CN105021553A - Arsenic-high density lipoprotein chelate and preparation method and application thereof - Google Patents
Arsenic-high density lipoprotein chelate and preparation method and application thereof Download PDFInfo
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- CN105021553A CN105021553A CN201510412253.4A CN201510412253A CN105021553A CN 105021553 A CN105021553 A CN 105021553A CN 201510412253 A CN201510412253 A CN 201510412253A CN 105021553 A CN105021553 A CN 105021553A
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Abstract
The invention provides an arsenic-high density lipoprotein chelate and its preparation method and application. The arsenic-high density lipoprotein chelate is formed by chelating arsenic ion and high density lipoprotein through a mercapto group or/and cysteine residues and can be used in preparation of a reagent for detecting the arsenic-high density lipoprotein chelate in human body. The invention has confirmed for the first time that arsenic ion can directly act on high density lipoprotein. A qualitative and quantitative detection method of the arsenic-high density lipoprotein chelate is established to detect the content of high density lipoprotein in bodies of people in a region so as to indirectly reflect the influence of the arsenic pollution level of a region on people's health. The arsenic-high density lipoprotein chelate quantitative detection method established in the invention has high accuracy and good repeatability.
Description
Technical field
The present invention relates to the immunology detection of heavy metal ion, be specifically related to a kind of arsenic-high-density lipoprotein (HDL) chelate 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), very low density lipoprotein (VLDL) (very lowdensity lipoprotein, VLDL), chylomicrons (chylomicron, CM).In blood, the cholesterol of about 30% is transported to liver by HDL and is converted into bile acid or is excreted by bile from histocyte.Angiography proves that HDL-C content and arterial lumen stenosis are significant negative correlation; therefore be called as " good cholesterol "; high-density lipoprotein (HDL) is a kind of antiatherogenic plasma lipoprotein; it is the protective factors of coronary heart disease; be commonly called as " blood vessel street cleaner "; its can inhibition cancer cell transfer diffusion, the prevention and control of cancer are played an important role.
But; research shows that the quantity of HDL-C can not be directly proportional to the degree of protection of angiocardiopathy, because the high-density lipoprotein (HDL) that wherein recurring structure has changed still can measure but lose normal function even can increase the weight of atherosclerotic progress.The quantity of HDL-C declines and then can significantly point out atherosclerotic protectiveness to weaken; thus HDL in quantitative serum is detected significant for coronary heart disease; not only may be used for the atherosclerotic danger of EARLY RECOGNITION, and can as one of index of the detection of lipid regulating drug therapeutic process and 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 cramp, 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 angiocardiopathy, causes maladjusted nervous system and cause diabetes and lung cancer, skin lung cancer, carcinoma of urinary bladder 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 dose 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, high-density lipoprotein (HDL) is a kind of protein of Special Significance, and whether arsenic can act on high-density lipoprotein (HDL), and high-density lipoprotein (HDL) structural change, content change, activity change, function are changed, 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-high-density lipoprotein (HDL) chelate and preparation method thereof, and set up the qualitative and quantitative analysis method of arsenic-high-density lipoprotein (HDL) chelate, quantitatively to detect the application of arsenic-high-density lipoprotein (HDL) chelate in the regional arsenic pollution level of evaluation one.By quantitatively detecting arsenic-high-density lipoprotein (HDL) chelate in regional crowd's serum, indirectly can reflect the situation that this regional crowd pollutes by arsenic, thus indirectly reflecting this regional arsenic pollution level.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of arsenic-high-density lipoprotein (HDL) chelate, this arsenic-high-density lipoprotein (HDL) chelate be arsenic ion and high-density lipoprotein (HDL) by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned arsenic-high-density lipoprotein (HDL) chelate, i.e. external synthetic method, comprises the following steps:
A) synthesis of arsenic-high-density lipoprotein (HDL): add arsenic ion and carry out chelatropic reaction purifying in the high-density lipoprotein (HDL) that comes from human body or the high-density lipoprotein (HDL) according to biological method restructuring, obtain reaction solution;
B) arsenic-high-density lipoprotein (HDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and arsenic ion in reaction solution, obtain arsenic-high-density lipoprotein (HDL) chelate.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-high-density lipoprotein (HDL) chelate of extraction be dissolved in physiological saline, obtain arsenic-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
Described can with the filler of high-density lipoprotein (HDL) specific binding be adsorbed with can with the silica gel of high-density lipoprotein (HDL) specific binding material or resin;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make high-density lipoprotein (HDL) and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
Described can with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-high-density lipoprotein (HDL) chelate.
As preferably, the preparation method of above-mentioned arsenic-high-density lipoprotein (HDL) chelate, also comprises the following authentication step to arsenic-high-density lipoprotein (HDL) chelate, specifically comprises the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-high-density lipoprotein (HDL) chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: 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 to detect the content whether containing arsenic and detection arsenic.
