CN105044009B - A kind of chromium VLDL chelate and its preparation method and application - Google Patents
A kind of chromium VLDL chelate and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of chromium VLDL chelate and its preparation method and application, the chromium VLDL chelate is that chromium ion is formed with VLDL by sulfydryl or/and cysteine residues chelating, available for the reagent for preparing detection human body chromium VLDL chelate.The present invention confirms that chromium ion can be done directly on VLDL first.The present invention establishes the method for qualitative and quantitative detection of chromium VLDL chelate, to detect the content of VLDL in regional crowd's body, so as to reflect a regional pollution of chromium degree and the health effect to crowd indirectly.Chromium VLDL chelate quantitative detecting method degree of accuracy Gao Chong that the present invention establishes, renaturation are good.
Description
Technical field
The present invention relates to the immunology detection of heavy metal ion, and in particular to a kind of chromium-VLDL chelate
And its preparation method and application.
Background technology
Lipoprotein (lipoproteins) is a kind of lipid-protein complex that protein is formed with lipid binding,
It is divided into HDL (high density lipoprotein, abbreviation HDL), intermediated-density lipoprotein by density
(intermediate density lipoprotein, abbreviation IDL), low-density lipoprotein (low density
Lipoprotein, abbreviation LDL), VLDL (very low density lipoprotein, abbreviation VLDL), breast
Rotten particle (chylomicron, CM).30 about percent cholesterol is to be transported by HDL from histocyte in blood
To liver it is converted into bile acid or is excreted by bile.About 70% cholesterol is as entrained by LDL and VLDL in blood,
Caused endogenous triglycerides (triglyceride, TG) also relies primarily on VLDL and transported to outside liver in liver.VLDL increasing
It is more closely bound up with hyperlipidemia, disorders of lipid metabolism, and dyslipidemia is the pathogenetic important risk factor of a variety of diseases, thus
The stabilization of VLDL contents levels plays an important role for the stabilization of human body each side function function.
The stabilization of VLDL contents levels and structure function is significant to body.Its too high levels and fatty liver, lipid metaboli
Disorder, angiocardiopathy, renal function damage etc. are closely bound up.Quantitatively detection VLDL can be used for the early diagnosis of these diseases
And therapeutic effect monitoring, or even one of evaluation index fine or not as prognosis.And for malnutrition, chronic anaemia, liver function
Patient, the serum VLDL contents such as energy sufferer can also decline, and quantitatively detect VLDL and can also be used for the early diagnosis of these diseases, control
One of therapeutic effect monitoring and the index as prognostic evaluation.
Chromium (chromium, Cr) is a kind of transition metal in the race of the periodic table of elements VI, and its quality is hard, and surface has light
Pool, there is higher fusing point, in the industrial production with great economic value, be widely used since a century, it is annual complete
Ball chromium output is up to 107 tons, thus chromium is a kind of qualified important meals pollutant.At present, chromium is widely used in electricity
The various aspects such as plating, tanning, pigment, paint, alloy, printing and dyeing, it is closely bound up with the life of people, but equally also to the mankind's
Health has an immense impact on.
Chromium mainly exists in the form of trivalent and sexavalence in nature, and it is generally believed that trivalent chromium (Cr3+) it is necessary for human
Trace element, and Cr VI (Cr6+) it is only obvious toxicant.Because Cr VI easily enters cell by cell membrane, and
And strong cytotoxicity and genotoxicity can be produced by series reaction, thus generally believe that the toxicity of Cr VI is high
In trivalent chromium, up to 10-100 times.The content of 6-valence Cr ions that scholar thinks to work as in water more than 0.05mg/L is poisonous.Cr VI
It can be drunk by skin contact, food intake, drinking water and enter human body into approach such as, respiratory tract suctions, for occupational exposure
Crowd mainly by respiratory tract suction Cr VI dust, and be then for general population by drink into polluted source, eat by
The modes such as the food of pollution, suction take in Cr VI.Cr VI enter in vivo after, can with initiated oxidation stress, developing body distortion,
Apoptosis, a variety of DNA damages (including single-strand break, DNA- protein cross, DNA- amino acid crosslinks, Cr-DNA complexs
Formed etc.), so as to cause body many places tissue, organ, system injury.
As it was previously stated, it is in close relations between chromium and multiple proteins, it can be combined with multiple proteins, suppress multiple protein
The activity of enzyme.With going deep into chromium toxicity research, people gradually recognize the interaction of chromium and albumen in its intoxicating process
In play important role, it is considered to be the key point of the toxicity mechanism of chromium is understood, therefore, to chromium and protein phase interaction
Research just seems ever more important.And the mechanism now concerning chromium and protein effect is only tip of the iceberg, also many eggs
Relation between white matter and chromium is not clear, thus strengthens most important for the relation between chromium and protein.
On chromium poisoning, the evaluation of particularly chronic chromium poisoning can not accurate evaluation human body by detecting blood chromium content
Interior circulation chromium content, degree of injury of the chromium for body function can not be further assessed, and with the hair at full speed of science and technology
The relation of exhibition, chromium and human body is also increasingly close, thus finds one kind and can be particularly slow from the evaluation chromium poisoning of immunologic function angle
Property chromium poisoning becomes more and more important for the evaluation method of the extent of damage of body.
The content of the invention
For pollution of chromium it is serious the problem of, it is an object of the invention to provide a kind of chromium-VLDL chelate
And preparation method thereof, and establish the qualitative and quantitative analysis method of chromium-VLDL chelate, so as to quantitative detection chromium-
VLDL chelate is evaluating the application of a regional pollution of chromium degree.Pass through one regional crowd's blood of quantitative detection
Chromium-VLDL chelate in clear, can reflect situation of this regional crowd by pollution of chromium indirectly, reversed so as between
Reflect this regional pollution of chromium degree.
The technical solution adopted for the present invention to solve the technical problems is：A kind of chromium-VLDL chelating is provided
Thing, the chromium-VLDL chelate are that chromium ion passes through sulfydryl or/and cysteine residues with VLDL
Chelating forms.
