CN105044008B

CN105044008B - A kind of cadmium HDL chelate and its preparation method and application

Info

Publication number: CN105044008B
Application number: CN201510413246.6A
Authority: CN
Inventors: 张积仁; 阳帆; 董欣敏; 吴婧; 蔡睿; 孙遥; 赵乙木
Original assignee: Shanghai Baihao Biotechnology Co Ltd
Current assignee: Guangzhou Jinhai Health Technology Co ltd
Priority date: 2015-07-14
Filing date: 2015-07-14
Publication date: 2018-01-05
Anticipated expiration: 2035-07-14
Also published as: CN105044008A

Abstract

The present invention provides a kind of cadmium HDL chelate and its preparation method and application, the cadmium HDL chelate is that cadmium ion is formed with HDL by sulfydryl or/and cysteine residues chelating, available for the reagent for preparing detection Cadmium Body HDL chelate.The present invention confirms that cadmium ion can be done directly on HDL first.The present invention establishes the method for qualitative and quantitative detection of cadmium HDL chelate, to detect the content of HDL in regional crowd's body, so as to reflect a regional cadmium pollution degree and the health effect to crowd indirectly.Cadmium HDL chelate quantitative detecting method degree of accuracy Gao Chong that the present invention establishes, renaturation are good.

Description

A kind of cadmium-HDL chelate and its preparation method and application

Technical field

The present invention relates to the immunology detection of heavy metal ion, and in particular to a kind of cadmium-HDL 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 (chylomiCdon, CM).30 about percent cholesterol is thin from tissue by HDL in blood Liver is transported in born of the same parents to be converted into bile acid or excrete by bile.HDL by 50% albumen, 30% Phosphatide, 25% cholesterol (wherein 70% is cholesteryl ester), 5% triacylglycerol composition.

HDL (abbreviation HDL) is one of major lipoprotein in blood plasma, its density highest, and particle diameter is minimum, The heterogeneous lipoprotein of albumen and lipid content approximate equality.By 50% albumen, 30% phosphatide, 25% cholesterol (its In 70% be cholesteryl ester), 5% triacylglycerol composition.

HDL is the major lipids protection factor of atherosclerosis occurrence and development, and it has very strong in haemocyanin Suppress Atherosclerosis, be the important protective factors of coronary artery disease, it is negatively correlated with coronary heart disease risk degree.

Cadmium is that one kind common are noxious metals and environmental contaminants, is widely used in industrial production environment, including plating, Process industry pigment, stabilizer for plastics, Ni-Cr battery etc., lived with people closely bound up.For general population, mainly pass through The air of cadmium pollution is sucked, and crops (such as rice containing cadmium) mode of the edible production of rural sewage disposal farm containing cadmium takes in cadmium. Research shows that cadmium is a kind of Systematic toxin, can cause a variety of organs and angiocarpy such as the lungs of humans and animals, liver, kidney, exempt from The multisystem function damage such as epidemic disease, nerve, also there is stronger carcinogenesis.1993 International Cancer Research Institute (IARC) will Cadmium and its compound are classified as the 1st class people's carcinogenic substance.It can be seen that influence of the cadmium to body and its important, very important.

Existing substantial amounts of document confirms the albumen that cadmium can be acted in each system of whole body at present, each so as to influence body The function of histoorgan.It is well known that HDL is a kind of protein of Special Significance, whether cadmium can act on height Density lipoprotein, and change HDL structural change, content change, activity change, function, these still lack correlation Research.

The content of the invention

For cadmium pollution it is serious the problem of, it is an object of the invention to provide a kind of cadmium-HDL chelate and Its preparation method, and establish the qualitative and quantitative analysis method of cadmium-HDL chelate, so as to quantitative detection cadmium-highly dense Spend lipoprotein chelate and evaluate the application of a regional cadmium pollution degree.By in one regional crowd's serum of quantitative detection Cadmium-HDL chelate, situation of this regional crowd by cadmium pollution can be reflected indirectly, so as to reflect this indirectly Regional cadmium pollution degree.

The technical solution adopted for the present invention to solve the technical problems is：A kind of cadmium-HDL chelate is provided, The cadmium-HDL chelate be cadmium ion with HDL by sulfydryl or/and cysteine residues chelate and Into.

The present invention also provides a kind of preparation method of above-mentioned cadmium-HDL chelate, i.e., external synthetic method, bag Include following steps：

A) the synthesis of cadmium-HDL：The HDL of human body is come from purification or according to biological method Cadmium ion is added in the HDL of restructuring and carries out chelatropic reaction, obtains reaction solution；

B) cadmium-HDL chelate purifying：Using immune-affinity chromatography, removal step A) in reaction solution not HDL, specific antibody and the cadmium ion of reaction, produce cadmium-HDL chelate.