The present invention also provides the application of a kind of arsenic described above-high-density lipoprotein (HDL) chelate in preparation human body in the reagent of arsenic-high-density lipoprotein (HDL) chelate or kit.
The present invention also provides a kind of and at least comprises the kit of arsenic described above-high-density lipoprotein (HDL) chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching high-density lipoprotein (HDL) or the material of catching arsenic.
The present invention also provides a kind of method of quantitative detection arsenic-high-density lipoprotein (HDL) chelate, using the above-mentioned arsenic-high-density lipoprotein (HDL) chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and enzyme linked immunological combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and atomic absorption spectrum combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Compared to existing technology, beneficial effect of the present invention is:
1. the present invention has synthesized arsenic-high-density lipoprotein (HDL) chelate first;
2. the present invention proposes arsenic-high-density lipoprotein (HDL) chelate first and can be used for preparing application in the reagent or kit detecting arsenic-high-density lipoprotein (HDL) chelate in blood sample;
3. present invention achieves-the specific recognition of high-density lipoprotein (HDL) chelate and quantitatively detecting, so that the content of arsenic-high-density lipoprotein (HDL) chelate in quantitative detection 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-high-density lipoprotein (HDL) chelate quantitative detecting method that the present invention sets up is high, reproducible.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-high-density lipoprotein (HDL) chelate;
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoresis strip of arsenic of the present invention-high-density lipoprotein (HDL) chelate.
Embodiment
Below in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention; Agents useful for same, material, as non-specified otherwise, are all considered as to buy from commercial mode:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
The material of catching high-density lipoprotein (HDL) is anti-high-density lipoprotein (HDL) antibody, by commercially available acquisition, as the anti-HDL antibody of " Abcam ab157595 " that Ai Bokang (Shanghai) trade Co., Ltd sells, in following embodiment, be describedly the material of catching high-density lipoprotein (HDL) with the material of high-density lipoprotein (HDL) specific binding, anti-high-density lipoprotein (HDL) antibody, anti-HDL antibody;
Enzyme labelled antibody is the one in the antibody containing the enzyme labeling such as horseradish peroxidase, alkaline phosphatase;
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example: the Na getting 1.5g
2cO
3with the NaHCO of 2.93g
3dissolving adds ddH
2o is settled to 1000mL;
Lavation buffer solution is the 0.15M PBS solution of pH7.4, compound method example: the KH getting 0.2g
2pO
4, 2.90g Na
2hPO
412H
2kCl, 0.5mL Tween-20 of NaCl, 0.2g of O, 8.0g, dissolves and adds ddH
2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin(BSA), adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2M H
2sO
4, compound method example: the ddH getting 178.3mL
2o, enriching H
2sO
4be settled to 200mL;
Substrate is methyl biphenyl amine (TMB) solution, compound method example: get the methyl biphenyl amine ethanolic solution that 0.5mL concentration is 2g/L, add substrate dilution and be diluted to 10mL;
Substrate buffer solution pH is 5.0, wherein Na
2hPO
4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, compound method example: get 1.42gNa
2hPO
4, 0.96g citric acid, then add ddH
2o to 50mL, to obtain final product;
The compound method example of eluent: papain pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/mL, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min, obtain eluent;
Sample-loading buffer can be formulated by the component of following ratio: Tris-HCl:1% bromophenol blue: ddH
2o: glycocoll=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
The compound method of electrophoretic buffer is as follows: get 3.0g Tris, 14.4g glycocoll, be dissolved in 800mLddH
2in O, after adjusting pH to 8.3, be settled to 1L;
The material of the catching arsenic mouse-anti As mAb that to be article No. be " Guangzhou Ran Ke company RK15728 "; 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-high-density lipoprotein (HDL) chelate, arsenic ion and high-density lipoprotein (HDL) by sulfydryl or/and cysteine residues chelating forms.
Particularly, arsenic-high-density lipoprotein (HDL) chelate is by the chelate of arsenic by combining in conjunction with at least one structure in zinc fingers, sulfydryl, cysteine residues and the apolipoprotein B in high-density lipoprotein (HDL) or cholesterol, triglyceride etc. and formed.
The present invention also provides the preparation method of arsenic-high-density lipoprotein (HDL) chelate, comprises the following steps:
A) synthesis of arsenic-high-density lipoprotein (HDL): add arsenic ion and carry out chelatropic reaction purifying in the high-density lipoprotein (HDL) that comes from human body or the high-density lipoprotein (HDL) according to biological method restructuring, obtain reaction solution;
B) arsenic-high-density lipoprotein (HDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and arsenic ion in reaction solution, obtain arsenic-high-density lipoprotein (HDL) chelate, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-high-density lipoprotein (HDL) chelate of extraction be dissolved in physiological saline, obtain arsenic-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
Described can with the filler of high-density lipoprotein (HDL) specific binding be adsorbed with can with the silica gel of high-density lipoprotein (HDL) specific binding material or resin;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make high-density lipoprotein (HDL) and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
Described can with the filler of arsenic specific binding be adsorbed with can with the silica gel of arsenic specific binding material or resin;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-high-density lipoprotein (HDL) chelate.