The present invention also provides a kind of preparation method of above-mentioned chromium-VLDL chelate, i.e., external synthetic method,
Comprise the following steps：
A) the synthesis of chromium-VLDL chelate：Purification come from human body VLDL or according to
Chromium ion is added in the VLDL of biological method restructuring and carries out chelatropic reaction, obtains reaction solution；
B) chromium-VLDL chelate purifying：Using immune-affinity chromatography, removal step A) in reaction solution
Unreacted VLDL, specific antibody and chromium ion, produce chromium-VLDL chelate.
Preferably, the step B) in specifically include following steps：
(1) sample dissolution：By above-mentioned steps A) chromium-VLDL chelate be dissolved in physiological saline, obtain
The solution of chromium-VLDL chelate；
(2) chromatographic column is balanced：Using dilution buffer rinse chromatographic column pipeline, in chromatographic column load can with it is extremely low close
The filler of lipoprotein specific binding is spent, after filling post, is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with the solution of dilution buffer dilution step (1), then upper prop, makes extremely low
Density lipoprotein is specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：By the eluent in step (10), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C
Dialysed overnight, sample is collected, produces chromium-VLDL chelate.
Preferably, the preparation method of above-mentioned chromium-VLDL chelate, in addition to below to chromium-extremely low close
The authentication step of lipoprotein chelate is spent, specifically includes following steps：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained chromium-VLDL chelate of extraction purification, add loading buffer
Liquid, and mix, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4)：The protein band containing chromium is found out on glue bed, the protein band is taken out, protein band is dissolved, Ran Houzai
The content of chromium is detected whether containing chromium and detected using ICP-MS or AAS.
The present invention also provides a kind of chromium-VLDL chelate described above and is preparing chromium-pole in detection human body
Application in the reagent or kit of low-density lipoprotein chelate.
The present invention, which also provides, a kind of comprises at least chromium described above-examination of the VLDL chelate as standard items
Agent box.
Preferably, also include coating buffer in the kit, the coating buffer include capture VLDL material or
Capture the material of crome metal.
The present invention also provides a kind of method for quantitatively detecting chromium-VLDL chelate, with the upper of known content
The chromium stated-VLDL chelate is detected as standard items using a pair of samples of following methods：Enzyme linked immunological
Method, enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-
VLDL chelate and enzyme linked immunological combined techniques, purification chromium-VLDL chelate and atomic absorption light
Spectrum combined techniques, purification chromium-VLDL chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis are exempted from enzyme-linked
Epidemic disease or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Compared with prior art, the beneficial effects of the present invention are：
1. the present invention has synthesized chromium-VLDL chelate first；
2. it can be used for preparing chromium-extremely low close in detection blood sample present invention firstly provides chromium-VLDL chelate
The application spent in the reagent or kit of lipoprotein chelate；
3. the present invention realizes the specific recognition of chromium-VLDL chelate and quantitative detection, so as to quantitative
The content of chromium-VLDL chelate in detection crowd's serum, to evaluate the application of a regional pollution of chromium degree, it is
The pollution of chromium of industrial area is horizontal to provide indirect indexes.The chromium that the present invention establishes-VLDL chelate quantitatively detects
The degree of accuracy of method is high, reproducible.
Brief description of the drawings
Fig. 1 is the non denatured electrophoretic band figure of chromium of the present invention-VLDL chelate；
Fig. 2 is the synchrotron radiation X line fluorescence point of the electrophoretic band of chromium of the present invention-VLDL chelate
Analysis figure.
Embodiment
With reference to embodiment, the present invention is described further：
Unless otherwise indicated, the laboratory operating procedures being related to are the conventional step in this area to the present invention；Agents useful for same, material
Material such as non-specified otherwise, is accordingly to be regarded as to buy from mode purchased in market：
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
The material for capturing VLDL is anti-VLDL antibody, can be real below by commercially available acquisition
Apply a mode, the anti-VLDL antibody used be article No. for " Genetex gtx16419 " VLDL antibody,
Material, anti-VLDL antibody, the anti-VLDL antibody with VLDL specific binding is capture
The material of VLDL；
Enzyme labelled antibody is one kind in the antibody containing the enzymes such as horseradish peroxidase, alkaline phosphatase mark；
Dilution buffer be pH 9.6 0.05M carbonate buffer solutions, compound method example：Take 1.5g Na2CO3With
2.93g NaHCO3Dissolving plus ddH2O is settled to 1000mL；
Lavation buffer solution be pH7.4 0.15M PBS solutions, compound method example：Take 0.2g KH2PO4, 2.90g
Na2HPO4·12H2O, 8.0g NaCl, 0.2g KCl, 0.5mLTween-20, dissolving plus ddH2O is settled to 1000mL；
Confining liquid is bovine serum albumin solution, compound method example：0.1g bovine serum albumin(BSA)s are taken, add washing buffer
Liquid dilution is settled to 100mL；
Terminate liquid is 2M H2SO4, compound method example：Take 178.3mL ddH2O, enriching H2SO4It is settled to 200mL；
Substrate is methyl biphenyl amine (TMB) solution, compound method example：Take the methyl biphenyl amine that 0.5mL concentration is 2g/L
Ethanol solution, substrate dilution is added to be diluted to 10mL；
Substrate buffer solution pH is 5.0, wherein Na2HPO4Molar concentration be 0.2M, the molar concentration of citric acid be 0.1M,
Compound method example：Take 1.42gNa2HPO4, 0.96g citric acids, then add ddH2O to 50mL, is produced；
The compound method example of eluent：By papain pH 8.0,0.1mol/L Tris-HCI buffers
Into 1-2mg/mL, 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT)s (DTT) are added, obtain eluent；
Sample-loading buffer can be formulated by the component of following ratio：Tris-HCl：1% bromophenol blue：ddH2O：Glycine
=15.5：2.5：7：25, wherein Tris-HCl pH is 6.8, molar concentration 1M；
The compound method of electrophoretic buffer is as follows：Take 3.0g Tris, 14.4g glycine, be dissolved in 800mLddH2In O, adjust
After pH to 8.3,1L is settled to；
The material of capture chromium is article No. for " Guangzhou Ran Ke companies RK10641 " the anti-Cr mAb of mouse, in implementation below
Described material, anti-Cr antibody, secondary antibody, anti-chromium antibody with chromium specific binding be capture chromium material；
The present invention provides a kind of chromium-VLDL chelate, and chromium ion passes through sulfydryl with VLDL
Or/and cysteine residues chelating forms.