Preferably, the step B) in specifically include following steps：

(1) sample dissolution：By above-mentioned steps A) cadmium-HDL chelate of extraction be dissolved in physiological saline In, obtain cadmium-HDL chelate solution；

(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and high density are loaded in chromatographic column The filler of lipoprotein specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；

The filler that can be specifically bound with HDL can be tied to be adsorbed with HDL specificity The silica gel or resin of compound matter；

(3) loading：After column equilibration is chromatographed, with dilution buffer dilution step (1) solution, then upper prop, makes height 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：The eluent of collection in step (5) is filled into bag filter, uses ddH₂O dialysis desalinations, after changing water three times, 4 DEG C of dialysed overnights, collect sample；

(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of cadmium specific binding, chromatographic column is balanced with dilution buffer again after filling post；

The filler that can be specifically bound with cadmium is to be adsorbed with silica gel or resin that material can be specifically bound with cadmium；

(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：The eluent of step (10) is filled into bag filter, uses ddH₂O dialysis desalinations, after changing water three times, 4 DEG C thoroughly Analysis overnight, collects sample, produces cadmium-HDL chelate.

Preferably, the preparation method of above-mentioned cadmium-HDL chelate, in addition to below to cadmium-high density fat The authentication step of protein chelates thing, 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 cadmium-HDL chelate of extraction purification, add sample-loading buffer, 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 cadmium is found out on glue bed, the protein band is taken out, protein band is dissolved, so The content of cadmium is detected whether containing cadmium and detected again afterwards using ICP-MS or AAS.

The present invention also provides a kind of cadmium-HDL chelate described above and is preparing cadmium in detection human body-highly dense The application spent in the reagent or kit of lipoprotein chelate.

The present invention, which also provides, a kind of comprises at least cadmium described above-reagent of the HDL chelate as standard items Box.

Preferably, coating buffer is also included in the kit, the material or catch that the coating buffer contains capture HDL Obtain the material of cadmium metal.

The present invention also provides a kind of method for quantitatively detecting cadmium-HDL chelate, with the above-mentioned of known content Cadmium-HDL chelate as standard items, detected using a pair of samples of following methods：ELISA, Enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification cadmium-highly dense Spend lipoprotein chelate and enzyme linked immunological combined techniques, purification cadmium-HDL chelate and atomic absorption spectrum combined techniques, Cadmium-HDL chelate is purified to inhale with inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atom Receive 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 cadmium-HDL chelate first；

2. it can be used for preparing cadmium-high density fat in detection blood sample present invention firstly provides cadmium-HDL chelate Application in the reagent or kit of protein chelates thing；

3. the present invention realizes-specific recognition of HDL chelate and quantitative detection, so as to quantitative detection The content of cadmium-HDL chelate in crowd's serum, to evaluate the application of a regional cadmium pollution degree, for industry ground The cadmium pollution in area is horizontal to provide indirect indexes.The standard of the cadmium that the present invention establishes-HDL chelate quantitative detecting method Exactness is high, reproducible.

Brief description of the drawings

Fig. 1 is the non denatured electrophoretic band figure of cadmium of the present invention-HDL chelate；

Fig. 2 is the fluorescence analysis of the synchrotron radiation X line electrophoresis strip of cadmium of the present invention-HDL chelate 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 HDL is anti-HDL antibody, can by commercially available acquisition, as Ai Bokang (on Sea) trade Co., Ltd sell " Abcam ab157595 " anti-HDL antibody, in implementation below, it is described with it is highly dense Material, anti-HDL antibody, the anti-HDL antibody of degree lipoprotein specific binding are the thing for capturing HDL Matter；

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 cadmium is that article No. is " bar proud anti-Cd mAb of mouse for obtaining AP7015 "；Wherein, it is of the present invention special with cadmium Material, anti-Cd antibody, secondary antibody, the Cadmium resistance antibody of opposite sex combination are the material for capturing cadmium；

The present invention provides a kind of cadmium-HDL chelate, cadmium ion and HDL by sulfydryl or/and Cysteine residues chelating forms.

Specifically, cadmium-HDL chelate is by combining zinc fingers, sulfydryl, cysteine residues by cadmium At least one of apolipoprotein B on structure and HDL or cholesterol, triglycerides etc. combine and the chela of formation Compound.

The present invention also provides the preparation method of cadmium-HDL chelate, comprises the following steps：

B) cadmium-HDL chelate purifying：Using immune-affinity chromatography, removal step A) in reaction solution not HDL, specific antibody and the cadmium ion of reaction, cadmium-HDL chelate is produced, is specifically included following Step：

C)：Identification to cadmium-HDL chelate, specifically includes following steps：

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 cadmium-HDL chelate in a kind of detection blood sample, including:Containing available for capturing high density The coating buffer of lipoprotein, in addition to confining liquid, lavation buffer solution, can capture the material of cadmium as secondary antibody, enzyme labelled antibody, substrate, Terminate liquid, dilution buffer, positive control, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including:Containing available for capturing high density The coating buffer of lipoprotein, in addition to confining liquid, lavation buffer solution, eluent, positive control, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including:Containing available for capturing high density The coating buffer of lipoprotein, in addition to confining liquid, lavation buffer solution, eluent, acidulant, hydrogen peroxide, standard items, negative control Deng.

