CN105021548B - 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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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, and in particular to a kind of arsenic-fibrinogen chelate and its preparation
Methods and applications.
Background technology
Fibrinogen (fibrinogen, Fg) its physiological function directly participates in blood coagulation primarily as factor I
Process.In blood coagulation common pathway, fibrin ferment first cracks two A α chain aminoterminals Arg16-Gly17 of fibrinogen and discharges one
To fibrin former peptide A, fibrinogen monomer I is formed；In cracking two B β chain aminoterminals Arg14-Gly15 of fibrinogen
A pair of fibrin former peptide B are discharged, fibrinogen monomer II is formed, the polymerization position of exposed fibers proteinogen monomer, leads to
Non-covalent binding is crossed, forms unstable soluble fibrin original monomer.In the factor XIII and Ca of activation2+Work
Under, fibrinogen monomer is crosslinked mutually, generates stable soluble fibrin original, and the visible component of blood is wrapped
Wherein, firm thrombus is formed.
In addition to blood coagulation is participated in, fibrinogen also has other multiple functions, is such as tied with a of platelet membrane glycoprotein Ⅱb/III
Close and mediate platelet aggregation reaction, participate in atherosclerosis and tumour hematogenous metastasis etc.；Fibrinogen level has an effect on
Blood viscosity, especially in recent years it is found that plasma fibrinogen level rise be cardiovascular and cerebrovascular, thrombotic diseases it is important
Hazards.Plasma fibrinogen is also acute phase protein, much stress be under situation, such as infection, severe trauma
It can be raised in short time.
Arsenic is that one kind common are noxious metals and environmental contaminants, and general population's contact or the main path for taking in arsenic have：
The air of arsenic pollution is sucked, arsenic container is used in intake or packs the beverage contained or food, intake medicine containing arsenic etc..Although arsenic is to machine
The infringement of body is fairly obvious, but its mechanism for causing body damage also waits to study.Positive trivalent and positive two kinds of pentavalent be present most in arsenic
Common oxidation state, inorganic arsenic can be aoxidized in the metabolic process of nature and organism, reduce, methylate, glycosylated
Etc. a series of changes, the arsenic of different shape is formed.Inorganic arsenic is considered as the very strong form of toxicity, is listed in carcinogen, its
Middle arsenite (iAsIII) toxicity be better than arsenate (iAsV)；The arsenic poison sex differernce of different shape is very big, monomethyl arsenious acid
(monomethylarsonous acid, MMAIII) and dimethyl arsenious acid (dimethylarsinous acid, DMAIII)
There is a severe toxicity, and its pentavalent form (MMAV, DMAV) then toxicity it is not strong.Research thinks, the toxicity of trivalent arsenic be by with protein mercapto
Base act on and it is caused.Arsenic can influence composition, structure and the function of protein with protein binding, influence the protein and other work(
Can protein combine, therefore study arsenic combined toxicity to understanding arsenic with protein and its damage mechanisms to body have it is important
Meaning.
More than 200 kinds of enzyme can be suppressed by being currently known arsenic, either trivalent arsenic or organo-arsenic (including oxidation arsenobenzene,
Phenylarsine (PAO)) there is inhibitory enzyme, these enzymes include glutathione reductase, glutathione transferase, paddy
The sweet peptide peroxidase of Guang, sulphur hydrogen reduction compound enzyme, DNA ligase, Arg-tRNA protein transferases, the gluathione of oxidized form two
Peptide spermidine reductase, I κ B activation β enzymes (IKK β), the enzyme of acetone acid active Galectins 1, tyrosine phosphatase protease, JNK phosphorus
Sour enzyme, Wip1 phosphatases, E3 connection c-CBL and SIAH1.Research thinks, arsenic is mainly by being incorporated on sulfydryl or protein
Or the cysteine residues in peptide chain combine, and play a role.
A large amount of methods both at home and abroad at present be present and enter the detection of promoting circulation of blood arsenic, including ELISA, atomic absorption spectrum (Atomic
Absorption Spectroscopy, AAS) quantitative assay, icp mses (inductively
Coupled plasma mass spectrometry, ICP-MS) quantitative assay etc..But these methods can only detect non-spy
Different in nature blood arsenic, majority can only be used as Disease activity prompting index, and lack disease specific value.
The content of the invention
For arsenic pollution it is serious the problem of, it is an object of the invention to provide a kind of arsenic-fibrinogen chelate and its
Preparation method, and the qualitative and quantitative analysis method of arsenic-fibrinogen chelate is established, so as to quantitative detection arsenic-fibrin
Former chelate is evaluating the application of a regional arsenic pollution degree.Pass through arsenic-fibrin in one regional crowd of quantitative detection
Former chelate, situation of this regional crowd by arsenic pollution can be reflected indirectly, so as to reflect this regional arsenic pollution journey indirectly
Degree.
The technical solution adopted for the present invention to solve the technical problems is：A kind of arsenic-fibrinogen chelate is provided, should
Arsenic-fibrinogen chelate is that arsenic ion is formed with fibrinogen by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned arsenic-fibrinogen chelate, i.e., external synthetic method, including
Following steps：
A) the synthesis of arsenic-fibrinogen chelant thing：The fibrinogen of human body is come from purification or according to biology side
Arsenic ion is added in the fibrinogen of method restructuring and carries out chelatropic reaction, obtains reaction solution；
B) arsenic-fibrinogen chelate purifying：Using immune-affinity chromatography, removal step A) it is not anti-in reaction solution
Fibrinogen, specific antibody and the arsenic ion answered, produce arsenic-fibrinogen chelate.