Specifically, chromium-VLDL chelate is residual by combining zinc fingers, sulfydryl, cysteine by chromium
At least one of base structure and the apoC I in VLDL, CⅡ and CⅢ, ApoC3, load fat
Albumen E or cholesterol, triglycerides etc. with reference to and formed chelate.
The present invention also provides the preparation method of chromium-VLDL chelate, comprises the following steps：
A) the synthesis of chromium-VLDL：The VLDL of human body is come from purification or according to biology
Chromium ion is added in the VLDL of method restructuring and carries out chelatropic reaction, obtains reaction solution；
B) chromium-VLDL chelate purifying：Using immune-affinity chromatography, removal step A) in reaction solution
Unreacted VLDL, specific antibody and chromium ion, chromium-VLDL chelate is produced, specific bag
Include following steps：
The step B) in specifically include following steps：
(1) sample dissolution：By above-mentioned steps A) chromium-VLDL chelate be dissolved in physiological saline, obtain
The solution of chromium-VLDL chelate；
(2) chromatographic column is balanced：Using dilution buffer rinse chromatographic column pipeline, in chromatographic column load can with it is extremely low close
The filler of lipoprotein specific binding is spent, after filling post, is continuing with dilution buffer balance chromatographic column；
The filler that can be specifically bound with VLDL can be special with VLDL to be adsorbed with
The silica gel or resin of property conjugate；
(3) loading：After column equilibration is chromatographed, with the solution of dilution buffer dilution step (1), then upper prop, makes extremely low
Density lipoprotein is specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling post；
The filler that can be specifically bound with chromium is to be adsorbed with silica gel or resin that material can be specifically bound with chromium；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces chromium-VLDL chelate.
C)：Identification to chromium-VLDL chelate, specifically includes following steps：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained chromium-VLDL chelate of extraction purification, add loading buffer
Liquid, and mix, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing chromium is found out on glue bed, the protein band is taken out, protein band is dissolved, so
The content of chromium is detected whether containing chromium and detected again afterwards using ICP-MS or AAS.
The present invention, which also provides, a kind of comprises at least chromium described above-examination of the VLDL chelate as standard items
Agent box.
In the present invention, can realize the kit of the object of the invention can list following several, but be not limited to this.
The kit of chromium-VLDL chelate in a kind of detection blood sample, including：Containing extremely low available for capturing
The coating buffer of the material of density lipoprotein, confining liquid, lavation buffer solution, the material of chromium can be captured as secondary antibody, enzyme labelled antibody, bottom
Thing, terminate liquid, dilution buffer, positive control, negative control etc..
The kit of chromium-VLDL chelate in a kind of detection blood sample, including：Containing extremely low available for capturing
The coating buffer of the material of density lipoprotein, confining liquid, lavation buffer solution, eluent, positive control, negative control etc..
The kit of chromium-VLDL chelate in a kind of detection blood sample, including：Containing extremely low available for capturing
Coating buffer, confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, the feminine gender of the material of density lipoprotein
Control etc..
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent, redissolution liquid, the coating buffer containing the material available for capture chromium, confining liquid, lavation buffer solution, enzyme needed for lipoprotein
Labeling antibody, substrate, terminate liquid, dilution buffer, standard items, negative control etc..
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent, redissolution liquid, positive control, negative control etc. needed for lipoprotein.
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc. needed for lipoprotein.
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent needed for lipoprotein, glue bed medium, redissolve liquid, sample loading buffer, liquid needed for the protein band containing chromium on dissolving glue bed
Body, coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, the positive containing the material available for capture chromium
Control, negative control etc..
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent needed for lipoprotein, glue bed medium, redissolve liquid, sample loading buffer, liquid needed for the protein band containing chromium on dissolving glue bed
Body, positive control, negative control etc..
Extra-low density in the kit of chromium-VLDL chelate in a kind of detection blood sample, including extraction whole blood
Extracts reagent needed for lipoprotein, glue bed medium, redissolve liquid, sample loading buffer, liquid needed for the protein band containing chromium on dissolving glue bed
Body, nitric acid, hydrogen peroxide, positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with the extra-low density fat egg of heavy metal chromium
White chelate or the BSA chelates for being chelated with heavy metal chromium；The negative control is dilution buffer.
Mentioned reagent box is used to detect chromium-VLDL chelate, to propose the accuracy of extremely low detection and repetition
Property, and be allowed to be promoted in clinic.
The present invention also provides a kind of method for quantitatively detecting chromium-VLDL chelate, with the upper of known content
The chromium stated-VLDL chelate is detected as standard items using a pair of samples of following methods：Enzyme linked immunological
Method, enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification chromium-
VLDL chelate and enzyme linked immunological combined techniques, purification chromium-VLDL chelate and atomic absorption light
Spectrum combined techniques, purification chromium-VLDL chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis are exempted from enzyme-linked
Epidemic disease or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, with detection chromium-extra-low density fat egg
It is following several that what the method for white chelate can be listed have, but is not limited to following several.
Method one：ELISA (ELISA method) detects chromium-VLDL chelate, examines in accordance with the following steps
Survey：
1) it is coated with：The material that can capture VLDL with dilution buffer dilution adds to 500-4000 times
In elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with lavation buffer solution, and after the completion of washing, elisa plate places 1 in 37 DEG C
Hour；
3) add sample to be tested, and incubate：Sampled from the circulatory system, make sample to be tested；With the chromium of known content-extremely low close
Degree lipoprotein chelate makees standard items；Sample to be tested is diluted with dilution buffer and standard items are diluted to 10-40 times, is added micro-
Kong Zhong, 37 DEG C of effect 1-2 hours；
4) material of chromium can be captured by adding, and be incubated：Sample to be tested is removed, and is washed with lavation buffer solution,
Wait after the completion of washing, addition dilutes 5000-40000 times of anti-chromium antibody with dilution buffer, 37 DEG C of effect 1-2 hours, makes to resist
Chromium antibody reacts with the crome metal in VLDL；
5) enzyme conjugates incubates：Anti- chromium antibody is removed, is washed, the HRP enzyme labelled antibodies that addition is diluted with dilution buffer, 37
DEG C effect 1-2 hours, react itself and HRP enzyme labelled antibodies；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds substrate,
37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction：Terminate liquid is to each micropore；
8) take wavelength 450nm, after adding terminate liquid, elisa plate is placed on ELIASA read respectively sample to be tested group and
The OD values of standard items, standard curve is drawn, by the content for compared with standard item group, trying to achieve sample to be tested.