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, redissolve liquid, the coating buffer containing the material available for capture cadmium, confining liquid, lavation buffer solution, enzyme labelled antibody, bottom Thing, terminate liquid, dilution buffer, standard items, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, redissolve liquid, positive control, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, liquid needed for the protein band containing cadmium on dissolving glue bed, containing available It is right in the coating buffer of the material of capture cadmium, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, positive control, feminine gender According to etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, be liquid needed for the protein band containing cadmium on dissolving glue bed, positive right According to, negative control etc..

The kit of cadmium-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, dissolve liquid, nitric acid, mistake needed for the protein band containing cadmium on glue bed Hydrogen oxide, positive control, negative control etc..

In above-mentioned several kits, the positive control is standard items, that is, is chelated with the HDL of heavy metal cadmium Chelate or the BSA chelates for being chelated with heavy metal cadmium；The negative control is the dilution eluent for not containing standard items.

Mentioned reagent box is used to detect cadmium-HDL chelate, to improve the accuracy and repeatability of detection, and It is allowed to be promoted in clinic.

The present invention also provides a kind of method for quantitatively detecting cadmium-HDL chelate, with the above-mentioned of known content Cadmium-HDL chelate as standard items, detected using a pair of samples of following methods：ELISA, Enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification cadmium-highly dense Spend lipoprotein chelate and enzyme linked immunological combined techniques, purification cadmium-HDL chelate and atomic absorption spectrum combined techniques, Cadmium-HDL chelate is purified to inhale with inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atom Receive spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, with the side of detection cadmium-HDL chelate It is following several that what method can be listed have, but is not limited to following several.

Dilution multiple proportions as described below is w/v.

Method one：ELISA (ELISA method) detects cadmium-HDL chelate, detects in accordance with the following steps：

1) it is coated with：The material that can capture HDL with dilution buffer dilution adds to 2000-16000 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 cadmium-high density of known content Lipoprotein chelate makees standard items；Sample to be tested and standard items are diluted to 10-40 times with dilution buffer, add to micropore In, 37 DEG C of effect 1-2 hours；

4) material of cadmium 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 12500-100000 times of Cadmium resistance antibody with dilution buffer, 37 DEG C of effect 1-2 hours, makes Cadmium resistance antibody and the metal cadmium reaction on HDL；

5) enzyme conjugates incubates：Cadmium resistance 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：Sample to be tested 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 added dropwise 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 staining conditions.

This method utilizes ELISA principles, can extract the specific HDL in whole blood, extract HDL upper part be chelated with heavy metal cadmium, and the cadmium on this part HDL can be by the special of Cadmium resistance Property antibody captured, afterwards can again by the enzymes such as horseradish peroxidase, alkaline phosphatase mark antibody capture the (antibody Nonrecognition coating protein), the antibody in capture can read OD values in the presence of developer and terminate liquid under instrument, and The HDL of chelated mineral cadmium is not contained, then will not be captured by the specific antibody of Cadmium resistance, also will not be with horseradish mistake The antibody of the enzymes such as oxide enzyme, alkaline phosphatase mark is captured, and cadmium metal (negative control group is not contained in agents useful for same yet As a result it is feminine gender), thus when the OD value results read are shown as the positive, you can prove to detect chela on HDL The cadmium metal of conjunction.

Method two：Enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection cadmium-HDL Chelate detects in accordance with the following steps：

1) it is coated with：The material of HDL will can be captured, such as anti-HDL antibody (anti-high density lipoprotein level Bai Kangti) it is coated on solid phase carrier, dilutes anti-HDL antibody to 2000-16000 times with dilution buffer, 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 cadmium of known content-highly dense Degree lipoprotein chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 hours；

4) elute：Sample to be tested is removed, is washed, adds the eluent that extension rate is 5-80, it is small that 1-3 is eluted at 37 DEG C When；

5) detect：Sample from ELISA micropores, chelated in Atomic Absorption Spectrometer detection on HDL Cadmium, read respective value；

The embodiment is captured using ELISA principles to the HDL in serum, and combines atomic absorption light Spectrum (AAS) instrument detection is chelated in the cadmium on HDL；Due to only containing HDL, and agents useful for same in solution In without any heavy metal (negative control group result for feminine gender), result will not be interfered, thus work as read result When being shown as the positive, you can prove to detect the cadmium metal chelated on HDL.

Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection cadmium- HDL chelate detects in accordance with the following steps：

1) it is coated with：The material of HDL will can be captured, such as anti-HDL antibody is coated in solid phase load On body, anti-HDL antibody is diluted to 2000-16000 times with dilution buffer, is added in elisa plate micropore, 4 DEG C of mistakes Hour at night 16-18, or 37 DEG C of water-bath 1-3 hours, store refrigerator；

3) add sample to be tested, and incubate：Whole blood is taken from the circulatory system, makees sample to be tested；With the cadmium of known content-highly dense Degree lipoprotein chelate makees standard items；10-40 times is diluted to dilution buffer, is added in micropore, 37 DEG C of effect 1-2 hours；

4) elute：Sample to be tested is removed, and is washed with corresponding lavation buffer solution, is waited after the completion of washing, adds dilution 5-80 times of eluent, 1-3 hours are eluted at 37-57 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 cadmium chelated in HDL 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 cadmium on HDL；I.e. first using ELISA principles by serum Cadmium-HDL chelate extracts, then using sense couple plasma mass spectrometer to chelating in HDL On cadmium carry out quantitative detection；Due to only containing HDL in solution, and it is (cloudy without any heavy metal in agents useful for same Property control group result be feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can card The bright cadmium metal for detecting to chelate on HDL.

