CN104987395A - Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof - Google Patents

Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof Download PDF

Info

Publication number
CN104987395A
CN104987395A CN201510412942.5A CN201510412942A CN104987395A CN 104987395 A CN104987395 A CN 104987395A CN 201510412942 A CN201510412942 A CN 201510412942A CN 104987395 A CN104987395 A CN 104987395A
Authority
CN
China
Prior art keywords
low
arsenic
density lipoprotein
inner complex
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510412942.5A
Other languages
Chinese (zh)
Inventor
张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Baihao Biotechnology Co Ltd
Original Assignee
Shanghai Baihao Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Baihao Biotechnology Co Ltd filed Critical Shanghai Baihao Biotechnology Co Ltd
Priority to CN201510412942.5A priority Critical patent/CN104987395A/en
Publication of CN104987395A publication Critical patent/CN104987395A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses an arsenic-low-density lipoprotein chelate compound, a preparation method and an application thereof. The arsenic-low-density lipoprotein chelate compound is formed by chelating arsenic ions and low-density lipoprotein by at least one structure in a zinc-finger structure, sulfhydryl and cysteine residues. The invention establishes a qualitative and quantitative detection method of the arsenic-low-density lipoprotein chelate compound so as to quantitatively detect an application of the arsenic-low-density lipoprotein chelate compound in evaluating the arsenic-pollution degree of a region. The condition of arsenic pollution to population groups in the region can be indirectly reflected by quantitatively detecting the arsenic-low-density lipoprotein chelate compound in serum of the population groups in the region, so that the arsenic-pollution degree of the region is indirectly reflected. The quantitative detection method of the arsenic-low-density lipoprotein chelate compound established by the invention has the advantages that the accuracy is greatly improved and the detection repeatability is greatly improved.

Description

Arsenic-low-density lipoprotein inner complex and its preparation method and application
Technical field
The present invention relates to medical field, be specifically related to arsenic-low-density lipoprotein inner complex and its preparation method and application.
Background technology
Lipoprotein (lipoproteins) is a kind of lipid-protein complex that protein and lipid binding are formed, high-density lipoprotein (HDL) (high density lipoprotein is divided into by density, HDL), intermediated-density lipoprotein (intermediate densitylipoprotein, IDL), low-density lipoprotein (low density lipoprotein, LDL), vldl (very lowdensity lipoprotein, VLDL), chylomicrons (chylomiAson, CM).The cholesterol of about 70% is entrained by LDL and VLDL in blood, and the cholesterol level entrained by LDL is greater than VLDL, because LDL is rich in cholesterol, thus be called as " bad cholesterol ", the generation of itself and various diseases, develop closely related, particularly coronary heart disease, the diseases such as diabetes, research finds that " bad cholesterol " also can promote development and the transfer of cancer cell.
LDL is the Major Risk Factors that atherosclerosis occurs and develops, and its change has very strong influence to atherosclerosis in serum protein, is the significant risk factor of coronary artery disease, is proportionate with cardiovascular disease risk degree.Thus in detection by quantitative serum, LDL is significant for cardiovascular disorder, not only may be used for the atherosclerotic danger of EARLY RECOGNITION, and can as one of index of the detection of lipid lowering drug treatment process and prognostic evaluation.In addition, nephrotic syndrome, chronic renal failure, hepatopathy and diabetes etc., also LDL content can be made in serum to increase, and detection by quantitative LDL also can be used for early diagnosis and the result for the treatment of monitoring of these diseases, even as one of the evaluation index of prognosis quality.And for patients such as malnutrition, chronic anaemia, myelomatosis, acute myocardial infarction, wound and severe liver disease, the content of serum LDL will decline, and detection by quantitative LDL also can be used for the early diagnosis of these diseases, result for the treatment of monitoring and one of the index as prognostic evaluation.
Arsenic is one of common environmental poisonous substance, extensively be present in arsenic among water, soil, air and food and can produce acute or chronic toxicity to human body, 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, and has caused in multiple countries and regions the arsenic of high dosage to expose, has become the global problem of environmental pollution and health risk.
Documents existing a large amount of at present confirms that arsenic can act on the albumen in each system of whole body, thus affects the function of Body organs.As everyone knows, low-density lipoprotein is a kind of protein of Special Significance, and whether arsenic can act on low-density lipoprotein, and makes low-density lipoprotein structural modification, content change, activity change, changing function, and these still lack correlative study.
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-low-density lipoprotein inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of arsenic-low-density lipoprotein inner complex, so that detection by quantitative arsenic-low-density lipoprotein inner complex is in the application of the regional arsenic pollution level of evaluation one.Indirectly can reflect by arsenic-low-density lipoprotein 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-low-density lipoprotein inner complex, this arsenic-low-density lipoprotein inner complex be arsonium ion and low-density lipoprotein by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned arsenic-low-density lipoprotein inner complex, i.e. external synthesis method, comprises the following steps:
The synthesis of A) arsenic-low-density lipoprotein inner complex: add arsonium ion and carry out chelatropic reaction purifying in the low-density lipoprotein that comes from human body or the low-density lipoprotein according to biological method restructuring, obtain reaction soln;
B) arsenic-low-density lipoprotein inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted low-density lipoprotein, specific antibody and arsonium ion in reaction soln, obtain arsenic-low-density lipoprotein inner complex.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-low-density lipoprotein inner complex of extraction be dissolved in physiological saline, obtain the solution of arsenic-low-density lipoprotein inner complex;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of low-density lipoprotein specific binding, after dress post, continue to use dilution buffer balance chromatography column;
Described can with the filler of low-density lipoprotein specific binding be adsorbed with can with the silica gel of low-density lipoprotein specific binding material or resin;
(3) loading: after chromatography column balance, with the solution of dilution buffer dilution step (1), then upper prop, make low-density lipoprotein and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of 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 with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after 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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of 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-low-density lipoprotein inner complex.