In this method, step 8) also directly can carry out qualitative detection without using ELIASA by the situation that develops the color.
This method utilizes ELISA principles, and the chromium in VLDL can be captured by the specific antibody of chromium, it
(antibody nonrecognition coating egg can be captured by the antibody that the enzymes such as horseradish peroxidase, alkaline phosphatase mark again afterwards
In vain), the antibody in capture can read OD values in the presence of developer and terminate liquid under instrument, and not contain chelating gold
Belong to the VLDL of chromium, then will not be captured by the specific antibody of anti-chromium, also will not be with horseradish peroxidase, alkali
The antibody of the enzymes such as acid phosphatase mark is captured, and crome metal is not contained in agents useful for same yet (negative control group result is cloudy
Property), thus when the OD value results read are shown as the positive, you can prove to detect the gold chelated in VLDL
Belong to chromium.
Method two：Enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection chromium-extra-low density fat egg
White chelate detects in accordance with the following steps：
1) it is coated with：The material of VLDL will can be captured, such as anti-VLDL antibody is (anti-extremely low close
Degree lipoprotein antibody) it is coated on solid phase carrier, anti-VLDL antibody is diluted to 500-4000 with dilution buffer
Times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
3) add sample to be tested, and incubate：Sampled from the circulatory system, make sample to be tested；With the chromium of known content-extremely low
Density lipoprotein chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 are small
When；
4) elute：Sample to be tested is removed, is washed, adds eluent, 1-3 hours are eluted at 37 DEG C；
5) detect：Sample from ELISA micropores, chelated in Atomic Absorption Spectrometer detection in VLDL
Chromium, read respective value；
The embodiment is captured using ELISA principles to the VLDL in serum, and combines Atomic absorption
The detection of spectrum (AAS) instrument is chelated in the chromium in VLDL；Due to only containing VLDL, and institute in solution
With any heavy metal (negative control group result is feminine gender) is free of in reagent, result will not be interfered, thus work as and read
Result when being shown as the positive, you can prove to detect the crome metal chelated in VLDL.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection chromium-
VLDL chelate detects in accordance with the following steps：
1) it is coated with：The material of VLDL will can be captured, such as anti-VLDL antibody is coated in solid
On phase carrier, anti-VLDL antibody is diluted to 500-4000 times with dilution buffer, is added in elisa plate micropore, 4
DEG C overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
3) add sample to be tested, and incubate：Whole blood is taken from the circulatory system, makees sample to be tested；With the chromium of known content-extremely low
Density lipoprotein chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 are small
When；
4) elute：Sample to be tested is removed, and is washed with corresponding lavation buffer solution, is waited after the completion of washing, adds elution
Liquid, 1-3 hours are eluted at 37 DEG C；
5) it is acidified：Add acidulant in solution in step 4) to be acidified solution, sealing overnight, is thoroughly acidified；
6) detect：Hydrogen peroxide is added, and heats and catches up with acid, and 0.5mL is taken in the solution eluted from ELISA agent plates
Liquid, the chromium chelated in VLDL is detected under icp mses, reads respective value.
This method is on the basis of using ELISA principles, with reference to sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption
Sense couple plasma mass spectrometer detection is chelated in the chromium in VLDL；I.e. first using ELISA principles by chromium-extremely low
Density lipoprotein chelate extracts, then using sense couple plasma mass spectrometer to chelating in VLDL
Chromium carries out quantitative detection；Due to only containing VLDL in solution, and it is (negative without any heavy metal in agents useful for same
Control group result is feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can prove
Detect the crome metal chelated in VLDL.
Method four：Chromium-VLDL chelate is purified to examine with enzyme linked immunological combined techniques (method of purification+ELISA method)
Chromium-VLDL chelate is surveyed, is detected in accordance with the following steps：
1) non-specific VLDL is extracted from whole blood：Using supercentrifugation, HPLC, coagulate
The methods of glue filtration chromatography, gel electrophoresis, VLDL, and the extra-low density that will be extracted are extracted from whole blood
Lipoprotein redissolves, and obtains the solution of VLDL；
2) it is coated with：Anti- chromium antibody is coated on solid phase carrier, dilutes anti-chromium antibody to 5000- with dilution buffer
40000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
3) close：Dilution buffer is removed, and is washed with lavation buffer solution, is waited after the completion of washing, adds confining liquid,
37 DEG C are placed 1 hour, remove confining liquid, and are washed with corresponding lavation buffer solution, and after the completion of washing, elisa plate is in 37 DEG C
Place 1 hour；
4) add sample to be tested, and incubate：Sampled from the solution of step 1), make sample to be tested；With the chromium of known content-
VLDL chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2
Hour；
5) enzyme conjugates incubates：Sample to be tested is removed, and is washed with lavation buffer solution, is waited after the completion of washing, is added
The enzyme labelled antibody diluted with dilution buffer, 37 DEG C of effect 1-2 hours, it is set to be reacted with enzyme labelled antibody；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with corresponding lavation buffer solution, is waited after the completion of washing, is added
Substrate, 37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction：Terminate liquid is to each micropore；
8) wavelength 450nm is taken, after adding terminate liquid, reads the OD of sample to be tested group and standard items respectively on ELIASA
Value, standard curve is drawn, by the content for compared with standard item group, trying to achieve sample to be tested.
In this method, also qualitative detection directly can be carried out by the situation that develops the color without using ELIASA.
Method five：Purify chromium-VLDL chelate and atomic absorption spectrum combined techniques (method of purification+AAS methods)
Chromium-VLDL chelate in blood sample is detected, is detected in accordance with the following steps：
1) non-specific VLDL is extracted from whole blood：Using supercentrifugation, HPLC, coagulate
The methods of glue filtration chromatography, gel electrophoresis, VLDL, and the extra-low density that will be extracted are extracted from whole blood
Lipoprotein redissolves, and obtains the solution of VLDL；
2) detect：Sample from the solution of step 1), chelated in Atomic Absorption Spectrometer detection in VLDL
On chromium, read respective value.
