenic material.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise to extract reagent needed for fibrinogen for purifying, redissolve liquid, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, standard items, negative control etc. of material that can be used for catching arsenic.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise and extract reagent, redissolution liquid, positive control, negative control etc. for purifying needed for fibrinogen.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise and extract reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc. for purifying needed for fibrinogen.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise to extract needed for fibrinogen for purifying reagent, glue bed medium, redissolve metal on liquid, sample loading buffer, dissolving glue bed protein band needed for liquid, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, positive control, negative control etc. of material that can be used for catching arsenic.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise to extract needed for fibrinogen for purifying reagent, glue bed medium, redissolve metal on liquid, sample loading buffer, dissolving glue bed protein band needed for liquid, positive control, negative control etc.
Detect a kit for arsenic in blood sample-fibrinogen chelate, comprise to extract needed for fibrinogen for purifying reagent, glue bed medium, redissolve metal on liquid, sample loading buffer, dissolving glue bed protein band needed for liquid, nitric acid, hydrogen peroxide, positive control, negative control etc.
In above-mentioned several kit, described positive control is standard items, is namely chelated with the fibrinogen chelate of heavy arsenic or is chelated with the BSA chelate of heavy arsenic; Described negative control is dilution buffer.
Mentioned reagent box, for detecting arsenic-fibrinogen chelate, to improve accuracy and the repeatability of detection, and makes it to be promoted in clinical.
The present invention also provides a kind of method of quantitative detection arsenic-fibrinogen chelate, using the above-mentioned arsenic-fibrinogen chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-fibrinogen chelate and enzyme linked immunological combined techniques, purification arsenic-fibrinogen chelate and atomic absorption spectrum combined techniques, purification arsenic-fibrinogen chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, following several with having of detecting that the method for arsenic-fibrinogen chelate can list, but be not limited to following several.
The following stated dilution multiple proportions is w/v.
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects arsenic-fibrinogen chelate, detects in accordance with the following steps:
1) bag quilt: can catch fibrinogenic material to 1000-8000 times with dilution buffer dilution, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with lavation buffer solution for 37 DEG C, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: from circulation system sampling, make sample to be tested; Standard items are made with the arsenic of known content-fibrinogen chelate; By dilution buffer, sample to be tested and standard items are all diluted to 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching arsenic is added, and incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, add with dilution buffer dilution 25000-200000 anti-arsenic antibody doubly, 37 DEG C of effect 1-2 hour, make the arsenic on anti-arsenic antibody and fibrinogen react;
5) enzyme conjugates incubation: remove anti-arsenic antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and HRP enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: add stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, elisa plate being placed in OD value microplate reader reading respectively sample to be tested group and standard items, drawing standard curve, by comparing with standard items group, trying to achieve the content of sample to be tested.
In this method, step 8) also can not use microplate reader, directly carry out qualitative detection by staining conditions.
The method utilizes ELISA principle, specificity fibrinogen in whole blood can be extracted, the fibrinogen upper part extracted is chelated with heavy arsenic, and arsenic on this part fibrinogen can catch by the specific antibody of anti-arsenic, afterwards can again by horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the fibrinogen not containing chelating arsenic, then can not catch by the specific antibody of anti-arsenic, also can not with horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase caught, and also not containing arsenic (negative control group result is negative) in agents useful for same, thus when read OD value result is shown as the positive, the i.e. provable arsenic detecting chelating on fibrinogen.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect arsenic-fibrinogen chelate and detect in accordance with the following steps:
1) bag quilt: fibrinogenic material can be caught, as antifibrin original antibody is coated on solid phase carrier: with dilution buffer dilution antifibrin original antibody to 1000-8000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: sample from the circulation system, make sample to be tested; Standard items are made with the arsenic of known content-fibrinogen chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds eluent, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the arsenic of chelating on fibrinogen in Atomic Absorption Spectrometer, read respective value;
This embodiment utilizes ELISA principle to catch fibrinogen, and detects the arsenic of chelating on fibrinogen in conjunction with atomic absorption spectrum (AAS) instrument; Owing to only containing fibrinogen in solution, and not containing arsenic (negative control group result is negative) in agents useful for same, interference can not be shone into result, thus when read result is shown as the positive, i.e. the provable arsenic detecting chelating on fibrinogen.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect arsenic-fibrinogen chelate and detect in accordance with the following steps:
1) bag quilt: fibrinogenic material can be caught, as antifibrin original antibody is coated on solid phase carrier, with dilution buffer dilution antifibrin original antibody to 1000-8000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: from circulation system sampling, make sample to be tested; Standard items are made with the arsenic of known content-fibrinogen chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C;
5) acidifying: in step 4) in solution in add acidulant acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in fibrinogenic arsenic, read respective value.