Method four：Cadmium-HDL chelate is purified to detect with enzyme linked immunological combined techniques (method of purification+ELISA method) Cadmium-HDL chelate, is detected in accordance with the following steps：

1) non-specific HDL is extracted from whole blood：Using supercentrifugation, HPLC, gel The methods of filtration chromatography, gel electrophoresis, HDL, and the HDL that will be extracted are extracted from whole blood Redissolve, obtain the solution of HDL；

2) it is coated with：Cadmium resistance antibody is coated on solid phase carrier, dilutes Cadmium resistance antibody to 12500- with dilution buffer 100000 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 cadmium of known content- HDL 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；

5) enzyme conjugates incubates：The redissolution liquid of HDL is removed, and is washed with lavation buffer solution, waits to wash After the completion of, enzyme labelled antibody that addition is diluted with dilution buffer, 37 DEG C of effect 1-2 hours, react itself and enzyme labelled antibody；

6) substrate incubates：Sample to be tested 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；

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 directly qualitative detection can be carried out by staining conditions without using ELIASA.

Method five：Cadmium-HDL chelate is purified to examine with atomic absorption spectrum combined techniques (method of purification+AAS methods) Cadmium-HDL chelate in blood sample is surveyed, is detected in accordance with the following steps：

2) detect：Sample from the redissolution liquid of step 1), chelated in Atomic Absorption Spectrometer detection in HDL On cadmium, read respective value.

Method six：Purify cadmium-HDL chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ ICP-MS methods) detection cadmium-HDL chelate, is detected in accordance with the following steps：

2) it is acidified：Sampled from the redissolution liquid of step 1), add nitric acid in the solution and solution is acidified, sealed Night, 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 cadmium on HDL under instrument, reads respective value.

Method four, method five and method six are to isolate HDL by whole blood extraction method, then using special Property detection method, determine the content of cadmium on cadmium-HDL chelate in HDL；First use physical separation Means, such as supercentrifugation, HPLC, gel-filtration chromatography, by HDL from test plasma sample Separate and redissolve in physiological saline in this, recycle ELISA principles, atomic absorption spectrum detection or carry out detection inductance Cadmium content on coupled plasma mass detection cadmium-HDL chelate.

Method seven：Electrophoresis+ELISA/AAS/ICP-MS methods detect cadmium-HDL chelate, specific as follows：

1) non-specific HDL is extracted from whole blood：Using supercentrifugation, HPLC, gel The methods of filtration chromatography, gel electrophoresis, HDL is extracted from whole blood, the HDL that will be extracted Redissolve in physiological saline, obtain the redissolution liquid of HDL；

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 redissolution liquid of step 1), with the cadmium of known content-HDL 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 cadmium is found out on glue bed, the band is taken out, the protein band is dissolved in liquid Among, the cadmium content that the detection of the principles such as ELISA, ICP-MS or AAS is dissolved in liquid is then utilized respectively again.

Further, it is also possible to isoelectric point, molecular weight and the content of cadmium-HDL chelate are detected using the method Deng.

In method seven, HDL is extracted from whole blood, then using gel electrophoresis to being extracted HDL is separated, then finds out the respective strap rich in cadmium, then detects the content of related HDL；It is i.e. high 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.), HDL out will be purified and redissolved in solution In, a certain amount of HDL is taken, using electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, solidifying Different bands can be run out of according to molecular weight, isoelectric point etc. are different on offset plate (different medium can be used as needed), searched out Respective strap rich in cadmium, the protein in gel is redissolved in solution with corresponding double solvents, you can with a particular wavelength The content of related HDL is detected, the principles such as ELISA, AAS, ICP-MS can also be utilized to detect to chelate in high density Cadmium content on lipoprotein, due to only containing HDL 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 cadmium metal chelated on HDL.

In method four, method five, method six and method seven, the example of non-specific HDL is extracted from whole blood It is as follows：

A) whole blood is taken, density is adjusted to 1.21g/mL, 50000rpm ultracentrifugation 24h with NaBr, it is light yellow to collect lower floor Component；

B) three kinds of solution that density is respectively d=1.006, d=1.182, d=1.478 are configured to neutral salt；

C) take 4mL step a) blood plasma to capacity be 10mL ultracentrifugation pipe in bottom, above bedding 2mLd=1.006 salt Solution, 4 DEG C of 2000rap/min centrifuge 30min, it is stand-by to collect lower floor's 4mL pooled plasmas；

D) 2mL1.006 salting liquids are added to step c) 4mL pooled plasmas, 18 DEG C of 40000rap/min centrifuge 17h, taken Layer 1mL, i.e. VLDL；

E) liquid on 1mL upper stratas is discarded, shifts residual mixed liquor, adds 2mLd=1.182 salting liquids, is mixed, 18 DEG C 40000rpm/min centrifuges 21h, takes out upper strata 1mL mixed liquors, this is low-density lipoprotein；

F) discard the liquid on 1mL upper stratas, shift residual mixed liquor, add 2mL1.478 salting liquids, 18 DEG C of 40kr/min from Heart 41h, take upper solution, i.e. HDL；

The neutral salt is more than one or both of NaBr, KBr, NaCl or KCl.