As preferably, the preparation method of above-mentioned arsenic-low-density lipoprotein inner complex, also comprises the following authentication step to arsenic-low-density lipoprotein inner complex, specifically comprises the following steps:
(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-low-density lipoprotein inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS whether to detect in this liquid containing arsenic and the content detecting arsenic.
The present invention also provides the application of a kind of arsenic described above-low-density lipoprotein inner complex in preparation human body in the reagent of arsenic-low-density lipoprotein inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of arsenic described above-low-density lipoprotein inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching low-density lipoprotein or the material of catching arsenic.
The present invention also provides the method for a kind of detection by quantitative arsenic-low-density lipoprotein inner complex, using the above-mentioned arsenic-low-density lipoprotein 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-low-density lipoprotein inner complex and enzyme linked immunological combined techniques, purification arsenic-low-density lipoprotein inner complex and atomic absorption spectrum combined techniques, purification arsenic-low-density lipoprotein 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.
Compared to existing technology, beneficial effect of the present invention is:
1. the present invention has synthesized arsenic-low-density lipoprotein inner complex first;
2. the present invention proposes arsenic-low-density lipoprotein inner complex first and can be used for preparing application in the reagent or test kit detecting arsenic-low-density lipoprotein inner complex in blood sample;
3. present invention achieves-specific recognition of low-density lipoprotein inner complex and detection by quantitative, so that the content of arsenic-low-density lipoprotein inner complex in detection by quantitative crowd serum, to evaluate the application of a regional arsenic pollution level, the arsenic polluted water for industrial area is flat provides indirect indexes.The accuracy of arsenic-low-density lipoprotein inner complex quantitative detecting method that the present invention sets up is high, reproducible.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-low-density lipoprotein inner complex; ;
Fig. 2 is the fluorometric analysis figure of the synchrotron radiation X line electrophoresis strip of arsenic of the present invention-low-density lipoprotein 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; Agents useful for same, material, as non-specified otherwise, are all considered as to buy from commercial mode:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
The material of catching low-density lipoprotein is anti-low-density lipoprotein antibody, by commercially available acquisition, as the Anti-LDL antibodies Antibodies of " Abcam ab157795 " that Ai Bokang (Shanghai) trade Co., Ltd sells, in following embodiment, be describedly the material of catching low-density lipoprotein with the material of low-density lipoprotein specific binding, anti-low-density lipoprotein antibody, anti-LDL antibody;
Enzyme labelled antibody is the one in the antibody containing the enzyme labelling such as horseradish peroxidase, alkaline phosphatase;
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example: the Na getting 1.5g 2cO 3with the NaHCO of 2.93g 3dissolving adds ddH 2o is settled to 1000mL;
Lavation buffer solution is the 0.15M PBS solution of pH7.4, compound method example: the KH getting 0.2g 2pO 4, 2.90g Na 2hPO 412H 2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH 2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin, adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2M H 2sO 4, compound method example: the ddH getting 178.3mL 2o, enriching H 2sO 4be settled to 200mL;
Substrate is methyl diphenyl amine (TMB) solution, compound method example: get the methyl diphenyl amine ethanolic soln that 0.5mL concentration is 2g/L, add substrate dilution and be diluted to 10mL;
Substrate buffer solution pH is 5.0, wherein Na 2hPO 4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, compound method example: get 1.42gNa 2hPO 4, 0.96g citric acid, then add ddH 2o to 50mL, to obtain final product;
The compound method example of elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/mL, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min, obtain elutriant;
Sample-loading buffer can be formulated by the component of following ratio: Tris-HCl:1% tetrabromophenol sulfonphthalein: ddH 2o: glycine=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
The compound method of electrophoretic buffer is as follows: get 3.0g Tris, 14.4g glycine, be dissolved in 800mLddH 2in O, after adjusting pH to 8.3, be settled to 1L;
The material of the catching arsenic mouse-anti As mAb that to be article No. be " Guangzhou Ran Ke company RK15728 "; Wherein, of the present invention with the material of arsenic specific binding, anti-As antibody, two anti-, anti-arsenic antibody is the material of catching arsenic;
The invention provides a kind of arsenic-low-density lipoprotein inner complex, arsonium ion and low-density lipoprotein by sulfydryl or/and cysteine residues chelating forms.
Particularly, arsenic-low-density lipoprotein inner complex is by the inner complex of arsenic by combining in conjunction with at least one structure in zinc fingers, sulfydryl, cysteine residues and the apolipoprotein B on low-density lipoprotein or cholesterol, triglyceride level etc. and formed.
The present invention also provides the preparation method of arsenic-low-density lipoprotein inner complex, comprises the following steps:
A) synthesis of arsenic-low-density lipoprotein: add arsonium ion and carry out chelatropic reaction purifying in the low-density lipoprotein that comes from human body or the low-density lipoprotein according to biological method restructuring, obtain reaction soln;
B) arsenic-low-density lipoprotein inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted low-density lipoprotein, specific antibody and arsonium ion in reaction soln, obtain arsenic-low-density lipoprotein inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-low-density lipoprotein inner complex of extraction be dissolved in physiological saline, obtain the solution of arsenic-low-density lipoprotein inner complex;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of low-density lipoprotein specific binding, after dress post, continue to use dilution buffer balance chromatography column;
Described can with the filler of low-density lipoprotein specific binding be adsorbed with can with the silica gel of low-density lipoprotein specific binding material or resin;
(3) loading: after chromatography column balance, with the solution of dilution buffer dilution step (1), then upper prop, make low-density lipoprotein and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: collect elutriant, after collection, make albumen restore immediately;
(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 with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after 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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: collect elutriant, after collection, make albumen restore immediately;
(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-low-density lipoprotein inner complex.
