CN104987389A - Arsenic-albumin chelate as well as preparation method and application thereof - Google Patents

Arsenic-albumin chelate as well as preparation method and application thereof Download PDF

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CN104987389A
CN104987389A CN201510412176.2A CN201510412176A CN104987389A CN 104987389 A CN104987389 A CN 104987389A CN 201510412176 A CN201510412176 A CN 201510412176A CN 104987389 A CN104987389 A CN 104987389A
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arsenic
albumin
inner complex
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chromatography column
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张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
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Shanghai Baihao Biotechnology Co Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA

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Abstract

The invention discloses an arsenic-albumin chelate as well as a preparation method and application thereof. The arsenic-albumin chelate is formed by chelating arsenic ions with albumin through sulfydryl or/and cysteine residues. According to the invention, a qualitative and quantitative detection method of the arsenic-albumin chelate is established for favorably quantitatively detecting the application of the arsenic-albumin chelate to the evaluation of the arsenic pollution degree of one region. By quantitatively detecting the arsenic-albumin chelates in serums of people in one region, the condition that people in the region are polluted by arsenic can be indirectly reflected, and further the arsenic pollution degree of the region is indirectly reflected. According to the quantitative detection method for the arsenic-albumin chelate, established by the invention, the accuracy is greatly improved, and the detection repeatability is greatly improved.

Description

A kind of arsenic-albumin inner complex and its preparation method and application
Technical field
The present invention relates to detection field, more particularly, relate to a kind of arsenic-albumin inner complex and its preparation method and application.
Background technology
Serum albumin (HSA) is the most rich in protein of content in mammalian plasma, and it can store and transport numerous endogenous and exogenous material, and this and its specificity structure are closely related.Serum albumin is a kind of simple protein synthesized by liver, is only made up of amino acid, does not have modification group and other appurtenants.According to data information display, bovine serum albumin (BSA) molecule is made up of 583 amino-acid residues.And human serum albumin (HSA) 2 amino acid more than BSA, i.e. 585 amino-acid residue compositions.Both difference is that BSA lacks the residue of HSA aminoacid sequence 116 and 585.The most significant feature of albumin aminoacid component is the tryptophane of low levels, respectively containing 1 or 2 residue in HSA and BSA per molecule.Phenylalanine and isoleucine content are also lower.Halfcystine, leucine, L-glutamic acid and lysine content are all very high.A large amount of ionization residues provides the very high electric charge of albumin, and under the condition of PH7, per molecule albumin, with 185 ions, so just forms albumin processable.
The unique constructional feature of albumin is disulfide linkage pattern.BSA sequence comprises 35 cysteine residues, forms 17 disulphide bridgeses, and residue nearest on adjacent halfcystine pair and chain links, and forms a pair ring of 10-47 residues in length.S-S key crossover adds rigidity and the stability of albumin structure.
Albuminous tertiary structure is considered to a kind of molecule of high degree of spiral, and learn that the residue of in HAS crystal about 67% participates in defining 28 α mono-spirochetes by X-ray diffraction, 10% residue participates in forming β-bend, and 23% residue forms extended chain.
On physiological mechanism, albumin maintains 80% of human plasma oncotic pressure.In addition, albumin is also the main carriers of various macromolecular substance in blood, plays an important role in metabolite transhipment, bio-transformation.It is also the component regulating coagulation function and Inflammatory response simultaneously.Human serum albumin is mainly used in research that is clinical, metabolic and genetic aspect.