Preferably, the step B) in specifically include following steps：
(1) sample dissolution：By above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibre
Fibrillarin original chelate solution；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with fiber egg
The filler of white former specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
The filler that can be specifically bound with fibrinogen can be with fibrinogen specificity junction mixture to be adsorbed with
The silica gel or resin of matter；
(3) loading：After column equilibration is chromatographed, with dilution buffer dilution step (1) solution, then upper prop, makes fibre
Fibrillarin is former to be specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：The eluent of collection in step (5) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times,
4 DEG C of dialysed overnights, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
The filler that can be specifically bound with arsenic is to be adsorbed with silica gel or resin that material can be specifically bound with arsenic；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：The eluent of step (10) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C thoroughly
Analysis overnight, collects sample, produces arsenic-fibrinogen chelate.
Preferably, the preparation method of above-mentioned arsenic-fibrinogen chelate, in addition to below to arsenic-fibrinogen
The authentication step of chelate, specifically includes following steps：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-fibrinogen chelate of extraction purification, add sample-loading buffer, and
Mix, be then loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band of metal is found out on glue bed, the protein band is taken out, protein band is dissolved, so
The content of arsenic is detected whether containing arsenic and detected again afterwards using ICP-MS or AAS.
The present invention also provides a kind of arsenic-fibrinogen chelate described above and is preparing arsenic-fiber egg in detection human body
Application in the reagent or kit of white former chelate.
The present invention, which also provides, a kind of comprises at least arsenic described above-reagent of the fibrinogen chelate as standard items
Box.
Preferably, coating buffer is also included in the kit, the coating buffer contains material or the capture of capture fibrinogen
The material of arsenic.
The present invention also provides a kind of method for quantitatively detecting arsenic-fibrinogen chelate, with the above-mentioned of known content
Arsenic-fibrinogen chelate is detected as standard items using a pair of samples of following methods：It is ELISA, enzyme-linked
Immune and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-fibrin
Former 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 electricity
Feel coupled plasma mass spectrometry combined techniques.
Compared with prior art, the beneficial effects of the present invention are：
1. arsenic-fibrinogen chelate is extracted in present invention synthesis in vitro first from whole blood；
2. it can be used for preparing arsenic-fibrinogen in detection blood sample present invention firstly provides arsenic-fibrinogen chelate
Application in the reagent or kit of chelate；
3. the present invention realizes the specific recognition of arsenic-fibrinogen chelate and quantitative detection, so as to quantitative detection
The content of arsenic-fibrinogen chelate in crowd, it is the arsenic of industrial area to evaluate the application of a regional arsenic pollution degree
Level of pollution provides indirect indexes.The degree of accuracy of the arsenic that the present invention establishes-fibrinogen chelate quantitative detecting method is high, again
Renaturation is good.
Brief description of the drawings
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 of the electrophoretic band of arsenic of the present invention-fibrinogen chelate
Figure.
Embodiment
With reference to embodiment, the present invention is described further：
Unless otherwise indicated, the laboratory operating procedures being related to are the conventional step in this area to the present invention；Agents useful for same, material
Material such as non-specified otherwise, is accordingly to be regarded as to buy from mode purchased in market：
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
The material for capturing fibrinogen is antifibrin original antibody, can be by commercially available acquisition, such as article No.
The human fibrin original antibody anti-human Fibrinogen of " NovusA-0577 ", in implementation below, it is described with it is fine
Material, antifibrin original antibody, the anti-Fg antibody of the former specific binding of fibrillarin are the material for capturing fibrinogen；
Enzyme labelled antibody is one kind in the antibody containing the enzymes such as horseradish peroxidase, alkaline phosphatase mark；
Dilution buffer be pH 9.6 0.05M carbonate buffer solutions, compound method example：Take 1.5g Na2CO3With
2.93g NaHCO3Dissolving plus ddH2O is settled to 1000mL；
Lavation buffer solution be pH7.4 0.15M PBS solutions, compound method example：Take 0.2g KH2PO4, 2.90g
Na2HPO4·12H2O, 8.0g NaCl, 0.2g KCl, 0.5mLTween-20, dissolving plus ddH2O is settled to 1000mL；
Confining liquid is bovine serum albumin solution, compound method example：0.1g bovine serum albumin(BSA)s are taken, add washing buffer
Liquid dilution is settled to 100mL；
Terminate liquid is 2M H2SO4, compound method example：Take 178.3mL ddH2O, enriching H2SO4It is settled to 200mL；
Substrate is methyl biphenyl amine (TMB) solution, compound method example：Take the methyl biphenyl amine that 0.5mL concentration is 2g/L
Ethanol solution, substrate dilution is added to be diluted to 10mL；
Substrate buffer solution pH is 5.0, wherein Na2HPO4Molar concentration be 0.2M, the molar concentration of citric acid be 0.1M,
Compound method example：Take 1.42gNa2HPO4, 0.96g citric acids, then add ddH2O to 50mL, is produced；
The compound method example of eluent：By papain pH 8.0,0.1mol/L Tris-HCI buffers
Into 1-2mg/mL, 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT)s (DTT) are added, obtain eluent；
Sample-loading buffer can be formulated by the component of following ratio：Tris-HCl：1% bromophenol blue：ddH2O：Glycine
=15.5：2.5：7：25, wherein Tris-HCl pH is 6.8, molar concentration 1M；
The compound method of electrophoretic buffer is as follows：Take 3.0g Tris, 14.4g glycine, be dissolved in 800mLddH2In O, adjust
After pH to 8.3,1L is settled to；
The material of capture arsenic is article No. for " Guangzhou Ran Ke companies RK15728 " the anti-As mAb of mouse, in implementation below
Described material, anti-As antibody, secondary antibody, anti-arsenic antibody with arsenic specific binding be capture arsenic material；
The present invention provides a kind of arsenic-fibrinogen chelate, and arsenic ion passes through sulfydryl or/and half Guang with fibrinogen
Histidine residue chelating forms.