Embodiment 1：Synthetic method synthesizes cadmium-HDL chelant thing, that is, comprises the following steps：

Cadmium-HDL chelant thing prepared by the present embodiment, is further separated, and pass through electricity by gel electrophoresis Sense coupled plasma mass spectrometry or atomic absorption spectrum carry out detection Qualitative Identification.

The method for preparing cadmium-HDL chelate, comprises the following steps：

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-NaHCO₃Mixed liquor, its preparation method are as follows：Take 1.86g EDTA2H₂O and 16.8g NaHCO₃, it is molten 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) HDL solution：Weigh 4.0mg HDLs and be dissolved in 4.0mL0.01M pH9.0 borates and delay In fliud flushing, fully vibration is dissolved, and is configured to 1.0mg/mL HDL 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 mixed liquor, 10min is boiled；Tipping EDTA-NaHCO₃Liquid, use ddH₂O is gently rinsed, then boils 10min with 500mL5mmol/L EDTA；Boiling liquid is discarded, is thoroughly used 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 substantial amounts of ddH₂Its surfaces externally and internally of O cleaning downs；

A) the synthesis of cadmium-HDL chelant thing：The HDL of human body is come from purification or according to biology Cadmium ion is added in the HDL of method restructuring and carries out chelatropic reaction, obtains reaction solution, concrete operation step is such as Under；

1) 2.0mg ITCBE are taken to be dissolved in 2mLDMSO；

2) liquid for slowly preparing step 1 is added in HDL 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 the ITCBE not combined with HDL；

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 cadmium-ion solutions, vibrated when being added dropwise, in case cadmium 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, obtains the reaction solution of cadmium-HDL chelate.

C) to the identification of cadmium-HDL 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 cadmium-HDL chelate of extraction purification, redissolve in physiological saline In, the redissolution liquid of cadmium-HDL chelate is obtained, takes the above-mentioned redissolution liquid of 8 μ L, adds 2 μ L sample-loading buffers, and mix, 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 degree；Stop electrophoresis when moving to glue bottom to bromophenol blue, Fig. 1 is cadmium of the present invention-HDL chelant thing Non denatured electrophoretic band figure；

D) qualification result

1) AAS testing results

Take step C) the protein band solution isolated, it is highly dense with graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination The content of heavy metal cadmium in lipoprotein is spent, it is as shown in the table, using NaCl solution, KBr solution, KCl solution as blank control；

The content of cadmium in the HDL of table 1

Sample name	Cd(μg/L)
		Sample to be tested	0.364
NaCl	0.023
		KBr	0.000
KCl	0.000

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 fluorescence analysis of the synchrotron radiation X line electrophoresis strip of cadmium of the present invention-HDL chelate Scheme, abscissa is protein band position in figure, and ordinate is each metal energy of the band (content) value.

The determination of testing conditions

1. the optimum diluting multiple of anti-HDL antibody, anti-Cd antibody best effort concentration and blood plasma is really It is fixed.

The method that ELISA detects cadmium-HDL chelate, specifically includes following steps：

1) it is coated with：Anti- HDL antibody protein is coated on solid phase carrier, will be wrapped with dilution buffer respectively By albumen with 1:2000、1:4000、1:8000、1:16000 doubling dilution, add in elisa plate micropore, each concentration coating 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 buffer solution dilution：10、1：20、1：40 doubling dilution, adds Into micropore, standard items are made with the cadmium of known content-HDL chelate, negative control and blank control are set, added Into micropore, 37 DEG C act on 1 hour；

4) anti-Cd antibody is added：Test plasma sample is removed, is washed, addition presses 1 with dilution buffer:12500、1:25000、 1:50000、1:The anti-Cd antibody of 100000 doubling dilution, 37 DEG C act on 1 hour, make itself and the metal on HDL Cadmium reaction；

5) enzyme-added mark：Anti- Cd antibody is removed, is washed, the HRP enzyme labelled antibodies that addition is diluted with dilution buffer, 37 DEG C of effects 1 hour, make itself and anti-Cd antibody responses；

6) substrate incubates：Sample to be tested 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, standard curve is drawn, tries to achieve the content of sample to be tested.