C): to the qualification of arsenic-high-density lipoprotein (HDL) chelate, specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-high-density lipoprotein (HDL) chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: 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 to detect the content whether containing arsenic and detection arsenic.
The present invention also provides a kind of and at least comprises the kit of arsenic described above-high-density lipoprotein (HDL) chelate as standard items.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
A kind of kit detecting arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise: containing the coating buffer that can be used for catching high-density lipoprotein (HDL), 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 kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprising: containing the coating buffer that can be used for catching high-density lipoprotein (HDL), also comprise confining liquid, lavation buffer solution, eluent, positive control, negative control etc.
Detect a kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprising: containing the coating buffer that can be used for catching high-density lipoprotein (HDL), also comprise confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, negative control etc.
Detect a kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise extract high-density lipoprotein (HDL) required extract reagent, redissolve liquid, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, standard items, negative control etc. of material that can be used for catching arsenic.
Detect a kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise and extract high-density lipoprotein (HDL) required extraction reagent, redissolution liquid, positive control, negative control etc.
Detect a kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise and extract high-density lipoprotein (HDL) required extraction reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc.
Detect a kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise and extract high-density lipoprotein (HDL) 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 kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise and extract high-density lipoprotein (HDL) 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 kit for arsenic in blood sample-high-density lipoprotein (HDL) chelate, comprise and extract high-density lipoprotein (HDL) 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 kit, described positive control is standard items, is namely chelated with the high-density lipoprotein (HDL) chelate of arsenic or is chelated with the BSA chelate of arsenic; Described negative control is the dilution eluent not containing standard items.
Mentioned reagent box, for detecting arsenic-high-density lipoprotein (HDL) chelate, to improve accuracy and the repeatability of detection, and makes it to be promoted in clinical.
The present invention also provides a kind of method of quantitative detection arsenic-high-density lipoprotein (HDL) chelate, using the above-mentioned arsenic-high-density lipoprotein (HDL) chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and enzyme linked immunological combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and atomic absorption spectrum combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, following several with having of detecting that the method for arsenic-high-density lipoprotein (HDL) chelate can list, but be not limited to following several.
The following stated dilution multiple proportions is w/v.
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects arsenic-high-density lipoprotein (HDL) chelate, detects in accordance with the following steps:
1) bag quilt: can catch the material of high-density lipoprotein (HDL) to 2000-16000 times with dilution buffer dilution, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with lavation buffer solution for 37 DEG C, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: from circulation system sampling, make sample to be tested; Standard items are made with the arsenic of known content-high-density lipoprotein (HDL) chelate; By dilution buffer, sample to be tested and standard items are all diluted to 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching arsenic is added, and incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, add with dilution buffer dilution 25000-200000 anti-arsenic antibody doubly, 37 DEG C of effect 1-2 hour, make the arsenic in anti-arsenic antibody and high-density lipoprotein (HDL) 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 sample to be tested, and wash with lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation 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 items, drawing standard curve, by comparing with standard items group, trying to achieve the content of sample to be tested.
In this method, step 8) also can not use microplate reader, directly carry out qualitative detection by staining conditions.
The method utilizes ELISA principle, specificity high-density lipoprotein (HDL) in whole blood can be extracted, the high-density lipoprotein (HDL) upper part extracted is chelated with arsenic, and arsenic in this part high-density lipoprotein (HDL) can catch by the specific antibody of anti-arsenic, afterwards can again by horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the high-density lipoprotein (HDL) not containing chelating arsenic, then can not catch by the specific antibody of anti-arsenic, also can not with horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase caught, and also not containing arsenic (negative control group result is negative) in agents useful for same, thus when read OD value result is shown as the positive, the i.e. provable arsenic detecting chelating in high-density lipoprotein (HDL).
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect arsenic-high-density lipoprotein (HDL) chelate and detect in accordance with the following steps:
1) bag quilt: the material that high-density lipoprotein (HDL) can be caught, as anti-high-density lipoprotein (HDL) antibody (anti-high-density lipoprotein (HDL) antibody) is coated on solid phase carrier, anti-high-density lipoprotein (HDL) antibody is diluted to 2000-16000 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 circulation system, make sample to be tested; Standard items are made with the arsenic of known content-high-density lipoprotein (HDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds the eluent that extension rate is 5-80, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the arsenic of chelating in high-density lipoprotein (HDL) in Atomic Absorption Spectrometer, read respective value;
This embodiment utilizes ELISA principle to catch the high-density lipoprotein (HDL) in serum, and detects the arsenic of chelating in high-density lipoprotein (HDL) in conjunction with atomic absorption spectrum (AAS) instrument; Owing to only containing high-density lipoprotein (HDL) 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 in high-density lipoprotein (HDL).