The present invention also provides the preparation method of arsenic-fibrinogen chelate, comprises the following steps：
A) the synthesis of arsenic-fibrinogen chelant thing：The fibrinogen of human body is come from purification or according to biology side
Arsenic ion is added in the fibrinogen of method restructuring and carries out chelatropic reaction, obtains reaction solution；
B) arsenic-fibrinogen chelate purifying：Using immune-affinity chromatography, removal step A) it is not anti-in reaction solution
Fibrinogen, specific antibody and the arsenic ion answered, arsenic-fibrinogen chelate is produced, specifically includes following steps：
(1) sample dissolution：By above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibre
Fibrillarin original chelate solution；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with fiber egg
The filler of white former specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
The filler that can be specifically bound with fibrinogen can be with fibrinogen specificity junction mixture to be adsorbed with
The silica gel or resin of matter；
(3) loading：After column equilibration is chromatographed, with dilution buffer dilution step (1) solution, then upper prop, makes fibre
Fibrillarin is former to be specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：The eluent of collection in step (5) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times,
4 DEG C of dialysed overnights, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
The filler that can be specifically bound with arsenic is to be adsorbed with silica gel or resin that material can be specifically bound with arsenic；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：The eluent of step (10) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C thoroughly
Analysis overnight, collects sample, produces arsenic-fibrinogen chelate.
C)：Identification to arsenic-fibrinogen chelate, specifically includes following steps：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-fibrinogen chelate of extraction purification, add sample-loading buffer, and
Mix, be then loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band of metal is found out on glue bed, the protein band is taken out, protein band is dissolved, so
The content of arsenic is detected whether containing arsenic and detected again afterwards using ICP-MS or AAS.
The present invention, which also provides, a kind of comprises at least arsenic described above-reagent of the fibrinogen chelate as standard items
Box.
In the present invention, can realize the kit of the object of the invention can list following several, but be not limited to this.
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including：Containing available for capturing fibrin
The coating buffer of former material, confining liquid, lavation buffer solution, the material of arsenic can be captured as secondary antibody, enzyme labelled antibody, substrate, termination
Liquid, dilution buffer, positive control, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including：Containing available for capturing fibrin
The coating buffer of former material, confining liquid, lavation buffer solution, eluent, positive control, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including：Containing available for capturing fibrin
The coating buffer of former material, confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, redissolve liquid, the coating buffer containing the material available for capture arsenic, confining liquid, lavation buffer solution, enzyme labelled antibody, bottom
Thing, terminate liquid, dilution buffer, standard items, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, redissolve liquid, positive control, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, liquid needed for the protein band of metal on dissolving glue bed, containing available
It is right in the coating buffer of the material of capture arsenic, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, positive control, feminine gender
According to etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, be liquid needed for the protein band of metal on dissolving glue bed, positive right
According to, negative control etc..
The kit of arsenic-fibrinogen chelate in a kind of detection blood sample, including for purifying needed for fibrinogen
Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, dissolve liquid, nitric acid, mistake needed for the protein band of metal on glue bed
Hydrogen oxide, positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with the fibrinogen chelate of weight arsenic
Or it is chelated with the BSA chelates of weight arsenic；The negative control is dilution buffer.
Mentioned reagent box is used to detect arsenic-fibrinogen chelate, to improve the accuracy and repeatability of detection, and makes
Be promoted in clinic.
The present invention also provides a kind of method for quantitatively detecting arsenic-fibrinogen chelate, with the above-mentioned of known content
Arsenic-fibrinogen chelate is detected as standard items using a pair of samples of following methods：It is ELISA, enzyme-linked
Immune and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-fibrin
Former 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 electricity
Feel coupled plasma mass spectrometry combined techniques.In the present invention, can be listed with the method for detection arsenic-fibrinogen chelate
Have following several, but be not limited to following several.
Dilution multiple proportions as described below is w/v.
Method one：ELISA (ELISA method) detects arsenic-fibrinogen chelate, detects in accordance with the following steps：
1) it is coated with：The material that can capture fibrinogen with dilution buffer dilution adds to 1000-8000 times
In elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with lavation buffer solution, and after the completion of washing, elisa plate places 1 in 37 DEG C
Hour；
3) add sample to be tested, and incubate：Sampled from the circulatory system, make sample to be tested；With the arsenic of known content-fiber egg
White former chelate makees standard items；Sample to be tested and standard items are diluted to 10-40 times with dilution buffer, added in micropore,
37 DEG C of effect 1-2 hours；
4) material of arsenic can be captured by adding, and be incubated：Sample to be tested is removed, and is washed with lavation buffer solution,
Wait after the completion of washing, addition dilutes 25000-200000 times of anti-arsenic antibody with dilution buffer, 37 DEG C of effect 1-2 hours, makes
Anti- arsenic antibody reacts with the arsenic on fibrinogen；
5) enzyme conjugates incubates：Anti- arsenic antibody is removed, is washed, the HRP enzyme labelled antibodies that addition is diluted with dilution buffer, 37
DEG C effect 1-2 hours, react itself and HRP enzyme labelled antibodies；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds substrate,
37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction：Terminate liquid is added to each micropore；
8) take wavelength 450nm, after adding terminate liquid, elisa plate is placed on ELIASA read respectively sample to be tested group and
The OD values of standard items, standard curve is drawn, by the content for compared with standard item group, trying to achieve sample to be tested.
In this method, step 8) also directly can carry out qualitative detection without using ELIASA by staining conditions.
