CN105044009A - Chromium-very low density lipoprotein chelate and preparation method and application thereof - Google Patents
Chromium-very low density lipoprotein chelate and preparation method and application thereof Download PDFInfo
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- CN105044009A CN105044009A CN201510413275.2A CN201510413275A CN105044009A CN 105044009 A CN105044009 A CN 105044009A CN 201510413275 A CN201510413275 A CN 201510413275A CN 105044009 A CN105044009 A CN 105044009A
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- chromium
- density lipoprotein
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a chromium-very low density lipoprotein chelate and a preparation method and application thereof, wherein the chromium-very low density lipoprotein chelate is formed by chelating chromium ions and very low density lipoprotein through sulfydryl or/and cysteine residues, and can be used for preparing a reagent for detecting the chromium-very low density lipoprotein chelate of a human body. The invention proves that the chromium ions can directly act on the very low density lipoprotein for the first time. The invention establishes a qualitative and quantitative detection method of chromium-very low density lipoprotein chelate to detect the content of very low density lipoprotein in a population in a region, thereby indirectly reflecting the chromium pollution degree in the region and the influence on the health of the population. The quantitative detection method of the chromium-very low density lipoprotein chelate established by the invention has high accuracy and repeatability 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 chromium-very low density lipoprotein (VLDL) 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) (highdensitylipoprotein is divided into by density, be called for short HDL), intermediated-density lipoprotein (intermediatedensitylipoprotein, be called for short IDL), low-density lipoprotein (lowdensitylipoprotein, be called for short LDL), very low density lipoprotein (VLDL) (verylowdensitylipoprotein, be called for short VLDL), chylomicrons (chylomicron, CM).In blood, about cholesterol of 30 percent is transported to liver by HDL and is converted into bile acid or is excreted by bile from histocyte.In blood, the cholesterol of about 70% is entrained by LDL and VLDL, and the endogenous triglyceride (triglyceride, TG) produced in liver also mainly relies on VLDL to be transported to outside liver.Increasing with hyperlipidemia, disorders of lipid metabolism of VLDL is closely bound up, and dyslipidemia is the important risk factor that various diseases occurs, thus stable stable the playing an important role for human body each side function function of VLDL contents level.
VLDL contents level and structure function stable significant to body.Its too high levels and fatty liver, disorders of lipid metabolism, angiocardiopathy, renal function damage etc. are closely bound up.Quantitative detection VLDL may be used for the early diagnosis of these diseases and result for the treatment of monitoring, even as one of the evaluation index of prognosis quality.And for patients such as malnutrition, chronic anaemia, liver function damage persons, serum VLDL content also can decline, quantitatively detect VLDL and 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.
Chromium (chromium, Cr) be a kind of transition metal in the periodic table of elements VI race, its quality is hard, surface has gloss, there is higher fusing point, there is great economic worth in the industrial production, be widely used since the century, annual global chromium turnout is up to 107 tons, and thus chromium is a kind of genuine important meals pollutant.At present, chromium is widely used in the various aspects such as plating, tanning, pigment, paint, alloy, printing and dyeing, closely bound up with the life of people, but equally also has an immense impact on to the healthy of the mankind.
Occurring in nature chromium mainly exists with the form of trivalent and sexavalence, and it is generally acknowledged, trivalent chromium (Cr
3+) be the trace element of necessary for human, and sexavalent chrome (Cr
6+) be only obvious toxicant.Because sexavalent chrome easily enters cell by cell membrane, and can series reaction be passed through, produce strong cytotoxicity and genotoxicity, thus generally believe that chromic toxicity is higher than trivalent chromium, reaches 10-100 times.Scholar thinks when namely the content of 6-valence Cr ions in water is poisonous more than 0.05mg/L.Sexavalent chrome can be drunk by skin contact, food intake, potable water and enter human body into approach such as, respiratory tract suctions, mainly sucking sexavalent chrome dust by respiratory tract for occupational exposed population group, is then take in sexavalent chrome by drinking into polluted source, the modes such as contaminated food, suction of eating for general population.Sexavalent chrome enters after in body, can initiated oxidation stress, developing body distortion, Apoptosis, multiple DNA damage (comprising single-strand break, DNA-protein cross, DNA-amino acid crosslinks, the formation of Cr-DNA complex etc.), thus cause body many places tissue, organ, system injury.