In the present embodiment, using cadmium provided by the invention-HDL chelate standard items as positive control, divide Not to be not added with test plasma as negative control 1, that is, sequentially added anti-HDL antibody, confining liquid, anti-Cd antibody, Enzyme mark and substrate；

Check experiment group to be not added with anti-Cd antibody has sequentially added anti-HDL and resisted as negative control 2 Body, confining liquid, test plasma, enzyme mark and substrate；

Using not enzyme-added target check experiment group as negative control 3, anti-HDL antibody, envelope are sequentially added Close liquid, test plasma, anti-Cd antibody and substrate；

Check experiment group to be not added with sample to be tested and anti-Cd antibody simultaneously has sequentially added anti-as negative control 4 HDL antibody, confining liquid, enzyme mark and substrate；

To be not added with check experiment group that anti-HDL antibody makees as blank control 1, that is, add confining liquid, to be measured Blood plasma, anti-Cd 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 the sample of multiple proportions, diluted plasma multiple proportions, anti-Cd antibody dilution multiple proportions for different anti-HDL antibody This OD Value Datas,

Testing result under the different anti-HDL antibody of table 2, anti-Cd antibody and diluted plasma multiple proportions

As known from Table 2, the dilution multiple proportions of anti-HDL antibody is 1:When 4000, sample OD values are more than parallel condition Under other anti-HDL antibody dilution multiple proportions；In this group of sample, diluted plasma multiple proportions is 1:When 20, anti-Cd antibody It is 1 to dilute multiple proportions:When 25000, OD values are maximum, are 0.591.

Table 3 is 1 for the dilution multiple proportions of anti-HDL antibody:4000th, diluted plasma multiple proportions is 1:20th, anti-Cd antibody It is 1 to dilute multiple proportions:The OD detected values of positive control, negative control and blank control when 25000,

The positive control of table 3, negative control and the testing result of blank control

As known from Table 3, tested under the conditions of determined by table 2, negative control group OD detected values are less than 0.1, explanation Under the optimal conditions, the systematic error of this method is small, meets analysis method requirement, so selecting the concentration conduct corresponding to this value Best effort concentration.

2.ELISA eluent best effort concentration and the determination of time

To seek optimum elution requirement, by ELISA after anti-Cd 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- Cd antibody is coated on solid phase carrier, 1 is pressed with dilution buffer：25000 doubling dilution, adds Enter 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-Cd 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:80、1:40、1:20、1:10、1:5, make at a temperature of being respectively placed in 37 DEG C With 1h, 2h, 3h；Eluent is removed, washs, waits after the completion of washing, adds substrate, 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-Cd antibody-hrp-antibody complex elution degree combined on ELISA hole walls, When OD values are minimum, anti-Cd 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, anti-Cd antibody-hrp-antibody complex elution degree reaches It is maximum；And action time, when being 1h, 2h, 3h, each group OD value changes are little, it is seen that with the extension of time, enzyme activity gradually subtracts It is weak, in the case where enzyme concentration is constant, extend digestion time can not improve digestibility, so in this experiment eluent effect Time be 1-3h all can, in summary, we select eluent 1:20 are used as most suitable working concentration, when 1-3h is as most suitable elution Between.

Application Example

Application Example 1

Cadmium-HDL chelate is extracted using whole blood extraction method, detected using ELISA (ELISA method) Cadmium-HDL chelate in 100 parts of sample blood plasma, i.e., the method recorded using specific embodiment method one are detected, Concrete operation step is as follows：

1) it is coated with：Anti- HDL antibody is coated on solid phase carrier, 4000 times are diluted to dilution buffer, 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, standard is made with the cadmium of known content-HDL chelate Product, diluted with dilution buffer and sample to be tested and standard items are diluted to 20 times, added in micropore, 37 DEG C act on 1 hour；

4) anti-Cd antibody is added：Sample to be removed, is washed, addition is diluted to 25000 times of anti-Cd antibody with dilution buffer, and 37 DEG C effect 1 hour, make itself and the metal cadmium reaction on HDL；

5) enzyme-added mark：Anti- Cd antibody is removed, is washed, adds the enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C of effects 1 are small When, make itself and anti-Cd antibody responses；

6) substrate incubates：Sample to be tested is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, enter terminate liquid；

7) detect：Read sample to be tested group and the OD values of standard items respectively on ELIASA under 450nm wavelength, cross and mark Quasi- product group compares, and the content of the middle cadmium of sample to be tested is tried to achieve according to normal equation, as a result as shown in table 5.