Method six：Purify chromium-VLDL chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification
+ ICP-MS methods) detection chromium-VLDL chelate, is detected in accordance with the following steps：
1) non-specific VLDL is extracted from whole blood：Using supercentrifugation, HPLC, coagulate
The methods of glue filtration chromatography, gel electrophoresis, VLDL, and the extra-low density that will be extracted are extracted from whole blood
Lipoprotein redissolves, and obtains the solution of VLDL；
2) it is acidified：Being sampled from the solution of step 1), add nitric acid in the solution and solution is acidified, sealing is stayed overnight,
Thoroughly acidifying；
3) detect：Add hydrogen peroxide, and heat catch up with acid after take 0.5mL solution, in inductivity coupled plasma mass spectrometry
Detection is chelated in the chromium in VLDL under instrument, reads respective value.
Method four, method five and method six are to isolate VLDL by whole blood extraction method, then using spy
Different in nature detection method, determine the content of chromium on chromium-VLDL chelate in VLDL；First use thing
Manage separation means, such as supercentrifugation, HPLC, gel-filtration chromatography, by VLDL from treating
Survey in plasma sample and separate and redissolve in physiological saline, recycle ELISA principles, atomic absorption spectrum detection or carry out
Detect the chromium content on inductively coupled plasma mass spectrometry detection chromium-VLDL chelate.
Method seven：Electrophoresis+ELISA/AAS/ICP-MS methods detect chromium-VLDL chelate, specific as follows：
1) non-specific VLDL is extracted from whole blood：Using supercentrifugation, HPLC, coagulate
The methods of glue filtration chromatography, gel electrophoresis, VLDL is extracted from whole blood, the extra-low density that will be extracted
Lipoprotein is redissolved in physiological saline, obtains the solution of VLDL；
2) glue bed is prepared：Suitable medium (such as Ago-Gel, polyacrylamide gel) is selected as needed, according to
Corresponding requirements prepare corresponding glue bed；
3) it is loaded：8 μ L solution are taken from the solution of step 1), with the chromium of known content-VLDL chelate
Make standard items, add 2 μ L sample-loading buffers, and mix, be then loaded onto in sample cell；
4) electrophoresis：Connect electrophoresis plate, add electrophoretic buffer, carry out electrophoresis, and according to demand by albumen according to molecular weight, etc.
The difference of the parameters such as electricity point is separated；
5) detect：The protein band containing chromium is found out on glue bed, the band is taken out, the protein band is dissolved, so
It is utilized respectively the principles such as ELISA or ICP-MS or AAS detection chromium content again afterwards.
Further, it is also possible to isoelectric point, molecular weight and the content of chromium-VLDL chelate are detected using the method
Deng.
In method seven, VLDL is extracted, then it is extremely low close to what is extracted using gel electrophoresis
Degree lipoprotein is separated, then finds out the respective strap rich in chromium, then detects the content of related VLDL；It is i.e. extremely low
After density lipoprotein discharges from red blood cell, can be come out with a variety of Methods For Purifications (such as supercentrifugation, high pressure liquid chromatography (HPLC)
Method, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), VLDL out will be purified and redissolved in molten
In liquid, a certain amount of VLDL is taken, using electric charge shifting principle, carries out electrophoresis (electrophoresis, EP),
Different bands can be run out of according to molecular weight, isoelectric point etc. are different on gel slab (different medium can be used as needed), sought
The respective strap rich in chromium is found out, the protein in gel is redissolved in solution, you can to detect correlation at a particular wavelength
The content of VLDL, the principles such as ELISA, AAS, ICP-MS can also be utilized to detect to chelate in extra-low density fat egg
Chromium content on white, due to only containing VLDL in solution, and it is (negative right without any heavy metal in agents useful for same
It is feminine gender according to group result), result will not be interfered, thus when the result read is shown as the positive, you can prove inspection
Measure the crome metal chelated in VLDL.
Embodiment 1：Synthetic method synthesizes chromium-VLDL chelant thing, that is, comprises the following steps：
Chromium-VLDL chelant thing prepared by the present embodiment, is further separated, and pass through by gel electrophoresis
Inductivity coupled plasma mass spectrometry or atomic absorption spectrum carry out detection Qualitative Identification.
Reagent is as follows used in the present embodiment：
1) borate buffer solution, its molar concentration are 0.01M, and its preparation method example is as follows：0.31g boric acid is weighed to be dissolved in
400mLddH2In O, pH to 9.0 is adjusted with 0.1mol/L NaOH, is settled to 500mL.
2)EDTA-NaHCO3Solution, its preparation method are as follows：Take 1.86g EDTA2H2O and 16.8g NaHCO3, it is dissolved in
900mLddH2In O, 1000mL, autoclaving, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
3) ITCBE is bought from Japanese colleague's chemistry institute, article No. M030；
4) VLDL solution：Weigh 4.0mg VLDL and be dissolved in 4.0mL0.01M pH9.0 boric acid
In salt buffer, fully vibration is dissolved, and is configured to 1.0mg/mL VLDL solution；
5) molecular cut off 14000 of bag filter, buy from Bioshop Inc；
The pretreatment of bag filter：Bag filter is put into 500mL EDTA-NaHCO3In solution, 10min is boiled；Tipping
EDTA-NaHCO3Solution, use ddH2O is gently rinsed, then boils 10min with 500mL5mmol/L EDTA；Boiling liquid is discarded, thoroughly
Use ddH2O is cleaned, and adds substantial amounts of ddH24 DEG C of O immersions bag filter is overnight.In use, put on one's gloves, bag filter is taken out, with a large amount of
DdH2Its surfaces externally and internally of O cleaning downs；
A) the synthesis of chromium-VLDL chelant thing：Purification come from human body VLDL or according to
Chromium ion is added in the VLDL of biological method restructuring and carries out chelatropic reaction, obtains reaction solution, concrete operations
Step is as follows；
1) 2.0mg ITCBE are taken to be dissolved in 2mLDMSO；
2) liquid for slowly preparing step 1 is added in VLDL solution, is shaken when being added dropwise, in 25 DEG C,
24h is acted in 100r/min shaking table, then with bag filter dialysis 24h, removes what is do not combined with VLDL
ITCBE；
3) liquid for gained of dialysing is adjusted into pH value to 7.0 with 1mol/L HCl, 80 μ l is then slowly gradually added dropwise
1mmol/L chromium ion solution, vibrated when being added dropwise, in case chromium ion precipitates albuminous degeneration；
4) solution added is reacted into 2h in 25 DEG C, 100r/min shaking table, dialysed with the bag filter handled well
24h；
5) liquid dialysed is preserved in -20 DEG C of packing, the reaction for obtaining chromium-VLDL chelate is molten
Liquid.
