CN104987391B - Mercury-albumin chelate and preparation method and application thereof - Google Patents

Mercury-albumin chelate and preparation method and application thereof Download PDF

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CN104987391B
CN104987391B CN201510413056.4A CN201510413056A CN104987391B CN 104987391 B CN104987391 B CN 104987391B CN 201510413056 A CN201510413056 A CN 201510413056A CN 104987391 B CN104987391 B CN 104987391B
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mercury
albumin
chelate
dilution buffer
solution
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CN104987391A (en
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张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
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Guangzhou Jinhai Health Technology Co ltd
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Shanghai Baihao Biotechnology Co ltd
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA

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Abstract

The invention discloses a mercury-albumin chelate and a preparation method and application thereof, wherein the mercury-albumin chelate is prepared by chelating mercury ions and albumin through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of mercury-albumin chelate so as to quantitatively detect the application of the mercury-albumin chelate in evaluating the mercury pollution degree of one area. The condition that the population in a region is polluted by mercury can be indirectly reflected by quantitatively detecting the mercury-albumin chelate in the serum of the population in the region, so that the mercury pollution degree in the region is indirectly reflected. The accuracy of the quantitative detection method of the mercury-albumin chelate is greatly improved, and the repeatability of detection is greatly improved.

Description

A kind of mercury-albumin chelate and its preparation method and application
Technical field
The present invention relates to detection field, more specifically to a kind of mercury-albumin chelate and preparation method thereof and answers With.
Background technology
Seralbumin (HSA) is the protein that content is most abundant in mammalian plasma, and it can store and transport crowd More endogenous and exogenous material, this is closely related with its specificity structure.Seralbumin is the one kind synthesized by liver Simple protein, only it is made up of amino acid, without modification group and other adjuncts.Shown according to data information, ox blood is pure Albumen (BSA) molecule is made up of 583 amino acid residues.And human serum albumins (HSA) 2 amino acid more than BSA, i.e., 585 Individual amino acid residue composition.Both difference is the residue of BSA missing HSA amino acid sequences 116 and 585.Albumin The most significant feature of aminoacid ingredient is the tryptophan of low content, contains 1 or 2 residue respectively in HSA and BSA per molecules.Benzene Alanine and isoleucine content are also than relatively low.Cysteine, leucine, glutamic acid and lysine content are all very high.Largely Ionization residue provide the very high electric charge of albumin, under conditions of PH7, per molecule albumin carries 185 ions, Thus form albumin ease of solubility.
Albumin it is most unique be structurally characterized in that disulfide bond pattern.BSA sequences include 35 cysteine residues, form 17 Individual disulphide bridges, adjacent cysteine pair link with residue nearest on chain, form a pair of rings of 10-47 residues in length.S-S Key is folded the rigidity and stability for adding albumin structure.
The tertiary structure of albumin is considered as a kind of molecule of high degree of spiral, is learnt by X-ray diffraction in HHG crystal About 67% residue participates in foring 28 conveyor screws of α mono-, and 10% residue participates in forming β-bend, and 23% residue forms stretching, extension Chain.
On physiological mechanism, albumin maintains the 80% of human plasma colloid osmotic pressure.In addition, albumin is also each in blood The main carriers of kind macromolecular substances, played an important role in metabolin transhipment, bioconversion.It is simultaneously and regulation is solidifying Blood function and the component of inflammatory reaction.Human serum albumins is mainly used in clinical, metabolism and the research of genetic aspect.
Mercury (Hgckel, Hg) is healthy closely bound up with human body, and it is that human body maintains micro member necessary to health first Element, it is present in a variety of hydrogenases, is catalyzed the redox reaction of hydrogen, participate in a variety of zymoproteins synthesis and cytohormone and The metabolism of pigment, there is the absorption for promoting iron and the growth of red blood cell, kinase to form coenzyme, enhancing insulin, hypoglycemic, guarantor Shield heart blood vessel etc. acts on, so if human body lacks mercury, can cause the generation of disease.But it is true in life, due to environment Pollute, mercury is seldom lacked in human body, the content of opposite mercury is usually excessive, frequently results in the generation of disease, or even threat to life.
Mercury common are noxious metals as one kind, among being widely used in living, producing, including production coin, jewelry, mercury Stainless steel alloy, manufacture Hg-Cd batteries etc., it is closely bound up with the life of people.With the development of science and technology people are using mercury as urging Among production of the agent for producing carbon nano-particle, influence of the mercury for the mankind is further deepened.For general population, Mercury mainly by eat, drink into, contact etc. mode take in mercury, certain smoking is also an important channel.Research is found, excessive Mercury can cause body many places tissue and organ damage, cause the generation of a variety of diseases, including respiratory disease (pneumonia, branch gas Guan Yan, pulmonary fibrosis, asthma, pulmonary edema etc.), angiocardiopathy, kidney trouble, disease of skin etc., or even also result in tumour Generation.