The method, on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, detects the arsenic of chelating on fibrinogen with icp ms; Namely first adopt ELISA principle to be extracted by the arsenic in whole blood-fibrinogen chelate, then adopt sense couple plasma mass spectrometer quantitatively to detect the arsenic of chelating on fibrinogen; Owing to only containing fibrinogen in solution, and not containing arsenic (negative control group result is negative) in agents useful for same, interference can not be shone into result, thus when read result is shown as the positive, i.e. the provable arsenic detecting chelating on fibrinogen.
method four:purification arsenic-fibrinogen chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detect arsenic-fibrinogen chelate, detect in accordance with the following steps:
1) from whole blood, non-specific fibrinogen is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved, obtain fibrinogenic solution;
2) bag quilt: be coated on solid phase carrier by anti-arsenic antibody, dilutes anti-arsenic antibody to 25000-200000 times by dilution buffer, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
3) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add sample to be tested, and incubation: from step 1) solution sample, make sample to be tested; Standard items are made with the arsenic of known content-fibrinogen chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, adds the enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: add stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, microplate reader reads the OD value of sample to be tested group and standard items respectively, drawing mark curve, by comparing with standard items group, trying to achieve the content of sample to be tested.
In this method, also can not use microplate reader, directly carry out qualitative detection by staining conditions.
method five:purification arsenic-fibrinogen chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detect arsenic in blood sample-fibrinogen chelate, detect in accordance with the following steps:
1) from whole blood, non-specific fibrinogen is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved, obtain fibrinogen solution;
2) detect: from step 1) solution sample, detect the arsenic of chelating on fibrinogen in Atomic Absorption Spectrometer, read respective value.
method six:purification arsenic-fibrinogen chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect arsenic-fibrinogen chelate, detect in accordance with the following steps:
1) from whole blood, non-specific fibrinogen is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved, obtain fibrinogenic solution;
2) acidifying: from step 1) solution sample, add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
3) detect: add hydrogen peroxide, and heating gets 0.5mL solution after catching up with acid, detects the arsenic of chelating on fibrinogen under icp ms, read respective value.
Method four, method five and method six are all isolate fibrinogen by whole blood extraction method, then adopt method for detecting specificity, measure the content of arsenic on arsenic-fibrinogen chelate in fibrinogen; Namely first physical separation means are adopted, as supercentrifugation, HPLC, gel-filtration chromatography etc., separated from test plasma sample by fibrinogen and redissolve in physiological saline, recycling ELISA principle, atomic absorption spectrum detect or carry out detecting the arsenic content on inductively coupled plasma mass spectrometry detection arsenic-fibrinogen chelate.
Method seven: electrophoresis+ELISA/AAS/ICP-MS method detects arsenic-fibrinogen chelate, specific as follows:
1) from whole blood, non-specific fibrinogen is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, the fibrinogen extracted is redissolved in physiological saline, obtains fibrinogenic solution;
2) glue bed is prepared: select suitable medium (as Ago-Gel, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) solution get 8 μ L solution, make standard items with the arsenic of known content-fibrinogen chelate, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, with electrophoretic buffer, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of isoelectric point;
5) detect: on glue bed, find out the protein band containing fibrinogen huge legendary turtle compound, this band is taken out, this protein band is dissolved among liquid, and then utilize the principle such as ELISA or ICP-MS or AAS to detect the arsenic content be dissolved in liquid respectively.