This method utilizes ELISA principles, can extract the specific fibrinogen in whole blood, extract
Fibrinogen upper part is chelated with weight arsenic, and the arsenic on this few fibers proteinogen can be caught by the specific antibody of anti-arsenic
Obtain, (antibody nonrecognition coating can be captured by the antibody that the enzymes such as horseradish peroxidase, alkaline phosphatase mark again afterwards
Albumen), the antibody in capture can read OD values, and do not contain chelating in the presence of developer and terminate liquid under instrument
The fibrinogen of arsenic, then it will not be captured by the specific antibody of anti-arsenic, also will not be with horseradish peroxidase, alkaline phosphatase
The antibody of the enzymes such as enzyme mark is captured, and arsenic (negative control group result is feminine gender) is not contained in agents useful for same yet, thus works as institute
When the OD value results of reading are shown as the positive, you can prove to detect the arsenic chelated on fibrinogen.
Method two：Enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection arsenic-fibrinogen chela
Compound detects in accordance with the following steps：
1) it is coated with：The material of fibrinogen will can be captured, as antifibrin original antibody is coated on solid phase carrier：
Antifibrin original antibody is diluted to 1000-8000 times with dilution buffer, is added in elisa plate micropore, 4 DEG C of overnight 16-18
Hour, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
3) add sample to be tested, and incubate：Sampled from the circulatory system, make sample to be tested；With arsenic-fiber of known content
Proteinogen chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 hours；
4) elute：Sample to be tested is removed, is washed, adds eluent, 1-3 hours are eluted at 37 DEG C；
5) detect：Sample from ELISA micropores, chelated in Atomic Absorption Spectrometer detection in the arsenic on fibrinogen,
Read respective value；
The embodiment is captured using ELISA principles to fibrinogen, and is combined atomic absorption spectrum (AAS) instrument and examined
Survey is chelated in the arsenic on fibrinogen；Due to only containing fibrinogen in solution, and it is (negative right without arsenic in agents useful for same
It is feminine gender according to group result), result will not be shone into interference, thus when the result read is shown as the positive, you can prove inspection
Measure the arsenic chelated on fibrinogen.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection arsenic-
Fibrinogen chelate detects in accordance with the following steps：
1) it is coated with：The material of fibrinogen will can be captured, as antifibrin original antibody is coated on solid phase carrier,
Antifibrin original antibody is diluted to 1000-8000 times with dilution buffer, is added in elisa plate micropore, 4 DEG C of overnight 16-18
Hour, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
3) add sample to be tested, and incubate：Sampled from the circulatory system, make sample to be tested；With the arsenic of known content-fiber egg
White former chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 hours；
4) elute：Sample to be tested is removed, and is washed with corresponding lavation buffer solution, is waited after the completion of washing, adds elution
Liquid, 1-3 hours are eluted at 37 DEG C；
5) it is acidified：Add acidulant in solution in step 4) to be acidified solution, sealing overnight, is thoroughly acidified；
6) detect：Hydrogen peroxide is added, and heats and catches up with acid, and 0.5mL is taken in the solution eluted from ELISA agent plates
Liquid, the arsenic chelated in fibrinogen is detected under icp mses, reads respective value.
This method is on the basis of using ELISA principles, with reference to sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption
Sense couple plasma mass spectrometer detection is chelated in the arsenic on fibrinogen；I.e. first using ELISA principles by the arsenic in whole blood-
Fibrinogen chelate extracts, then is entered using sense couple plasma mass spectrometer to chelating in the arsenic on fibrinogen
The quantitative detection of row；Due to only containing fibrinogen in solution, and without arsenic, (negative control group result is the moon in agents useful for same
Property), result will not be shone into interference, thus when the result read is shown as the positive, you can prove to detect fibrin
The arsenic chelated in original.
Method four：Purify arsenic-fibrinogen chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detection arsenic-
Fibrinogen chelate, is detected in accordance with the following steps：
1) non-specific fibrinogen is extracted from whole blood：Using supercentrifugation, HPLC, gel mistake
The methods of filtering chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved,
Obtain the solution of fibrinogen；
2) it is coated with：Anti- arsenic antibody is coated on solid phase carrier, dilutes anti-arsenic antibody to 25000- with dilution buffer
200000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
3) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
4) add sample to be tested, and incubate：Sampled from the solution of step 1), make sample to be tested；With the arsenic of known content-
Fibrinogen chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 are small
When；
5) enzyme conjugates incubates：Sample to be tested is removed, and is washed with lavation buffer solution, is waited after the completion of washing, is added
The enzyme labelled antibody diluted with dilution buffer, 37 DEG C of effect 1-2 hours, it is set to be reacted with enzyme labelled antibody；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with corresponding lavation buffer solution, is waited after the completion of washing, is added
Substrate, 37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction：Terminate liquid is added to each micropore；
8) wavelength 450nm is taken, after adding terminate liquid, reads the OD of sample to be tested group and standard items respectively on ELIASA
Value, mark song line is drawn, by the content for compared with standard item group, trying to achieve sample to be tested.
In this method, also directly qualitative detection can be carried out by staining conditions without using ELIASA.
Method five：Arsenic-fibrinogen chelate is purified to detect with atomic absorption spectrum combined techniques (method of purification+AAS methods)
Arsenic-fibrinogen chelate, is detected in accordance with the following steps in blood sample：
1) non-specific fibrinogen is extracted from whole blood：Using supercentrifugation, HPLC, gel mistake
The methods of filtering chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved,
Obtain fibrinogen solution；
2) detect：Sample from the solution of step 1), chelated in Atomic Absorption Spectrometer detection on fibrinogen
Arsenic, read respective value.