As previously mentioned, in close relations between chromium and multiple proteins, can be combined with multiple proteins, suppress the activity of multiple protein enzyme.Along with going deep into chromium toxicity research, people recognize that the interaction of chromium and albumen plays important role in its intoxicating process gradually, be considered to the key point of toxicity mechanism understanding chromium, therefore, the research of chromium and protein interaction just seemed ever more important.And the present mechanism about chromium and protein effect is only tip of the iceberg, also has the relation between a lot of protein and chromium not clear, thus strengthen for the relation between chromium and protein most important.
About chromium poisoning, the particularly evaluation of chronic chromium poisoning, can not circulation chromium content in accurate evaluation human body by detecting blood chromium content, the degree of injury of chromium for body function cannot be assessed further, and along with the develop rapidly of science and technology, the relation of chromium and human body is also day by day tight, and thus finding one can become more and more important from the evaluation of immunologic function angle chromium poisoning, particularly chronic chromium poisoning for the evaluation method of the degree of damage of body.
Summary of the invention
For the problem that pollution of chromium is serious, the object of the present invention is to provide a kind of chromium-very low density lipoprotein (VLDL) chelate and preparation method thereof, and set up the qualitative and quantitative analysis method of chromium-very low density lipoprotein (VLDL) chelate, quantitatively to detect the application of chromium-very low density lipoprotein (VLDL) chelate in the regional pollution of chromium degree of evaluation one.By quantitatively detecting chromium-very low density lipoprotein (VLDL) chelate in regional crowd's serum, indirectly can reflect the situation of this regional crowd by pollution of chromium, thus indirectly reflecting this regional pollution of chromium degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of chromium-very low density lipoprotein (VLDL) chelate, this chromium-very low density lipoprotein (VLDL) chelate be chromium ion and very low density lipoprotein (VLDL) by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned chromium-very low density lipoprotein (VLDL) chelate, i.e. external synthetic method, comprises the following steps:
The synthesis of A) chromium-very low density lipoprotein (VLDL) chelate: add chromium ion and carry out chelatropic reaction purifying in the very low density lipoprotein (VLDL) that comes from human body or the very low density lipoprotein (VLDL) according to biological method restructuring, obtain reaction solution;
B) chromium-very low density lipoprotein (VLDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted very low density lipoprotein (VLDL), specific antibody and chromium ion in reaction solution, obtain chromium-very low density lipoprotein (VLDL) chelate.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-very low density lipoprotein (VLDL) chelate be dissolved in physiological saline, obtain the solution of chromium-very low density lipoprotein (VLDL) chelate;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of very low density lipoprotein (VLDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with the solution of dilution buffer dilution step (1), then upper prop, make very low density lipoprotein (VLDL) 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, uses 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 chromium 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 in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-very low density lipoprotein (VLDL) chelate.
As preferably, the preparation method of above-mentioned chromium-very low density lipoprotein (VLDL) chelate, also comprises the following authentication step to chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) 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): on glue bed, find out the protein band containing chromium, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing chromium and detection chromium.
The present invention also provides the application of a kind of chromium described above-very low density lipoprotein (VLDL) chelate in preparation human body in the reagent of chromium-very low density lipoprotein (VLDL) chelate or kit.
The present invention also provides a kind of and at least comprises the kit of chromium described above-very low density lipoprotein (VLDL) chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer comprises the material of catching very low density lipoprotein (VLDL) or the material of catching crome metal.