Arsenic is one of common environmental poisonous substance, is extensively present among water, soil, air and food.Due to its toxicity and universal existence, U.S.'s poisonous substance and disease registration administration (UAsted States Agency for ToxicSubstances and Disease Registry, ATSDR) be classified as first-class objectionable impurities (http://www.atsdr.Asc.gov/SPL/index.html) always, arsenic also (is comprised international cancer research institution (International Agency for Research on Cancer by many international organizations simultaneously, and Environmental Protection Agency (U.S.Environmental Protection Agency IARC), EPA)) delimit as one of human carcinogen.Arsenic can produce acute or chronic toxicity to human body, and acute arsenic poisoning: Report can cause the exacerbated event such as vomiting, dry, muscle spasm, stomachache, trick shouting pain, and arsenicalism has also become a significant problem of people's concern on the impact of human health.Be two kinds and contact the most direct mode by drinking water and air with arsenic in environment, it can bring out cardiovascular disorder, causes maladjusted nervous system and cause diabetes and lung cancer, skin lung cancer, bladder cancer etc.And epidemiology display at present, the underground drinking water caused at the arsenic of nature existence pollutes, in Taiwan, Argentina, Chile, West Bengal, India, Bangladesh, the Inner Mongol, multiple countries and regions such as China cause the arsenic of high dosage to expose, and become the global problem of environmental pollution and health risk.The current whole world has and is among the threat of arsenic exposure and arsenicalism more than 50,000,000 people.
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 carcinogenic substance, wherein arsenite (iAs iII) toxicity be better than arsenate (iAs v); The arsenic Difference In Toxicity of different shape is very large, monomethyl arsenus acid (monomethylarsonousacid, MMA iII) and dimethyl arsenus 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 binding 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.
Epidemiologic data shows, the workman of Long Term Contact arsenic, and it suffers from probability of tumor in respiratory system increases greatly, and it also increases greatly because of the probability that tumor in respiratory system is lethal.The test of animal model, external model also finds, arsenic can the generation of induced tumor.Nineteen ninety, international cancer research institution (International Agency forResearch on Cancer, IARC) using arsenic compound as first kind carcinogenic substance (Group 1), using arsenic as 2B class carcinogenic substance (Group 2B).Visible, the health of arsenic serious threat human body.
Arsenic can be combined with multiple proteins, suppresses the activity of multiple protein enzyme.Along with going deep into arsenic toxicity research, people recognize that the interaction of arsenic and albumen plays important role in its intoxicating process gradually, be considered to the key point of toxicity mechanism understanding arsenic, therefore, the research of arsenic and protein interaction just seemed ever more important.And the present mechanism about arsenic and protein effect is only tip of the iceberg, also has the relation between a lot of protein and arsenic not clear, thus strengthen for the relation between arsenic and protein most important.
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-albumin inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of arsenic-albumin inner complex, so that detection by quantitative arsenic-albumin inner complex is in the application of the regional arsenic pollution level of evaluation one.Indirectly can reflect by arsenic-albumin inner complex in the regional crowd's serum of detection by quantitative one situation that this regional crowd pollutes by arsenic, thus indirectly reflect this regional arsenic pollution level.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of arsenic-albumin inner complex, arsonium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned arsenic-albumin inner complex, comprises the following steps:
A) arsenic and albuminous chelatropic reaction: add arsonium ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying arsenic-albumin inner complex: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary arsonium ion in reaction soln, obtains arsenic-albumin inner complex.
Wherein, step B described in external synthesis method) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains arsenic-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of arsenic specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-albumin inner complex.
Wherein, the preparation method of above-mentioned arsenic-albumin inner complex, also comprises step C): to the qualification of arsenic-albumin inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing arsenic and detection arsenic.
The present invention also provides the application of a kind of arsenic described above-albumin inner complex in preparation human body in the reagent of arsenic-albumin inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of arsenic described above-albumin inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching albuminous material or catching arsenic.
The present invention also provides the method for a kind of detection by quantitative arsenic-albumin inner complex, using the above-mentioned arsenic-albumin inner complex of known content as standard substance, 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-albumin inner complex and enzyme linked immunological combined techniques, purification arsenic-albumin inner complex and atomic absorption spectrum combined techniques, purification arsenic-albumin inner complex 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.
Implement arsenic of the present invention-albumin inner complex and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis arsenic-albumin inner complex first;
2. the present invention proposes arsenic-albumin inner complex first and can be used for preparing application in the reagent or test kit detecting arsenic-albumin inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of arsenic-albumin inner complex, so that detection by quantitative arsenic-albumin inner complex is in the application of the regional arsenic pollution level of evaluation one.Indirectly can reflect by arsenic-albumin inner complex in the regional crowd's serum of detection by quantitative one situation that this regional crowd pollutes by arsenic, thus indirectly reflect this regional arsenic pollution level.Arsenic-its accuracy of albumin inner complex quantitative detecting method that the present invention sets up greatly improves, and the repeatability of detection is greatly enhanced.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-albumin inner complex;
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of arsenic of the present invention-albumin inner complex.