C): to the qualification of arsenic-low-density lipoprotein inner complex, specifically comprise the following steps:
(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-low-density lipoprotein inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS whether to detect in this liquid containing arsenic and the content detecting arsenic.
The present invention also provides a kind of and at least comprises the test kit of arsenic described above-low-density lipoprotein inner complex as standard substance.
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.
A kind of test kit detecting arsenic in blood sample-low-density lipoprotein inner complex, comprise: containing the coating buffer that can be used for catching low-density lipoprotein, also comprise confining liquid, lavation buffer solution, the material of arsenic can be caught as two anti-, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative controls etc.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprising: containing the coating buffer that can be used for catching low-density lipoprotein, also comprise confining liquid, lavation buffer solution, elutriant, positive control, negative control etc.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprising: containing the coating buffer that can be used for catching low-density lipoprotein, also comprise confining liquid, lavation buffer solution, elutriant, souring agent, hydrogen peroxide, standard substance, negative control etc.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprise extract low-density lipoprotein required extract reagent, redissolve liquid, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, standard substance, negative control etc. of material that can be used for catching arsenic.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprise and extract low-density lipoprotein required extraction reagent, redissolution liquid, positive control, negative control etc.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprise and extract low-density lipoprotein required extraction reagent, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprise and extract low-density lipoprotein and required extract reagent, glue bed medium, to redissolve on liquid, sample loading buffer, dissolving glue bed containing liquid needed for the protein band of arsenic, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, positive control, negative control etc. of material that can be used for catching arsenic.
Detect a test kit for arsenic in blood sample-low-density lipoprotein inner complex, comprise and extract low-density lipoprotein and required extract reagent, glue bed medium, redissolve on liquid, sample loading buffer, 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-low-density lipoprotein inner complex, comprise and extract low-density lipoprotein and required extract reagent, glue bed medium, redissolve on liquid, sample loading buffer, dissolving glue bed containing liquid, nitric acid, 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 low-density lipoprotein inner complex of arsenic or is chelated with the BSA inner complex of arsenic; Described negative control is the dilution elutriant not containing standard substance.
Mentioned reagent box, for detecting arsenic-low-density lipoprotein inner complex, to improve accuracy and the repeatability of detection, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative arsenic-low-density lipoprotein inner complex, using the above-mentioned arsenic-low-density lipoprotein 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-low-density lipoprotein inner complex and enzyme linked immunological combined techniques, purification arsenic-low-density lipoprotein inner complex and atomic absorption spectrum combined techniques, purification arsenic-low-density lipoprotein 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-low-density lipoprotein inner complex can list, but be not limited to following several.
method one: euzymelinked immunosorbent assay (ELISA) detects arsenic-low-density lipoprotein inner complex, detects according to the following steps:
1) bag quilt: the material can catching low-density lipoprotein by dilution buffer is diluted to 250 ~ 2000 times, adds in elisa plate micropore, and spend the night at 4 DEG C 16-18 hour, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
2) close: remove dilution buffer, and wash with washings, after washing completes, add 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with corresponding washings for 37 DEG C.After having washed, elisa plate is placed 1 hour in 337 DEG C;
3) sample to be tested is added, and incubation: from recycle system sampling, make sample to be tested; Standard substance are made with the arsenic of known content-low-density lipoprotein inner complex; By dilution buffer, sample to be tested and standard substance are all diluted to 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching arsenic is added, and incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, add the anti-arsenic antibody with dilution buffer dilution 25000 ~ 200000 times, 37 DEG C of effect 1-2 hour, make the arsenic on anti-arsenic antibody and low-density lipoprotein react;
5) enzyme conjugates incubation: remove anti-arsenic antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and HRP enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: drip stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, elisa plate being placed in OD value microplate reader reading respectively sample to be tested group and standard substance, drawing standard curve, by comparing with standard substance group, trying to achieve the content of sample to be tested.
In present method, step 8) also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
The method utilizes ELISA principle, specificity low-density lipoprotein in whole blood can be extracted, the low-density lipoprotein upper part extracted is chelated with arsenic, and arsenic on this part low-density lipoprotein can catch by the specific antibody of anti-arsenic, 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 low-density lipoprotein 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 on low-density lipoprotein.
method two: enzyme linked immunological (ELISA) and atomic absorption spectrum (AAS) combined techniques detect arsenic-low-density lipoprotein inner complex, detect according to the following steps:
1) bag quilt: the material that low-density lipoprotein can be caught, as anti-low-density lipoprotein antibody (anti-low-density lipoprotein antibody) is coated on solid phase carrier, anti-low-density lipoprotein antibody is diluted to 250-2000 times by dilution buffer, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: sample from the recycle system, make sample to be tested; Standard substance are made with the arsenic of known content-low-density lipoprotein inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds elutriant, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the arsenic of chelating on low-density lipoprotein in Atomic Absorption Spectroscopy AAS, read respective value;
This embodiment utilizes ELISA principle to catch the low-density lipoprotein in serum, and detects the arsenic of chelating on low-density lipoprotein in conjunction with atomic absorption spectrum (AAS) instrument; Owing to only containing low-density lipoprotein in solution, and not containing 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 on low-density lipoprotein.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect arsenic-low-density lipoprotein inner complex and detect in accordance with the following steps:
1) bag quilt: the material that low-density lipoprotein can be caught, as anti-low-density lipoprotein antibody is coated on solid phase carrier, anti-low-density lipoprotein antibody is diluted to 250-2000 times by dilution buffer, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: get whole blood from the recycle system, make sample to be tested; Standard substance are made with the arsenic of known content-low-density lipoprotein inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add elutriant, wash-out 1-3 hour at 37-57 DEG C;
5) acidifying: in step 4) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in the arsenic of low-density lipoprotein, read respective value.