The present invention also provides a kind of method of quantitative detection chromium-very low density lipoprotein (VLDL) chelate, using the above-mentioned chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate and enzyme linked immunological combined techniques, purification chromium-very low density lipoprotein (VLDL) chelate and atomic absorption spectrum combined techniques, purification chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate first;
2. the present invention proposes chromium-very low density lipoprotein (VLDL) chelate first and can be used for preparing application in the reagent or kit detecting chromium-very low density lipoprotein (VLDL) chelate in blood sample;
3. present invention achieves the specific recognition of chromium-very low density lipoprotein (VLDL) chelate and quantitatively detect, so that the content of chromium-very low density lipoprotein (VLDL) chelate in quantitative detection crowd serum, to evaluate the application of a regional pollution of chromium degree, for the pollution of chromium level of industrial area provides indirect indexes.The accuracy of chromium-very low density lipoprotein (VLDL) 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 chromium of the present invention-very low density lipoprotein (VLDL) chelate;
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium of the present invention-very low density lipoprotein (VLDL) 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 very low density lipoprotein (VLDL) is anti-very low density lipoprotein (VLDL) antibody, by commercially available acquisition, following examples mode, the anti-very low density lipoprotein (VLDL) antibody used is the VLDLantibody of " Genetexgtx16419 " for article No., is describedly the material of catching very low density lipoprotein (VLDL) with the material of very low density lipoprotein (VLDL) specific binding, anti-very low density lipoprotein (VLDL) antibody, anti-VLDL 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 pH9.6, 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 0.15MPBS solution of pH7.4, compound method example: the KH getting 0.2g
2pO
4, 2.90g Na
2hPO
412H
2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH
2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin(BSA), adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2MH
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 pH8.0,0.1mol/LTris-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 3.0gTris, 14.4g glycocoll, be dissolved in 800mLddH
2in O, after adjusting pH to 8.3, be settled to 1L;
The material of the catching chromium mouse-anti CrmAb that to be article No. be " Guangzhou Ran Ke company RK10641 ", being with the material of chromium specific binding, anti-Cr antibody, two anti-, anti-chromium antibody described in following embodiment catches to obtain the material of chromium;
The invention provides a kind of chromium-very low density lipoprotein (VLDL) chelate, chromium ion and very low density lipoprotein (VLDL) by sulfydryl or/and cysteine residues chelating forms.
Particularly, chromium-very low density lipoprotein (VLDL) chelate is by the chelate of chromium by combining in conjunction with at least one structure in zinc fingers, sulfydryl, cysteine residues and the apoC I in very low density lipoprotein (VLDL), CⅡ and CⅢ, ApoC3, apo E or cholesterol, triglyceride etc. and formed.
The present invention also provides the preparation method of chromium-very low density lipoprotein (VLDL) chelate, comprises the following steps:
A) synthesis of chromium-very low density lipoprotein (VLDL): add chromium ion and carry out chelatropic reaction purifying in the very low density lipoprotein (VLDL) that comes from human body or the very low density lipoprotein (VLDL) according to biological method restructuring, obtain reaction solution;
B) chromium-very low density lipoprotein (VLDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted very low density lipoprotein (VLDL), specific antibody and chromium ion in reaction solution, obtain chromium-very low density lipoprotein (VLDL) chelate, specifically comprise the following steps:
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-very low density lipoprotein (VLDL) chelate be dissolved in physiological saline, obtain the solution of chromium-very low density lipoprotein (VLDL) chelate;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of very low density lipoprotein (VLDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
Described can with the filler of very low density lipoprotein (VLDL) specific binding be adsorbed with can with the silica gel of very low density lipoprotein (VLDL) specific binding material or resin;
(3) loading: after chromatographic column balance, with the solution of dilution buffer dilution step (1), then upper prop, make very low density lipoprotein (VLDL) 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, uses 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 chromium specific binding, dress post after balance chromatographic column by dilution buffer again;
Described can with the filler of chromium specific binding be adsorbed with can with the silica gel of chromium 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 the collection in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-very low density lipoprotein (VLDL) chelate.
C): to the qualification of chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) 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 chromium, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing chromium and detection chromium.
The present invention also provides a kind of and at least comprises the kit of chromium described above-very low density lipoprotein (VLDL) chelate as standard items.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprising: containing can be used for catching very low density lipoprotein (VLDL) the coating buffer of material, confining liquid, lavation buffer solution, the material of chromium can be caught as two anti-, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative controls etc.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprising: containing the coating buffer, confining liquid, lavation buffer solution, eluent, positive control, negative control etc. of material that can be used for catching very low density lipoprotein (VLDL).
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprising: containing the coating buffer, confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, negative control etc. of material that can be used for catching very low density lipoprotein (VLDL).
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise extract very low density lipoprotein (VLDL) in whole blood requiredly 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 chromium.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise and extract very low density lipoprotein (VLDL) required extraction reagent, redissolution liquid, positive control, negative control etc. in whole blood.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise and extract very low density lipoprotein (VLDL) required extraction reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc. in whole blood.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise and extract very low density lipoprotein (VLDL) in whole blood and requiredly extract reagent, glue bed medium, to redissolve on liquid, sample loading buffer, dissolving glue bed containing liquid needed for the protein band of chromium, 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 chromium.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise and extract very low density lipoprotein (VLDL) in whole blood and requiredly 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 chromium.