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, and agents useful for same, material are as cited by following, and the reagent being this area conventional not enumerating out in the present invention maybe can be obtained by commercial mode:
Phosphate solution is 0.05-0.1mol/L Na 2hPO 4solution or 0.5-1mol/L Na 2hPO 4solution;
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Glue bed medium is the one in sepharose, polyacrylamide gel;
To catch albuminous material be the model that Abcam company produces is the Anti-mankind SerumAlbumin antibody of ab106349; Wherein, be of the present inventionly catch albuminous material with the material of albumin specific binding, anti-HSA antibody, anti-albumin antibodies;
The material purchase of catching arsenic is the mouse-anti As mAb of RK15728 from the model of Ran Ke bio tech ltd, Guangzhou; Wherein, of the present inventionly resist with the material of arsenic specific binding, anti-As antibody, anti-arsenic antibody, arsenic the material being and catching arsenic;
Enzyme labelled antibody is the one in the antibody containing the enzyme labelling such as horseradish peroxidase, alkaline phosphatase;
Substrate is methyl diphenyl amine (TMB) solution;
Washings is for containing KH 2pO 40.2mg/ml, Na 2hPO 412H 2the pH of O 2.90mg/ml, NaCl8.0mg/ml, KCl 0.2mg/ml, 0.5%Tween-20 is the 0.15M PBS solution of 7.4;
Confining liquid is 1%-5% bovine serum albumin or skim-milk;
Dilution buffer is for containing 1.5mg/mL Na 2cO 3, 2.93mg/ml NaHCO 3pH be 9.6 0.05M carbonate buffer solution;
Enzyme labelled antibody is HRP enzyme labelled antibody;
Stop buffer is: by the 2M H of 21.7ml 2sO 4be settled to the ddH of 200ml 2in O;
Elutriant be containing the PH of papoid be 8.0 0.1mol/L Tris-HCL damping fluid;
Souring agent is nitric acid;
Sample-loading buffer is for containing 1M Tris-HCl (pH 6.8) 15.5mL, 1% tetrabromophenol sulfonphthalein 2.5mL, ddH 2the Sample buffer (5X) of O7mL, glycine 25mL;
Electrophoretic buffer be containing Tris 3mg/ml, glycine 14.4mg/ml PH be the ddH of 6.8 2o solution.
The invention provides a kind of arsenic-albumin inner complex, arsonium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of arsenic-albumin inner complex, comprises the following steps:
A) arsenic and albuminous chelatropic reaction: add arsonium ion and carry out chelatropic reaction in the albumin in people source, obtain reaction soln;
B) extraction of purifying arsenic-albumin inner complex: adopt immune-affinity chromatography, remove unreacted albumin and unnecessary arsonium ion in reaction soln, obtain arsenic-albumin inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains arsenic-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column; Described can be the silica gel or the resin that contain anti-albumin antibodies with the filler of albumin specific binding;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of arsenic specific binding, dress post after balance chromatography column by dilution buffer again; Described can be the silica gel or the resin that contain anti-arsenic antibody with the filler of arsenic specific binding;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-albumin inner complex;
C) to the qualification of arsenic-albumin inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing arsenic and detection arsenic.
The present invention also provides a kind of and at least comprises the test kit of arsenic described above-albumin inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching albuminous material or catching arsenic.
In the present invention, the test kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, material, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught arsenic.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, elutriant, positive control, negative control etc.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, elutriant, souring agent, hydrogen peroxide, positive control, negative control etc.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise as extracting reagent in purification whole blood needed for albumin, redissolve liquid, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching arsenic.
Detecting a test kit for arsenic in blood sample-albumin inner complex, comprising as extracting reagent, redissolution liquid, positive control, negative control etc. in purification whole blood needed for albumin.