B) chromium-VLDL chelate purifying：Using immune-affinity chromatography, removal step A) in reaction solution
Unreacted VLDL, specific antibody and chromium ion, chromium-VLDL chelate is produced, specific bag
Include following steps：
(1) sample dissolution：By above-mentioned steps A) chromium-VLDL chelate be dissolved in physiological saline, obtain
The solution of chromium-VLDL chelate；
(2) chromatographic column is balanced：Using dilution buffer rinse chromatographic column pipeline, in chromatographic column load can with it is extremely low close
The filler of lipoprotein specific binding is spent, after filling post, is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with the solution of dilution buffer dilution step (1), then upper prop, makes extremely low
Density lipoprotein is specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L's
Na2HPO4Solution is eluted；
(5) collect：The eluent of collection step (4), restores albumen immediately after collection；
(6) dialyse：By the eluent of step (5), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C of dialysis
Overnight, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L's
Na2HPO4Solution is eluted；
(10) collect：The eluent of collection step (9), restores albumen immediately after collection；
(11) dialyse：By the eluent of step (10), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C thoroughly
Analysis overnight, collects sample, produces chromium-VLDL chelate.
C) to the identification of chromium-VLDL chelant thing, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using Ago-Gel as medium；
(2) it is loaded：Take step B) in the obtained chromium-VLDL chelate of extraction purification, redissolve in physiology salt
In water, the solution of chromium-VLDL chelate is obtained, takes 8 μ L previous solus, adds 2 μ L sample-loading buffers, and is mixed,
Then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, adds electrophoretic buffer to carry out electrophoresis；In electrophoresis process, electric current is 22mA constant currents, ring
Border temperature is 4 DEG C；Stop electrophoresis when moving to glue bottom to bromophenol blue, Fig. 1 is chromium of the present invention-VLDL chelant
The non denatured electrophoretic band figure of thing, M swimming lanes are denaturation Marker, and VLDL swimming lanes are VLDL；
(4) detect：The protein band containing chromium is found out on glue bed, the protein band is taken out, protein band is dissolved, so
The content of chromium is detected whether containing chromium and detected again afterwards using ICP-MS or AAS.
D) qualification result
1) AAS testing results
Take step C) the protein band solution isolated, it is extremely low with graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination
The content of heavy metal chromium, as shown in the table in density lipoprotein, using NaCl solution, KBr solution, KCl solution as blank control；
The content of chromium in the VLDL of table 1
| 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
4W1 " of the SRXRF analyses of micronutrient levels in BEPC (BEPC) is synchronous in protein band
Completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200
Type, Beijing are stood upright Han Guang companies) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into
Penetrate facula position, moving step length 0.0025mm.From the X ray that electromagnetic radiation goes out by Si (Li) detectors (PGT
Inc.LS30143-DS) detect, probe is with incident SR lines copline and being mutually perpendicular to, away from sample irradiation point 20mm, signal PGT
Multi-channel analyzer (MCA 4000) obtains output.Sample is excited with 11.5keV monochromatic synchrotron radiation light, adjusts launching spot
(1mmx 3mm) position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always,
Hot spot moves on to the band other end at the end of counting.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings,
And normalization is carried out to other element peaks with the Ar signal peaks from air and content constant, to offset beam intensity change
On influence caused by signal strength.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way under the same conditions.
Fig. 2 is the synchrotron radiation X line fluorescence point of the electrophoretic band of chromium of the present invention-VLDL chelate
Analysis is schemed, and abscissa is protein band position in figure, and ordinate is band chromium metal energy (content) value.
The determination of testing conditions
1. the optimum diluting multiple of anti-VLDL antibody, anti-Cr antibody best effort concentration and blood plasma is really
It is fixed.
The method that ELISA detects chromium-VLDL chelate, specifically includes following steps：
1) it is coated with：Anti- VLDL antibody protein is coated on solid phase carrier, respectively will with dilution buffer
Coating protein is with 1：500、1：1000、1：2000 and 1：4000 doubling dilution, add in elisa plate micropore, each concentration bag
By three rows, 4 DEG C preserve 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed；
3) sample to be tested is added：Test plasma sample is pressed 1 with dilution buffer：10、1：20、1：40 doubling dilution, adds
Enter in micropore, standard items made with the chromium of known content-VLDL chelate, negative control and blank control are set,
Add in micropore, 37 DEG C act on 1 hour；
4) secondary antibody is added：Test plasma sample is removed, is washed, addition presses 1 with dilution buffer：5000、1：10000、1：
20000、1：The anti-Cr antibody of 40000 doubling dilution, 37 DEG C act on 1 hour, make itself and the metal in VLDL
Chromium reacts；
5) enzyme-added mark：Anti- Cr antibody is removed, is washed, it is 2 μ g/mL's that addition is diluted to antibody content with dilution buffer
HRP enzyme labelled antibodies, 37 DEG C act on 1 hour, make itself and anti-Cr antibody responses；
6) substrate incubates：Enzyme labelled antibody is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, add terminate liquid；
7) detect：Read on ELIASA standard items, test plasma, positive control, negative right respectively under 450nm wavelength
According to the OD values with blank control sample.
