CN105001322A - Chromium-high density lipoprotein chelate as well as preparation method and application thereof - Google Patents
Chromium-high density lipoprotein chelate as well as preparation method and application thereof Download PDFInfo
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- CN105001322A CN105001322A CN201510413239.6A CN201510413239A CN105001322A CN 105001322 A CN105001322 A CN 105001322A CN 201510413239 A CN201510413239 A CN 201510413239A CN 105001322 A CN105001322 A CN 105001322A
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- 239000013522 chelant Substances 0.000 title claims abstract description 10
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- 239000011651 chromium Substances 0.000 claims abstract description 154
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims abstract description 134
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
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- Gastroenterology & Hepatology (AREA)
- Genetics & Genomics (AREA)
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Abstract
The invention provides a chromium-high density lipoprotein chelate and a preparation method and application thereof, wherein the chromium-high density lipoprotein chelate is formed by chelating chromium ions and high density lipoprotein through sulfydryl or/and cysteine residues, and can be used for preparing a reagent for detecting the chromium-high density lipoprotein chelate of a human body. The invention proves that the chromium ions can directly act on the high-density lipoprotein for the first time. The invention establishes a qualitative and quantitative detection method of chromium-high density lipoprotein chelate to detect the content of high 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-high density lipoprotein chelate, which is 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-high-density lipoprotein (HDL) inner complex and preparation method thereof 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, be called for short HDL), intermediated-density lipoprotein (intermediate densitylipoprotein, be called for short IDL), low-density lipoprotein (low density lipoprotein, be called for short LDL), vldl (very low density lipoprotein, be called for short VLDL), chylomicrons (chylomicron, CM).In blood, about cholesterol of 30 percent is transported to liver by high-density lipoprotein (HDL) and is converted into bile acide or is excreted by bile from histocyte.High-density lipoprotein (HDL) is made up of the albumen of 50%, phosphatide, the cholesterol (wherein 70% is cholesteryl ester) of 25%, the triacylglycerol of 5% of 30%.
High-density lipoprotein (HDL) (being called for short HDL) is one of major lipoprotein in blood plasma, and its density is the highest, and particle diameter is minimum, the heterogeneous lipoprotein of albumen and lipid content approximate equality.Albumen by 50%, the phosphatide of 30%, the cholesterol (wherein 70% is cholesteryl ester) of 25%, the triacylglycerol of 5% form.
HDL is that atherosclerosis occurs and the major lipids of development protects factor, and it has very strong suppression Atherosclerosis in serum protein, and being the important protective factors of coronary artery disease, is negative correlation with coronary heart disease risk degree.
But; research shows that the quantity of high density lipoprotein cholesterol can not be directly proportional to the degree of protection of cardiovascular disorder, 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 high density lipoprotein cholesterol 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.
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 pollutent.At present, chromium is widely used in all respects 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 is the trace element of necessary for human, and sexavalent chrome is only obvious toxicant.Because sexavalent chrome easily enters cell by cytolemma, 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, tap 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 title 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-high-density lipoprotein (HDL) inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of chromium-high-density lipoprotein (HDL) inner complex, so that detection by quantitative chromium-high-density lipoprotein (HDL) inner complex is in the application of the regional pollution of chromium degree of evaluation one.By chromium-high-density lipoprotein (HDL) inner complex in the regional crowd's serum of detection by quantitative one, indirectly can reflect the situation of this regional crowd by pollution of chromium, thus indirectly reflect 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-high-density lipoprotein (HDL) inner complex, this chromium-high-density lipoprotein (HDL) inner complex be chromium 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 chromium-high-density lipoprotein (HDL) inner complex, i.e. external synthesis method, comprises the following steps:
A) synthesis of chromium-high-density lipoprotein (HDL): add chromium 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 soln;
B) chromium-high-density lipoprotein (HDL) inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and chromium ion in reaction soln, obtain chromium-high-density lipoprotein (HDL) inner complex.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-high-density lipoprotein (HDL) inner complex be dissolved in physiological saline, obtain chromium-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatography 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 chromatography 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 chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium specific binding, dress post after balance chromatography 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 chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-high-density lipoprotein (HDL) inner complex.
