CN105021550B

CN105021550B - A kind of mercury HDL chelate and its preparation method and application

Info

Publication number: CN105021550B
Application number: CN201510411417.1A
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: CN105021550A

Abstract

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

Description

A kind of mercury-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 mercury-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 (chylomicron, 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.

But research shows that the quantity of HDL-C can not be with the degree of protection of angiocardiopathy into just Than that still can measure because wherein having occurred and that the HDL of structural change but lose normal function even The progress of atherosclerosis can be aggravated.And the quantity decline of HDL-C then can significantly prompt Atherosclerosis The protectiveness of change weakens, thus HDL is significant for coronary heart disease in the quantitative serum of detection, can be not only used for knowing in early days Do not move the danger of pulse atherosclerosis, and can be as the detection of lipid regulating drug therapeutic process and the finger of prognostic evaluation One of mark.

Mercury (Mercury, Hg) is a kind of ubiquitous heavy metal, is widely used in industry, agricultural, pharmaceutical sector, is one The very important environmental contaminants of kind.2011, (United States Agency for were affixed one's name in U.S.'s poisonous substance and disease registration Toxic Substances and Disease Registry, ATSDR) it has been included in material preferred list (Substance Priority List), and the World Health Organization (World Health Organization, WHO) is also already It is classified as one of chemicals of 10 big easily attractive health problems.

Mercury is considered as to human body and its harmful, can influence multiple tissues, organ and the system of human body, including angiocarpy System, respiratory system, nervous system, internal system, digestive system, urinary system, immune system etc., harm to the human body pole Greatly.The mechanism and its complexity of body injury are caused on mercury, is the research center of many scholars and expert.Generally research is thought at present Mercury, which is one, has the metal of its extremely strong sulfydryl binding ability, therefore speculates that it is in close relations between multiple proteins.It is many It is well known, executor of the protein as body multiple functions, respectively organized with body, organ dysfunction stablize it is closely bound up, and Mercury is all in close relations with the multiple proteins in body as a kind of metal, it is believed that correlation and mercury between mercury and protein Cause body injury closely bound up, the relation of research mercury and protein will be helpful to us and understand the specific machine that mercury causes body injury System.

Mercury is a kind of strong sulfydryl bonding agent, and mercury also shows huge to amino, phosphoryl, carboxyl and hydroxyl groups Affinity.Therefore, mercury can widely combine multiple proteins, and influence their function, suppress their activity.With right Hg toxicity research is goed deep into, and people gradually recognize that the interaction of mercury and albumen plays important angle during its intoxicating Color, it is considered to be understand the key point of the toxicity mechanism of mercury, therefore, the research to mercury and protein interaction just seems more Send out important.And the mechanism now concerning mercury and protein effect is only tip of the iceberg, the pass between also many protein and mercury It is not clear, thus it is most important for the relation between mercury and protein to strengthen research.

The content of the invention

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

The technical solution adopted for the present invention to solve the technical problems is：A kind of mercury-HDL chelate is provided, The mercury-HDL chelate be mercury 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 mercury-HDL chelate, i.e., external synthetic method, bag Include following steps：

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

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

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

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

(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；

(3) loading：After column equilibration is chromatographed, with the solution of dilution buffer dilution step (1), then upper prop, makes highly dense Lipoprotein is spent to specifically bind 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：Collection step 4) eluent, restore 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 mercury 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：Collection step 9) eluent, restore 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 mercury-HDL chelate.

Preferably, the preparation method of above-mentioned mercury-HDL chelate, in addition to below to mercury-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 mercury-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：Mercurous protein band is found out on glue bed, the protein band is taken out, protein band is dissolved, so The content of mercury is detected whether containing mercury and detected again afterwards using ICP-MS or AAS.

The present invention also provides a kind of mercury-HDL chelate described above and is preparing mercury 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 mercury described above-reagent of the HDL chelate as standard items Box.

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

The present invention also provides a kind of method for quantitatively detecting mercury-HDL chelate, with the above-mentioned of known content Mercury-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 mercury-highly dense Spend lipoprotein chelate and enzyme linked immunological combined techniques, purification mercury-HDL chelate and atomic absorption spectrum combined techniques, Mercury-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 mercury-HDL chelate first；

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

3. the present invention realizes the specific recognition of chelating type HDL and quantitative detection, so as to quantitative detection people The content of mercury chelating type HDL, is industrial area to evaluate the application of a regional mercury pollution degree in group's serum Mercury pollution horizontal provide indirect indexes.The mercury that the present invention establishes-HDL chelate quantitative detecting method it is accurate Spend high, reproducible.