Method six：Purify arsenic-fibrinogen chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+
ICP-MS methods) detection arsenic-fibrinogen chelate, is detected in accordance with the following steps：
1) non-specific fibrinogen is extracted from whole blood：Using supercentrifugation, HPLC, gel mistake
The methods of filtering chromatography, gel electrophoresis, fibrinogen is extracted from whole blood, and the fibrinogen extracted is redissolved,
Obtain the solution of fibrinogen；
2) it is acidified：Being sampled from the solution of step 1), add nitric acid in the solution and solution is acidified, sealing is stayed overnight,
Thoroughly acidifying；
3) detect：Add hydrogen peroxide, and heat catch up with acid after take 0.5mL solution, in inductivity coupled plasma mass spectrometry
Detection is chelated in the arsenic on fibrinogen under instrument, reads respective value.
Method four, method five and method six are to isolate fibrinogen by whole blood extraction method, then using specificity
Detection method, determine the content of arsenic on arsenic-fibrinogen chelate in fibrinogen；Physical separation means are first used,
Such as supercentrifugation, HPLC, gel-filtration chromatography, fibrinogen is separated from test plasma sample
Out and redissolve in physiological saline, recycle ELISA principles, atomic absorption spectrum detection or carry out detect inductive etc. from
Arsenic content on daughter mass spectrography detection arsenic-fibrinogen chelate.
Method seven：Electrophoresis+ELISA/AAS/ICP-MS methods detect arsenic-fibrinogen chelate, specific as follows：
1) non-specific fibrinogen is extracted from whole blood：Using supercentrifugation, HPLC, gel mistake
Filter chromatography, gel electrophoresis the methods of, fibrinogen is extracted from whole blood, by the fibrinogen extracted redissolve in
In physiological saline, the solution of fibrinogen is obtained；
2) glue bed is prepared：Suitable medium (such as Ago-Gel, polyacrylamide gel) is selected as needed, according to
Corresponding requirements prepare corresponding glue bed；
3) it is loaded：8 μ L solution are taken from the solution of step 1), are marked with the arsenic of known content-fibrinogen chelate
Quasi- product, 2 μ L sample-loading buffers are added, and mixed, is then loaded onto in sample cell；
4) electrophoresis：Connect electrophoresis plate, add electrophoretic buffer, carry out electrophoresis, and according to demand by albumen according to molecular weight, etc.
The difference of the parameters such as electricity point is separated；
5) detect：The protein band containing fibrinogen chelant thing is found out on glue bed, the band is taken out, by the egg
Informal voucher band is dissolved among liquid, is then utilized respectively the arsenic that the detection of the principles such as ELISA or ICP-MS or AAS is dissolved in liquid again and is contained
Amount.
Further, it is also possible to isoelectric point, molecular weight and the content of arsenic-fibrinogen chelate are detected using the method etc..
In method seven, fibrinogen is extracted from whole blood, then using gel electrophoresis to the fibre that is extracted
Fibrillarin original is separated, then finds out the respective strap rich in arsenic, then detects the content of associated fiber proteinogen；That is fibrin
Original can with a variety of Methods For Purifications come out (such as supercentrifugation, HPLC, gel-filtration chromatography, gel electricity
Swimming method, ELISA method etc.), fibrinogen out will be purified and redissolved in solution, take a certain amount of fibrinogen, profit
With electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, gel slab (different medium can be used as needed)
On can run out of different bands according to molecular weight, isoelectric point etc. are different, the respective strap rich in arsenic is searched out, by the egg in gel
White matter is redissolved in solution, you can to detect the content of associated fiber proteinogen at a particular wavelength, can also utilize ELISA,
The principles such as AAS, ICP-MS detect to chelate in the arsenic content on fibrinogen, due to only containing fibrinogen in solution,
And result will not be shone in agents useful for same without arsenic (negative control group result is feminine gender) into interference, thus work as read knot
When fruit is shown as the positive, you can prove to detect the arsenic chelated on fibrinogen.
Embodiment 1：Synthetic method synthesizes arsenic-fibrinogen chelant thing, comprises the following steps：
Arsenic-fibrinogen chelant thing prepared by the present embodiment, is further separated, and pass through inductance by gel electrophoresis
Coupled plasma mass spectrometry or atomic absorption spectrum carry out detection Qualitative Identification.
Reagent is as follows used in the present embodiment：
1) borate buffer solution, its molar concentration are 0.01M, and its preparation method example is as follows：0.31g boric acid is weighed to be dissolved in
400mLddH2In O, pH to 9.0 is adjusted with 0.1mol/L NaOH, is settled to 500mL.
2)EDTA-NaHCO3Solution, its preparation method are as follows：Take 1.86g EDTA2H2O and 16.8g NaHCO3, it is dissolved in
900mLddH2In O, 1000mL, autoclaving, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
3) ITCBE is bought from Japanese colleague's chemistry institute, article No. M030；
4) fibrinogen solution：Weigh 4.0mg fibrinogens and be dissolved in 4.0mL0.01M pH9.0 borate buffer solutions
In, fully vibration is dissolved, and is configured to 1.0mg/mL fibrinogen solution；
5) molecular cut off 14000 of bag filter, buy from Bioshop Inc；
The pretreatment of bag filter：Bag filter is put into 500mL EDTA-NaHCO3In solution, 10min is boiled；Tipping
EDTA-NaHCO3Solution, use ddH2O is gently rinsed, then boils 10min with 500mL5mmol/L EDTA；Boiling liquid is discarded, thoroughly
Use ddH2O is cleaned, and adds substantial amounts of ddH24 DEG C of O immersions bag filter is overnight.In use, put on one's gloves, bag filter is taken out, with a large amount of
DdH2Its surfaces externally and internally of O cleaning downs；
The method for preparing arsenic-fibrinogen chelate, comprises the following steps：
A) the synthesis of arsenic-fibrinogen chelant thing：Arsenic ion is added in the fibrinogen in people source chelate instead
Should, obtain reaction solution；
1) 2.0mg ITCBE are taken to be dissolved in 2mLDMSO；
2) liquid for slowly preparing step 1 is added in fibrinogen solution, is shaken when being added dropwise, in 25 DEG C, 100r/
24h is acted in min shaking table, then with bag filter dialysis 24h, removes the ITCBE not combined with fibrinogen；
3) liquid for gained of dialysing is adjusted into pH value to 7.0 with 1mol/L HCl, 80 μ l is then slowly gradually added dropwise
1mmol/L arsenic ion solns, vibrated when being added dropwise, in case arsenic ion precipitates albuminous degeneration；
4) solution added is reacted into 2h in 25 DEG C, 100r/min shaking table, dialysed with the bag filter handled well
24h；
5) liquid dialysed is preserved in -20 DEG C of packing, obtains the reaction solution of arsenic-fibrinogen chelate.