As preferably, the preparation method of above-mentioned chromium-high-density lipoprotein (HDL) inner complex, also comprises the following authentication step to chromium-high-density lipoprotein (HDL) inner complex, specifically comprises the following steps:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-high-density lipoprotein (HDL) inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing 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-high-density lipoprotein (HDL) inner complex in preparation human body in the reagent of chromium-high-density lipoprotein (HDL) inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of chromium described above-high-density lipoprotein (HDL) inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the albumen of catching high-density lipoprotein (HDL) or the material of catching chromium metal.
The present invention also provides the method for a kind of detection by quantitative chromium-high-density lipoprotein (HDL) inner complex, using the above-mentioned chromium-high-density lipoprotein (HDL) inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and enzyme linked immunological combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and atomic absorption spectrum combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Compared to existing technology, beneficial effect of the present invention is:
1. the present invention has synthesized extraction chromium-high-density lipoprotein (HDL) inner complex first;
2. the present invention proposes chromium-high-density lipoprotein (HDL) inner complex first and can be used for preparing application in the reagent or test kit detecting chromium-high-density lipoprotein (HDL) inner complex in blood sample;
3. present invention achieves specific recognition and the detection by quantitative of chelating type high-density lipoprotein (HDL), so that the content of chromium-high-density lipoprotein (HDL) inner complex in detection by quantitative 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-high-density lipoprotein (HDL) inner complex quantitative detecting method that the present invention sets up is high, reproducible.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-high-density lipoprotein (HDL) inner complex;
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of chromium of the present invention-high-density lipoprotein (HDL) inner complex.
Embodiment
Below in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention; Agents useful for same, material, as non-specified otherwise, are all considered as to buy from commercial mode:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
The albumen 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 albumen 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 labelling such as horseradish peroxidase, alkaline phosphatase;
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example: the Na getting 1.5g
2cO
3with the NaHCO of 2.93g
3dissolving adds ddH
2o is settled to 1000mL;
Lavation buffer solution is the 0.15M PBS solution of pH7.4, compound method example: the KH getting 0.2g
2pO
4, 2.90g Na
2hPO
412H
2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH
2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin, adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2M H
2sO
4, compound method example: the ddH getting 178.3mL
2o, enriching H
2sO
4be settled to 200mL;
Substrate is methyl diphenyl amine (TMB) solution, compound method example: get the methyl diphenyl amine ethanolic soln that 0.5mL concentration is 2g/L, add substrate dilution and be diluted to 10mL;
Substrate buffer solution pH is 5.0, wherein Na
2hPO
4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, compound method example: get 1.42gNa
2hPO
4, 0.96g citric acid, then add ddH
2o to 50mL, to obtain final product;
The compound method example of elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/mL, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min, obtain elutriant;
Sample-loading buffer can be formulated by the component of following ratio: Tris-HCl:1% tetrabromophenol sulfonphthalein: ddH
2o: glycine=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
The compound method of electrophoretic buffer is as follows: get 3.0g Tris, 14.4g glycine, be dissolved in 800mLddH
2in O, after adjusting pH to 8.3, be settled to 1L;
The material of the catching chromium mouse-anti Cr mAb 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-high-density lipoprotein (HDL) inner complex, this chromium-high-density lipoprotein (HDL) inner complex be chromium ion and high-density lipoprotein (HDL) by sulfydryl or/and cysteine residues chelating forms.
Particularly, chromium-high-density lipoprotein (HDL) inner complex is by the inner complex of chromium 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 level etc. and formed.
The present invention also provides the preparation method of chromium-high-density lipoprotein (HDL) inner complex, comprises the following steps:
A) synthesis of chromium-high-density lipoprotein (HDL): add chromium 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 soln;
B) chromium-high-density lipoprotein (HDL) inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and chromium ion in reaction soln, obtain chromium-high-density lipoprotein (HDL) inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-high-density lipoprotein (HDL) inner complex be dissolved in physiological saline, obtain chromium-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatography 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 chromatography 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 chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium specific binding, dress post after balance chromatography 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 chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-high-density lipoprotein (HDL) inner complex.
C): to the qualification of chromium-high-density lipoprotein (HDL) inner complex, specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-high-density lipoprotein (HDL) inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing 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 test kit of chromium described above-high-density lipoprotein (HDL) inner complex as standard substance.
In the present invention, the test kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, comprising: containing can be used for catching the coating buffer of high-density lipoprotein (HDL), confining liquid, lavation buffer solution, the material of chromium can being caught as two anti-, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative controls etc.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, comprising: containing the coating buffer, confining liquid, lavation buffer solution, elutriant, positive control, negative control etc. that can be used for catching high-density lipoprotein (HDL).