Detect a kit for chromium in blood sample-very low density lipoprotein (VLDL) chelate, comprise and extract very low density lipoprotein (VLDL) in whole blood and requiredly 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 chromium.
In above-mentioned several kit, described positive control is standard items, is namely chelated with the very low density lipoprotein (VLDL) chelate of heavy metal chromium or is chelated with the BSA chelate of heavy metal chromium; Described negative control is dilution buffer.
Mentioned reagent box, for detecting chromium-very low density lipoprotein (VLDL) chelate, to put forward accuracy and the repeatability of extremely low detection, and makes it to be promoted in clinical.
The present invention also provides a kind of method of quantitative detection chromium-very low density lipoprotein (VLDL) chelate, using the above-mentioned chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate and enzyme linked immunological combined techniques, purification chromium-very low density lipoprotein (VLDL) chelate and atomic absorption spectrum combined techniques, purification chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate can list, but be not limited to following several.
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-very low density lipoprotein (VLDL) chelate, detects in accordance with the following steps:
1) bag quilt: can catch the material of very low density lipoprotein (VLDL) to 500-4000 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 chromium of known content-very low density lipoprotein (VLDL) chelate; All be diluted to 10-40 doubly with dilution buffer dilution sample to be tested and standard items, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching chromium is added, and incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, add with dilution buffer dilution 5000-40000 anti-chromium antibody doubly, 37 DEG C of effect 1-2 hour, make the crome metal in anti-chromium antibody and very low density lipoprotein (VLDL) react;
5) enzyme conjugates incubation: remove anti-chromium antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and HRP enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: stop buffer is 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 colour developing situation.
The method utilizes ELISA principle, chromium in very low density lipoprotein (VLDL) can catch by the specific antibody of chromium, 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 very low density lipoprotein (VLDL) not containing chelated mineral chromium, then can not catch by the specific antibody of anti-chromium, also can not with horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase caught, and also not containing crome metal (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 crome metal detecting chelating in very low density lipoprotein (VLDL).
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect chromium-very low density lipoprotein (VLDL) chelate and detect in accordance with the following steps:
1) bag quilt: the material that very low density lipoprotein (VLDL) can be caught, as anti-very low density lipoprotein (VLDL) antibody (anti-very low density lipoprotein (VLDL) antibody) is coated on solid phase carrier, anti-very low density lipoprotein (VLDL) antibody is diluted to 500-4000 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 chromium of known content-very low density lipoprotein (VLDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds eluent, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the chromium of chelating in very low density lipoprotein (VLDL) in Atomic Absorption Spectrometer, read respective value;
This embodiment utilizes ELISA principle to catch the very low density lipoprotein (VLDL) in serum, and detects the chromium of chelating in very low density lipoprotein (VLDL) in conjunction with atomic absorption spectrum (AAS) instrument; Owing to only containing very low density lipoprotein (VLDL) in solution, and not containing any heavy metal (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 crome metal detecting chelating in very low density lipoprotein (VLDL).
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect chromium-very low density lipoprotein (VLDL) chelate and detect in accordance with the following steps:
1) bag quilt: the material that very low density lipoprotein (VLDL) can be caught, as anti-very low density lipoprotein (VLDL) antibody is coated on solid phase carrier, anti-very low density lipoprotein (VLDL) antibody is diluted to 500-4000 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 chromium of known content-very low density lipoprotein (VLDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C;
5) acidifying: in step 4) in solution in add acidulant acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in the chromium of very low density lipoprotein (VLDL), read respective value.
The method, on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, detects the chromium of chelating in very low density lipoprotein (VLDL) with icp ms; Namely first adopt ELISA principle to be extracted by chromium-very low density lipoprotein (VLDL) chelate, then adopt sense couple plasma mass spectrometer quantitatively to detect the chromium of chelating in very low density lipoprotein (VLDL); Owing to only containing very low density lipoprotein (VLDL) in solution, and not containing any heavy metal (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 crome metal detecting chelating in very low density lipoprotein (VLDL).