In addition, the isoelectric point of the method detection arsenic-fibrinogen chelate, molecular weight and content etc. can also be utilized.
In method seven, fibrinogen is extracted from whole blood, then adopt gel electrophoresis to be separated extracted fibrinogen, then find out the respective strap being rich in arsenic, then detect the content of associated fiber proteinogen, namely fibrinogen can by multiple Methods For Purification out (such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the fibrinogen of purifying out is redissolved in solution, get a certain amount of fibrinogen, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different medium can be adopted as required), the difference such as isoelectric point runs out of different bands, find out the respective strap being rich in arsenic, protein in gel is redissolved in solution, namely the content of associated fiber proteinogen can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the arsenic content of chelating on fibrinogen, owing to only containing fibrinogen in solution, and not containing arsenic (negative control group result is negative) in agents useful for same, interference can not be shone into result, thus when read result is shown as the positive, the i.e. provable arsenic detecting chelating on fibrinogen.
embodiment 1:synthetic method synthesis arsenic-fibrinogen huge legendary turtle compound, comprises the following steps:
Arsenic prepared by the present embodiment-fibrinogen huge legendary turtle compound, is separated further by gel electrophoresis, and carries out detection Qualitative Identification by inductivity coupled plasma mass spectrometry or atomic absorption spectrum.
The reagent that the present embodiment uses is as follows:
1) borate buffer solution, its volumetric molar concentration is 0.01M, and its preparation method example is as follows: take 0.31g boric acid and be dissolved in 400mLddH
2in O, regulate pH to 9.0 with the NaOH of 0.1mol/L, be settled to 500mL.
2) EDTA-NaHCO
3solution, its preparation method is as follows: get 1.86g EDTA2H
2o and 16.8g NaHCO
3, be dissolved in 900mLddH
2in O, adjust pH to 8.0 with 1.0M NaOH and be settled to 1000mL, autoclaving, room temperature preservation;
3) ITCBE buys from Japanese colleague's chemistry institute, article No. M030;
4) fibrinogen solution: take 4.0mg fibrinogen and be dissolved in 4.0mL0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the fibrinogen solution of 1.0mg/mL;
5) molecular cut off 14000 of bag filter, buys from Bioshop Inc;
The pre-service of bag filter: EDTA-NaHCO bag filter being put into 500mL
3in solution, boil 10min; Tipping EDTA-NaHCO
3solution, uses ddH
2o is rinsing gently, then boils 10min with 500mL5mmol/L EDTA; Discard boiling liquid, thoroughly use ddH
2o cleans, and adds a large amount of ddH
2o soaks bag filter 4 DEG C and spends the night.During use, put on one's gloves, take out bag filter, with a large amount of ddH
2its surfaces externally and internally of O cleaning down;
Prepare the method for arsenic-fibrinogen chelate, comprise the following steps:
The synthesis of A) arsenic-fibrinogen huge legendary turtle compound: add arsenic ion and carry out chelatropic reaction in the fibrinogen in people source, obtain reaction solution;
1) getting 2.0mg ITCBE is dissolved in 2mLDMSO;
2) liquid slowly step 1 prepared adds in fibrinogen solution, and dropping limit, limit is shaken, and in 25 DEG C, acts on 24h in the shaking table of 100r/min, then to dialyse 24h with bag filter, removes the ITCBE be not combined with fibrinogen;
3) by the liquid 1mol/L HCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l 1mmol/L arsenic ion solns gradually, dropping limit, limit vibrates, in order to avoid arsenic ion makes albuminous degeneration precipitate;
4) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the bag filter handled well;
5) liquid of having dialysed is preserved in-20 DEG C of packing, obtain the reaction solution of arsenic-fibrinogen chelate.