Epidemiologic data shows that the worker of Long Term Contact mercury, it is suffered from the probability of tumor in respiratory system and greatly increased, and It is also greatly increased because of the lethal probability of tumor in respiratory system.Animal model, external model experiment, it was also found that mercury can induce it is swollen The generation of knurl.Nineteen ninety, international cancer research institution (International Agency for Research on Cancer, IARC) using mercury compound as first kind carcinogen (Group 1), using mercury metal as 2B class carcinogens (Group 2B).It can be seen that the health of mercury serious threat human body.
It is in close relations between mercury and multiple proteins, it can be combined with multiple proteins, suppress the activity of multiple protein enzyme. As what Hg toxicity was studied gos deep into, people gradually recognize that the interaction of mercury and albumen is played the part of emphatically during its intoxicating The role wanted, it is considered to be understand the key point of the toxicity mechanism of mercury, therefore, the research to mercury and protein interaction is just Seem ever more important.And the mechanism now concerning mercury and protein effect is only tip of the iceberg, also many protein and mercury it Between relation it is not clear, thus strengthen it is most important for the relation between mercury and protein.
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-albumin chelate and its preparation Method, and the qualitative and quantitative analysis method of mercury-albumin chelate is established, so that quantitative detection mercury-albumin chelate is being commented The application of the regional mercury pollution degree of valency one.Can be with by mercury-albumin chelate in one regional crowd's serum of quantitative detection Reflect the mercury contaminated situation of this regional crowd indirectly, so as to reflect this regional mercury pollution degree indirectly.
The technical solution adopted for the present invention to solve the technical problems is：A kind of mercury-albumin chelate, mercury ion are provided Formed with albumin by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned mercury-albumin chelate, comprises the following steps：
A) the chelatropic reaction of mercury and albumin：The albumin of human body is come from purification or according to the white of biological method restructuring Mercury ion is added in albumen and carries out chelatropic reaction, obtains reaction solution；
B the extraction of mercury-albumin chelate) is purified：Using immune-affinity chromatography, remove unreacted in reaction solution Albumin and unnecessary mercury ion, produce mercury-albumin chelate.
Wherein, step B described in external synthetic method) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution makes mercury-albumin chelate Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of 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 phosphoric acid Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces mercury-albumin chelate.
Wherein, the preparation method of above-mentioned mercury-albumin chelate, in addition to step C)：To the mirror of mercury-albumin chelate It is fixed；
Wherein, step C) in it is specific as follows：
(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-albumin chelate of extraction purification, add dilution buffer, and mix It is even, 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 and dissolved, is then used again ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
The present invention also provides a kind of mercury-albumin chelate described above and is preparing mercury in detection human body-albumin chelating Application in the reagent or kit of thing.
The present invention, which also provides, a kind of comprises at least mercury described above-kit of the albumin chelate as standard items.
Preferably, coating buffer is also included in the kit, the material or capture mercury that the coating buffer contains capture albumin Material.
The present invention also provides a kind of method for quantitatively detecting mercury-albumin chelate, with the above-mentioned mercury of known content-white Protein chelates thing is detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and atom Absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification mercury-albumin chelate with it is enzyme-linked Immune combined techniques, purification mercury-albumin chelate and atomic absorption spectrum combined techniques, purification mercury-albumin chelate and inductance Coupled plasma mass spectrometry combined techniques, electrophoresis are combined with enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry Method.
Implement mercury-albumin chelate of the present invention and its preparation method and application, have the advantages that：
1. the present invention synthesizes mercury-albumin chelate in vitro first；
2. present invention firstly provides mercury-albumin chelate can be used for preparing mercury-albumin chelate in detection blood sample Application in reagent or kit.
3. the present invention establishes the method for qualitative and quantitative detection of mercury-albumin chelate, so as to quantitative detection mercury-albumin Chelate is evaluating the application of a regional mercury pollution degree.Pass through mercury-albumin in one regional crowd's serum of quantitative detection Chelate can reflect the mercury contaminated situation of this regional crowd indirectly, so as to reflect this regional mercury pollution degree indirectly. Its degree of accuracy of the mercury that the present invention establishes-albumin chelate quantitative detecting method greatly improves, and makes the repeated of detection To greatly improving.
Brief description of the drawings
Fig. 1 is the non denatured electrophoretic band figure of mercury of the present invention-albumin chelate；
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of mercury of the present invention-albumin chelate.