method four:purification chromium-very low density lipoprotein (VLDL) chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detect chromium-very low density lipoprotein (VLDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific very low density lipoprotein (VLDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, very low density lipoprotein (VLDL) is extracted from whole blood, and the very low density lipoprotein (VLDL) extracted is redissolved, obtain the solution of very low density lipoprotein (VLDL);
2) bag quilt: be coated on solid phase carrier by anti-chromium antibody, dilutes anti-chromium antibody to 5000-40000 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 chromium of known content-very low density lipoprotein (VLDL) chelate; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, adds the enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: stop buffer is 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 colour developing situation.
method five:purification chromium-very low density lipoprotein (VLDL) chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detect chromium in blood sample-very low density lipoprotein (VLDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific very low density lipoprotein (VLDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, very low density lipoprotein (VLDL) is extracted from whole blood, and the very low density lipoprotein (VLDL) extracted is redissolved, obtain the solution of very low density lipoprotein (VLDL);
2) detect: from step 1) solution sample, detect the chromium of chelating in very low density lipoprotein (VLDL) in Atomic Absorption Spectrometer, read respective value.
method six:purification chromium-very low density lipoprotein (VLDL) chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect chromium-very low density lipoprotein (VLDL) chelate, detect in accordance with the following steps:
1) from whole blood, non-specific very low density lipoprotein (VLDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, very low density lipoprotein (VLDL) is extracted from whole blood, and the very low density lipoprotein (VLDL) extracted is redissolved, obtain the solution of very low density lipoprotein (VLDL);
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 chromium of chelating in very low density lipoprotein (VLDL) under icp ms, read respective value.
Method four, method five and method six are all isolate very low density lipoprotein (VLDL) by whole blood extraction method, then adopt method for detecting specificity, measure the content of chromium on chromium-very low density lipoprotein (VLDL) chelate in very low density lipoprotein (VLDL); Namely first physical separation means are adopted, as supercentrifugation, HPLC, gel-filtration chromatography etc., very low density lipoprotein (VLDL) separated from test plasma sample and redissolve in physiological saline, recycling ELISA principle, atomic absorption spectrum detect or carry out detecting the chromium content on inductively coupled plasma mass spectrometry detection chromium-very low density lipoprotein (VLDL) chelate.
Method seven: electrophoresis+ELISA/AAS/ICP-MS method detects chromium-very low density lipoprotein (VLDL) chelate, specific as follows:
1) from whole blood, non-specific very low density lipoprotein (VLDL) is extracted: adopt the methods such as supercentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, very low density lipoprotein (VLDL) is extracted from whole blood, the very low density lipoprotein (VLDL) extracted is redissolved in physiological saline, obtains the solution of very low density lipoprotein (VLDL);
2) glue bed is prepared: select suitable medium (as Ago-Gel, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) solution get 8 μ L solution, make standard items with the chromium of known content-very low density lipoprotein (VLDL) 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 chromium, this band is taken out, this protein band is dissolved, and then utilize the principle such as ELISA or ICP-MS or AAS to detect chromium content respectively.
In addition, the isoelectric point of the method detection chromium-very low density lipoprotein (VLDL) chelate, molecular weight and content etc. can also be utilized.
In method seven, very low density lipoprotein (VLDL) is extracted, then adopt gel electrophoresis to be separated extracted very low density lipoprotein (VLDL), then find out the respective strap being rich in chromium, then detect the content of relevant pole low-density lipoprotein, namely after very low density lipoprotein (VLDL) 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 very low density lipoprotein (VLDL) of purifying out is redissolved in solution, get a certain amount of very low density lipoprotein (VLDL), 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 chromium, protein in gel is redissolved in solution, namely the content of relevant pole low-density lipoprotein can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the chromium content of chelating in very low density lipoprotein (VLDL), owing to only containing very low density lipoprotein (VLDL) in solution, and not containing any heavy metal (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 crome metal detecting chelating in very low density lipoprotein (VLDL).
embodiment 1:synthetic method synthesis chromium-very low density lipoprotein (VLDL) huge legendary turtle compound, namely comprises the following steps:
Chromium prepared by the present embodiment-very low density lipoprotein (VLDL) huge legendary turtle compound, is separated further by gel electrophoresis, and carries out detection Qualitative Identification by inductivity coupled plasma mass spectrometry or atomic absorption spectrum.
The reagent that the present embodiment uses is as follows:
1) borate buffer solution, its volumetric molar concentration is 0.01M, and its preparation method example is as follows: take 0.31g boric acid and be dissolved in 400mLddH
2in O, regulate pH to 9.0 with the NaOH of 0.1mol/L, be settled to 500mL.