B) arsenic-fibrinogen chelate purifying：Using immune-affinity chromatography, removal step A) it is not anti-in reaction solution
Fibrinogen, specific antibody and the arsenic ion answered, arsenic-fibrinogen chelate is produced, specifically includes following steps：
(1) sample dissolution：By above-mentioned steps A) arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fibre
Fibrillarin original chelate solution；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with fiber egg
The filler of white former specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with dilution buffer dilution step (1) solution, then upper prop, makes fibre
Fibrillarin is former to be specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：The eluent of collection in step (5) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times,
4 DEG C of dialysed overnights, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：The eluent of step (10) is filled into bag filter, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C thoroughly
Analysis overnight, collects sample, produces arsenic-fibrinogen chelate.
C) to the identification of arsenic-fibrinogen chelant thing, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using Ago-Gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-fibrinogen chelate of extraction purification, redissolve in physiological saline,
8 μ L are taken out, add 2 μ L sample-loading buffers, and are mixed, are then loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, adds electrophoretic buffer to carry out electrophoresis；In electrophoresis process, electric current is 22mA constant currents, ring
Border temperature is 4 DEG C；Stop electrophoresis when moving to glue bottom to bromophenol blue, Fig. 1 is arsenic of the present invention-fibrinogen chelant thing
Non denatured electrophoretic band figure；
(4) detect：The protein band of metal is found out on glue bed, the protein band is taken out, protein band is dissolved, so
The content of arsenic is detected whether containing arsenic and detected again afterwards using ICP-MS or AAS.
D) qualification result
1) AAS testing results
Take step C) the protein band solution isolated, with graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination fiber
The content of weight arsenic, as shown in the table in proteinogen, with (NH4)2SO4Solution is blank control；
The content of arsenic in the fibrinogen of table 1
2) Synchrotron Radiation X-Ray Fluorescence Anal ysis
4W1 " of the SRXRF analyses of micronutrient levels in BEPC (BEPC) is synchronous in protein band
Completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200
Type, Beijing are stood upright Han Guang companies) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into
Penetrate facula position, moving step length 0.0025mm.From the X ray that electromagnetic radiation goes out by Si (Li) detectors (PGT
Inc.LS30143-DS) detect, probe is with incident SR lines copline and being mutually perpendicular to, away from sample irradiation point 20mm, signal PGT
Multi-channel analyzer (MCA 4000) obtains output.Sample is excited with 11.5keV monochromatic synchrotron radiation light, adjusts launching spot
(1mmx 3mm) position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always,
Hot spot moves on to the band other end at the end of counting.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings,
And normalization is carried out to other element peaks with the Ar signal peaks from air and content constant, to offset beam intensity change
On influence caused by signal strength.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way under the same conditions.
Fig. 2 is the synchrotron radiation X line fluorescence analysis of the electrophoretic band of arsenic of the present invention-fibrinogen chelate
Scheme, abscissa is protein band position in figure, and ordinate is energy (content) value of the 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.
The method that ELISA detects arsenic-fibrinogen chelate, specifically includes following steps：
1) it is coated with：Antifibrin original antibody albumen is coated on solid phase carrier, will be coated with dilution buffer respectively
Albumen is with 1：1000、1：2000、1：4000 and 1：8000 doubling dilution, add in elisa plate micropore, each concentration coating three
Row, 4 DEG C preserve 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed；
3) sample to be tested is added：Test plasma sample is pressed 1 with dilution buffer：10、1：20、1：40 doubling dilution, adds
Into micropore, standard items are made with the arsenic of known content-fibrinogen chelate, negative control and blank control are set, added to
In micropore, 37 DEG C act on 1 hour；
4) anti-As antibody is added：Test plasma sample is removed, is washed, addition presses 1 with dilution buffer：25000、1：50000、
1：The anti-As antibody of 100000 and 1,200000 doubling dilution, 37 DEG C act on 1 hour, make it anti-with the arsenic on fibrinogen
Should；
5) enzyme-added mark：Anti- As antibody is removed, is washed, the HRP enzyme labelled antibodies that addition is diluted with dilution buffer, 37 DEG C of effects
1 hour, make itself and anti-As antibody responses；
6) substrate incubates：Enzyme labelled antibody is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, add terminate liquid；
7) detect：Read on ELIASA standard items, test plasma, positive control, negative right respectively under 450nm wavelength
According to the OD values with blank control sample.