Detecting a test kit for arsenic in blood sample-albumin inner complex, comprising as extracting reagent, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc. in purification whole blood needed for albumin.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid needed for the protein band of arsenic, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching arsenic.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, positive control, negative control etc. needed for the protein band of arsenic.
Detect a test kit for arsenic in blood sample-albumin inner complex, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, souring agent, hydrogen peroxide, positive control, negative control etc. needed for the protein band of arsenic.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the albumin inner complex of heavy arsenic or is chelated with the BSA inner complex of heavy arsenic; Described negative control is the dilution buffer not containing standard substance.
Mentioned reagent box is for detecting the albumin of chelating arsenic, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative arsenic-albumin inner complex, using the above-mentioned arsenic-albumin inner complex of known content as standard substance, 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-albumin inner complex and enzyme linked immunological combined techniques, purification arsenic-albumin inner complex and atomic absorption spectrum combined techniques, purification arsenic-albumin inner complex 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-albumin inner complex can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative arsenic-albumin inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects arsenic-albumin inner complex, detects in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching arsenic is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and be diluted to 25000-200000 anti-As Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the arsenic in anti-As Ab and albumin react and form immune complex;
6) enzyme conjugates incubation: remove anti-arsenic antibody, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
The method utilizes ELISA principle, specificity albumin in whole blood can be extracted, the albumin upper part extracted is chelated with heavy arsenic, and the arsenic in this part albumin catch by anti-arsenic antibody, afterwards can again by horseradish peroxidase, the antibody of the enzyme labellings 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 albumin 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 labellings 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 in albumin.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect arsenic-albumin inner complex and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) detect: sample from the micropore of elisa plate, detect the arsenic of chelating in albumin in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value;
In conjunction with atomic absorption spectrum (AAS) principle on the basis that the method utilizes ELISA principle, Atomic Absorption Spectroscopy AAS is utilized to detect the arsenic of chelating in albumin, owing to only containing albumin in solution, and not containing any arsenic (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable arsenic detecting chelating in albumin.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect arsenic-albumin inner complex and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) acidifying: in step 5) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) elisa plate solution get 0.5ml liquid, under icp ms, detect chelating in albuminous arsenic, and drawing standard curve, readout value.
The method is on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, the arsenic of chelating in albumin is detected with icp ms, owing to only containing albumin in solution, and not containing any arsenic (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable arsenic detecting chelating in albumin.
method four:purification arsenic-albumin inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect arsenic-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) anti-As Ab is coated on solid phase carrier: with sample diluting liquid, anti-As Ab is diluted to 25000-200000 doubly, 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, store refrigerator;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: from step 1) solution sample, make measuring samples; Standard substance are made with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove step 4) in measuring samples, and to wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: stop buffer is dropped to each micropore;
8) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
method five:purification arsenic-albumin inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect arsenic in blood sample-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) detect: from step 1) sample the solution that obtains, detect the arsenic of chelating in albumin in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value.
method six:purification arsenic-albumin inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect arsenic-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) acidifying: from step 1) sample the solution that obtains, add souring agent (as 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;
3) detect: from step 2) get 0.5ml liquid the solution that obtains, under icp ms, detect the arsenic of chelating in albumin, and drawing standard curve, readout value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) or atomic absorption spectrometry or inductivity coupled plasma mass spectrometry combined techniques (electrophoretic method-ELISA method/AAS method/ICP-MS method) detect arsenic-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare glue bed;
3) application of sample: get step 1) the solution 8 μ L that obtains adds 2 μ L sample-loading buffers, mixing, of short duration centrifugal; (noticing that step can not be boiled herein)
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: on glue bed, find out the protein band containing arsenic, this band is taken out, this protein band is dissolved among liquid, and then utilize ELISA or ICP-MS or AAS to detect the arsenic content be dissolved in liquid respectively.In addition, the albuminous iso-electric point of this method detection chelating arsenic, molecular weight and content etc. can also be utilized.