The method, on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, detects the arsenic of chelating on low-density lipoprotein with icp ms; Namely first adopt ELISA principle to be extracted by the arsenic in serum-low-density lipoprotein inner complex, then adopt sense couple plasma mass spectrometer to carry out detection by quantitative to the arsenic of chelating on low-density lipoprotein; Owing to only containing low-density lipoprotein in solution, and not containing arsenic in agents useful for same, negative control group result is negative, can not cause interference to result, thus when read result is shown as the positive, i.e. and the provable arsenic detecting chelating on low-density lipoprotein.
method four:purification arsenic-low-density lipoprotein inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect arsenic-low-density lipoprotein inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific low-density lipoprotein is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, low-density lipoprotein is extracted from whole blood, and the low-density lipoprotein extracted is redissolved, obtain the solution of low-density lipoprotein;
2) bag quilt: be coated on solid phase carrier by anti-arsenic antibody, dilutes anti-arsenic antibody to 25000 ~ 200000 times by dilution buffer, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
3) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add sample to be tested, and incubation: from step 1) solution sample, make sample to be tested; Standard substance are made with the arsenic of known content-low-density lipoprotein inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, adds the enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: drip stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, microplate reader reading the OD value of sample to be tested group and standard substance respectively, drawing standard curve, by comparing with standard substance group, trying to achieve the content of sample to be tested.
In present method, also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
method five:purification arsenic-low-density lipoprotein inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect arsenic in blood sample-low-density lipoprotein inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific low-density lipoprotein is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, low-density lipoprotein is extracted from whole blood, and the low-density lipoprotein extracted is redissolved, obtain the solution of low-density lipoprotein;
2) detect: from step 1) solution sample, detect the arsenic of chelating on low-density lipoprotein in Atomic Absorption Spectroscopy AAS, read respective value.
method six:purification arsenic-low-density lipoprotein inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect arsenic-low-density lipoprotein inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific low-density lipoprotein is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, low-density lipoprotein is extracted from whole blood, and the low-density lipoprotein extracted is redissolved, obtain the solution of low-density lipoprotein;
2) acidifying: from step 1) solution sample, add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
3) detect: add hydrogen peroxide, and heating gets 0.5mL solution after catching up with acid, detects the arsenic of chelating on low-density lipoprotein under icp ms, read respective value.
Method four, method five and method six are all isolate low-density lipoprotein by whole blood extraction method, then adopt method for detecting specificity, measure the content of arsenic on arsenic-low-density lipoprotein inner complex in low-density lipoprotein; Namely first physical sepn means are adopted, as ultracentrifugation, HPLC, gel-filtration chromatography etc., separated from test plasma sample by low-density lipoprotein and redissolve in physiological saline, recycling ELISA principle, atomic absorption spectrum detect or carry out detecting the arsenic content on inductively coupled plasma mass spectrometry detection arsenic-low-density lipoprotein inner complex.
Method seven: electrophoretic method+ELISA/AAS/ICP-MS method detects arsenic-low-density lipoprotein inner complex, specific as follows:
1) from whole blood, non-specific low-density lipoprotein is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, low-density lipoprotein is extracted from whole blood, the low-density lipoprotein extracted is redissolved in physiological saline, obtains the solution of low-density lipoprotein;
2) glue bed is prepared: select suitable medium (as sepharose, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) solution get 8 μ L solution, make standard substance with the arsenic of known content-low-density lipoprotein inner complex, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, with electrophoretic buffer, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of iso-electric point;
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 the principles such as ELISA, ICP-MS or AAS to detect the arsenic content be dissolved in liquid respectively.
In addition, the iso-electric point of the immunocomplex of this method detection arsenic-low-density lipoprotein inner complex, molecular weight and content etc. can also be utilized.
In method seven, low-density lipoprotein is extracted from whole blood, then adopt gel electrophoresis to be separated extracted low-density lipoprotein, then find out the respective strap being rich in arsenic, then detect the content of relevant low-density lipoprotein, namely after low-density lipoprotein discharges from red corpuscle, can by multiple Methods For Purification out (such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the low-density lipoprotein of purifying out is redissolved in solution, get a certain amount of low-density lipoprotein, 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 the content of relevant low-density lipoprotein can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the arsenic content of chelating on low-density lipoprotein, owing to only containing low-density lipoprotein in solution, and not containing 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 on low-density lipoprotein.
embodiment 1:synthesis method synthesis arsenic-low-density lipoprotein huge legendary turtle compound, comprises the following steps:
Arsenic prepared by the present embodiment-low-density lipoprotein huge legendary turtle compound, is separated further by gel electrophoresis, and carries out detection Qualitative Identification by inductivity coupled plasma mass spectrometry or atomic absorption spectrum.
Prepare the method for arsenic-low-density lipoprotein inner complex, comprise the following steps:
The reagent that the present embodiment uses is as follows:
1) borate buffer solution, its volumetric molar concentration is 0.01M, and its preparation method example is as follows: take 0.31g boric acid and be dissolved in 400mLddH 2in O, regulate pH to 9.0 with the NaOH of 0.1mol/L, be settled to 500mL.