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect arsenic-high-density lipoprotein (HDL) chelate and detect in accordance with the following steps:
1) bag quilt: the material that high-density lipoprotein (HDL) can be caught, as anti-high-density lipoprotein (HDL) antibody is coated on solid phase carrier, anti-high-density lipoprotein (HDL) antibody is diluted to 2000-16000 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 circulation system, make sample to be tested; Standard items are made with the arsenic of known content-high-density lipoprotein (HDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add dilution 5-80 eluent doubly, wash-out 1-3 hour at 37-57 DEG C;
5) acidifying: in step 4) in solution in add acidulant acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in the arsenic of high-density lipoprotein (HDL), 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 in high-density lipoprotein (HDL) with icp ms; Namely first adopt ELISA principle to be extracted by the arsenic in serum-high-density lipoprotein (HDL) chelate, then adopt sense couple plasma mass spectrometer quantitatively to detect the arsenic of chelating in high-density lipoprotein (HDL); Owing to only containing high-density lipoprotein (HDL) 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 in high-density lipoprotein (HDL).
method four:purification arsenic-high-density lipoprotein (HDL) chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detect arsenic-high-density lipoprotein (HDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, high-density lipoprotein (HDL) is extracted from whole blood, and the high-density lipoprotein (HDL) extracted is redissolved, obtain the solution of high-density lipoprotein (HDL);
2) bag quilt: be coated on solid phase carrier by anti-arsenic antibody, dilutes anti-arsenic antibody to 25000-200000 times by dilution buffer, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
3) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add sample to be tested, and incubation: from step 1) solution sample, make sample to be tested; Standard items are made with the arsenic of known content-high-density lipoprotein (HDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: the redissolution liquid removing high-density lipoprotein (HDL), and wash with lavation buffer solution, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove sample to be tested, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation 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 items respectively, drawing standard curve, by comparing with standard items group, trying to achieve the content of sample to be tested.
In this method, also can not use microplate reader, directly carry out qualitative detection by staining conditions.
method five:purification arsenic-high-density lipoprotein (HDL) chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detect arsenic in blood sample-high-density lipoprotein (HDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, high-density lipoprotein (HDL) is extracted from whole blood, and the high-density lipoprotein (HDL) extracted is redissolved, obtain the solution of high-density lipoprotein (HDL);
2) detect: from step 1) redissolution liquid sample, detect the arsenic of chelating in high-density lipoprotein (HDL) in Atomic Absorption Spectrometer, read respective value.
method six:purification arsenic-high-density lipoprotein (HDL) chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect arsenic-high-density lipoprotein (HDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, high-density lipoprotein (HDL) is extracted from whole blood, and the high-density lipoprotein (HDL) extracted is redissolved, obtain the solution of high-density lipoprotein (HDL);
2) acidifying: from step 1) redissolution liquid 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 in high-density lipoprotein (HDL) under icp ms, read respective value.
Method four, method five and method six are all isolate high-density lipoprotein (HDL) by whole blood extraction method, then adopt method for detecting specificity, measure the content of arsenic on arsenic-high-density lipoprotein (HDL) chelate in high-density lipoprotein (HDL); Namely first physical separation means are adopted, as supercentrifugation, HPLC, gel-filtration chromatography etc., high-density lipoprotein (HDL) separated from test plasma sample 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-high-density lipoprotein (HDL) chelate.
Method seven: electrophoresis+ELISA/AAS/ICP-MS method detects arsenic-high-density lipoprotein (HDL) chelate, specific as follows:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, high-density lipoprotein (HDL) is extracted from whole blood, the high-density lipoprotein (HDL) extracted is redissolved in physiological saline, obtains the redissolution liquid of high-density lipoprotein (HDL);
2) glue bed is prepared: select suitable medium (as Ago-Gel, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) redissolution liquid get 8 μ L solution, make standard items with the arsenic of known content-high-density lipoprotein (HDL) chelate, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, with electrophoretic buffer, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of isoelectric point;
5) detect: on glue bed, find out the protein band containing 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 isoelectric point of the method detection arsenic-high-density lipoprotein (HDL) chelate, molecular weight and content etc. can also be utilized.