In the present embodiment, using arsenic provided by the invention-fibrinogen chelate standard items as positive control, difference
To be not added with test plasma as negative control 1, that is, antifibrin original antibody, confining liquid, anti-As antibody, enzyme mark are sequentially added
And substrate；
To be not added with the check experiment group of anti-As antibody as negative control 2, that is, sequentially added antifibrin original antibody,
Confining liquid, test plasma, enzyme mark and substrate；
Using not enzyme-added target check experiment group as negative control 3, antifibrin original antibody, closing are sequentially added
Liquid, test plasma, anti-As antibody and substrate；
Check experiment group to be not added with sample to be tested and anti-As antibody simultaneously has sequentially added anti-as negative control 4
Antibody against fibrinogen, confining liquid, enzyme mark and substrate；
Using the check experiment group for being not added with antifibrin original antibody work as blank control 1, that is, add confining liquid, blood to be measured
Slurry, anti-As antibody, enzyme mark and substrate；
And only to add the check experiment group of substrate as blank control 2, only to add PBS check experiment group as blank control
3。
Table 2 is that different antifibrin original antibodies dilute the sample that multiple proportions, diluted plasma multiple proportions, anti-As antibody dilute multiple proportions
OD Value Datas,
Testing result under the different antifibrin original antibodies of table 2, anti-As antibody and diluted plasma multiple proportions
As known from Table 2, the dilution multiple proportions of antifibrin original antibody is 1:When 2000, sample OD values are more than under parallel condition
Other antifibrin original antibodies dilution multiple proportions；In this group of sample, diluted plasma multiple proportions is 1:When 10, anti-As antibody dilution
Multiple proportions is 1:When 25000, OD values are maximum, are 0.756.
Table 3 is that the dilution multiple proportions of antifibrin original antibody is 1:2000th, diluted plasma multiple proportions is 1:10th, anti-As antibody is dilute
Multiple proportions is released as 1:Positive control, negative control and the OD of blank control detected values corresponding to 25000 phases,
The positive control of table 3, negative control and the testing result of blank control
As known from Table 3, negative control group OD detected values are less than 0.1, illustrate under the optimal conditions, the systematic error of this method
Property it is small, meet analysis method requirement, so selecting the concentration corresponding to this value as best effort concentration.
2. ELISA eluent best effort concentration and elution time determine
To seek optimum elution requirement, by ELISA after anti-As antibody and enzyme labelled antibody incubate, with not
Eluent with concentration is eluted, then detects OD values by ELIASA, is comprised the following steps that：
(1) it is coated with：Anti- As antibody is coated on solid phase carrier, 1 is pressed with dilution buffer：25000 doubling dilution, adds
Enter in elisa plate micropore, 4 DEG C preserve 16 hours；
(2) close：Dilution buffer is removed, after washing, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and wash
Wash；
(3) enzyme-added labeling antibody：Confining liquid is removed, after washing, it is 2ng/ that addition is diluted to antibody concentration with dilution buffer
ML HRP enzyme labelled antibodies, 37 DEG C act on 2 hours, make itself and anti-As antibody responses；
(4) elute：Enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes Papain in eluent
The concentration of enzyme：Concentration=1 of antibody in enzyme labelled antibody:80、1:40、1:20、1:10、1:5, make at a temperature of being respectively placed in 37 DEG C
With 1h, 2h, 3h；Eluent is removed, washs, waits after the completion of washing, adds substrate, 37 DEG C of lucifuges act on 30 minutes, add terminate liquid
Terminating reaction；
(5) the OD values of each micropore are read respectively on ELIASA under 450nm Detection wavelength, concrete outcome is referring to table
4,
Testing result under the different eluent extension rates of table 4
By comparing OD values, to judge the anti-As antibody-hrp-antibody complex elution degree combined on ELISA hole walls,
When OD values are minimum, anti-As antibody-hrp-antibody complex elution degree reaches maximum.As shown in table 4, when pawpaw in eluent
The concentration of protease：Concentration=1 of antibody in enzyme labelled antibody:When 20, elution degree is maximum；And action time is 1h, 2h, 3h
When, each group OD value changes are little, it is seen that with the extension of time, enzyme activity gradually weakens, in the case where enzyme concentration is constant, prolong
Long digestion time can not improve digestibility, thus in this experiment eluent action time for 1-3h all can, in summary, I
Select eluent 1:20 are used as most suitable working concentration, and 1-3h is used as most suitable elution time.
Application Example
Application Example 1
Arsenic-fibrinogen chelate in 100 parts of sample blood plasma is detected using ELISA (ELISA method), that is, adopted
The method recorded with specific embodiment method one detects, and concrete operation step is as follows：
1) it is coated with：Antifibrin original antibody is coated on solid phase carrier, 2000 times is diluted to dilution buffer, adds
Enter in elisa plate micropore, 37 DEG C preserve 1 hour after 4 DEG C of storages in refrigerator；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
37 DEG C of elisa plate places 1 hour after 4 DEG C of storages in refrigerator；
3) it is loaded：Using sample blood plasma as sample to be tested, standard is made with the arsenic of known content-fibrinogen chelate
Product, sample to be tested and standard items are diluted to 10 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
4) anti-As antibody is added：Sample to be removed, is washed, addition is diluted to 25000 times of anti-As antibody with dilution buffer, and 37
DEG C effect 1 hour, reacts itself and arsenic on fibrinogen；
5) enzyme-added mark：Anti- As antibody is removed, is washed, adds the enzyme labelled antibody that antibody concentration is 2ng/mL, 37 DEG C of effects 1 are small
When, make itself and anti-As antibody responses；
6) substrate incubates：Enzyme labelled antibody is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, add terminate liquid extremely
Each micropore；
7) detect：Read sample to be tested group and the OD values of standard items respectively on ELIASA under 450nm wavelength, as a result such as
Shown in table 5.
The measured result of 5 method of table, a pair of 100 parts of sample blood plasma
It in this application embodiment 1, in step 7), can also detect without using ELIASA, but directly be carried out by dyeing
Qualitative detection.