2) EDTA-NaHCO 3mixed solution, its preparation method is as follows: get 1.86g EDTA2H 2o and 16.8g NaHCO 3, be dissolved in 900mLddH 2in O, adjust pH to 8.0 with 1.0M NaOH and be settled to 1000mL, autoclaving, room temperature preservation;
3) ITCBE buys from Japanese colleague's chemistry institute, article No. M030;
4) low-density lipoprotein solution: take 4.0mg low-density lipoprotein and be dissolved in 4.0mL0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, be mixed with the low-density lipoprotein solution of 1.0mg/mL;
5) molecular weight cut-off 14000 of dialysis tubing, buys from Bioshop Inc;
The pre-treatment of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500mL 3in mixed solution, boil 10min; Tipping EDTA-NaHCO 3liquid, uses ddH 2o is rinsing gently, then boils 10min with 500mL5mmol/L EDTA; Discard boiling liquid, thoroughly use ddH 2o cleans, and adds a large amount of ddH 2o soaks dialysis tubing 4 DEG C and spends the night.During use, put on one's gloves, take out dialysis tubing, with a large amount of ddH 2its surfaces externally and internally of O cleaning down;
The synthesis of A) arsenic-low-density lipoprotein huge legendary turtle compound: add arsonium ion and carry out chelatropic reaction purifying in the low-density lipoprotein that comes from human body or the low-density lipoprotein according to biological method restructuring, obtain reaction soln, concrete operation step is as follows;
1) getting 2.0mg ITCBE is dissolved in 2mLDMSO;
2) liquid slowly step 1 prepared adds in low-density lipoprotein solution, and dropping limit, limit is shaken, and in 25 DEG C, acts on 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, removes the ITCBE be not combined with low-density lipoprotein;
3) by the liquid 1mol/L HCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l 1mmol/L arsenic ion solns gradually, dropping limit, limit vibrates, in order to avoid arsonium ion makes protein denaturation precipitate;
4) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the dialysis tubing handled well;
5) liquid of having dialysed is preserved in-20 DEG C of packing, obtain the reaction soln of arsenic-low-density lipoprotein inner complex.
B) arsenic-low-density lipoprotein inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted low-density lipoprotein, specific antibody and arsonium ion in reaction soln, obtain arsenic-low-density lipoprotein inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-low-density lipoprotein inner complex of extraction be dissolved in physiological saline, obtain the solution of arsenic-low-density lipoprotein inner complex;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of low-density lipoprotein specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with the solution of dilution buffer dilution step (1), then upper prop, make low-density lipoprotein and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: collect elutriant, after collection, make albumen restore immediately;
(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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: collect elutriant, after collection, make albumen restore immediately;
(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-low-density lipoprotein inner complex.
C) to the qualification of arsenic-low-density lipoprotein huge legendary turtle compound, 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-low-density lipoprotein inner complex of obtaining of extraction purification, redissolve in physiological saline, obtain the solution of arsenic-low-density lipoprotein inner complex, get the above-mentioned solution of 8 μ L, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 degree; Stop electrophoresis when moving to bottom glue to tetrabromophenol sulfonphthalein, Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-low-density lipoprotein huge legendary turtle compound;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS whether to detect in this liquid containing arsenic and the content detecting arsenic.
D) qualification result
1) AAS detected result
Get step C) isolated protein band solution, with the content of arsenic in graphite furnace atomic absorption spectrometry (AAS) rough determination low-density lipoprotein, as shown in the table, with NaCl solution, KBr solution, KCl solution for blank;
The content of arsenic in table 1 arsenic-low-density lipoprotein inner complex
Sample name As(μg/L)
Sample to be tested 4.705
NaCl 0.109
KBr 0.002
KCl 0.032
2) Synchrotron Radiation X-Ray Fluorescence Anal ysis
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of 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.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA 4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx 3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band 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 fluorometric analysis figure of the synchrotron radiation X line electrophoresis strip of arsenic of the present invention-low-density lipoprotein inner complex, and in figure, X-coordinate is protein band position, and ordinate zou is this band each arsenic energy (content) value.
The determination of testing conditions:
1. the determination of the optimum diluting multiple of anti-low-density lipoprotein antibody, anti-As antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for arsenic-low-density lipoprotein inner complex, specifically comprises the following steps:
1) bag quilt: anti-low-density lipoprotein antibody albumen is coated on solid phase carrier, respectively by dilution buffer by coating protein with the doubling dilution of 1:250,1:500,1:10000 and 1:2000, add in elisa plate micropore, each concentration bag, by three rows, is preserved 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) sample to be tested is added: by dilution buffer by the doubling dilution of test plasma sample by 1:10,1:20,1:40, add in micropore, make standard substance with the arsenic of known content-low-density lipoprotein inner complex, negative control and blank are set, add in micropore, 37 DEG C act on 1 hour;
4) add anti-As antibody: remove test plasma sample, washing, adds with the anti-As antibody of dilution buffer by the doubling dilution of 1:25000,1:50000,1:10000,1:200000, and 37 DEG C act on 1 hour, the arsenic on itself and low-density lipoprotein is reacted;
5) enzyme-added mark: remove anti-As antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, make itself and anti-As antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer;
7) detect: the OD value reading standard substance, test plasma, positive control, negative control and blank sample under 450nm wavelength in microplate reader respectively, drawing standard curve, tries to achieve the content of sample to be tested.