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, comprising: containing the coating buffer, confining liquid, lavation buffer solution, elutriant, souring agent, hydrogen peroxide, standard substance, negative control etc. that can be used for catching high-density lipoprotein (HDL).
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, 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 substance, negative control etc. of material that can be used for catching chromium.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, comprise and extract high-density lipoprotein (HDL) required extraction reagent, redissolution liquid, positive control, negative control etc.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, comprise and extract high-density lipoprotein (HDL) required extraction reagent, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, 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 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 test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, 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 chromium.
Detect a test kit for chromium in blood sample-high-density lipoprotein (HDL) inner complex, 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 chromium.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the high-density lipoprotein (HDL) inner complex of heavy metal chromium or is chelated with the BSA inner complex of heavy metal chromium; Described negative control is dilution buffer.
Mentioned reagent box, for detecting chromium-high-density lipoprotein (HDL) inner complex, to improve accuracy and the repeatability of detection, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative chromium-high-density lipoprotein (HDL) inner complex, using the above-mentioned chromium-high-density lipoprotein (HDL) inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and enzyme linked immunological combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and atomic absorption spectrum combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, following several with having of detecting that the method for chromium-high-density lipoprotein (HDL) inner complex can list, but be not limited to following several.
The following stated dilution multiple proportions is weightmeasurement ratio.
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-high-density lipoprotein (HDL) inner complex, detects in accordance with the following steps:
1) bag quilt: can catch the albumen 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 recycle system sampling, make sample to be tested; Standard substance are made with the chromium of known content-high-density lipoprotein (HDL) inner complex; By dilution buffer, sample to be tested and standard substance are all diluted to 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching 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 chromium metal in anti-chromium antibody and high-density lipoprotein (HDL) 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) termination reaction: add stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, elisa plate is placed in OD value microplate reader reading respectively sample to be tested and standard substance, drawing standard curve, tries to achieve the content of sample to be tested.
In present method, step 8) also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
The method utilizes ELISA principle, chromium in high-density lipoprotein (HDL) can catch by the specific antibody of anti-chromium, afterwards can again by horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the high-density lipoprotein (HDL) 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 labellings such as alkaline phosphatase caught, and also not containing chromium 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 chromium metal detecting chelating in high-density lipoprotein (HDL).
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect chromium-high-density lipoprotein (HDL) inner complex and detect in accordance with the following steps:
1) bag quilt: the albumen 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: sample from the recycle system, make sample to be tested; Standard substance are made with the chromium of known content-high-density lipoprotein (HDL) inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds elutriant, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the chromium of chelating in high-density lipoprotein (HDL) in Atomic Absorption Spectroscopy AAS, read respective value;
This embodiment utilizes ELISA principle to catch the high-density lipoprotein (HDL) in whole blood, and detects the chromium 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 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 chromium metal 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 chromium-high-density lipoprotein (HDL) inner complex and detect in accordance with the following steps:
1) bag quilt: the albumen 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 recycle system, make sample to be tested; Standard substance are made with the chromium of known content-high-density lipoprotein (HDL) inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C;
5) acidifying: in step 4) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in the chromium 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 chromium of chelating in high-density lipoprotein (HDL) with icp ms; Namely first adopt ELISA principle to be extracted by chromium-high-density lipoprotein (HDL) inner complex, then adopt sense couple plasma mass spectrometer to carry out detection by quantitative to the chromium of chelating in high-density lipoprotein (HDL); Owing to only containing high-density lipoprotein (HDL) 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 chromium metal detecting chelating in high-density lipoprotein (HDL).
method four:purification chromium-high-density lipoprotein (HDL) inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect chromium-high-density lipoprotein (HDL) inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as ultracentrifugation, 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-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 substance are made with the chromium of known content-high-density lipoprotein (HDL) inner complex; Be diluted to 10-40 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, adds the enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: drip stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, microplate reader reading the OD value of sample to be tested and standard substance respectively, drawing standard curve, by comparing with standard substance group, trying to achieve the content of sample to be tested.