Embodiment
Below, 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 as following cited, do not include in the present invention come be reagent commonly used in the art or can be by mode purchased in market Obtain：
Phosphate solution is 0.05-0.1mol/L Na2HPO4Solution or 0.5-1mol/L Na2HPO4Solution；
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
Glue bed medium is one kind in Ago-Gel, polyacrylamide gel；
The material for capturing albumin is the model ab106349 of Abcam companies production Anti- mankind Serum Albumin antibody；Wherein, it is of the present invention equal with material, anti-HSA antibody, the anti-albumin antibodies of albumin specific binding To capture the material of albumin；
The material purchase of mercury is captured from the model AP7014 of the Ba Ao get bio tech ltd anti-HgmAb of mouse；Its In, anti-material, anti-Hg antibody, anti-mercury antibody, the mercury of the specific binding of of the present invention and mercury are the material for capturing mercury；
Enzyme labelled antibody is one kind in the antibody containing the enzymes such as horseradish peroxidase, alkaline phosphatase mark；
Substrate is methyl biphenyl amine (TMB) solution；
Cleaning solution is to contain KH2PO40.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl8.0mg/ml、KCl 0.2mg/ml, 0.5%Tween-20 pH are 7.4 0.15M PBS solutions；
Confining liquid is 1%-5% bovine serum albumin(BSA)s or skimmed milk power；
Dilution buffer is Na containing 1.5mg/mL2CO3、2.93mg/ml NaHCO3PH be 9.6 0.05M carbonate delay Fliud flushing；
Enzyme labelled antibody is HRP enzyme labelled antibodies；
Terminate liquid is：By 21.7ml 2M H2SO4It is settled to 200ml ddH2In O；
Eluent is the 0.1mol/L Tris-HCL buffer solutions that the PH containing papain is 8.0；
Acidulant is nitric acid；
Sample-loading buffer is to contain 1M Tris-HCl (pH 6.8) 15.5mL, 1% bromophenol blue 2.5mL, ddH2It is O7mL, sweet Propylhomoserin 25mL Sample buffer (5X)；
Electrophoretic buffer is the ddH that 3mg/ml containing Tris, glycine 14.4mg/ml PH are 6.82O solution.
The present invention provides a kind of mercury-albumin chelate, and mercury ion passes through sulfydryl or/and cysteine residues with albumin Chelating forms.
The present invention also provides a kind of preparation method of mercury-albumin chelate, comprises the following steps：
A) the chelatropic reaction of mercury and albumin：Mercury ion is added in the albumin in people source and carries out chelatropic reaction, is obtained anti- Answer solution；
B the extraction of mercury-albumin chelate) is purified：Using immune-affinity chromatography, remove unreacted in reaction solution Albumin and unnecessary mercury ion, mercury-albumin chelate is produced, is comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution makes mercury-albumin chelate Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；It is described to be specifically bound with albumin Filler be silica gel or resin containing anti-albumin antibodies；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of mercury specific binding, chromatographic column is balanced with dilution buffer again after filling post；It is described to be filled out with what mercury was specifically bound Expect for silica gel or resin containing anti-mercury antibody；
(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 phosphoric acid Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water After three times, 4 DEG C of dialysed overnights, sample is collected, produces mercury-albumin chelate；
C) to the identification of mercury-albumin chelate, comprise the following steps that：
(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-albumin chelate of extraction purification, add dilution buffer, and mix It is even, 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 and dissolved, is then used again ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
The present invention, which also provides, a kind of comprises at least mercury described above-kit of the albumin chelate as standard items.
Preferably, coating buffer is also included in the kit, the material or capture mercury that the coating buffer contains capture albumin Material.
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-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin Liquid, confining liquid, cleaning solution, the material for capturing mercury, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, sun as secondary antibody Property control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin Liquid, confining liquid, cleaning solution, eluent, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin Liquid, confining liquid, cleaning solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood Reagent is taken, liquid is redissolved, is the coating buffer of material containing capture mercury, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilute Release buffer solution, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood Take reagent, redissolve liquid, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood Take reagent, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin Liquid, glue bed medium, redissolve liquid, sample-loading buffer, dissolve liquid needed for protein band mercurous on glue bed, the thing containing capture mercury The coating buffer of matter, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood Take reagent, glue bed medium, redissolve liquid, sample-loading buffer, liquid needed for mercurous protein band on dissolving glue bed, positive control, Negative control etc..
The kit of mercury-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood Reagent, glue bed medium are taken, liquid, sample-loading buffer is redissolved, dissolves liquid, acidulant, mistake needed for protein band mercurous 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 albumin chelate of heavy metal Hg Or it is chelated with the BSA chelates of heavy metal Hg；The negative control is the dilution buffer for not containing standard items.
Mentioned reagent box is used for the albumin for detecting chelating mercury, to improve the accuracy of detection, repeatability, and is allowed to facing It is promoted in bed.