B) arsenic-fibrinogen chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted fibrinogen, specific antibody and arsenic ion in reaction solution, obtain arsenic-fibrinogen chelate, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibrinogen chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-fibrinogen chelate.
C) to the qualification of arsenic-fibrinogen huge legendary turtle compound, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using Ago-Gel as medium;
(2) application of sample: get step B) in arsenic-fibrinogen chelate of obtaining of extraction purification, redissolve in physiological saline, take out 8 μ L, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer; In electrophoresis process, electric current is 22mA constant current, and environment temperature is 4 DEG C; Stop electrophoresis when moving to bottom glue to bromophenol blue, Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-fibrinogen huge legendary turtle compound;
(4) detect: the protein band finding out metal on glue bed, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing arsenic and detection arsenic.
D) qualification result
1) AAS testing result
Get step C) isolated protein band solution, with the content of arsenic heavy in GFAAS (graphite furnace atomic absorption spectrometry) (AAS) Preliminary Determination fibrinogen, as shown in the table, with (NH
4)
2sO
4solution is blank;
The content of arsenic in table 1 fibrinogen
2) Synchrotron Radiation X-Ray Fluorescence Anal ysis
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of BEPC (BEPC).In storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA 4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx 3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of arsenic of the present invention-fibrinogen chelate, and in figure, horizontal ordinate is protein band position, and ordinate is energy (content) value of this band arsenic.
the determination of testing conditions
1. the determination of the optimum diluting multiple of antifibrin original antibody, anti-As antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for arsenic-fibrinogen chelate, specifically comprises the following steps:
1) bag quilt: antifibrin original antibody albumen is coated on solid phase carrier, respectively by dilution buffer by coating protein with the doubling dilution of 1:1000,1:2000,1:4000 and 1:8000, add in elisa plate micropore, each concentration bag, by three rows, is preserved 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) sample to be tested is added: by dilution buffer by the doubling dilution of test plasma sample by 1:10,1:20,1:40, add in micropore, make standard items with the arsenic of known content-fibrinogen chelate, negative control and blank are set, add in micropore, 37 DEG C act on 1 hour;
4) anti-As antibody is added: remove test plasma sample, washing, add with dilution buffer by 1:25000,1:50000,1:100000 and 1,200000 the anti-As antibody of doubling dilution, 37 DEG C of effects 1 hour, make the arsenic on itself and fibrinogen react;
5) enzyme-added mark: remove anti-As antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, make itself and anti-As antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer;
7) detect: the OD value reading standard items, test plasma, positive control, negative control and blank sample under 450nm wavelength in microplate reader respectively.
In the present embodiment, adopt arsenic provided by the invention-fibrinogen chelate standard items as positive control, respectively not add test plasma as negative control 1, namely add antifibrin original antibody, confining liquid, anti-As antibody, enzyme mark and substrate successively;
Not add the control test group of anti-As antibody as negative control 2, namely add antifibrin original antibody, confining liquid, test plasma, enzyme mark and substrate successively;
Antifibrin original antibody, confining liquid, test plasma, anti-As antibody and substrate is added successively using not enzyme-added target control test group as negative control 3, namely;
Not add the control test group of sample to be tested and anti-As antibody as negative control 4 simultaneously, namely add antifibrin original antibody, confining liquid, enzyme mark and substrate successively;
Not add the control test group of antifibrin original antibody work for blank 1, namely add confining liquid, test plasma, anti-As antibody, enzyme mark and substrate;
And with the control test group only adding substrate for blank 2, only to add the control test group of PBS for blank 3.