In method seven, high-density lipoprotein (HDL) is extracted from whole blood, then adopt gel electrophoresis to be separated extracted high-density lipoprotein (HDL), then find out the respective strap being rich in arsenic, then detect the content of relevant high-density lipoprotein (HDL), namely after high-density lipoprotein (HDL) discharges from red blood cell, can by multiple Methods For Purification out (such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the high-density lipoprotein (HDL) of purifying out is redissolved in solution, get a certain amount of high-density lipoprotein (HDL), utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different medium can be adopted as required), the difference such as isoelectric point runs out of different bands, find out the respective strap being rich in arsenic, the corresponding double solvents of protein in gel is redissolved in solution, namely the content of relevant high-density lipoprotein (HDL) 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 high-density lipoprotein (HDL), owing to only containing high-density lipoprotein (HDL) 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 in high-density lipoprotein (HDL).
In method four, method five, method six and method seven, the example extracting non-specific high-density lipoprotein (HDL) from whole blood is as follows:
A) get whole blood, with NaBr regulating density to 1.21g/mL, 50000rpm ultracentrifugation 24h, collect the light yellow component of lower floor;
B) be configured to neutral salt three kinds of solution that density is respectively d=1.006, d=1.182, d=1.478;
C) get 4mL step a) blood plasma to capacity be the bottom in the ultracentrifugation pipe of 10mL, above bedding 2mLd=1.006 salt solusion, 4 DEG C of centrifugal 30min of 2000rap/min, collect lower floor 4mL pooled plasma stand-by;
D) to step c) 4mL pooled plasma adds 2mL1.006 salt solusion, and 18 DEG C of centrifugal 17h of 40000rap/min, get upper strata 1mL, i.e. very low density lipoprotein (VLDL);
E) discard the liquid on 1mL upper strata, transfer residual mixed liquor, adds 2mLd=1.182 salt solusion, mixing, and 18 DEG C of centrifugal 21h of 40000rpm/min take out upper strata 1mL mixed liquor, and this i.e. low-density lipoprotein;
F) discard the liquid on 1mL upper strata, transfer residual mixed liquor, add 2mL1.478 salt solusion, 18 DEG C of centrifugal 41h of 40kr/min, get upper solution, i.e. high-density lipoprotein (HDL);
Described neutral salt is one or more in NaBr, KBr, NaCl or KCl.
embodiment 1:synthetic method synthesis arsenic-high-density lipoprotein (HDL) huge legendary turtle compound, namely comprises the following steps:
Arsenic prepared by the present embodiment-high-density lipoprotein (HDL) 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-high-density lipoprotein (HDL) chelate, 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 liquor, 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) high-density lipoprotein (HDL) solution: take 4.0mg high-density lipoprotein (HDL) and be dissolved in 4.0mL0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, be mixed with the high-density lipoprotein (HDL) solution of 1.0mg/mL;
5) molecular cut off 14000 of bag filter, buys from Bioshop Inc;
The pre-service of bag filter: EDTA-NaHCO bag filter being put into 500mL
3in mixed liquor, 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 bag filter 4 DEG C and spends the night.During use, put on one's gloves, take out bag filter, with a large amount of ddH
2its surfaces externally and internally of O cleaning down;
The synthesis of A) arsenic-high-density lipoprotein (HDL) huge legendary turtle compound: add arsenic ion and carry out chelatropic reaction purifying in the high-density lipoprotein (HDL) that comes from human body or the high-density lipoprotein (HDL) according to biological method restructuring, obtain reaction solution, concrete operation step is as follows;
1) getting 2.0mg ITCBE is dissolved in 2mLDMSO;
2) liquid slowly step 1 prepared adds in high-density lipoprotein (HDL) solution, and dropping limit, limit is shaken, and in 25 DEG C, acts on 24h in the shaking table of 100r/min, then to dialyse 24h with bag filter, removes the ITCBE be not combined with high-density lipoprotein (HDL);
3) by the liquid 1mol/L HCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l 1mmol/L arsenic ion solns gradually, dropping limit, limit vibrates, in order to avoid arsenic ion makes albuminous degeneration precipitate;
4) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the bag filter handled well;
5) liquid of having dialysed is preserved in-20 DEG C of packing, obtain the reaction solution of arsenic-high-density lipoprotein (HDL) chelate.
B) arsenic-high-density lipoprotein (HDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and arsenic ion in reaction solution, obtain arsenic-high-density lipoprotein (HDL) chelate, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-high-density lipoprotein (HDL) chelate of extraction be dissolved in physiological saline, obtain arsenic-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make high-density lipoprotein (HDL) and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-high-density lipoprotein (HDL) chelate.