In present method, also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
method five:purification chromium-high-density lipoprotein (HDL) inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect chromium in blood sample-high-density lipoprotein (HDL) inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as ultracentrifugation, 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 high-density lipoprotein (HDL) solution;
2) detect: from step 1) solution sample, detect the chromium of chelating in high-density lipoprotein (HDL) in Atomic Absorption Spectroscopy AAS, read respective value.
method six:purification chromium-high-density lipoprotein (HDL) inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect chromium-high-density lipoprotein (HDL) inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as ultracentrifugation, 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) 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 high-density lipoprotein (HDL) under icp ms, read respective value.
Method four, method five and method six are all isolate high-density lipoprotein (HDL), then adopt method for detecting specificity, measure the content of chromium on chromium-high-density lipoprotein (HDL) inner complex in high-density lipoprotein (HDL); Namely first physical sepn means are adopted, as ultracentrifugation, 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 chromium content on inductively coupled plasma mass spectrometry detection chromium-high-density lipoprotein (HDL) inner complex.
Method seven: electrophoretic method+ELISA/AAS/ICP-MS method detects chromium-high-density lipoprotein (HDL) inner complex, specific as follows:
1) from whole blood, non-specific high-density lipoprotein (HDL) is extracted: adopt the methods such as ultracentrifugation, 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 solution of high-density lipoprotein (HDL);
2) glue bed is prepared: select suitable medium (as sepharose, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) solution get 8 μ L, make standard substance with the chromium of known content-high-density lipoprotein (HDL) inner complex, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, with electrophoretic buffer, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of iso-electric point;
5) detect: on glue bed, find out the protein band containing 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 iso-electric point of this method detection chromium-high-density lipoprotein (HDL) inner complex, molecular weight and content etc. can also be utilized.
In method seven, high-density lipoprotein (HDL) is extracted, then adopt gel electrophoresis to be separated extracted high-density lipoprotein (HDL), then find out the respective strap being rich in chromium, then detect the content of relevant high-density lipoprotein (HDL), high-density lipoprotein (HDL) can by multiple Methods For Purification out (such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the high-density lipoprotein (HDL) of purifying out is redissolved, 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 media can be adopted as required), the difference such as iso-electric point runs out of different bands, find out the respective strap being rich in chromium, protein in gel is redissolved, 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 chromium content of chelating in high-density lipoprotein (HDL), owing to only containing high-density lipoprotein (HDL) 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 chromium metal detecting chelating in high-density lipoprotein (HDL).
embodiment 1:synthesis method synthesis chromium-high-density lipoprotein (HDL) huge legendary turtle compound, comprises the following steps:
Chromium 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 chromium-high-density lipoprotein (HDL) inner complex, comprise the following steps:
The reagent that the present embodiment uses is as follows:
1) borate buffer solution, its volumetric molar concentration is 0.01M, and its preparation method example is as follows: take 0.31g boric acid and be dissolved in 400mLddH
2in O, regulate pH to 9.0 with the NaOH of 0.1mol/L, be settled to 500mL.
2) EDTA-NaHCO
3solution, 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 weight cut-off 14000 of dialysis tubing, buys from Bioshop Inc;
The pre-treatment of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500mL
3in solution, boil 10min; Tipping EDTA-NaHCO
3solution, uses ddH
2o is rinsing gently, then boils 10min with 500mL5mmol/L EDTA; Discard boiling liquid, thoroughly use ddH
2o cleans, and adds a large amount of ddH
2o soaks dialysis tubing 4 DEG C and spends the night.During use, put on one's gloves, take out dialysis tubing, with a large amount of ddH
2its surfaces externally and internally of O cleaning down;
The synthesis of A) chromium-high-density lipoprotein (HDL) huge legendary turtle compound: add chromium 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 soln, 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 dialysis tubing, 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 chromium ion solution gradually, dropping limit, limit vibrates, in order to avoid chromium ion makes protein denaturation precipitate;
4) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the dialysis tubing handled well;
5) liquid of having dialysed is preserved in-20 DEG C of packing, obtain the reaction soln of chromium-high-density lipoprotein (HDL) inner complex.
B) chromium-high-density lipoprotein (HDL) inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and chromium ion in reaction soln, obtain chromium-high-density lipoprotein (HDL) inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-high-density lipoprotein (HDL) inner complex be dissolved in physiological saline, obtain chromium-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography 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 chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-high-density lipoprotein (HDL) inner complex.