The present invention also provides a kind of method for quantitatively detecting mercury-albumin chelate, with the above-mentioned mercury of known content-white Protein chelates thing is detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and atom Absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification mercury-albumin chelate with it is enzyme-linked Immune combined techniques, purification mercury-albumin chelate and atomic absorption spectrum combined techniques, purification mercury-albumin chelate and inductance Coupled plasma mass spectrometry combined techniques, electrophoresis are combined with enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry Method.In the present invention, the having of can listing of method with detection mercury-albumin chelate is following several, but is not limited to following It is several.
Wherein, the reagent used in the above-mentioned method for quantitatively detecting mercury-albumin chelate is as follows：
Method one：ELISA (ELISA method) detects mercury-albumin chelate, detects in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With Anti- HSA Ab are diluted to 1000-8000 times by dilution buffer, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp 5-8 minutes are centrifuged, centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) material of mercury can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed After the completion of washing, addition is diluted to 2500-20000 times of anti-HgAb with dilution buffer, 37 DEG C of effect 1-2 hours, makes anti-Hg Ab React to form antigen antibody complex with the mercury metal on albumin；
6) enzyme conjugates incubates：Anti- mercury antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute Release the enzyme labelled antibody of buffer solution dilution, the concentration for making the enzyme labelled antibody of dilution is 2 μ g/mL, 37 DEG C of effect 1-2 hours, make its with Enzyme labelled antibody reacts；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore；
9) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA Colour developing situation carries out qualitative detection).
This method utilizes ELISA principles, can extract the specific albumin in whole blood, the white egg extracted White upper part is chelated with heavy metal Hg, and the mercury on this part albumin is captured by anti-mercury antibody, afterwards can be again by horseradish The antibody of the enzymes such as peroxidase, alkaline phosphatase mark captures (the antibody nonrecognition coating protein), the antibody in capture In the presence of developer and terminate liquid, OD values can be read under instrument, and do not contain the albumin of chelated mineral mercury, then not It can be captured by the specific antibody of anti-mercury, the antibody institute that will not be also marked with enzymes such as horseradish peroxidase, alkaline phosphatases Capture, and mercury metal (negative control group result is feminine gender) is not contained in agents useful for same yet, thus work as read OD value results When being shown as the positive, you can prove to detect the mercury metal chelated on albumin.
Method two：Enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection mercury-albumin chelate Detect in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With Anti- HSA Ab are diluted to 1000-8000 times by dilution buffer, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp 5-8 minutes are centrifuged, centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C Lower elution 1-3 hours.
6) detect：Sampled from the micropore of elisa plate, chelated in Atomic Absorption Spectrometer detection in the mercury on albumin, And standard curve is drawn, readout value；
This method combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principles, utilizes atomic absorption spectrum Instrument detection is chelated in the mercury on albumin, due to only containing albumin 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 mercury metal for detecting to chelate on albumin.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection mercury- Albumin chelate detects in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With Dilution buffer is anti-to be diluted to 1000-8000 times by HSA Ab, adds in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp 5-8 minutes are centrifuged, centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C Lower elution 1-3 hours.
6) it is acidified：Acidulant is added in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified, Hydrogen peroxide is added, and heats and catches up with acid；
7) detect：0.5ml liquid is taken from the solution of the elisa plate of step 6), in icp mses Lower detection chelates the mercury in albumin, and draws standard curve, readout 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 albumin, due to only containing albumin, 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 mercury metal chelated on albumin.
Method four：Purify mercury-albumin chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detection mercury-white egg White chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) anti-Hg Ab are coated on solid phase carrier：Anti- Hg Ab are diluted to 2500-20000 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；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Sampled in solution from step 1), make measuring samples；With known content Mercury-albumin chelate makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of works With 1-2 hours；
5) enzyme conjugates incubates：The measuring samples in step 4) are removed, and are washed with cleaning solution, treat that washing is completed Afterwards, the enzyme labelled antibody that addition is diluted with dilution buffer, the concentration for making the enzyme labelled antibody of dilution are 2 μ g/mL, 37 DEG C of effect 1-2 Hour, it is reacted with enzyme labelled antibody；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
7) terminating reaction：Terminate liquid is added dropwise to each micropore；
8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA Colour developing situation carries out qualitative detection).
Method five：Purify mercury-albumin chelate and atomic absorption spectrum combined techniques (method of purification+AAS methods) detection blood sample Middle mercury-albumin chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) detect：Sample in the solution obtained from step 1), chelated in Atomic Absorption Spectrometer detection on albumin Mercury, and standard curve is drawn, readout value.