C) to the qualification of arsenic-high-density lipoprotein (HDL) huge legendary turtle compound, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using Ago-Gel as medium;
(2) application of sample: get step B) in arsenic-high-density lipoprotein (HDL) chelate of obtaining of extraction purification, redissolve in physiological saline, obtain the redissolution liquid of arsenic-high-density lipoprotein (HDL) chelate, get the above-mentioned redissolution liquid 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 environment temperature is 4 degree; Stop electrophoresis when moving to bottom glue to bromophenol blue, Fig. 1 is the non denatured electrophoretic band figure of arsenic of the present invention-high-density lipoprotein (HDL) 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 to detect the content whether containing arsenic and detection arsenic.
D) qualification result
1) AAS testing result
Get step C) isolated protein band solution, with the content of arsenic in GFAAS (graphite furnace atomic absorption spectrometry) (AAS) Preliminary Determination high-density lipoprotein (HDL), as shown in the table, with NaCl solution, KBr solution, KCl solution for blank;
The content of arsenic in table 1 high-density lipoprotein (HDL)
| Sample name | As(μg/L) |
| Sample to be tested | 2.548 |
| 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 BEPC (BEPC).In storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA 4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx 3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoresis strip of arsenic of the present invention-high-density lipoprotein (HDL) chelate, and in figure, horizontal ordinate is protein band position, and ordinate is this band each arsenic energy (content) value.
the determination of testing conditions
1. the determination of the optimum diluting multiple of anti-high-density lipoprotein (HDL) antibody, anti-As antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for arsenic-high-density lipoprotein (HDL) chelate, specifically comprises the following steps:
1) bag quilt: anti-high-density lipoprotein (HDL) antibody protein is coated on solid phase carrier, respectively by dilution buffer by coating protein with the doubling dilution of 1:2000,1:4000,1:8000,1:16000, add in elisa plate micropore, each concentration bag, by three rows, is preserved 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) sample to be tested is added: by dilution buffer by the doubling dilution of test plasma sample by 1:10,1:20,1:40, add in micropore, make standard items with the arsenic of known content-high-density lipoprotein (HDL) chelate, 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 in itself and high-density lipoprotein (HDL) 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 sample to be tested, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer;
7) detect: the OD value reading standard items, test plasma, positive control, negative control and blank sample under 450nm wavelength in microplate reader respectively, drawing standard curve, tries to achieve the content of sample to be tested.
In the present embodiment, adopt arsenic provided by the invention-high-density lipoprotein (HDL) chelate standard items as positive control, respectively not add test plasma as negative control 1, namely add anti-high-density lipoprotein (HDL) antibody, confining liquid, anti-As antibody, enzyme mark and substrate successively;
Not add the control test group of anti-As antibody as negative control 2, namely add anti-high-density lipoprotein (HDL) antibody, confining liquid, test plasma, enzyme mark and substrate successively;
Anti-high-density lipoprotein (HDL) antibody, confining liquid, test plasma, anti-As antibody and substrate is added successively using not enzyme-added target control test group as negative control 3, namely;
Not add the control test group of sample to be tested and anti-As antibody as negative control 4 simultaneously, namely add anti-high-density lipoprotein (HDL) antibody, confining liquid, enzyme mark and substrate successively;
Not add the control test group of anti-high-density lipoprotein (HDL) antibody work for blank 1, namely add confining liquid, test plasma, anti-As antibody, enzyme mark and substrate;
And with the control test group only adding substrate for blank 2, only to add the control test group of PBS for blank 3.
Table 2 is the sample OD Value Data of different anti-high-density lipoprotein (HDL) antibody dilution multiple proportions, diluted plasma multiple proportions, anti-As antibody dilution multiple proportions,
Testing result under the different anti-high-density lipoprotein (HDL) antibody of table 2, anti-As antibody and diluted plasma multiple proportions
As known from Table 2, when the dilution multiple proportions of anti-high-density lipoprotein (HDL) antibody is 1:4000, sample OD value is greater than the dilution multiple proportions of other the anti-high-density lipoprotein (HDL) antibody under parallel condition; In this group sample, when diluted plasma multiple proportions is 1:20, when anti-As antibody dilution multiple proportions is 1:25000, OD value is maximum, is 0.752.
The OD detected value of positive control, negative control and blank when the dilution multiple proportions that table 3 is anti-high-density lipoprotein (HDL) antibody is 1:4000, diluted plasma multiple proportions is 1:20, anti-As antibody dilution multiple proportions is 1:25000,
The testing result of table 3 positive control, negative control and blank
As known from Table 3, test under the determined condition of table 2, negative control group OD detected value is less than 0.1, and under this optimal conditions is described, the systematic error of this method is little, meets analytical approach requirement, so select concentration corresponding to this value as best effort concentration.