C) to the qualification of chromium-high-density lipoprotein (HDL) huge legendary turtle compound, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using sepharose as medium;
(2) application of sample: get step B) in chromium-high-density lipoprotein (HDL) inner complex of obtaining of extraction purification, redissolve in physiological saline, take out 8 μ L, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 DEG C; Stop electrophoresis when moving to bottom glue to tetrabromophenol sulfonphthalein, Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-high-density lipoprotein (HDL) huge legendary turtle compound, and M swimming lane is sex change Marker, HDL swimming lane is high-density lipoprotein (HDL);
(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 detected result
Get step C) isolated protein band solution, with the content of heavy metal chromium in graphite furnace atomic absorption spectrometry (AAS) rough determination high-density lipoprotein (HDL), as shown in the table, with NaCl solution, KBr solution, KCl solution for blank;
The content of chromium in table 1 high-density lipoprotein (HDL)
| Sample name | Cr(μg/L) |
| Sample to be tested | 10.548 |
| 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 Beijing electron positron collider (BEPC).In storage ring, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X-ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA 4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx 3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band the other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar fignal center of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the fluorometric analysis figure of the synchrotron radiation X line electrophoretic band of the electrophoretic band of chromium of the present invention-high-density lipoprotein (HDL) inner complex, and in figure, X-coordinate is protein band position, and ordinate zou is this band chromium metal energy (content) value.
the determination of testing conditions
1. the determination of the optimum diluting multiple of anti-high-density lipoprotein (HDL) antibody, anti-Cr antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for chromium-high-density lipoprotein (HDL) inner complex, 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 and 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 substance with the chromium of known content-high-density lipoprotein (HDL) inner complex, negative control and blank are set, add in micropore, 37 DEG C act on 1 hour;
4) anti-Cr antibody is added: 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 chromium metal in itself and high-density lipoprotein (HDL) are reacted;
5) enzyme-added mark: remove anti-Cr antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, 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 substance, 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-high-density lipoprotein (HDL) inner complex standard substance as positive control, respectively not add test plasma as negative control 1, namely add anti-high-density lipoprotein (HDL) antibody, confining liquid, anti-Cr antibody, enzyme mark and substrate successively;
Not add the controlled trial group of anti-Cr 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-Cr antibody and substrate is added successively using not enzyme-added target controlled trial group as negative control 3, namely;
Not add the controlled trial group of sample to be tested and anti-Cr antibody as negative control 4 simultaneously, namely add anti-high-density lipoprotein (HDL) antibody, confining liquid, enzyme mark and substrate successively;
Not add the controlled trial group of anti-high-density lipoprotein (HDL) antibody work for blank 1, namely add confining liquid, test plasma, anti-Cr antibody, enzyme mark and substrate;
And with the controlled trial group only adding substrate for blank 2, only to add the controlled trial group of PBS for blank 3.
Table 2 is the sample OD Value Data of different anti-high-density lipoprotein (HDL) antibody dilution multiple proportions, diluted plasma multiple proportions, anti-Cr antibody dilution multiple proportions,
Detected result under the different anti-high-density lipoprotein (HDL) antibody of table 2, anti-Cr antibody and diluted plasma multiple proportions
As known from Table 2, when the dilution multiple proportions of anti-high-density lipoprotein (HDL) antibody is 1:8000, 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-Cr antibody dilution multiple proportions is 1:5000, OD value is maximum, is 0.758.
The OD detected value of positive control, negative control and blank corresponding when the dilution multiple proportions that table 3 is anti-high-density lipoprotein (HDL) antibody is 1:8000, diluted plasma multiple proportions is 1:20, anti-Cr antibody dilution multiple proportions is 1:5000,
The detected 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 present method is little, meets analytical procedure requirement, so select concentration corresponding to this value as best effort concentration.