Table 2 is the sample OD Value Data of different antifibrin original antibody dilution multiple proportions, diluted plasma multiple proportions, anti-As antibody dilution multiple proportions,
Testing result under table 2 different antifibrin original antibody, anti-As antibody and diluted plasma multiple proportions
As known from Table 2, when the dilution multiple proportions of antifibrin original antibody is 1:2000, sample OD value is greater than the dilution multiple proportions of other antifibrin original antibody under parallel condition; In this group sample, when diluted plasma multiple proportions is 1:10, when anti-As antibody dilution multiple proportions is 1:25000, OD value is maximum, is 0.756.
The OD detected value of positive control, negative control and blank corresponding when the dilution multiple proportions that table 3 is antifibrin original antibody is 1:2000, diluted plasma multiple proportions is 1:10, anti-As antibody dilution multiple proportions is 1:25000,
The testing result of table 3 positive control, negative control and blank
As known from Table 3, negative control group OD detected value is less than 0.1, and under this optimal conditions is described, the systematic error of this method is little, meets analytical approach requirement, so select concentration corresponding to this value as best effort concentration.
2. ELISA eluent best effort concentration and elution time are determined
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-As antibody and enzyme labelled antibody incubation, carry out wash-out with the eluent of variable concentrations, then detect OD value by microplate reader, concrete steps are as follows:
(1) bag quilt: be coated on solid phase carrier by anti-As antibody, press the doubling dilution of 1:25000 by dilution buffer, adds in elisa plate micropore, preserves 16 hours for 4 DEG C;
(2) close: remove dilution buffer, after washing, add confining liquid, place 1 hour, remove confining liquid, and wash for 37 DEG C;
(3) add enzyme labelled antibody: remove confining liquid, after washing, add and be diluted to by dilution buffer the HRP enzyme labelled antibody that antibody concentration is 2ng/mL, 37 DEG C act on 2 hours, make itself and anti-As antibody response;
(4) wash-out: remove enzyme labelled antibody, by dilution buffer, eluent is diluted, make the concentration of papain in eluent: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove eluent, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer cessation reaction;
(5) under the determined wavelength of 450nm, in microplate reader, read the OD value of each micropore respectively, concrete outcome see table 4,
Testing result under the different eluent extension rate of table 4
By comparing OD value, to judge anti-As antibody-hrp-antibody complex wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-As antibody-hrp-antibody complex wash-out degree reaches maximum.As shown in table 4, the concentration when papain in eluent: in enzyme labelled antibody during the concentration=1:20 of antibody, wash-out degree is maximum; And action time is when being 1h, 2h, 3h, each group of OD value change is little, the visible prolongation along with the time, enzyme activity weakens gradually, when enzyme concentration is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of eluent be that 1-3h all can, in sum, we select eluent 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
Application Example
application Example 1
Employing euzymelinked immunosorbent assay (ELISA) (ELISA method) detects the arsenic-fibrinogen chelate in 100 parts of sample blood plasma, and the method namely adopting specific embodiment method one to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by antifibrin original antibody, is diluted to 2000 times by dilution buffer, adds in elisa plate micropore, preserves 4 DEG C of storages in refrigerator after 1 hour for 37 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, and place 1 hour, remove confining liquid for 37 DEG C, washing, elisa plate 37 DEG C places 4 DEG C of storages in refrigerator after 1 hour;
3) application of sample: using sample blood plasma as sample to be tested, makes standard items with the arsenic of known content-fibrinogen chelate, by dilution buffer, sample to be tested and standard items is all diluted to 10 times, adds in micropore, and 37 DEG C act on 1 hour;
4) add anti-As antibody: remove sample, washing, add the anti-As antibody being diluted to 25000 times by dilution buffer, 37 DEG C of effects 1 hour, make the arsenic on itself and fibrinogen react;
5) enzyme-added mark: remove anti-As antibody, washing, adds the enzyme labelled antibody that antibody concentration is 2ng/mL, and 37 DEG C act on 1 hour, make itself and anti-As antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer to each micropore;
7) detect: the OD value reading sample to be tested group and standard items under 450nm wavelength in microplate reader respectively, result is as shown in table 5.