2.ELISA eluent best effort concentration and the determination of time
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-As antibody and enzyme labelled antibody incubation, carry out wash-out with the eluent of variable concentrations, then detect OD value by microplate reader, concrete steps are as follows:
(1) bag quilt: be coated on solid phase carrier by anti-As antibody, press the doubling dilution of 1:25000 by dilution buffer, adds in elisa plate micropore, preserves 16 hours for 4 DEG C;
(2) close: remove dilution buffer, after washing, add confining liquid, place 1 hour, remove confining liquid, and wash for 37 DEG C;
(3) add enzyme labelled antibody: remove confining liquid, after washing, add and be diluted to by dilution buffer the HRP enzyme labelled antibody that antibody concentration is 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, eluent is diluted, make the concentration of papain in eluent: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove eluent, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer cessation reaction;
(5) under the determined wavelength of 450nm, in microplate reader, read the OD value of each micropore respectively, concrete outcome see table 4,
Testing result under the different eluent extension rate of table 4
By comparing OD value, to judge anti-As antibody-hrp-antibody complex wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-As antibody-hrp-antibody complex wash-out degree reaches maximum.As shown in table 4, the concentration when papain in eluent: in enzyme labelled antibody during the concentration=1:20 of antibody, 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 concentration is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of eluent be that 1-3h all can, in sum, we select eluent 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
Application Example
application Example 1
Whole blood extraction method is adopted to extract arsenic-high-density lipoprotein (HDL) chelate, employing euzymelinked immunosorbent assay (ELISA) (ELISA method) detects the arsenic-high-density lipoprotein (HDL) chelate in 100 parts of sample blood plasma, namely the method adopting specific embodiment method one to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by anti-high-density lipoprotein (HDL) antibody, be diluted to 4000 times by dilution buffer, adds in elisa plate micropore, preserves 4 DEG C of storages in refrigerator after 1 hour for 37 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, and place 1 hour, remove confining liquid for 37 DEG C, washing, elisa plate 37 DEG C places 4 DEG C of storages in refrigerator after 1 hour;
3) application of sample: using sample blood plasma as sample to be tested, makes standard items with the arsenic of known content-high-density lipoprotein (HDL) chelate, with dilution buffer dilution, sample to be tested and standard items is all diluted to 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 in itself and high-density lipoprotein (HDL) 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 sample to be tested, 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 items under 450nm wavelength in microplate reader respectively, cross and compare with standard items group, try to achieve the content of arsenic in sample to be tested according to standard equation, result is as shown in table 5.
The measured result of table 5 method a pair 100 parts of plasma samples
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD | 0.402 | 0.285 | 0.336 | 0.259 | 0.587 | 0.241 | 0.284 | 0.596 | 0.575 | 0.669 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| OD | 0.254 | 0.635 | 0.338 | 0.39 | 0.408 | 0.38 | 0.384 | 0.277 | 0.29 | 0.579 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| OD | 0.638 | 0.671 | 0.572 | 0.426 | 0.327 | 0.641 | 0.58 | 0.384 | 0.587 | 0.201 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| OD | 0.694 | 0.598 | 0.372 | 0.258 | 0.595 | 0.306 | 0.463 | 0.377 | 0.309 | 0.32 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| OD | 0.463 | 0.543 | 0.421 | 0.45 | 0.465 | 0.485 | 0.207 | 0.649 | 0.668 | 0.567 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| OD | 0.439 | 0.565 | 0.629 | 0.553 | 0.58 | 0.656 | 0.313 | 0.402 | 0.452 | 0.415 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| OD | 0.269 | 0.393 | 0.262 | 0.649 | 0.289 | 0.687 | 0.389 | 0.398 | 0.347 | 0.542 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| OD | 0.448 | 0.624 | 0.338 | 0.527 | 0.351 | 0.215 | 0.331 | 0.353 | 0.458 | 0.386 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| OD | 0.225 | 0.544 | 0.24 | 0.394 | 0.538 | 0.605 | 0.608 | 0.372 | 0.385 | 0.544 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| OD | 0.693 | 0.535 | 0.291 | 0.48 | 0.677 | 0.39 | 0.582 | 0.596 | 0.442 | 0.648 |
In this application embodiment 1, step 7) in, microplate reader also can not be used to detect, but directly carry out qualitative detection by dyeing.
application Example 2
Adopt enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) to detect arsenic-high-density lipoprotein (HDL) chelate in 100 parts of sample blood plasma, the method namely adopting specific embodiment method two to record detects, and concrete operation step is as follows:
1) bag quilt: anti-high-density lipoprotein (HDL) antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 4000 times, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, preserve at elisa plate 4 DEG C;
3) application of sample: make sample to be tested with sample blood plasma, makes standard items with the arsenic of known content-high-density lipoprotein (HDL) chelate, by dilution buffer, sample to be tested and standard items is diluted to 20 times, add in micropore, 37 DEG C of effect 1-2 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 in high-density lipoprotein (HDL) in Atomic Absorption Spectrometer, result is as shown in table 6.