2.ELISA elutriant 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 elutriant of different concns, then detect OD value by microplate reader, concrete steps are as follows:
(1) bag quilt: be coated on solid phase carrier by anti-Cr antibody, 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, elutriant is diluted, make the concentration of papoid in elutriant: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove elutriant, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer termination reaction;
(5) under the determined wavelength of 450nm, in microplate reader, read the OD value of each micropore respectively, concrete outcome see table 4,
Detected result under the different elutriant extension rate of table 4
By comparing OD value, to judge anti-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 papoid in elutriant: in enzyme labelled antibody during the concentration=1:20 of antibody, anti-Cr antibody-hrp-antibody complex wash-out degree reaches maximum; And action time is when being 1h, 2h, 3h, each group of OD value change is little, the visible prolongation along with the time, enzyme activity weakens gradually, when enzyme concn is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of elutriant be that 1-3h all can, in sum, we select elutriant 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
Application Example
application Example 1
Employing euzymelinked immunosorbent assay (ELISA) (ELISA method) detects the chromium-high-density lipoprotein (HDL) inner complex 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-high-density lipoprotein (HDL) antibody, be diluted to 8000 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 substance with the chromium of known content-high-density lipoprotein (HDL) inner complex, by dilution buffer, sample to be tested and standard substance 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 chromium metal in itself and high-density lipoprotein (HDL) react;
5) enzyme-added mark: remove anti-Cr 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-Cr antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, are added dropwise to stop buffer;
7) detect: the OD value reading sample to be tested and standard substance 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-high-density lipoprotein (HDL) inner complex in 100 parts of sample blood plasma, the method namely adopting specific embodiment method two to record detects, and concrete operation step is as follows:
1) bag quilt: anti-high-density lipoprotein (HDL) antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 8000 times, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, preserve at elisa plate 4 DEG C;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the chromium of known content-high-density lipoprotein (HDL) inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 20 times, add in micropore, and 37 DEG C act on 2 hours;
4) wash-out: remove 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 high-density lipoprotein (HDL) in Atomic Absorption Spectroscopy AAS, 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-high-density lipoprotein (HDL) inner complex in 100 parts of sample blood plasma, namely the method adopting specific embodiment method three to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by anti-high-density lipoprotein (HDL) antibody, with dilution buffer dilution coating protein to 8000 times, adds in elisa plate micropore, preserves 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, elisa plate 4 DEG C preservation;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the chromium of known content-high-density lipoprotein (HDL) inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 20 times, add in micropore, and 37 DEG C act on 2 hours;
4) wash-out: remove sample to be tested and standard substance, washing, adds the Na of 0.8mol/L
2hPO
4solution, 37 DEG C of wash-outs 2 hours;
5) acidifying: add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
6) detect: sampling, under icp ms, detect the chromium of chelating in high-density lipoprotein (HDL), 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-high-density lipoprotein (HDL) inner complex, is characterized in that, this chromium-high-density lipoprotein (HDL) inner complex be chromium ion and high-density lipoprotein (HDL) by sulfydryl or/and cysteine residues chelating forms.
2. prepare the method for chromium as claimed in claim 1-high-density lipoprotein (HDL) inner complex, comprise the following steps:
A) synthesis of chromium-high-density lipoprotein (HDL): add chromium 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 soln;
B) chromium-high-density lipoprotein (HDL) inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted high-density lipoprotein (HDL), specific antibody and chromium ion in reaction soln, obtain chromium-high-density lipoprotein (HDL) inner complex.
3. method as claimed in claim 2, is characterized in that,
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) chromium-high-density lipoprotein (HDL) inner complex be dissolved in physiological saline, obtain chromium-high-density lipoprotein (HDL) chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of high-density lipoprotein (HDL) specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography 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 chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L
2hPO
4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.5-1.0mol/L
2hPO
4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-high-density lipoprotein (HDL) inner complex.
4. the preparation method of chromium according to claim 2-high-density lipoprotein (HDL) inner complex, is characterized in that, step B) after also comprise step C): to the qualification of chromium-high-density lipoprotein (HDL) inner complex;
Wherein, step C) in specifically comprise the following steps:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in chromium-high-density lipoprotein (HDL) inner complex of obtaining of extraction purification, add sample-loading buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing 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-high-density lipoprotein (HDL) inner complex in the reagent or test kit preparing to detect chromium-high-density lipoprotein (HDL) inner complex in blood sample.
6. one kind at least comprises the test kit of chromium as claimed in claim 1-high-density lipoprotein (HDL) inner complex as standard substance.
7. test kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the albumen of catching high-density lipoprotein (HDL) or the material of catching chromium metal.
8. the method for detection by quantitative chromium-high-density lipoprotein (HDL) inner complex, it is characterized in that, using the chromium according to claim 1-high-density lipoprotein (HDL) inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and enzyme linked immunological combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and atomic absorption spectrum combined techniques, purification chromium-high-density lipoprotein (HDL) inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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