2) EDTA-NaHCO
3solution, its preparation method is as follows: get 1.86gEDTA2H
2o and 16.8gNaHCO
3, be dissolved in 900mLddH
2in O, adjust pH to 8.0 with 1.0MNaOH and be settled to 1000mL, autoclaving, room temperature preservation;
3) ITCBE buys from Japanese colleague's chemistry institute, article No. M030;
4) very low density lipoprotein (VLDL) solution: take 4.0mg very low density lipoprotein (VLDL) and be dissolved in 4.0mL0.01MpH9.0 borate buffer solution, dissolving of fully vibrating, be mixed with the very low density lipoprotein (VLDL) solution of 1.0mg/mL;
5) molecular cut off 14000 of bag filter, buys from BioshopInc;
The pre-service of bag filter: EDTA-NaHCO bag filter being put into 500mL
3in solution, boil 10min; Tipping EDTA-NaHCO
3solution, uses ddH
2o is rinsing gently, then boils 10min with 500mL5mmol/LEDTA; 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) chromium-very low density lipoprotein (VLDL) huge legendary turtle compound: add chromium ion and carry out chelatropic reaction purifying in the very low density lipoprotein (VLDL) that comes from human body or the very low density lipoprotein (VLDL) according to biological method restructuring, obtain reaction solution, concrete operation step is as follows;
1) getting 2.0mgITCBE is dissolved in 2mLDMSO;
2) liquid slowly step 1 prepared adds in very low density lipoprotein (VLDL) 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 very low density lipoprotein (VLDL);
3) by the liquid 1mol/LHCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l1mmol/L chromium ion solution gradually, dropping limit, limit vibrates, in order to avoid chromium 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 chromium-very low density lipoprotein (VLDL) chelate.
B) chromium-very low density lipoprotein (VLDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted very low density lipoprotein (VLDL), specific antibody and chromium ion in reaction solution, obtain chromium-very low density lipoprotein (VLDL) chelate, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-very low density lipoprotein (VLDL) chelate be dissolved in physiological saline, obtain the solution of chromium-very low density lipoprotein (VLDL) chelate;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of very low density lipoprotein (VLDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with the solution of dilution buffer dilution step (1), then upper prop, make very low density lipoprotein (VLDL) 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 step (5), dress bag filter, uses 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 chromium 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, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-very low density lipoprotein (VLDL) chelate.
C) to the qualification of chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate of obtaining of extraction purification, redissolve in physiological saline, obtain the solution of chromium-very low density lipoprotein (VLDL) chelate, get 8 μ L previous solu, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer; In electrophoresis process, electric current is 22mA constant current, and environment temperature is 4 DEG C; Stop electrophoresis when moving to bottom glue to bromophenol blue, Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-very low density lipoprotein (VLDL) huge legendary turtle compound, and M swimming lane is sex change Marker, VLDL swimming lane is very low density lipoprotein (VLDL);
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing chromium and detection chromium.
D) qualification result
1) AAS testing result
Get step C) isolated protein band solution, with the content of heavy metal chromium in GFAAS (graphite furnace atomic absorption spectrometry) (AAS) Preliminary Determination very low density lipoprotein (VLDL), as shown in the table, with NaCl solution, KBr solution, KCl solution for blank;
The content of chromium in table 1 very low density lipoprotein (VLDL)
| Sample name | Cr(μg/L) |
| Sample to be tested | 7.489 |
| NaCl | 0.077 |
| KBr | 0.000 |
| KCl | 0.065 |
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 (PGTInc.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AXIL software data processing, and with deriving from air and the Ar signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium of the present invention-very low density lipoprotein (VLDL) chelate, and in figure, horizontal ordinate is protein band position, and ordinate is this band chromium metal energy (content) value.