In the present embodiment, adopt arsenic provided by the invention-low-density lipoprotein inner complex standard substance as positive control, respectively not add test plasma as negative control 1, namely add anti-low-density lipoprotein antibody, confining liquid, anti-As antibody, enzyme mark and substrate successively;
Not add the controlled trial group of anti-As antibody as negative control 2, namely add anti-low-density lipoprotein antibody, confining liquid, test plasma, enzyme mark and substrate successively;
Anti-low-density lipoprotein antibody, confining liquid, test plasma, anti-As antibody and substrate is added successively using not enzyme-added target controlled trial group as negative control 3, namely;
Not add the controlled trial group of sample to be tested and anti-As antibody as negative control 4 simultaneously, namely add anti-low-density lipoprotein antibody, confining liquid, enzyme mark and substrate successively;
Not add the controlled trial group of anti-low-density lipoprotein antibody work for blank 1, namely add confining liquid, test plasma, anti-As antibody, enzyme mark and substrate;
And with the controlled trial group only adding substrate for blank 2, only to add the controlled trial group of PBS for blank 3.
Table 2 is the sample OD Value Data of different anti-low-density lipoprotein antibody dilution multiple proportions, diluted plasma multiple proportions, anti-As antibody dilution multiple proportions.
The determination of table 2 anti-LDL antibody and anti-As antibody best effort concentration and blood plasma optimum diluting multiple
Table 3 ELISA positive control and negative control ELISA detected result
From table 2 and table 3, when the extent of dilution of anti-LDL antibody be 1:500, dilution rate of blood plasma is 1:20, anti-As antibody dilution is 1:25000 time, OD value is maximum, although its OD value is less than 0.8, the negative control group OD value corresponding to it is all less than 0.1, and the OD value of the positive control of its correspondence is greater than 0.8, so select concentration corresponding to this value as best effort concentration, namely the extent of dilution of anti-LDL antibody is 1:500, and dilution rate of blood plasma is 1:20, and anti-As antibody dilution is 1:25000.
2.ELISA elutriant best effort concentration and time are determined
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-As antibody and enzyme labelled antibody incubation, carry out wash-out with the elutriant of different concns, then detect OD value by microplate reader, concrete steps are as follows:
(1) bag quilt: be coated on solid phase carrier by anti-low-density lipoprotein antibody albumen, press the doubling dilution of 1:500 by dilution buffer, adds in elisa plate micropore, preserves 16 hours for 4 DEG C;
(2) close: remove dilution buffer, after washing, add confining liquid, place 1 hour, remove confining liquid, and wash for 37 DEG C;
(3) add enzyme labelled antibody: remove confining liquid, after washing, add and be diluted to by dilution buffer the HRP enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C act on 2 hours, make itself and anti-As antibody response;
(4) wash-out: remove enzyme labelled antibody, by dilution buffer, elutriant is diluted, make the concentration of papoid in elutriant: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove elutriant, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer termination reaction;
(5) under the determined wavelength of 450nm, in microplate reader, read the OD value of each micropore respectively, concrete outcome see table 4,
Detected result under the different elutriant extension rate of table 4
By comparing OD value, to judge anti-As antibody-hrp-antibody complex wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-As antibody-hrp-antibody complex wash-out degree reaches maximum.As shown in table 4, the concentration when papoid in elutriant: in enzyme labelled antibody during the concentration=1:20 of antibody, anti-As antibody-hrp-antibody complex wash-out degree reaches maximum.And action time is when being 1h, 2h, 3h, each group of OD value change is little, the visible prolongation along with the time, enzyme activity weakens gradually, when enzyme concn is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of elutriant be that 1-3h all can, in sum, we select elutriant 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
application Example 1:
Employing euzymelinked immunosorbent assay (ELISA) detects the arsenic-low-density lipoprotein inner complex in 100 parts of sample blood plasma, detects according to following steps:
1) anti-LDL antibody protein is coated on solid phase carrier: by dilution buffer with the dilution proportion coating protein of 1:500, add in elisa plate micropore, 37 DEG C of water-baths 1 hour, stores refrigerator;
2) close: remove dilution buffer, washing, adds confining liquid, and place 1 hour, remove confining liquid for 37 DEG C, washing, elisa plate 37 DEG C places 4 DEG C of storages in refrigerator after 1 hour;
3) application of sample: using sample blood plasma as sample to be tested, makes standard substance with the arsenic of known content-low-density lipoprotein inner complex, by dilution buffer, sample to be tested and standard substance is all diluted 20 times, adds in micropore, and 37 DEG C act on 1 hour;
4) add anti-As antibody: remove sample, washing, add the anti-As antibody being diluted to 25000 times by dilution buffer, 37 DEG C of effects 1 hour, make the arsenic on itself and low-density lipoprotein react;
5) enzyme-added mark: remove anti-As antibody, washing, adds the enzyme labelled antibody that antibody concentration is 2 μ g/mL, and 37 DEG C act on 1 hour, make itself and anti-As antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, enter stop buffer;
7) detect: the OD value reading sample to be tested group and standard substance under 450nm wavelength in microplate reader respectively, cross and compare with standard substance group, try to achieve the content of arsenic in sample to be tested according to standard equation, result is as shown in table 5.