Albumin in method seven can with multiple Methods For Purification out (such as ultracentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the albumin of purifying out is redissolved in solution, get a certain amount of albumin, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different media can be adopted as required), the difference such as iso-electric point runs out of different bands, find out the respective strap being rich in arsenic, protein in gel is redissolved in solution, namely relevant albuminous content 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 in albumin, owing to only containing albumin in solution, and not containing any arsenic (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, the i.e. provable arsenic detecting chelating in albumin.
embodiment 1: the preparation method of arsenic-albumin inner complex, comprises the following steps:
A) arsenic and albuminous chelatropic reaction: add arsonium ion and carry out chelatropic reaction in the albumin in people source, obtain reaction soln;
Preparation of reagents:
1) borate buffer solution (0.01M): take 0.31g boric acid and be dissolved in 400ml ultrapure water, regulates pH to 9.0 with the NaOH of 0.1M, is settled to 500mL.
2) albumin solution: take 4.0mg albumin and be dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the protein solution of 1.0mg/mL;
3) EDTA-NaHCO3 mixing solutions: take EDTA2H 2o 1.86g, NaHCO 316.8g is dissolved in 900mL ultrapure water, adjusts pH to 8.0 be settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH;
4) ITCBE buys from Japanese colleague's chemistry institute, and article No. is M030;
5) dialysis tubing is purchased from Bioshop Inc, molecular weight cut-off 14000.
The chelation step of arsenic-albumin inner complex:
1) process of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500ml 3in mixing solutions, boil 10min; Tipping EDTA/NaHCO 3liquid, with ultrapure water rinsing gently, then boils 10min with the 5mmol/LEDTA solution of 500ml; Discard boiling liquid, thoroughly clean with ultrapure water, add ultrapure water immersion dialysis tubing 4 DEG C and spend the night.During use, put on one's gloves, take out dialysis tubing, with its surfaces externally and internally of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg albumin is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the albumin solution prepared, dropping limit, limit is shaken, and being placed in temperature is 25 DEG C, and rotating speed is react 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, removing not with the ITCBE of albumin bound;
5) by step 4) the liquid 1mol/L HCl adjust ph to 7.0 of gained, then slowly drip the 1mmol/L arsenic ion soln of 80 μ l gradually, dropping limit, limit vibrates, in order to avoid arsonium ion makes protein denaturation precipitate;
6) by step 5) to be placed in temperature be 25 DEG C for the solution of gained, rotating speed is react 2h in the shaking table of 100r/min, carries out dialysis 24h with dialysis tubing;
7) by step 6) dialysis after liquid preserve in-20 DEG C of packing, obtain reaction soln.B) extraction of purifying arsenic-albumin inner complex: adopt immune-affinity chromatography, remove reaction soln (the i.e. step 7) reaction soln that obtains) in unreacted albumin and unnecessary arsonium ion, obtain arsenic-albumin inner complex, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction soln that obtains arsenic-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of arsenic specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-albumin inner complex;
C) to the qualification of arsenic-albumin inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using sepharose as medium;
(2) application of sample: get step B) in arsenic-albumin inner complex of obtaining of 8 μ L extraction purification, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, add electrophoretic buffer and carry out electrophoresis; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 DEG C; Electrophoresis is stopped when electrophoresis to tetrabromophenol sulfonphthalein moves to bottom glue;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing arsenic and detection arsenic.
D) detected result
(1) arsenic content in graphite furnace atomic absorption spectrometry (AAS) rough determination HSA: the results are shown in Table 1
Table 1 is arsenic content in albumin (μ g/L)
Sample name As(μg/L)
HSA 169.212
(NH 4) 2SO 4 0.077
N.S 0
N.S 0.014
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-albumin inner complex.
Native polyacrylamide gel electrophoresis (Native-PAGE) or be called that active electrophoresis is under the condition not adding the denaturing agent such as SDS and thin base ethanol, to keeping active protein to carry out polyacrylamide gel electrophoresis, be usually used in the qualification of enzyme, Isozyme Analysis and purification.The native polyacrylamide gel electrophoresis not adding SDS can make biomacromolecule in electrophoresis process, keep its natural shape and electric charge, their separation is the molecular sieve effect of difference according to its electrophoretic mobility and gel, thus higher resolving power can be obtained, especially after electrophoretic separation, still can keep the biological activity of the biomacromolecule such as protein and enzyme, therefore the right swimming lane M is sex change Marker, only for detecting, do not mark effect, swimming lane 1 is albuminous band.