Brief description of the drawings

Fig. 1 is the non denatured electrophoresis protein band figure of mercury of the present invention-HDL chelate；

Fig. 2 is the synchrotron radiation X line fluorescence analysis of the electrophoretic band of mercury 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 albumen 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 egg for capturing HDL In vain；

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 ox blood pure protein solution in vain, compound method example：0.1g ox bloods pure albumen in vain is taken, adds washing Buffer solution 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 mercury is to buy the anti-Hg of mouse from the article No. of Ba Ao get bio tech ltd is " AP7014 " MAb, the material specifically bound with mercury, anti-Hg antibody, secondary antibody, anti-mercury antibody described in implementation below are to capture The material of mercury；

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

Specifically, mercury-HDL chelate is by combining zinc fingers, sulfydryl, cysteine residues by mercury 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 mercury-HDL chelate, comprises the following steps：

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

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；

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

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

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

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

The kit of mercury-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 mercury, confining liquid, lavation buffer solution, enzyme labelled antibody, bottom Thing, terminate liquid, dilution buffer, standard items, negative control etc..

The kit of mercury-HDL chelate in a kind of detection blood sample, including extraction whole blood middle-high density fat egg Bai Suoxu extracts reagents, redissolve liquid, positive control, negative control etc..

The kit of mercury-HDL chelate in a kind of detection blood sample, including extraction whole blood middle-high density fat egg Bai Suoxu extracts reagents, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..

The kit of mercury-HDL chelate in a kind of detection blood sample, including extraction whole blood middle-high density fat egg Bai Suoxu extracts reagents, glue bed medium, liquid, sample loading buffer are redissolved, liquid needed for protein band mercurous on glue bed is dissolved, contains Have the coating buffer of material available for capture mercury, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, positive control, Negative control etc..

The kit of mercury-HDL chelate in a kind of detection blood sample, including needed for extraction HDL Extracts reagent, glue bed medium, liquid, sample loading buffer are redissolved, liquid needed for protein band mercurous on glue bed is dissolved, is positive right According to, negative control etc..

The kit of mercury-HDL chelate in a kind of detection blood sample, including as extraction HDL Required extracts reagent, glue bed medium, redissolve liquid, sample loading buffer, dissolve liquid, nitre needed for protein band mercurous on glue bed 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 HDL of heavy metal Hg Chelate or the BSA chelates for being chelated with heavy metal Hg；The negative control is dilution buffer.

Mentioned reagent box is used to detect mercury-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 mercury-HDL chelate, with the above-mentioned of known content Mercury-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 mercury-highly dense Spend lipoprotein chelate and enzyme linked immunological combined techniques, purification mercury-HDL chelate and atomic absorption spectrum combined techniques, Mercury-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 mercury-HDL chelate It is following several that what method can be listed have, but is not limited to following several.

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

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

4) material of mercury 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 2500-20000 times of anti-mercury antibody with dilution buffer, 37 DEG C of effect 1-2 hours, makes to resist Mercury antibody reacts with the mercury metal on HDL；

5) enzyme conjugates incubates：Anti- mercury 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 enzyme labelled antibody；

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 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 the situation that develops the color.

This method utilizes ELISA principles, can extract the specific HDL in whole blood, extract HDL upper part be chelated with heavy metal Hg, and the mercury on this part HDL can be by the special of anti-mercury 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 mercury is not contained, then will not be captured by the specific antibody of anti-mercury, also will not be with horseradish mistake The antibody of the enzymes such as oxide enzyme, alkaline phosphatase mark is captured, and mercury 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 mercury metal of conjunction.

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

1) it is coated with：The albumen 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, is diluted to 2000-16000 times with dilution buffer, adds in elisa plate micropore, 4 DEG C 16-18 hours overnight, 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 mercury 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 eluent, 1-3 hours are eluted at 37 DEG C；

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

The embodiment is captured using ELISA principles to HDL, and combines atomic absorption spectrum (AAS) instrument Detection is chelated in the mercury on HDL；Due to only containing HDL in solution, and be free of and appoint in agents useful for same What heavy metal (negative control group result is feminine gender), will not be interfered to result, thus is worked as read result and be shown as sun During property, you can prove to detect the mercury metal chelated on HDL.

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

1) it is coated with：The albumen of HDL will can be captured, such as anti-HDL antibody is coated in solid phase load On body, 2000-16000 times is diluted to dilution buffer, is added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C 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 mercury 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 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 mercury 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 mercury on HDL；ELISA principles are first used by mercury-high density Lipoprotein chelate extracts, then is carried out using sense couple plasma mass spectrometer to chelating in the mercury on HDL Quantitative detection；Due to only containing HDL in solution, and any heavy metal (negative control group knot is free of in agents useful for same Fruit is feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can prove to detect height The mercury metal chelated on density lipoprotein.