Method six：Purify mercury-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS Method) detection mercury-albumin chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) it is acidified：Sampled in the solution obtained from step 1), add acidulant (such as nitric acid) in the solution and solution is carried out Acidifying, sealing overnight, thoroughly acidifying, add hydrogen peroxide, and heat and catch up with acid；
3) detect：0.5ml liquid is taken in the solution obtained from step 2), is detected under icp mses Chelate in the mercury on albumin, and draw standard curve, readout value.
Method seven：Electrophoresis and ELISA or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ELISA method/AAS methods/ICP-MS methods) detects mercury-albumin chelate, detects in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) glue bed is prepared：Selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gels as needed Deng medium is used as, glue bed is conventionally prepared；
3) it is loaded：The μ L of solution 8 for taking step 1) to obtain add 2 μ L sample-loading buffers, mix, of short duration centrifugation；(pay attention to herein Step can not boil)
4) electrophoresis：Electrophoresis plate is connected, is separated by electrophoresis；
5) detect：The protein band containing mercury is found out on glue bed, the band is taken out, the protein band is dissolved in liquid Among, ELISA or ICP-MS are then utilized respectively again or AAS detects the mercury content being dissolved in liquid.Further, it is also possible to utilize this Isoelectric point, molecular weight and content of albumin of method detection chelating mercury etc..
Albumin in method seven can come out (such as supercentrifugation or HPLC with a variety of Methods For Purifications Or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), albumin out will be purified and redissolved in solution, taken A certain amount of albumin, using electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, (can basis in gel slab Need to use different medium) on can run out of different bands according to molecular weight, isoelectric point etc. are different, search out rich in the corresponding of mercury Band, the protein in gel is redissolved in solution, you can to detect the content of related albumin at a particular wavelength, also may be used It is white due to only containing in solution to detect to chelate in the mercury content on albumin using principles such as ELISA or AAS or ICP-MS Albumen, and result will not be interfered without any heavy metal (negative control group result is feminine gender) in agents useful for same, thus When the result read is shown as the positive, you can prove to detect the mercury metal chelated on albumin.
Embodiment 1：The preparation method of mercury-albumin chelate, comprises the following steps：
A) the chelatropic reaction of mercury and albumin：Mercury ion is added in the albumin in people source and carries out chelatropic reaction, is obtained anti- Answer solution；
Preparation of reagents：
1) borate buffer solution (0.01M)：Weigh 0.31g boric acid to be dissolved in 400ml ultra-pure waters, adjusted with 0.1M NaOH PH to 9.0, is settled to 500mL.
2) albumin solution：Weigh 4.0mg albumin to be dissolved in 4.0mL 0.01M pH9.0 borate buffer solutions, fully Vibration dissolving, it is configured to 1.0mg/mL protein solution；
3) EDTA-NaHCO3 mixed solutions：Weigh EDTA2H2O 1.86g、NaHCO316.8g is dissolved in 900mL ultra-pure waters In, it is settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH adjustment pH to 8.0；
4) ITCBE is bought from Japanese colleague's chemistry institute, article No. M030；
5) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of mercury-albumin chelate：
1) processing of bag filter：Bag filter is put into the 5mmol/L of 500ml (according to the convertible dosage of beaker volume) volume EDTA+200mmol/L NaHCO3In solution, 10min is boiled；Tipping EDTA/NaHCO3Liquid, gently rinsed, then used with ultra-pure water 500ml 5mmol/L EDTA boil 10min；Boiling liquid is discarded, is thoroughly cleaned with ultra-pure water, adds substantial amounts of ultra-pure water immersion 4 DEG C of bag filter is overnight.In use, putting on one's gloves, bag filter is taken out, with substantial amounts of its surfaces externally and internally of ultra-pure water cleaning down；
2) 2.0mg ITCBE are taken to be dissolved in 2ml DMSO；
3) 4.0mg albumin is taken to be dissolved in 4.0ml borate buffer solutions in (0.01M pH9.0)；
4) liquid for slowly preparing step 2) is added in albumin solution, is shaken when being added dropwise, in 25 DEG C, 100r/min Shaking table in act on 24h, then dialysed 24h with bag filter, remove the not ITCBE with albumin combination；
5) 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；
6) solution added is reacted into 2h in 25 DEG C, 100r/min shaking table, dialysed with the bag filter handled well 24h；
7) liquid after dialysis is preserved in -20 DEG C of packing.