Table 6 method two is to the measured result of 100 parts of plasma samples
application Example 3:
Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) is adopted to detect arsenic-high-density lipoprotein (HDL) chelate in 100 parts of sample blood plasma, namely the method adopting specific embodiment method three to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by anti-high-density lipoprotein (HDL) antibody, with dilution buffer dilution coating protein to 4000 times, adds in elisa plate micropore, preserves 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, elisa plate 4 DEG C preservation;
3) application of sample: make sample to be tested with sample blood plasma, makes standard items with the arsenic of known content-high-density lipoprotein (HDL) chelate, by dilution buffer, sample to be tested and standard items 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 items, washing, 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 in high-density lipoprotein (HDL) under icp ms, result is as shown in table 7.
Table 7 method three is to the measured result of 100 parts of samples
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μg/L | 2.951 | 1.816 | 1.251 | 2.084 | 1.427 | 2.276 | 2.144 | 2.109 | 2.179 | 1.718 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 1.438 | 1.809 | 2.643 | 1.29 | 2.896 | 1.633 | 1.912 | 2.185 | 1.546 | 2.647 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 1.917 | 1.363 | 1.73 | 2.671 | 1.486 | 2.283 | 2.875 | 2.634 | 1.548 | 2.271 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 2.832 | 1.2 | 2.414 | 1.662 | 2.451 | 2.189 | 2.122 | 1.637 | 1.34 | 1.888 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 2.776 | 1.82 | 2.456 | 2.678 | 1.243 | 1.822 | 2.05 | 1.879 | 2.662 | 2.937 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 2.706 | 2.846 | 2.238 | 2.752 | 2.071 | 1.327 | 1.251 | 2.112 | 2.676 | 2.417 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 1.979 | 1.604 | 2.968 | 1.204 | 2.317 | 2.812 | 1.887 | 1.807 | 1.471 | 1.957 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 1.64 | 1.778 | 1.637 | 2.377 | 1.524 | 1.657 | 1.482 | 2.684 | 2.201 | 2.578 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 2.977 | 2.983 | 1.643 | 1.735 | 2.856 | 1.971 | 2.28 | 2.418 | 1.424 | 2.913 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 2.705 | 2.162 | 1.805 | 2.115 | 1.779 | 1.574 | 1.257 | 2.816 | 1.921 | 1.802 |
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-high-density lipoprotein (HDL) chelate, is characterized in that, this arsenic-high-density lipoprotein (HDL) chelate be arsenic ion and high-density lipoprotein (HDL) by sulfydryl or/and cysteine residues chelating forms.
2. the preparation method of arsenic-high-density lipoprotein (HDL) chelate as claimed in claim 1, comprises the following steps:
A) synthesis of arsenic-high-density lipoprotein (HDL): add arsenic ion and carry out chelatropic reaction purifying in the high-density lipoprotein (HDL) that comes from human body or the high-density lipoprotein (HDL) according to biological method restructuring, obtain reaction solution;
B) arsenic-high-density lipoprotein (HDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and arsenic ion in reaction solution, obtain arsenic-high-density lipoprotein (HDL) chelate.
3. method as claimed in claim 2, is characterized in that,
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) the arsenic-high-density lipoprotein (HDL) chelate of extraction be dissolved in physiological saline, obtain arsenic-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with dilution buffer dilution step (1) described solution, then upper prop, make high-density lipoprotein (HDL) and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the eluent collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the eluent of the collection in step (5) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of arsenic specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the eluent collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the eluent of step (10) dress bag filter, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain arsenic-high-density lipoprotein (HDL) chelate.
4. the preparation method of arsenic according to claim 2-high-density lipoprotein (HDL) chelate, is characterized in that, step B) after also comprise step C): to the qualification of arsenic-high-density lipoprotein (HDL) chelate;
Wherein, step C) in specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in arsenic-high-density lipoprotein (HDL) chelate of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: 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 to detect the content whether containing arsenic and detection arsenic.
5. the application of arsenic as claimed in claim 1-high-density lipoprotein (HDL) chelate in the reagent or kit preparing to detect arsenic-high-density lipoprotein (HDL) chelate in blood sample.
6. one kind at least comprises the kit of arsenic as claimed in claim 1-high-density lipoprotein (HDL) chelate as standard items.
7. kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching high-density lipoprotein (HDL) or the material of catching arsenic.
8. one kind is quantitatively detected the method for arsenic-high-density lipoprotein (HDL) chelate, it is characterized in that, using the arsenic according to claim 1-high-density lipoprotein (HDL) chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and enzyme linked immunological combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and atomic absorption spectrum combined techniques, purification arsenic-high-density lipoprotein (HDL) chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
| WO2006123343A2 (en) * | 2005-05-18 | 2006-11-23 | Ramot At Tel Aviv University Ltd. | Biologically active metal-coated proteins |
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