Method four：Mercury-HDL chelate is purified to detect with enzyme linked immunological combined techniques (method of purification+ELISA method) Mercury-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：Anti- mercury antibody is coated on solid phase carrier, dilutes anti-mercury antibody to 2500- with dilution buffer 20000 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 lavation buffer solution, and after the completion of washing, elisa plate places 1 in 37 DEG C Hour；

4) add sample to be tested, and incubate：Sampled from the solution of step 1), make sample to be tested；With the mercury 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：Testing sample 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；

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

2) detect：Sample from the solution of step 1), chelated in Atomic Absorption Spectrometer detection on HDL Mercury, read respective value.

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

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 mercury on HDL under instrument, reads respective value.

Method four, method five and method six are to isolate HDL, then using method for detecting specificity, measure In HDL on mercury-HDL chelate mercury content；First use physical separation means, such as ultracentrifugation Method, HPLC, gel-filtration chromatography etc., HDL is separated and answered from test plasma sample It is dissolved in physiological saline, recycles ELISA principles, atomic absorption spectrum detection or carry out detection inductivity coupled plasma mass spectrometry Mercury content on method detection mercury-HDL chelate.

Method seven：Electrophoresis+ELISA/AAS/ICP-MS methods detect mercury-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 solution 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 are taken from the solution of step 1), standard is made with the mercury of known content-HDL chelate Product, 2 μ L sample-loading buffers are added, and mixed, is 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：Mercurous protein band is found out on glue bed, the band is taken out, the protein band is dissolved, then The principles such as ELISA or ICP-MS or AAS detection mercury content is utilized respectively again.

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

In method seven, HDL is extracted, then using gel electrophoresis to the high density fat that is extracted Albumen is separated, then finds out the respective strap rich in mercury, then detects the content of related HDL；That is high density lipoprotein level Can be come out in vain with a variety of Methods For Purifications (such as supercentrifugation, HPLC, gel-filtration chromatography, gel electricity Swimming method, ELISA method etc.), HDL out will be purified and redissolved in solution, take a certain amount of high density lipoprotein level In vain, using electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, (can be as needed using difference in gel slab Medium) on can run out of different bands according to molecular weight, isoelectric point etc. are different, the respective strap rich in mercury is searched out, by gel In protein redissolve in solution, you can to detect the content of related HDL at a particular wavelength, can also profit Detect to chelate in the mercury content on HDL with principles such as ELISA, AAS, ICP-MS, due to only containing height in solution Density lipoprotein, and result will not be caused to do without any heavy metal (negative control group result is feminine gender) in agents useful for same Disturb, thus when the result read is shown as the positive, you can prove to detect the mercury metal chelated on HDL.

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

Mercury-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 mercury-HDL chelate, comprises the following steps：

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 0.1M 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) 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 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 mercury-HDL chelant thing：The HDL of human body is come from purification or according to biology Mercury 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 mercury ion solution, vibrated when being added dropwise, in case mercury 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 mercury-HDL chelate.

C) to the identification of mercury-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 mercury-HDL chelate of extraction purification, redissolve in physiological saline In, the solution of mercury-HDL chelate is obtained, takes the 8 above-mentioned solution of 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 mercury of the present invention-HDL chelant thing Non denatured electrophoretic band figure, M swimming lanes for denaturation Marker, HDL swimming lanes are HDL；

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 Hg in lipoprotein is spent, it is as shown in the table, using NaCl solution, KBr solution, KCl solution as blank control；

The content of mercury in the HDL of table 1

Sample name	Hg(μg/L)
		Sample to be tested	0.149
NaCl	0.000
		KBr	0.055
KCl	0.031

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 analysis of the electrophoretic band of mercury of the present invention-HDL chelate Scheme, abscissa is protein band position in figure, and ordinate is band mercury metal energy (content) value.

The determination of testing conditions

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

The method that ELISA detects mercury-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 and 1：16000 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 are made with the mercury of known content-HDL chelate, negative control and blank control are set, added Enter 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：2500、1：5000、1： 10000、1：The anti-Hg antibody of 20000 doubling dilution, 37 DEG C act on 1 hour, make itself and the mercury metal on HDL Reaction；

5) enzyme-added mark：Anti- Hg antibody is removed, is washed, it is 2 μ g/mL's that addition is diluted to antibody concentration with dilution buffer HRP enzyme labelled antibodies, 37 DEG C act on 1 hour, make itself and anti-Hg 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 mercury 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-Hg antibody, Enzyme mark and substrate；

Check experiment group to be not added with anti-Hg 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-Hg antibody and substrate；

Check experiment group to be not added with sample to be tested and anti-Hg 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-Hg 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-Hg 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-Hg antibody and diluted plasma multiple proportions

As known from Table 2, the dilution multiple proportions of anti-HDL antibody is 1:When 8000, 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-Hg antibody It is 1 to dilute multiple proportions:When 5000, OD values are maximum, are 0.731.