Table 5 100 parts of sample serum arsenic-low-density lipoprotein inner complex ELISA detected result
Numbering 1 2 3 4 5 6 7 8 9 10
OD 405 0.371 0.325 0.52 0.675 0.532 0.561 0.783 0.181 0.187 0.803
Numbering 11 12 13 14 15 16 17 18 19 20
OD 405 0.614 0.17 0.76 0.735 0.403 0.751 0.84 0.584 0.223 0.688
Numbering 21 22 23 24 25 26 27 28 29 30
OD 405 0.516 0.759 0.495 0.752 0.211 0.541 0.492 0.645 0.698 0.672
Numbering 31 32 33 34 35 36 37 38 39 40
OD 405 0.665 0.311 0.656 0.815 0.682 0.816 0.741 0.515 0.263 0.201
Numbering 41 42 43 44 45 46 47 48 49 50
OD 405 0.596 0.736 0.765 0.627 0.772 0.547 0.771 0.4 0.668 0.795
Numbering 51 52 53 54 55 56 57 58 59 60
OD 405 0.445 0.224 0.23 0.154 0.179 0.352 0.313 0.225 0.734 0.16
Numbering 61 62 63 64 65 66 67 68 69 70
OD 405 0.792 0.523 0.231 0.709 0.47 0.56 0.75 0.427 0.658 0.349
Numbering 71 72 73 74 75 76 77 78 79 80
OD 405 0.822 0.16 0.196 0.777 0.587 0.285 0.808 0.647 0.468 0.413
Numbering 81 82 83 84 85 86 87 88 89 90
OD 405 0.464 0.491 0.175 0.518 0.501 0.745 0.179 0.734 0.431 0.689
Numbering 91 92 93 94 95 96 97 98 99 100
OD 405 0.731 0.758 0.203 0.644 0.384 0.508 0.326 0.81 0.48 0.48
In this application embodiment 1, step 7) in, microplate reader also can not be used to detect, but directly carry out qualitative detection by colour developing.
application Example 2
Adopt enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) to detect arsenic-low-density lipoprotein inner complex in 100 parts of sample blood plasma, the method namely adopting specific embodiment method two to record detects, and concrete operation step is as follows:
1) bag quilt: anti-low-density lipoprotein antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 500 times, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, preserve at elisa plate 4 DEG C;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the arsenic of known content-low-density lipoprotein inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 20 times, add in micropore, and 37 DEG C act on 1 hour;
4) wash-out: remove test plasma sample, washing, adds the Na of 0.8mol/L 2hPO 4solution, 37 DEG C act on 2 hours;
5) detect: from ELISA micropore, get appropriate amount of fluid, detect the arsenic of chelating on low-density lipoprotein in Atomic Absorption Spectroscopy AAS, result is as shown in table 6.
Table 6 100 parts of sample blood plasma arsenic-low-density lipoprotein inner complex AAS detected result
application Example 3:
Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) is adopted to detect arsenic-low-density lipoprotein inner complex in 100 parts of sample blood plasma, namely the method adopting specific embodiment method three to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by anti-low-density lipoprotein antibody, with dilution buffer dilution coating protein to 500 times, adds in elisa plate micropore, preserves 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, elisa plate 4 DEG C preservation;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the arsenic of known content-low-density lipoprotein inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 20 times, add in micropore, and 37 DEG C act on 2 hours;
4) wash-out: remove sample to be tested and standard substance, washing, adds the Na of 0.8mol/L 2hPO 4solution, 37 DEG C of wash-outs 2 hours;
5) acidifying: add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
6) detect: sample and detect the arsenic of chelating on low-density lipoprotein under icp ms, result is as shown in table 7.
Table 7 100 parts of sample serum arsenic-low-density lipoprotein inner complex ICP-MS detected result
Numbering 1 2 3 4 5 6 7 8 9 10
μg/L 3.149 3.432 2.348 3.652 4.272 2.525 3.949 3.302 3.464 2.727
Numbering 11 12 13 14 15 16 17 18 19 20
μg/L 4.37 3.062 4.253 2.03 3.613 3.462 3.089 2.917 4.186 3.083
Numbering 21 22 23 24 25 26 27 28 29 30
μg/L 2.794 3.638 3.119 3.698 4.389 4.703 3.308 3.624 4.791 4.905
Numbering 31 32 33 34 35 36 37 38 39 40
μg/L 3.655 2.601 3.604 4.292 2.64 2.54 2.28 3.957 2.032 2.482
Numbering 41 42 43 44 45 46 47 48 49 50
μg/L 3.807 2.359 4.371 3.85 4.867 3.879 4.35 3.699 4.787 4.122
Numbering 51 52 53 54 55 56 57 58 59 60
μg/L 3.276 2.236 3.954 3.239 4.465 2.574 2.526 2.017 3.154 4.088
Numbering 61 62 63 64 65 66 67 68 69 70
μg/L 4.133 3.907 3.838 4.679 3.602 2.409 2.131 4.444 3.78 4.226
Numbering 71 72 73 74 75 76 77 78 79 80
μg/L 3.203 2.279 2.702 2.15 3.268 2.484 4.182 3.311 3.942 2.282
Numbering 81 82 83 84 85 86 87 88 89 90
μg/L 3.256 3.695 3.076 2.36 3.052 4.429 3.514 2.695 3.045 2.114
Numbering 91 92 93 94 95 96 97 98 99 100
μg/L 3.478 2.893 2.812 2.6 3.279 2.406 2.279 3.346 4.958 3.479
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.

Claims (8)

1. arsenic-low-density lipoprotein inner complex, is characterized in that, this arsenic-low-density lipoprotein inner complex be arsonium ion and low-density lipoprotein by sulfydryl or/and cysteine residues chelating forms.
2. the preparation method of arsenic-low-density lipoprotein inner complex as claimed in claim 1, comprises the following steps:
The synthesis of A) arsenic-low-density lipoprotein inner complex: add arsonium ion and carry out chelatropic reaction purifying in the low-density lipoprotein that comes from human body or the low-density lipoprotein according to biological method restructuring, obtain reaction soln;
B) arsenic-low-density lipoprotein inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted low-density lipoprotein, specific antibody and arsonium ion in reaction soln, obtain arsenic-low-density lipoprotein inner complex.
3. method as claimed in claim 2, is characterized in that,
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-low-density lipoprotein inner complex of extraction be dissolved in physiological saline, obtain the solution of arsenic-low-density lipoprotein inner complex;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of low-density lipoprotein specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with the solution of dilution buffer dilution step (1), then upper prop, make low-density lipoprotein and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: collect step (4) elutriant, after collection, make albumen restore immediately;
(6) dialyse: by the elutriant of 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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of 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-low-density lipoprotein inner complex.