The measured result of table 5 method a pair 100 parts of sample blood plasma
In this application embodiment 1, step 7) in, microplate reader also can not be used to detect, but directly carry out qualitative detection by dyeing.
application Example 2
Adopt enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) to detect arsenic-fibrinogen chelate in 100 parts of sample blood plasma, the method namely adopting specific embodiment method two to record detects, and concrete operation step is as follows:
1) bag quilt: antifibrin original antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 2000 times, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, preserve at elisa plate 4 DEG C;
3) application of sample: make sample to be tested with sample blood plasma, makes standard items with the arsenic of known content-fibrinogen chelate, by dilution buffer, sample to be tested and standard items is diluted to 10 times, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove test plasma sample, washing, adds and is diluted to by dilution buffer the eluent that Papain enzyme concentration is 100ng/mL, and 37 DEG C act on 2 hours;
5) detect: sample from ELISA micropore, detect the arsenic of chelating on fibrinogen in Atomic Absorption Spectrometer, result is as shown in table 6.
Table 6 method two is to the measured result of 100 parts of sample blood plasma
application Example 3:
Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) is adopted to detect arsenic-fibrinogen chelate in 100 parts of sample blood plasma, namely the method adopting specific embodiment method three to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by antifibrin original antibody, with dilution buffer dilution coating protein to 2000 times, adds in elisa plate micropore, preserves 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, elisa plate 4 DEG C preservation;
3) application of sample: make sample to be tested with sample blood plasma, makes standard items with the arsenic of known content-fibrinogen chelate, by dilution buffer, sample to be tested and standard items is diluted to 10 times, add in micropore, and 37 DEG C act on 2 hours;
4) wash-out: remove sample to be tested and standard items, washing, adds and is diluted to by dilution buffer the eluent that Papain enzyme concentration is 100ng/mL, 37 DEG C of wash-outs 2 hours;
5) acidifying: add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
6) detect: sampling, under icp ms, detect the arsenic of chelating on fibrinogen, result is as shown in table 7.
Table 7 method three is to the measured result of 100 parts of sample blood plasma
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (8)
1. arsenic-fibrinogen chelate, is characterized in that, this arsenic-fibrinogen chelate be arsenic ion and fibrinogen by sulfydryl or/and cysteine residues chelating forms.
2. the preparation method of arsenic-fibrinogen chelate as claimed in claim 1, comprises the following steps:
The synthesis of A) arsenic-fibrinogen huge legendary turtle compound: add arsenic ion and carry out chelatropic reaction purifying in the fibrinogen that comes from human body or the fibrinogen according to biological method restructuring, obtain reaction solution;
B) arsenic-fibrinogen chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted fibrinogen, specific antibody and arsenic ion in reaction solution, obtain arsenic-fibrinogen chelate.
3. method as claimed in claim 2, is characterized in that,
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibrinogen chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-fibrinogen chelate.
4. the preparation method of arsenic according to claim 2-fibrinogen chelate, is characterized in that, step B) after also comprise step C): to the qualification of arsenic-fibrinogen chelate;
Wherein, step C) in specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-fibrinogen chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: the protein band finding out metal on glue bed, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing arsenic and detection arsenic.
5. the application of arsenic as claimed in claim 1-fibrinogen chelate in the reagent or kit preparing to detect arsenic-fibrinogen chelate in blood sample.
6. one kind at least comprises the kit of arsenic as claimed in claim 1-fibrinogen chelate as standard items.
7. kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching fibrinogenic material or catching arsenic.
8. one kind is quantitatively detected the method for arsenic-fibrinogen chelate, it is characterized in that, using the arsenic according to claim 1-fibrinogen chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-fibrinogen chelate and enzyme linked immunological combined techniques, purification arsenic-fibrinogen chelate and atomic absorption spectrum combined techniques, purification arsenic-fibrinogen chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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