In the present embodiment, using chromium provided by the invention-VLDL chelate standard items as positive control,
Respectively to be not added with test plasma as negative control 1, that is, anti-VLDL antibody, confining liquid, anti-Cr are sequentially added
Antibody, enzyme mark and substrate；
Check experiment group to be not added with anti-Cr antibody has sequentially added anti-VLDL as negative control 2
Antibody, confining liquid, test plasma, enzyme mark and substrate；
Using not enzyme-added target check experiment group as negative control 3, sequentially added anti-VLDL antibody,
Confining liquid, test plasma, anti-Cr antibody and substrate；
Check experiment group to be not added with sample to be tested and anti-Cr antibody simultaneously has sequentially added anti-as negative control 4
VLDL antibody, confining liquid, enzyme mark and substrate；
Using the check experiment group for being not added with anti-VLDL antibody work as blank control 1, that is, add confining liquid, treat
Survey blood plasma, anti-Cr antibody, enzyme mark and substrate；
And only to add the check experiment group of substrate as blank control 2, only to add PBS check experiment group as blank control
3。
Table 2 dilutes multiple proportions, diluted plasma multiple proportions, anti-Cr antibody for different anti-VLDL antibody and dilutes multiple proportions
Sample OD Value Datas,
Testing result under the different anti-VLDL antibody of table 2, anti-Cr antibody and diluted plasma multiple proportions
As known from Table 2, the dilution multiple proportions of anti-VLDL antibody is 1:When 1000, sample OD values are more than parallel strip
The dilution multiple proportions of other anti-VLDL antibody under part；In this group of sample, diluted plasma multiple proportions is 1:When 20, anti-Cr
Antibody dilution multiple proportions is 1:When 5000, OD values are maximum, are 0.707.
Table 3 is 1 for the dilution multiple proportions of anti-VLDL antibody:1000th, diluted plasma multiple proportions is 1:20th, anti-Cr resists
Body dilution multiple proportions is 1:Positive control, negative control and the OD of blank control detected values corresponding to 5000 phases,
The positive control of table 3, negative control and the testing result of blank control
As known from Table 3, negative control group OD detected values are less than 0.1, illustrate under the optimal conditions, the systematic error of this method
Property it is small, meet analysis method requirement, so selecting the concentration corresponding to this value as best effort concentration.
2.ELISA eluent best effort concentration and elution time determine
To seek optimum elution requirement, by ELISA after anti-Cr antibody and enzyme labelled antibody incubate, with not
Eluent with concentration is eluted, then detects OD values by ELIASA, is comprised the following steps that：
(1) it is coated with：Anti- Cr antibody proteins are coated on solid phase carrier, 1 is pressed with dilution buffer：5000 multiple proportions is dilute
Release, add in elisa plate micropore, 4 DEG C preserve 16 hours；
(2) close：Dilution buffer is removed, after washing, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and wash
Wash；
(3) enzyme-added labeling antibody：Confining liquid is removed, after washing, it is 2 μ g/ that addition is diluted to antibody concentration with dilution buffer
ML HRP enzyme labelled antibodies, 37 DEG C act on 2 hours, make itself and anti-Cr antibody responses；
(4) elute：Enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes Papain in eluent
The concentration of enzyme：Concentration=1 of antibody in enzyme labelled antibody:5、1:10、1:20、1:40、1:Eluent is added micropore by 80 multiple proportions
In, each concentration makees 3 multiple holes, is respectively placed in effect 1h, 2h, 3h at 37 DEG C；Eluent is removed, is washed, treats that washing is completed
Afterwards, substrate is added, 37 DEG C of lucifuges act on 30 minutes, add terminate liquid terminating reaction；
(5) the OD values of each micropore are read respectively on ELIASA under 450nm Detection wavelength, concrete outcome is referring to table
4,
Testing result under the different eluent extension rates of table 4
By comparing OD values, to judge the anti-Cr antibody-hrp-antibody complex elution degree combined on ELISA hole walls,
When OD values are minimum, anti-Cr antibody-hrp-antibody complex elution degree reaches maximum.As shown in table 4, when pawpaw in eluent
The concentration of protease：Concentration=1 of antibody in enzyme labelled antibody:When 20, elution degree is maximum；And action time is 1h, 2h, 3h
When, each group OD value changes are little, it is seen that with the extension of time, enzyme activity gradually weakens, in the case where enzyme concentration is constant, prolong
Long digestion time can not improve digestibility, thus in this experiment eluent action time for 1-3h all can, in summary, I
Select eluent 1:20 are used as most suitable working concentration, and 1-3h is used as most suitable elution time.
Application Example
Application Example 1
Chromium-VLDL chelate in 100 parts of sample blood plasma is detected using ELISA (ELISA method),
The method detection recorded using specific embodiment method one, concrete operation step are as follows：
1) it is coated with：Anti- VLDL antibody is coated on solid phase carrier, 1000 are diluted to dilution buffer
Times, add in elisa plate micropore, 37 DEG C preserve 1 hour after 4 DEG C of storages in refrigerator；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
37 DEG C of elisa plate places 1 hour after 4 DEG C of storages in refrigerator；
3) it is loaded：Using sample blood plasma as sample to be tested, marked with the chromium of known content-VLDL chelate
Quasi- product, sample to be tested and standard items are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
4) anti-Cr antibody is added：Sample to be removed, is washed, addition is diluted to 5000 times of anti-Cr antibody with dilution buffer, and 37
DEG C effect 1 hour, reacts itself and crome metal in VLDL；
5) enzyme-added mark：Anti- Cr antibody is removed, is washed, addition is diluted to the enzyme that antibody concentration is 2 μ g/mL with dilution buffer
Labeling antibody, 37 DEG C act on 1 hour, make itself and anti-Cr antibody responses；
6) substrate incubates：Enzyme labelled antibody is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, with adding substrate solution
Same speed and order are added dropwise to terminate liquid；
7) detect：The OD values of sample to be tested and standard items are read respectively on ELIASA under 450nm wavelength, as a result such as table
Shown in 5.
The measured result of 5 method of table, a pair of 100 parts of sample blood plasma
It in this application embodiment 1, in step 7), can also detect without using ELIASA, but directly be carried out by developing the color
Qualitative detection.