4. the preparation method of arsenic according to claim 2-low-density lipoprotein inner complex, is characterized in that, step B) after also comprise step C): to the qualification of arsenic-low-density lipoprotein inner complex;
Wherein, step C) in specifically comprise the following steps:
(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-low-density lipoprotein inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing arsenic, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS whether to detect in this liquid containing arsenic and the content detecting arsenic.
5. the application of arsenic as claimed in claim 1-low-density lipoprotein inner complex in the reagent or test kit preparing to detect arsenic-low-density lipoprotein inner complex in blood sample.
6. one kind at least comprises the test kit of arsenic as claimed in claim 1-low-density lipoprotein 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 low-density lipoprotein or the material of catching arsenic.
8. the method for detection by quantitative arsenic-low-density lipoprotein inner complex, it is characterized in that, using the arsenic according to claim 1-low-density lipoprotein 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-low-density lipoprotein inner complex and enzyme linked immunological combined techniques, purification arsenic-low-density lipoprotein inner complex and atomic absorption spectrum combined techniques, purification arsenic-low-density lipoprotein 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.
CN201510412942.5A 2015-07-14 2015-07-14 Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof Pending CN104987395A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510412942.5A CN104987395A (en) 2015-07-14 2015-07-14 Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510412942.5A CN104987395A (en) 2015-07-14 2015-07-14 Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN104987395A true CN104987395A (en) 2015-10-21

Family

ID=54299310

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510412942.5A Pending CN104987395A (en) 2015-07-14 2015-07-14 Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN104987395A (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands
US6323354B1 (en) * 2001-03-16 2001-11-27 Agri-Nutrients Technology Group, Inc. Amino acid chelates from lipoproteins
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins
CN101084237A (en) * 2004-10-11 2007-12-05 希尔蛋白质有限公司 Ubiquitin or gamma-crystalline conjugates for use in therapy, diagnosis and chromatography
CN101082045A (en) * 2007-01-22 2007-12-05 耿永健 Preparation method of apolipoprotein-J
CN102766188A (en) * 2012-07-24 2012-11-07 上海交通大学 Cholesterol derivative, chelate, recombinant high density lipoprotein and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands
US6323354B1 (en) * 2001-03-16 2001-11-27 Agri-Nutrients Technology Group, Inc. Amino acid chelates from lipoproteins
CN101084237A (en) * 2004-10-11 2007-12-05 希尔蛋白质有限公司 Ubiquitin or gamma-crystalline conjugates for use in therapy, diagnosis and chromatography
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins
CN101082045A (en) * 2007-01-22 2007-12-05 耿永健 Preparation method of apolipoprotein-J
CN102766188A (en) * 2012-07-24 2012-11-07 上海交通大学 Cholesterol derivative, chelate, recombinant high density lipoprotein and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
刘冬 等: "《生物分离技术》", 31 December 2007, 高等教育出版社 *
刘功良等: "铅螯合物人工抗原的制备与鉴定", 《生物技术通报》 *
吴士良 等: "《生物化学与分子生物学实验教程》", 28 February 2009, 科学出版社 *
王琦光: "高密度脂蛋白抗氧化作用及意义", 《中国分子心脏病学杂质》 *

Similar Documents

Publication Publication Date Title
CN104961824A (en) Lead and high-density lipoprotein chelate as well as preparation method and application thereof
CN104987395A (en) Arsenic-low-density lipoprotein chelate compound, preparation method and application thereof
CN104987399A (en) Arsenic-very low density lipoprotein chelate as well as preparation method and application thereof
CN105021553A (en) Arsenic-high density lipoprotein chelate as well as preparation method and application thereof
CN105044008B (en) Cadmium-high density lipoprotein chelate as well as preparation method and application thereof
CN105044009B (en) Chromium-very low density lipoprotein chelate and preparation method and application thereof
CN104987393A (en) Cadmium-very low density lipoprotein chelate as well as preparation method and application thereof
CN104987397A (en) Mercury-low density lipoprotein chelate as well as preparation method and application thereof
CN105115914B (en) Cadmium-low density lipoprotein chelate as well as preparation method and application thereof
CN105037528A (en) Nickel-low density lipoprotein chelate as well as preparation method and application thereof
CN105021552A (en) Nickel-high density lipoprotein chelate as well as preparation method and application thereof
CN105001322A (en) Chromium-high density lipoprotein chelate as well as preparation method and application thereof
CN104987394A (en) Mercury-very low density lipoprotein chelate as well as preparation method and application thereof
CN105001323A (en) Nickel-very low density lipoprotein chelate and preparation method and application thereof
CN104987396A (en) Lead-very low density lipoprotein chelate as well as preparation method and application thereof
CN105021550A (en) Mercury-high density lipoprotein chelate and preparation method and application thereof
CN104987398A (en) Chromium-low density lipoprotein chelate as well as preparation method and application thereof
CN104987409A (en) Lead-IgG chelate as well as preparation method and application thereof
CN105021548A (en) Arsenic-fibrinogen chelate as well as preparation method and application thereof
CN105004686A (en) Chromium-albumin chelate as well as preparation method and application thereof
CN105004684A (en) Nickel-IgE chelate as well as preparation method and application thereof
CN104987400A (en) Mercury-hemoglobin chelate as well as preparation method and application thereof
CN104987390A (en) Cadmium-albumin chelate as well as preparation method and application thereof
CN105021554B (en) arsenic-IgM chelate as well as preparation method and application thereof
CN105004683B (en) chromium-IgE chelate as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20151021

RJ01 Rejection of invention patent application after publication