the determination of testing conditions
1. the determination of the optimum diluting multiple of anti-very low density lipoprotein (VLDL) antibody, anti-Cr antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for chromium-very low density lipoprotein (VLDL) chelate, specifically comprises the following steps:
1) bag quilt: anti-very low density lipoprotein (VLDL) antibody protein is coated on solid phase carrier, respectively by dilution buffer by coating protein with the doubling dilution of 1:500,1:1000,1:2000 and 1:4000, 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, standard items are made with the chromium of known content-very low density lipoprotein (VLDL) chelate, negative control and blank are set, add in micropore, 37 DEG C act on 1 hour;
4) add two to resist: remove test plasma sample, washing, add with the anti-Cr antibody of dilution buffer by the doubling dilution of 1:5000,1:10000,1:20000,1:40000,37 DEG C act on 1 hour, the crome metal in itself and very low density lipoprotein (VLDL) are reacted;
5) enzyme-added mark: remove anti-Cr antibody, washing, adds and is diluted to by dilution buffer the HRP enzyme labelled antibody that antibody content is 2 μ g/mL, and 37 DEG C act on 1 hour, make itself and anti-Cr antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer;
7) detect: the OD value reading standard items, test plasma, positive control, negative control and blank sample under 450nm wavelength in microplate reader respectively.
In the present embodiment, adopt chromium provided by the invention-very low density lipoprotein (VLDL) chelate standard items as positive control, respectively not add test plasma as negative control 1, namely add anti-very low density lipoprotein (VLDL) antibody, confining liquid, anti-Cr antibody, enzyme mark and substrate successively;
Not add the control test group of anti-Cr antibody as negative control 2, namely add anti-very low density lipoprotein (VLDL) antibody, confining liquid, test plasma, enzyme mark and substrate successively;
Anti-very low density lipoprotein (VLDL) antibody, confining liquid, test plasma, anti-Cr 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-Cr antibody as negative control 4 simultaneously, namely add anti-very low density lipoprotein (VLDL) antibody, confining liquid, enzyme mark and substrate successively;
Not add the control test group of anti-very low density lipoprotein (VLDL) antibody work for blank 1, namely add confining liquid, test plasma, anti-Cr 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-very low density lipoprotein (VLDL) antibody dilution multiple proportions, diluted plasma multiple proportions, anti-Cr antibody dilution multiple proportions,
Testing result under the different anti-very low density lipoprotein (VLDL) antibody of table 2, anti-Cr antibody and diluted plasma multiple proportions
As known from Table 2, when the dilution multiple proportions of anti-very low density lipoprotein (VLDL) antibody is 1:1000, sample OD value is greater than the dilution multiple proportions of other the anti-very low density lipoprotein (VLDL) antibody under parallel condition; In this group sample, when diluted plasma multiple proportions is 1:20, when anti-Cr antibody dilution multiple proportions is 1:5000, OD value is maximum, is 0.707.
The OD detected value of positive control, negative control and blank corresponding when the dilution multiple proportions that table 3 is anti-very low density lipoprotein (VLDL) antibody is 1:1000, diluted plasma multiple proportions is 1:20, anti-Cr antibody dilution multiple proportions is 1:5000,
The testing result of table 3 positive control, negative control and blank
As known from Table 3, negative control group OD detected value is less than 0.1, and under this optimal conditions is described, the systematic error of this method is little, meets analytical approach requirement, so select concentration corresponding to this value as best effort concentration.
2.ELISA eluent best effort concentration and elution time are determined
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-Cr 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-Cr antibody protein, press the doubling dilution of 1:5000 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-Cr antibody response;
(4) wash-out: remove enzyme labelled antibody, by dilution buffer, eluent is diluted, make the concentration of papain in eluent: in enzyme labelled antibody, eluent adds in micropore by the multiple proportions of the concentration=1:5,1:10,1:20,1:40,1:80 of antibody, each concentration does 3 multiple holes, acts on 1h, 2h, 3h at being positioned over 37 DEG C 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-Cr antibody-hrp-antibody complex wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-Cr antibody-hrp-antibody complex wash-out degree reaches maximum.As shown in table 4, the concentration when papain in eluent: in enzyme labelled antibody during the concentration=1:20 of antibody, wash-out degree is maximum; And action time is when being 1h, 2h, 3h, each group of OD value change is little, the visible prolongation along with the time, enzyme activity weakens gradually, when enzyme concentration is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of eluent be that 1-3h all can, in sum, we select eluent 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
Application Example
application Example 1
Employing euzymelinked immunosorbent assay (ELISA) (ELISA method) detects the chromium-very low density lipoprotein (VLDL) chelate in 100 parts of sample blood plasma, and the method namely adopting specific embodiment method one to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by anti-very low density lipoprotein (VLDL) antibody, be diluted to 1000 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 chromium of known content-very low density lipoprotein (VLDL) chelate, by dilution buffer, 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-Cr antibody: remove sample, washing, add the anti-Cr antibody being diluted to 5000 times by dilution buffer, 37 DEG C of effects 1 hour, make the crome metal in itself and very low density lipoprotein (VLDL) react;
5) enzyme-added mark: remove anti-Cr antibody, washing, adds and is diluted to by dilution buffer the enzyme labelled antibody that antibody concentration is 2 μ g/mL, and 37 DEG C act on 1 hour, make itself and anti-Cr antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, adds substrate, 37 DEG C of lucifuge effect 30min, is added dropwise to stop buffer with the speed identical with adding substrate solution and order;
7) detect: the OD value reading sample to be tested and standard items under 450nm wavelength in microplate reader respectively, result is as shown in table 5.