Application Example 2
Using enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detect chromium in 100 parts of sample blood plasma-
VLDL chelate, i.e., the method recorded using specific embodiment method two are detected, and concrete operation step is as follows：
1) it is coated with：Anti- VLDL antibody is coated on solid phase carrier, dilutes coating egg with dilution buffer
In vain to 1000 times, add in elisa plate micropore, 4 DEG C are stayed overnight 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
Preserved at 4 DEG C of elisa plate；
3) it is loaded：Sample to be tested is made with sample blood plasma, standard is made with the chromium of known content-VLDL chelate
Product, sample to be tested and standard items are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
4) elute：Test plasma sample is removed, is washed, adds 0.8mol/L Na2HPO4Solution, 37 DEG C act on 2 hours；
5) detect：Sampled from ELISA micropores, in Atomic Absorption Spectrometer detect chelated in sample to be tested and standard items in
Chromium in VLDL, as a result as shown in table 6.
Measured result of the method two of table 6 to 100 parts of sample blood plasma
Application Example 3：
100 parts of marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods)
Chromium-VLDL chelate in this blood plasma, i.e., the method recorded using specific embodiment method three are detected, concrete operations
Step is as follows：
1) it is coated with：Anti- VLDL antibody is coated on solid phase carrier, dilutes coating egg with dilution buffer
In vain to 1000 times, add in elisa plate micropore, 4 DEG C preserve 18 hours；
2) close：Dilution buffer is removed, is washed, adds confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, are washed,
4 DEG C of preservations of elisa plate；
3) it is loaded：Sample to be tested is made with sample blood plasma, standard is made with the chromium of known content-VLDL chelate
Product, sample to be tested and standard items are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 2 hours；
4) elute：Sample to be tested and standard items are removed, is washed, adds 0.8mol/L Na2HPO4Solution, 37 DEG C of elutions 2 are small
When；
5) it is acidified：Add nitric acid in the solution to be acidified solution, sealing overnight, is thoroughly acidified；
6) detect：Hydrogen peroxide is added, and heats and catches up with acid, samples to detect under icp mses and treats
Chelated in test sample sheet and standard items in the chromium in VLDL, as a result as shown in table 7.
Measured result of the method three of table 7 to 100 parts of sample blood plasma
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed scope.
Claims (6)
- A kind of 1. chromium-VLDL chelate, it is characterised in that the chromium-VLDL chelate be chromium from Son is formed with VLDL by sulfydryl or/and cysteine residues chelating；The preparation method of the chromium-VLDL chelate, comprises the following steps：A）The synthesis of chromium-VLDL：The VLDL of human body is come from purification or according to biological method Chromium ion is added in the VLDL of restructuring and carries out chelatropic reaction；B）Chromium-VLDL chelate purifying：Using immune-affinity chromatography, removal step A）It is not anti-in reaction solution VLDL, specific antibody and the chromium ion answered, produce chromium-VLDL chelate；The step B）In specifically include following steps：（1）Sample dissolution：By above-mentioned steps A）Chromium-VLDL chelate be dissolved in physiological saline, obtain chromium-pole The solution of low-density lipoprotein chelate；（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with extra-low density fat The filler that protein-specific combines, after filling post, it is continuing with dilution buffer balance chromatographic column；（3）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（1）Solution, then upper prop, makes extra-low density Lipoprotein is specifically bound with filler；（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.10mol/L Na2HPO4 Solution is eluted；（5）Collect：Collection step（4）Eluent, restore albumen immediately after collection；（6）Dialysis：By step（5）Eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C of dialysed overnights, Collect sample；（7）Balance chromatographic column：Using new chromatographic column, with dilution buffer flushing line, energy and chromium are loaded in the chromatographic column The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；（8）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（6）Middle sample, then upper prop；（9）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L Na2HPO4It is molten Liquid is eluted；（10）Collect：Collection step（9）Eluent, restore albumen immediately after collection；（11）Dialysis：By step（10）Eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C were dialysed At night, sample is collected, produces chromium-VLDL chelate.
- 2. chromium according to claim 1-VLDL chelate, it is characterised in that the step of its preparation method B）Also include step C afterwards）：Identification to chromium-VLDL chelate；Wherein, step C）In specifically include following steps：（1）Prepare glue bed：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；（2）Sample-adding：Take step B）The chromium that middle extraction purification obtains-VLDL chelate, sample-loading buffer is added, and Mix, be then loaded onto in sample cell；（3）Electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；（4）Detection：The protein band containing chromium is found out on glue bed, the protein band is taken out, protein band is dissolved, Ran Houzai The content of chromium is detected whether containing chromium and detected using ICP-MS or AAS.
- A kind of 3. chromium-VLDL chelate as claimed in claim 1 chromium-extra-low density in detection blood sample is prepared Application in the reagent or kit of lipoprotein chelate.
- 4. a kind of comprise at least chromium as claimed in claim 1-kit of the VLDL chelate as standard items.
- 5. kit according to claim 4, it is characterised in that also including coating buffer, it is extremely low that the coating buffer includes capture The material of density lipoprotein or the material for capturing crome metal.
- A kind of 6. method for quantitatively detecting chromium-VLDL chelate, it is characterised in that will with the right of known content Chromium-VLDL chelate described in 1 is asked to be detected as standard items using a pair of samples of following methods：Enzyme Linked immunosorbent assay, enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, carry Pure chromium-VLDL chelate is inhaled with enzyme linked immunological combined techniques, purification chromium-VLDL chelate and atom Receive spectrum combined techniques, purification chromium-VLDL chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme Connection is immunized or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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| CN201510413275.2A CN105044009B (en) | 2015-07-14 | 2015-07-14 | A kind of chromium VLDL chelate and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
| CN101281164A (en) * | 2008-02-27 | 2008-10-08 | 南京大学 | Method for preparing dimolecular modified nanometer probe as well as application thereof |
| CN102276719A (en) * | 2010-06-09 | 2011-12-14 | 中国科学院生物物理研究所 | Avian influenza virus single domain antibody, pentameric antibody and preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
| CN101281164A (en) * | 2008-02-27 | 2008-10-08 | 南京大学 | Method for preparing dimolecular modified nanometer probe as well as application thereof |
| CN102276719A (en) * | 2010-06-09 | 2011-12-14 | 中国科学院生物物理研究所 | Avian influenza virus single domain antibody, pentameric antibody and preparation and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 《六价铬间接竞争ELISA检测试剂盒的研制及新型一步法竞争ELISA的建立》;邹军辉;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20121015(第10期);正文第13-16页 * |
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