Application Example 2
Using enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detect arsenic in 100 parts of sample blood plasma-
Fibrinogen chelate, i.e., the method recorded using specific embodiment method two are detected, and concrete operation step is as follows：
1) it is coated with：Antifibrin original antibody is coated on solid phase carrier, dilutes coating protein extremely with dilution buffer
2000 times, add in elisa plate micropore, 4 DEG C are stayed overnight 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
Preserved at 4 DEG C of elisa plate；
3) it is loaded：Sample to be tested is made with sample blood plasma, standard items are made with the arsenic of known content-fibrinogen chelate,
Sample to be tested and standard items are diluted to 10 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
4) elute：Test plasma sample is removed, is washed, addition is diluted to Papain enzyme concentration with dilution buffer and is
100ng/mL eluent, 37 DEG C act on 2 hours；
5) detect：Sample from ELISA micropores, chelated in Atomic Absorption Spectrometer detection in the arsenic on fibrinogen,
As a result it is as shown in table 6.
Measured result of the method two of table 6 to 100 parts of sample blood plasma
Application Example 3：
100 parts of marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods)
Arsenic-fibrinogen chelate in this blood plasma, i.e., the method recorded using specific embodiment method three are detected, concrete operation step
It is as follows：
1) it is coated with：Antifibrin original antibody is coated on solid phase carrier, dilutes coating protein extremely with dilution buffer
2000 times, add in elisa plate micropore, 4 DEG C preserve 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
4 DEG C of preservations of elisa plate；
3) it is loaded：Sample to be tested is made with sample blood plasma, standard items are made with the arsenic of known content-fibrinogen chelate,
Sample to be tested and standard items are diluted to 10 times with dilution buffer, added in micropore, 37 DEG C act on 2 hours；
4) elute：Sample to be tested and standard items are removed, is washed, addition is diluted to Papain enzyme concentration with dilution buffer
For 100ng/mL eluent, 37 DEG C elute 2 hours；
5) it is acidified：Nitric acid is added in the solution to be acidified solution, sealing overnight, thoroughly acidifying, adds hydrogen peroxide,
And heat and catch up with acid；
6) detect：Sampling, under icp mses detection chelate in the arsenic on fibrinogen, as a result
As shown in table 7.
Measured result of the method three of table 7 to 100 parts of sample blood plasma
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (6)
1. a kind of arsenic-fibrinogen chelate, it is characterised in that the arsenic-fibrinogen chelate is arsenic ion and fiber
Proteinogen is formed by sulfydryl or/and cysteine residues chelating；
The arsenic-fibrinogen chelate is through the following steps that be prepared：
A）The synthesis of arsenic-fibrinogen chelant thing：The fibrinogen of human body is come from purification or according to biological method weight
Arsenic ion is added in the fibrinogen of group and carries out chelatropic reaction, obtains reaction solution；
B）Arsenic-fibrinogen chelate purifying：Using immune-affinity chromatography, removal step A）It is unreacted in reaction solution
Fibrinogen, specific antibody and arsenic ion, produce arsenic-fibrinogen chelate, and concrete operations are as follows：
（1）Sample dissolution：By above-mentioned steps A）Arsenic-fibrinogen chelate be dissolved in physiological saline, obtain arsenic-fiber egg
White former chelate solution；
（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, energy and fibrinogen are loaded in chromatographic column
The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
（3）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（1）The solution, then upper prop, makes fiber egg
White former and filler is specifically bound；
（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L Na2HPO4
Solution is eluted；
（5）Collect：Collection step（4）Eluent, restore albumen immediately after collection；
（6）Dialysis：By step（5）In collection eluent dress bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C thoroughly
Analysis overnight, collects sample；
（7）Balance chromatographic column：Using new chromatographic column, with dilution buffer flushing line, energy and arsenic are loaded in the chromatographic column
The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；
（8）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（6）Middle sample, then upper prop；
（9）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L Na2HPO4It is molten
Liquid is eluted；
（10）Collect：Collection step（9）Eluent, restore albumen immediately after collection；
（11）Dialysis：By step（10）Eluent dress bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C were dialysed
At night, sample is collected, produces arsenic-fibrinogen chelate.
2. arsenic according to claim 1-fibrinogen chelate, the step B of its preparation method）Also include step afterwards
C）：Identification to arsenic-fibrinogen chelate；
Wherein, step C）In specifically include following steps：
（1）Prepare glue bed：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
（2）Sample-adding：Take step B）The arsenic that middle extraction purification obtains-fibrinogen chelate, sample-loading buffer is added, and mixed
It is even, then it is loaded onto in sample cell；
（3）Electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
（4）Detection：The protein band of metal is found out on glue bed, the protein band is taken out, protein band is dissolved, Ran Houzai
The content of arsenic is detected whether containing arsenic and detected using ICP-MS or AAS.
A kind of 3. arsenic-fibrinogen chelate as claimed in claim 2 arsenic-fibrinogen chela in detection blood sample is prepared
Application in the reagent or kit of compound.
4. a kind of comprise at least arsenic as claimed in claim 1-kit of the fibrinogen chelate as standard items.
5. kit according to claim 4, it is characterised in that also contain capture fiber including coating buffer, the coating buffer
The material of proteinogen or the material for capturing arsenic.
A kind of 6. method for quantitatively detecting arsenic-fibrinogen chelate, it is characterised in that with the claim 1 of known content
Described arsenic-fibrinogen chelate is detected as standard items using a pair of samples of following methods：Enzyme linked immunological
Method, 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,
Purify 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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| 六价铬间接竞争ELISA检测试剂盒的研制及新型一步法竞争ELISA的建立;邹军辉;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20121015;正文第13-16页第2.1节 * |
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