The measured result of table 5 method a pair 100 parts of sample blood plasma
In this application embodiment 1, step 7) in, microplate reader also can not be used to detect, but directly carry out qualitative detection by colour developing.
application Example 2
Adopt enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) to detect chromium-very low density lipoprotein (VLDL) 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-very low density lipoprotein (VLDL) antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 1000 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 chromium of known content-very low density lipoprotein (VLDL) 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 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: sample from ELISA micropore, detect the chromium of chelating in very low density lipoprotein (VLDL) in sample to be tested and standard items in Atomic Absorption Spectrometer, result is as shown in table 6.
Table 6 method two is to the measured result of 100 parts of sample blood plasma
application Example 3:
Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) is adopted to detect chromium-very low density lipoprotein (VLDL) 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-very low density lipoprotein (VLDL) antibody, with dilution buffer dilution coating protein to 1000 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 chromium of known content-very low density lipoprotein (VLDL) 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, 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, thorough acidifying;
6) detect: add hydrogen peroxide, and acid is caught up with in heating, sample and detect the chromium of chelating in very low density lipoprotein (VLDL) in sample to be tested and standard items under icp ms, result is as shown in table 7.
Table 7 method three is to the measured result of 100 parts of sample blood plasma
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (8)
1. chromium-very low density lipoprotein (VLDL) chelate, is characterized in that, this chromium-very low density lipoprotein (VLDL) chelate be chromium ion and very low density lipoprotein (VLDL) by sulfydryl or/and cysteine residues chelating forms.
2. the preparation method of chromium-very low density lipoprotein (VLDL) chelate as claimed in claim 1, comprises the following steps:
A) synthesis of chromium-very low density lipoprotein (VLDL): add chromium ion and carry out chelatropic reaction purifying in the very low density lipoprotein (VLDL) that comes from human body or the very low density lipoprotein (VLDL) according to biological method restructuring;
B) chromium-very low density lipoprotein (VLDL) chelate purifying: adopt immune-affinity chromatography, removal step A) unreacted very low density lipoprotein (VLDL), specific antibody and chromium ion in reaction solution, obtain chromium-very low density lipoprotein (VLDL) 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) chromium-very low density lipoprotein (VLDL) chelate be dissolved in physiological saline, obtain the solution of chromium-very low density lipoprotein (VLDL) chelate;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of very low density lipoprotein (VLDL) specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with the solution of dilution buffer dilution step (1), then upper prop, make very low density lipoprotein (VLDL) 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 step (5), dress bag filter, uses 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 chromium 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, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-very low density lipoprotein (VLDL) chelate.
4. the preparation method of chromium according to claim 2-very low density lipoprotein (VLDL) chelate, is characterized in that, step B) after also comprise step C): to the qualification of chromium-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) 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 chromium, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS to detect the content whether containing chromium and detection chromium.
5. the application of chromium as claimed in claim 1-very low density lipoprotein (VLDL) chelate in the reagent or kit preparing to detect chromium-very low density lipoprotein (VLDL) chelate in blood sample.
6. one kind at least comprises the kit of chromium as claimed in claim 1-very low density lipoprotein (VLDL) chelate as standard items.
7. kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer comprises the material of catching very low density lipoprotein (VLDL) or the material of catching crome metal.
8. one kind is quantitatively detected the method for chromium-very low density lipoprotein (VLDL) chelate, it is characterized in that, using the chromium according to claim 1-very low density lipoprotein (VLDL) 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 chromium-very low density lipoprotein (VLDL) chelate and enzyme linked immunological combined techniques, purification chromium-very low density lipoprotein (VLDL) chelate and atomic absorption spectrum combined techniques, purification chromium-very low density lipoprotein (VLDL) 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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