B the extraction of mercury-albumin chelate) is purified：Using immune-affinity chromatography, reaction solution (i.e. step 7) is removed Obtained reaction solution) in unreacted albumin and unnecessary mercury ion, produce mercury-albumin chelate, specific steps It is as follows：
(1) sample dissolution：Physiological saline is added into the reaction solution obtained by step 7) makes mercury-albumin chelate Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of 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 phosphoric acid Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water After three times, 4 DEG C of dialysed overnights, sample is collected, produces mercury-albumin chelate；
C) to the identification of mercury-albumin chelate, 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-albumin chelate of 8 μ L extraction purifications, add 2 μ L sample-loading buffers, And mix, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, electrophoretic buffer is added and carries out electrophoresis；In electrophoresis process, electric current is 22mA constant currents, Environment temperature is 4 DEG C；Electrophoresis to bromophenol blue stops electrophoresis when moving to glue bottom；
(4) detect：Mercurous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
D) testing result
(1) content of beary metal in graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination HSA：It the results are shown in Table 1
Table 1 is content of beary metal (μ g/L) in albumin
Sample name Hg(μg/L)
HSA 63.071
(NH4)2SO4 0.369
N.S 0
N.S 0
(2) electrophoresis result：
Wherein, Fig. 1 is the non denatured electrophoretic band figure of mercury of the present invention-albumin chelate.
Native polyacrylamide gel electrophoresis (Native-PAGE) or for active electrophoresis be be added without SDS and dredge Under conditions of the denaturants such as base ethanol, polyacrylamide gel electrophoresis is carried out to the protein for keeping activity, is usually used in the mirror of enzyme Fixed, Isozyme Analysis and purification.Not plus SDS native polyacrylamide gel electrophoresis can make large biological molecule in electrophoresis process Middle its natural shape of holding and electric charge, their separation are made according to the difference of its electrophoretic mobility and the molecular sieve of gel With, thus higher resolution ratio can be obtained, especially remain to keep the large biological molecule such as protein and enzyme after electrophoretic separation Bioactivity, therefore the right swimming lane M is denaturation Marker, is served only for detecting, does not mark effect, and swimming lane 1 is albumin Band.
(3) 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) detector (PGT Inc.LS 30143-DS) detect, probe is with incident SR lines copline and being mutually perpendicular to, and away from sample irradiation point 20mm, signal is divided with PGT multiple tracks Analyzer (MCA4000) obtains output.Sample, regulation launching spot (1mmx3mm) are excited with 11.5keV monochromatic synchrotron radiation light Position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always, at the end of counting Hot spot moves on to the band other end.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings, and with deriving from Air and the Ar signal peaks of content constant carry out normalization to other element peaks, to offset beam intensity change to signal strength Caused influence.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 figure of the electrophoretic band of mercury of the present invention-albumin chelate, is schemed Middle abscissa is protein band position, and ordinate is each metal energy of the band (content) value.
A kind of determination of the testing conditions for the method for quantitatively detecting mercury-albumin chelate of the present invention：
1. the determination of the optimum diluting multiple of anti-albumin antibodies, anti-mercury antibody best effort concentration and blood plasma
Step is as follows：
(1) it is 1 according to anti-HSA Ab and the mass volume ratio of dilution buffer by anti-HSA Ab dilution buffers: 1000、1:2000、1:4000、1:8000 are diluted, and add in elisa plate micropore, anti-HSA Ab are coated in into solid phase carrier On, each concentration is coated with three rows, and 4 DEG C are stayed overnight 18 hours；
(2) dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is made with 2% bovine serum albumin(BSA) For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) measuring samples are 1 by measuring samples and the mass volume ratio of dilution buffer:10、1:20、1:40 progress are dilute Release, add in micropore, according to above-mentioned coated anti-HSA Ab concentration, being separately added into for the anti-HSAAb of same concentration is different dilute Degree of releasing blood plasma, 37 DEG C act on 1 hour；
(4) measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, add anti-Hg Ab, anti-Hg Ab are pressed Anti- Hg Ab and the mass volume ratio of dilution buffer are 1:2500、1:5000、1:10000、1:20000 are diluted, according to every One identical anti-HSA Ab, serum-dilution concentration, the anti-Hg Ab of various concentrations respectively add 2 holes, and 37 DEG C act on 1 hour, make anti-Hg Ab Reacted with the mercury metal on HSA；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2 μ g/mL, removes anti-Hg antibody, and is washed with cleaning solution, Wait after the completion of washing, addition dilution buffer dilutes HRP enzyme labelled antibodies, and 37 DEG C act on 1 hour, make HRP enzyme labelled antibodies and mercury- Albumin chelate reacts；
(6) enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects 30 minutes；
(7) terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads each hole OD respectively Value.According to each hole OD value numerical value, anti-HSA Ab, anti-Hg Ab best effort concentration and blood plasma optimum diluting multiple are selected.