(3) 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 Beijing electron positron collider (BEPC).In storage ring, 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.LS 30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx3mm) 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 the 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 fignal center 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 fluorometric analysis figure of the electrophoretic band of arsenic of the present invention-albumin inner complex, and in figure, X-coordinate is protein band position, and ordinate zou is energy (content) value of this band arsenic.
the determination of the testing conditions of the method for a kind of detection by quantitative arsenic-albumin inner complex of the present invention:
1. the determination of the optimum diluting multiple of anti-albumin antibodies, anti-arsenic antibody best effort concentration and blood plasma
Step is as follows:
(1) be that 1:1000,1:2000,1:4000,1:8000 dilute by anti-HSA Ab dilution buffer according to the mass volume ratio of anti-HSA Ab and dilution buffer, add in elisa plate micropore, anti-HSA Ab is coated on solid phase carrier, each concentration bag is by three rows, and 4 DEG C are spent the night 18 hours;
(2) remove sample diluting liquid, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) measuring samples is that 1:10,1:20,1:40 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HSA Ab concentration of above-mentioned bag quilt, the anti-HSAAb of same concentration adds different extent of dilution blood plasma respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with washings, after washing completes, add anti-As Ab, anti-As Ab is that 1:25000,1:50000,1:100000,1:200000 dilute by the mass volume ratio of anti-As Ab and dilution buffer, and according to each identical anti-HSA Ab, serum-dilution concentration, the anti-As Ab of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the arsenic on anti-As Ab and HSA are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-As antibody, and wash with washings, after washing completes, add with dilution buffer dilution HRP enzyme labelled antibody, 37 DEG C act on 1 hour, HRP enzyme labelled antibody and arsenic-albumin inner complex are reacted;
(6) remove enzyme labelled antibody, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and detects, read each hole OD value respectively.According to each hole OD value numerical value, select anti-HSA Ab, the best effort concentration of anti-As Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made standard substance as positive control in test, select anti-HSA Ab+ close+anti-As Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HSA Ab+ closes+and blood plasma+enzyme mark+substrate (namely not adding anti-As Ab) is as negative control 2; Anti-HSA Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-As Ab) is as negative control 3; Anti-HSA Ab+ closes+and blood plasma+anti-As Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+blood plasma+anti-As Ab+ enzyme mark+substrate (namely do not add anti-HSA Ab and catch HSA) is as blank 1; Only add substrate as blank 2 and PBS as blank 3.Detected result is in Table 2-3.
Table 2: Checkerboard titration method determines the data (each data are all mean value) of anti-HSA Ab, the best effort concentration of anti-As Ab and the optimum diluting multiple of blood plasma
Table 3:ELISA positive control and negative control ELISA detected result
By table 2-3 data presentation, when we can find out, and the extent of dilution as anti-HSA Ab is 1:2000, whole blood extent of dilution be the anti-extent of dilution of 1:20, As is 1:25000, OD value is maximum, so select concentration corresponding to this value as best effort concentration.
2. the determination of elutriant best effort concentration and elution time in method two, three in the method for detection by quantitative arsenic-albumin inner complex
Step is as follows: (1) bag quilt: anti-As Ab dilution buffer be diluted to 25000 times (mass volume ratios), add in elisa plate micropore, 37 DEG C of water-baths 3 hours, stores refrigerator;
(2) close: remove sample diluting liquid, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) remove confining liquid, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, it is 2 μ g/mL that enzyme labelled antibody is diluted to concentration, and 37 DEG C act on 2 hours, itself and anti-As Ab are reacted;
(4) prepare elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 100ng/ml, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min;
(5) enzyme labelled antibody is removed, by dilution buffer, elutriant is diluted, make the concentration of the papoid in elutriant and the ratio of the concentration of enzyme labelled antibody be 1:80,1:40,1:20,1:10,1:5 (wherein each concentration does 3 multiple holes), be positioned over wash-out 1h, 2h, 3h at 37 DEG C of temperature respectively;
(6) remove elutriant, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get 405nm wavelength, after adding stop buffer, be placed in by elisa plate in microplate reader and detect, read respectively and often organize OD value, by comparing with PBS damping fluid group, compare optimal concentration and the elution time of elutriant, concrete outcome is see table 4.