The measured result of 5 method of table, a pair of 100 parts of plasma samples

Numbering	1	2	3	4	5	6	7	8	9	10
											OD	0.534	0.562	0.403	0.582	0.524	0.418	0.592	0.308	0.596	0.504
Numbering	11	12	13	14	15	16	17	18	19	20
											OD	0.326	0.589	0.454	0.462	0.529	0.627	0.426	0.319	0.517	0.511
Numbering	21	22	23	24	25	26	27	28	29	30
											OD	0.487	0.365	0.594	0.443	0.327	0.518	0.513	0.534	0.219	0.515
Numbering	31	32	33	34	35	36	37	38	39	40
											OD	0.566	0.458	0.611	0.122	0.529	0.53	0.426	0.322	0.505	0.508
Numbering	41	42	43	44	45	46	47	48	49	50
											OD	0.513	0..411	0.512	0.509	0.416	0.329	0.23	0.523	0.543	0.556
Numbering	51	52	53	54	55	56	57	58	59	60
											OD	0.615	0.358	0.374	0.412	0.53	0.529	0.491	0.439	0.732	0.36
Numbering	61	62	63	64	65	66	67	68	69	70
											OD	0.514	0.526	0.379	0.213	0.532	0.434	0.524	0.516	0.552	0.611
Numbering	71	72	73	74	75	76	77	78	79	80
											OD	0.545	0.458	0.46	0.402	0.461	0.514	0.581	0.569	0.31	0.561
Numbering	81	82	83	84	85	86	87	88	89	90
											OD	0.359	8	0.546	0.415	0.148	0.334	0.555	0.587	0.3569	0.61
Numbering	91	92	93	94	95	96	97	98	99	100
											OD	0.597	0.576	0.4768	0.323	0.562	0.619	0.403	0.577	0.227	0.32

It in this application embodiment 1, in step 7), can also detect without using ELIASA, but directly be carried out by dyeing Qualitative detection.

Application Example 2

Using enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detect cadmium in 100 parts of sample blood plasma- HDL 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- HDL antibody is coated on solid phase carrier, dilutes coating protein with dilution buffer To 4000 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 cadmium of known content-HDL chelate Product, diluted with dilution buffer and sample to be tested and standard items are diluted to 20 times, added in micropore, 37 DEG C of effect 1-2 hours；

4) elute：Test plasma sample is removed, is washed, adds 0.8mol/L Na₂HPO₄Solution, 37 DEG C act on 2 hours；

5) detect：Appropriate amount of fluid is taken from ELISA micropores, is chelated in Atomic Absorption Spectrometer detection in HDL On cadmium, as a result as shown in table 6.

Measured result of the method two of table 6 to 100 parts of plasma samples

Numbering	1	2	3	4	5	6	7	8	9	10
											μg/L	0.244	0.206	0.177	0.129	0.1	0.29	0.259	0.293	0.184	0.215
Numbering	11	12	13	14	15	16	17	18	19	20
											μg/L	0.234	0.071	0.165	0.145	0.334	0.126	0.261	0.203	0.117	0.129
Numbering	21	22	23	24	25	26	27	28	29	30
											μg/L	0.109	0,205	0.268	0.236	0.265	0.254	0.275	0.104	0.183	0.085
Numbering	31	32	33	34	35	36	37	38	39	40
											μg/L	0.232	0.131	0.247	0.372	0.276	0.237	0.169	0.26	0.274	0.319
Numbering	41	42	43	44	45	46	47	48	49	50
											μg/L	0.153	0.224	0.171	0.146	0.141	0.195	0.288	0.241	0.212	0.295
Numbering	51	52	53	54	55	56	57	58	59	60
											μg/L	0.333	0.231	0.205	0.213	0.239	0.236	0.167	0.137	0.177	0.258
Numbering	61	62	63	64	65	66	67	68	69	70
											μg/L	0.242	0.273	0.175	0.188	0.319	0.228	0.257	0.296	0.214	0.237
Numbering	71	72	73	74	75	76	77	78	79	80
											μg/L	0.201	0.108	0.122	0.121	0.314	0.203	0.178	0.126	0.232	0.244
Numbering	81	82	83	84	85	86	87	88	89	90
											μg/L	0.343	0.398	0.183	0.235	0.323	0.285	0.227	0.146	0.157	0.127
Numbering	91	92	93	94	95	96	97	98	99	100
											μg/L	0.266	0.297	0.264	0.112	0.144	0.137	0.396	0.324	0.266	0.185

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) Cadmium-HDL chelate in this blood plasma, i.e., the method recorded using specific embodiment method three are detected, concrete operations step It is rapid as follows：

1) it is coated with：Anti- HDL antibody is coated on solid phase carrier, dilutes coating protein with dilution buffer To 4000 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 cadmium of known content-HDL chelate Product, diluted with buffer solution and sample to be tested and standard items are diluted to 20 times, added in micropore, 37 DEG C act on 2 hours；

4) elute：Sample to be tested and standard items are removed, is washed, 0.8mol/L Na₂HPO₄Solution, 37 DEG C elute 2 hours；

5) it is acidified：Nitric acid is added in the solution to be acidified solution, sealing overnight, thoroughly acidifying, adds hydrogen peroxide, And heat and catch up with acid；

6) detect：The detection under icp mses is sampled to chelate in the cadmium on HDL, knot Fruit is as shown in table 7.