Simultaneously using made standard items as positive control in experiment, anti-HSA Ab+ closings+anti-Hg Ab+ enzymes mark+bottom is selected Thing (being not added with measuring samples) is used as negative control 1；Anti- HSA Ab+ closings+blood plasma+enzyme mark+substrate (being not added with anti-Hg Ab) is made For negative control 2；Anti- HSA Ab+ closings+enzyme mark+substrate (being not added with measuring samples and anti-Hg Ab) are used as negative control 3；It is anti- HSA Ab+ closings+blood plasma+anti-Hg Ab+ substrates (i.e. not enzyme-added mark) are used as negative control 4；Closing+blood plasma+anti-Hg Ab+ enzyme marks + substrate (being not added with anti-HSA Ab captures HSA) is used as blank control 1；Only add substrate as blank control 2 and PBS as blank Control 3.Testing result is shown in Table 2-3.
Table 2：Checkerboard titration method determines anti-HSA Ab, anti-Hg Ab
The data of the optimum diluting multiple of best effort concentration and blood plasma (each data are all average value)
Table 3：ELISA positive controls and negative control ELISA testing results
Shown by table 2-3 data, we can see that when anti-HSA Ab dilution factor is 1:2000th, whole blood dilution factor is 1: 20th, the anti-dilution factors of Hg are 1:When (2500-5000), OD values are maximum, so selecting the concentration corresponding to this value dense as best effort Degree.
2. quantitatively detect eluent best effort concentration and elution in method two, three in the method for mercury-albumin chelate The determination of time
Step is as follows：(1) it is coated with：Anti- Hg Ab are diluted to 2500-5000 times (mass volume ratio) with dilution buffer, Add in elisa plate micropore, 37 DEG C of water-baths 3 hours, store refrigerator；
(2) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure with 2% ox blood As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and washed with cleaning solution；
(3) confining liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, addition is diluted with dilution buffer Enzyme labelled antibody, enzyme labelled antibody are diluted to concentration as 2 μ g/mL, and 37 DEG C act on 2 hours, it is reacted with anti-Hg Ab；
(4) eluent is prepared：By papain with pH 8.0,0.1mol/L Tris-HCI buffers into 100ng/ml, add 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT)s (DTT)；
(5) enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes papain in eluent The ratio between concentration and the concentration of enzyme labelled antibody are 1:80、1:40、1:20、1:10、1:5 (wherein each concentration makees 3 multiple holes), respectively 1h, 2h, 3h are eluted at a temperature of being positioned over 37 DEG C；
(6) eluent is removed, and is washed with cleaning solution, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects 30 Minute；
(7) terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads every group of OD respectively Value, by compared with PBS group, comparing the optimum concentration and elution time of eluent, concrete outcome is referring to table 4.
Table 4：ELISA eluent best effort concentration and elution time determine
We are it can be found that when the concentration of the papain in eluent and the ratio between the concentration of enzyme labelled antibody from table 4 =1:When 20, each group OD values are below other several groups, illustrate that the concentration elution effect is optimal (i.e. by ELISA hole walls With reference to anti-Hg Ab- enzyme marks compound elution degree reach maximum, thus OD values are minimum)；And elution time either 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 the elution time of eluent all may be used for 1-3h in this experiment.
Application Example 1
Take using mercury-albumin chelate in 100 parts of sample blood plasma of ELISA method detection, follow the steps below inspection Survey：ELISA (ELISA method) detects mercury-albumin chelate, detects in accordance with the following steps：
1) anti-HSA Ab are coated on solid phase carrier：Anti- HSA Ab are diluted to 2000 times with dilution buffer, added In elisa plate micropore, 37 DEG C of water-baths 1 hour, refrigerator is stored；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp 5-8 minutes are centrifuged, centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, adds 2% bovine serum albumin It is white be used as confining liquid, 37 DEG C of placements 1 hour, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate in 37 DEG C are placed 1 hour；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) material of mercury can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed After the completion of washing, addition dilution buffer dilutes 2500-5000 times, and 37 DEG C act on 1 hour, make on anti-Hg Ab and albumin Mercury metal reacts；
6) enzyme conjugates incubates：Anti- mercury antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute The HRP enzyme labelled antibodies of buffer solution dilution are released, 37 DEG C act on 1 hour, it is reacted with enzyme labelled antibody；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore；
9) it is 405nm to take wavelength, after adding terminate liquid, elisa plate is placed on ELIASA and detected, and reads treat sample respectively Product OD values (also directly can carry out qualitative detection) without using ELIASA by the situation that develops the color, and specific detected value is as shown in table 5.
Table 5：The ELISA testing results of mercury-albumin chelate in 100 parts of blood samples
Application Example 2
Using in enzyme linked immunological and minute mark this blood sample of atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection 100 Mercury-albumin chelate, is detected in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With Anti- HSA Ab are diluted to 2000 times by dilution buffer, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp 5-8 minutes are centrifuged, centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood Albumen or skimmed milk power are as confining liquid, and 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, and washing is completed Afterwards, elisa plate is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain The eluent for 100ng/ml is spent, elutes 1-3 hours；
6) detect：Sampled from the micropore of elisa plate, chelated in Atomic Absorption Spectrometer detection in the mercury on albumin, Detected value is as shown in table 6.