Table 4:ELISA elutriant best effort concentration and elution time are determined
From table 4, we can find, as the ratio=1:20 of the concentration of the papoid in elutriant with the concentration of enzyme labelled antibody, each group of OD value is all lower than other several groups, this concentration elution effect optimum (reach maximum by the anti-As Ab-enzyme mark mixture wash-out degree that ELISA hole wall combines, thus OD value is minimum) is described; And no matter elution time is 1h, 2h, 3h, the change of each group OD value is all little, as seen along with the prolongation of time, enzyme activity weakens gradually, when enzyme concn is constant, extends digestion time and can not improve digestibility, so the elution time of elutriant is that 1-3h all can in this test.
application Example 1
Get and adopt ELISA method to detect arsenic-albumin inner complex in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects arsenic-albumin inner complex, detect in accordance with the following steps:
1) anti-HSA Ab is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 37 DEG C of water-baths 1 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) material can catching arsenic is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and dilute 25000 times by dilution buffer, 37 DEG C act on 1 hour, the arsenic in anti-As Ab and albumin are reacted;
6) enzyme conjugates incubation: remove anti-arsenic antibody, and wash with washings, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, itself and enzyme labelled antibody are reacted;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) getting wavelength is 405nm, after adding stop buffer, is placed in by elisa plate in microplate reader and detects, and read measuring samples OD value (also can not use microplate reader, directly carry out qualitative detection by colour developing situation) respectively, concrete detected value is as shown in table 5.
The ELISA detected result of arsenic-albumin inner complex in table 5:100 part blood sample
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the arsenic-albumin inner complex in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add 2% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) detect: sample from the micropore of elisa plate, detect the arsenic of chelating in albumin in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 6.
The AAS detected result of arsenic-albumin inner complex in table 6:100 part blood sample
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the arsenic-albumin chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: with sample diluting liquid, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) 100 parts of standard blood samples are got as measuring samples;
3) close: remove sample diluting liquid, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the arsenic of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) sample the solution that obtains, under icp ms, detect chelating in albuminous arsenic, detected value is as shown in table 7.
The ICP-MS detected result of arsenic-albumin inner complex in table 7:100 part blood sample
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.

Claims (8)

1. arsenic-albumin inner complex, is characterized in that, arsonium ion and albumin by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for arsenic as claimed in claim 1-albumin inner complex, is characterized in that, comprise the following steps:
A) arsenic and albuminous chelatropic reaction: add arsonium ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying arsenic-albumin inner complex: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary arsonium ion in reaction soln, obtains arsenic-albumin inner complex.
3. preparation method according to claim 2, is characterized in that,
Described step B) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains arsenic-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of arsenic specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-albumin inner complex.
4. the preparation method of arsenic according to claim 2-albumin inner complex, is characterized in that, also comprise step C): to the qualification of arsenic-albumin inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing arsenic and detection arsenic.
5. the application of arsenic as claimed in claim 1-albumin inner complex in the reagent or test kit preparing to detect arsenic-albumin inner complex in blood sample.
6. one kind at least comprises the test kit of arsenic as claimed in claim 1-albumin inner complex as standard substance.
7. test kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching albuminous material or catching arsenic.
8. the method for detection by quantitative arsenic-albumin inner complex, it is characterized in that, using the arsenic according to claim 1-albumin inner complex of known content as standard substance, 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-albumin inner complex and enzyme linked immunological combined techniques, purification arsenic-albumin inner complex and atomic absorption spectrum combined techniques, purification arsenic-albumin inner complex 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.
CN201510412176.2A 2015-07-14 2015-07-14 Arsenic-albumin chelate as well as preparation method and application thereof Pending CN104987389A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins

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Title
刘冬等: "《生物分离技术》", 31 December 2007 *
吴士良等: "《生物化学与分子生物学实验教程》", 28 February 2009 *

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Application publication date: 20151021