Table 3 is 1 for the dilution multiple proportions of anti-HDL antibody:8000th, diluted plasma multiple proportions is 1:20th, anti-Hg antibody It is 1 to dilute multiple proportions: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-Hg 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- Hg antibody is coated on solid phase carrier, 1 is pressed with dilution buffer：5000 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, addition is diluted to 2 μ g/mL HRP enzyme marks with dilution buffer Antibody, 37 DEG C act on 2 hours, make itself and anti-Hg antibody responses；

(4) elute：Enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes the dense of papain in eluent Degree：Concentration=1 of antibody in enzyme labelled antibody:5、1:10、1:20、1:40、1:80, eluent is added in micropore, each concentration Make 3 multiple holes, be respectively placed in effect 1h, 2h, 3h at 37 DEG C；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-Hg antibody-hrp-antibody complex elution degree combined on ELISA hole walls, When OD values are minimum, anti-Hg antibody-hrp-antibody complex elution degree reaches maximum.As shown in table 4, when pawpaw in eluent The concentration of protease：The multiple proportions of the concentration of antibody is 1 in enzyme labelled antibody:20, OD values are less than other test groups, and elution degree is most Greatly.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 weakens, In the case where enzyme concentration is constant, digestibility can not be improved by extending digestion time, so in this experiment during the effect of eluent Between for 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

Mercury-HDL chelate in 100 parts of sample blood plasma is detected using ELISA (ELISA method), i.e., The method recorded using specific embodiment method one is detected, and concrete operation step is as follows：

1) it is coated with：Anti- HDL antibody is coated on solid phase carrier, 8000 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 mercury of known content-HDL 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) secondary antibody is added：Sample is removed, is washed, addition is diluted to 5000 times of anti-Hg antibody, 37 DEG C of works with dilution buffer With 1 hour, it is set to be reacted with the mercury metal on HDL；

5) enzyme-added mark：Anti- Hg antibody is removed, is washed, addition is diluted to the enzyme mark that concentration is 2 μ g/mL with dilution buffer and resisted Body, 37 DEG C act on 1 hour, make itself and anti-Hg antibody responses；

6) substrate incubates：Enzyme labelled antibody is removed, is washed, adds substrate, 37 DEG C of lucifuges act on 30min, are added dropwise to terminate liquid；

7) detect：Read sample to be tested group and the OD values of standard items respectively on ELIASA under 450nm wavelength, as a result such as Shown in table 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 mercury 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 8000 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 mercury of known content-HDL 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：Test plasma sample is removed, is washed, adds 0.8mol/L Na₂HPO₄Solution, 37 DEG C act on 2 hours；

5) detect：Sample from ELISA micropores, chelated in Atomic Absorption Spectrometer detection on HDL Mercury, 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) Mercury-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 8000 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；

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：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：Sampling, detect and chelated in the mercury on HDL, knot under icp mses Fruit is 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

A kind of 1. mercury-HDL chelate, it is characterised in that the mercury-HDL chelate be mercury ion with HDL is formed by sulfydryl or/and cysteine residues chelating；The method of the mercury-HDL chelate, Comprise the following steps：

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

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

The step B）In specifically include following steps：

（1）Sample dissolution：By above-mentioned steps A）Mercury-HDL chelate be dissolved in physiological saline, obtain mercury-highly dense Spend the solution of lipoprotein chelate；

（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）Solution, then upper prop, makes high density fat Albumen 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 mercury 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 mercury-HDL chelate.
2. mercury according to claim 1-HDL chelate, it is characterised in that the step B of its preparation method） Also include step C afterwards）：Identification to mercury-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 mercury 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：Mercurous protein band is found out on glue bed, the protein band is taken out, protein band is dissolved, Ran Houzai The content of mercury is detected whether containing mercury and detected using ICP-MS or AAS.
A kind of 3. mercury-HDL chelate as claimed in claim 1 mercury-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 mercury 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 albumen of lipoprotein or capture the material of mercury metal.
A kind of 6. method for quantitatively detecting mercury-HDL chelate, it is characterised in that with the claim of known content Mercury-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 Mercury-HDL chelate and enzyme linked immunological combined techniques, purification mercury-HDL chelate and atomic absorption spectrum Combined techniques, purification mercury-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.