Table 6：The AAS testing results of mercury-albumin chelate in 100 parts of blood samples
Application Example 3
100 minute marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) Mercury-albumin chelate content in this blood sample, is detected in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With Anti- HSA Ab are diluted to 2000 times by dilution buffer, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) 100 parts of standard blood samples are taken as measuring samples；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and are washed with cleaning solution, after the completion of washing, elisa plate Placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the mercury of known content-albumin chela Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain The eluent for 100ng/ml is spent, elutes 1-3 hours；
6) it is acidified：Add nitric acid in solution in step 5) to be acidified solution, overnight, thoroughly acidifying, adds for sealing Enter hydrogen peroxide, and heat and catch up with acid；
7) detect：Sampled in the solution obtained from step 6), under icp mses detection chelate in The mercury of albumin, detected value are as shown in table 7.
Table 7：The ICP-MS testing results of mercury-albumin chelate in 100 parts of blood samples
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention Within.

Claims (5)

1. a kind of mercury-albumin chelate, it is characterised in that mercury ion passes through sulfydryl or/and cysteine residues with albumin Chelating forms；
Described mercury-albumin chelate through the following steps that prepare：
A）The chelatropic reaction of mercury and albumin：In the albumin that purification comes from the albumin of human body or recombinated according to biological method Middle addition mercury ion carries out chelatropic reaction, obtains reaction solution；
1）The processing of bag filter：Bag filter is put into the 5mmol/L EDTA+200mmol/L NaHCO of 500ml volumes3In solution, Boil 10min；Tipping EDTA/NaHCO3Liquid, gently rinsed with ultra-pure water, then 10min is boiled with 500ml 5mmol/L EDTA； Boiling liquid is discarded, is thoroughly cleaned with ultra-pure water, adds substantial amounts of 4 DEG C of ultra-pure water immersion bag filter overnight；In use, put on hand Set, bag filter is taken out, with substantial amounts of its surfaces externally and internally of ultra-pure water cleaning down；
2）2.0mg ITCBE are taken to be dissolved in 2ml DMSO；
3）It is in the borate buffer solution that 0.01M, pH are 9.0 to take 4.0mg albumin to be dissolved in 4.0ml concentration；
4）Slowly by step 2）The liquid of preparation is added in albumin solution, is shaken when being added dropwise, in 25 DEG C, 100r/min's shakes 24h is acted in bed, then with bag filter dialysis 24h, removes the not ITCBE with albumin combination；
5）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 Mercury ion solution, vibrated when being added dropwise, in case mercury ion precipitates albuminous degeneration；
6）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；
7）Liquid after dialysis is preserved in -20 DEG C of packing;B）Purify the extraction of mercury-albumin chelate：Using affine in immunity Chromatography, unreacted albumin and unnecessary mercury ion in reaction solution are removed, produces mercury-albumin chelate；
The step B）It is specific as follows：
（1）Sample dissolution：To by step A）Physiological saline is added in obtained reaction solution answers mercury-albumin chelate It is molten；
（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with albumin Property combine filler, fill post after, be continuing with dilution buffer balance chromatographic column；
（3）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（1）Middle sample, then upper prop, albumin is with filling out Material specific binding；
（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate Solution is eluted, and obtains eluent I；
（5）Collect：Collect eluent I；
（6）Dialysis：By step（5）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C Dialysed overnight, 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, it is then molten using 0.5-1.0mol/L phosphate Liquid is eluted, and obtains eluent II；
（10）Collect：Collect eluent II；
（11）Dialysis：By step（10）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C dialysed overnight, sample is collected, produces mercury-albumin chelate.
2. mercury according to claim 1-albumin chelate, it is characterised in that also including step C）：To mercury-albumin The identification of chelate；
Wherein, step C）In it is specific as follows：
（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-albumin chelate, dilution buffer is added, and mixed, so After be 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 and dissolved, then uses ELISA again Method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
A kind of 3. examination of mercury-albumin chelate as claimed in claim 1 mercury-albumin chelate in detection blood sample is prepared Application in agent or kit.
4. a kind of comprise at least mercury as claimed in claim 1-kit of the albumin chelate as standard items.
5. kit according to claim 4, it is characterised in that also including coating buffer, the coating buffer contains the white egg of capture White material or the material of capture mercury.
CN201510413056.4A 2015-07-14 2015-07-14 Mercury-albumin chelate and preparation method and application thereof Expired - Fee Related CN104987391B (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103472231A (en) * 2013-09-28 2013-12-25 郑州大学 Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103472231A (en) * 2013-09-28 2013-12-25 郑州大学 Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof

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