Measured result of the method three of table 7 to 100 parts of samples

Numbering	1	2	3	4	5	6	7	8	9	10
											μg/L	0.273	0.222	0.265	0.143	0.158	0.176	0.236	0.375	0.207	0.263
Numbering	11	12	13	14	15	16	17	18	19	20
											μg/L	0.291	0.149	0.338	0.134	0.265	0.209	0.264	0.299	0.259	0.146
Numbering	21	22	23	24	25	26	27	28	29	30
											μg/L	0.103	0.147	0.154	0.244	0.256	0.223	0.225	0.182	0.187	0.159
Numbering	31	32	33	34	35	36	37	38	39	40
											μg/L	0.189	0.108	0.124	0.163	0.224	0.296	0.255	0.324	0.217	0.299
Numbering	41	42	43	44	45	46	47	48	49	50
											μg/L	0.216	0.247	0.219	0.161	0.194	0.221	0.171	0.233	0.245	0.276
Numbering	51	52	53	54	55	56	57	58	59	60
											μg/L	0.123	0.137	0.152	0.157	0.291	0.285	0.231	0.269	0.213	0.111

Numbering	61	62	63	64	65	66	67	68	69	70
											μg/L	0.234	0.235	0.192	0.154	0.165	0.215	0.216	0.247	0.172	0.167
Numbering	71	72	73	74	75	76	77	78	79	80
											μg/L	0.288	0.176	0.141	0.228	0.246	0.241	0.144	0.157	0.851	0.179
Numbering	81	82	83	84	85	86	87	88	89	90
											μg/L	0.243	0.248	0.256	0.173	0.241	0.266	0.209	0.199	0.28	0.278
Numbering	91	92	93	94	95	96	97	98	99	100
											μg/L	0.122	0.134	0.175	0.244	0.24	0.287	0.26	0.137	0.145	0.377

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

A kind of 1. cadmium-HDL chelate, it is characterised in that the cadmium-HDL chelate be cadmium ion with HDL is formed by sulfydryl or/and cysteine residues chelating；

The preparation method of described cadmium-HDL chelate, comprises the following steps：

A）The synthesis of cadmium-HDL：The HDL of human body is come from purification or is recombinated according to biological method HDL in add cadmium ion carry out chelatropic reaction, obtain reaction solution；

Concrete operation step is as follows；

1）2.0mg ITCBE are taken to be dissolved in 2mLDMSO；

2）Slowly by step 1）The liquid of preparation is added in HDL solution, is shaken when being added dropwise, in 25 DEG C, 100r/ 24h is acted in min shaking table, then with bag filter dialysis 24h, removes the ITCBE not combined with HDL；

3）The liquid for gained of dialysing is adjusted into pH value to 7.0 with 1mol/L HCl, 80 μ l 1mmol/L are then slowly gradually added dropwise Cadmium-ion solution, vibrated when being added dropwise, in case cadmium ion precipitates albuminous degeneration；

4）The solution added is reacted into 2h in 25 DEG C, 100r/min shaking table, dialysis 24h is carried out with the bag filter handled well；

5）The liquid dialysed is preserved in -20 DEG C of packing, obtains the reaction solution of cadmium-HDL chelate；

B）Cadmium-HDL chelate purifying：Using immune-affinity chromatography, removal step A）Unreacted in reaction solution HDL, specific antibody and cadmium ion, produce cadmium-HDL chelate；

The step B）In specifically include following steps：

（1）Sample dissolution：By above-mentioned steps A）Cadmium-HDL chelate of extraction be dissolved in physiological saline, obtain Cadmium-HDL chelate solution；

（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, energy and high density lipoprotein level are loaded in chromatographic column The filler specifically bound in vain, 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）The solution, then upper prop, makes high 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）In collection eluent dress bag filter, use ddH₂O dialysis desalinations, after changing water three times, 4 DEG C thoroughly Analysis overnight, collects sample；

（7）Balance chromatographic column：Using new chromatographic column, with dilution buffer flushing line, energy and cadmium 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 dress bag filter, use ddH₂O dialysis desalinations, after changing water three times, 4 DEG C were dialysed At night, sample is collected, produces cadmium-HDL chelate.
2. cadmium according to claim 1-HDL chelate, it is characterised in that step B）Also include step afterwards C）：Identification to cadmium-HDL 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 cadmium that middle extraction purification obtains-HDL chelate, sample-loading buffer is added, and mixed It is even, then it is loaded onto in sample cell；

（3）Electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；

（4）Detection：The protein band containing cadmium is found out on glue bed, the protein band is taken out, protein band is dissolved, Ran Houzai The content of cadmium is detected whether containing cadmium and detected using ICP-MS or AAS.
A kind of 3. cadmium-HDL chelate as claimed in claim 1 cadmium-high density lipoprotein level in detection blood sample is prepared Application in the reagent or kit of white chelate.
4. a kind of comprise at least cadmium as claimed in claim 1-kit of the HDL chelate as standard items.
5. kit according to claim 4, it is characterised in that also including coating buffer, it is highly dense that the coating buffer contains capture Spend the material of lipoprotein or capture the material of cadmium metal.
A kind of 6. method for quantitatively detecting cadmium-HDL chelate, it is characterised in that with the claim of known content Cadmium-HDL chelate described in 1 is detected as standard items using a pair of samples of following methods：It is enzyme-linked to exempt from Epidemic disease method, enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification Cadmium-HDL chelate and enzyme linked immunological combined techniques, purification cadmium-HDL chelate and atomic absorption spectrum Combined techniques, purification cadmium-HDL chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or Atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.