CN105044006B - Mercury-IgG chelate and preparation method and application thereof - Google Patents
Mercury-IgG chelate and preparation method and application thereof Download PDFInfo
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- CN105044006B CN105044006B CN201510412924.7A CN201510412924A CN105044006B CN 105044006 B CN105044006 B CN 105044006B CN 201510412924 A CN201510412924 A CN 201510412924A CN 105044006 B CN105044006 B CN 105044006B
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- 239000013522 chelant Substances 0.000 title abstract description 10
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Abstract
The invention discloses a mercury-IgG chelate and a preparation method and application thereof, wherein the mercury-IgG chelate is formed by chelating mercury ions and IgG through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of mercury-IgG chelate so as to quantitatively detect the application of the mercury-IgG chelate in evaluating the mercury pollution degree of one area. The mercury-IgG chelate in the serum of people in one region can be quantitatively detected to indirectly reflect the mercury pollution condition of the people in the region, so that the mercury pollution degree of the region can be indirectly reflected. The accuracy of the quantitative detection method of the mercury-IgG chelate is greatly improved, and the repeatability of detection is greatly improved.
Description
Technical field
The present invention relates to detection field, more specifically to a kind of mercury-IgG chelates and its preparation method and application.
Background technology
IgG is mainly synthesized and secreted by the thick liquid cell in spleen, lymph node, is existed with monomeric form.In ontogenetic process
Middle body synthesis IgG age will be later than IgM, start to synthesize within 3rd month after birth, 3~5 years old horizontal close to adult.IgG
It is antibody component main in serum, accounts for the 75% of serum total Ig.IgG is uniquely can be by the Ig of placenta, naturally passive
Played an important role in immune.In addition IgG also has opsonophagocytosis, ADCC and combines the effect such as SPA.Due to IgG These characteristics,
IgG plays main effect in immunity of organism protection, and most of antibacterials, antiviral, t antibody belong to IgG classes and resisted
Body.It is artificial passive using that can be carried out to third kind of the immune puerpera such as measles, hepatitis A or normal person or placental globulin
It is immune, prevent corresponding communicable disease with having suburb.
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.In the U.S., Cr and Hg, Cd, Pb are considered as four overall situation
Pollutant, it drastically influence the health of people.Common mercury exposed population group includes being engaged in metal smelt, exploitation gold
Ore deposit, the crowd for burning coal, electric industry and wood producing, and for general population mainly by eating the seafood of mercury pollution, drinking into mercury
The mercury vapour of polluted source, suction fuel combustion or evaporation.In addition, the contact of mercury cosmetic, filling mercury alloy dental filler
The contact of thing, ethyl mercury, mercurous insecticide in contact vaccine preservative is all common mercury exposure chamber, the life of mercury and people
Work is closely related.2009, national health in nutrition test investigation with finding (National Health and Nutrition
Examination Study, NHANES), the mercury in 2% U.S.'s Women of Childbearing Age body is all exceeded.Mercury is considered as to human body
And its it is harmful, can influence multiple tissues, organ and the system of human body, including cardiovascular system, respiratory system, nervous system,
Internal system, digestive system, urinary system, immune system etc., harm to the human body is very big.
A variety of methods detection bleeding mercury content, including ELISA, atomic absorption spectrum (Atomic can be used at present
Absorption Spectroscopy, AAS) quantitative assay, icp mses (inductively
Coupled plasma mass spectrometry, ICP-MS) quantitative assay etc., but still suffer from certain defect.
On the evaluation of mercury poisoning, particularly chronic mercury poisoning, it is only capable of by detecting blood mercury content, in indirect reaction human body
Circulation mercury content, can not further assess degree of injury of the mercury for body function, and with the rapid development of science and technology,
The relation of mercury and human body is also increasingly close, thus searching one kind can evaluate mercury poisoning from body function angle, particularly chronic
Mercury poisoning becomes more and more important for the evaluation method of the extent of damage of body.
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-IgG chelates and its preparation side
Method, and the qualitative and quantitative analysis method of mercury-IgG chelates is established, so that quantitative detection mercury-IgG chelates are evaluating a ground
The application of area's mercury pollution degree.This can be reflected indirectly by mercury-IgG chelates in one regional crowd's serum of quantitative detection
The mercury contaminated situation of regional crowd, 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-IgG chelates are provided, mercury ion with
IgG is formed by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned mercury-IgG chelates, comprises the following steps：
A) mercury and IgG chelatropic reaction：Mercury ion is added in the IgG in people source and carries out chelatropic reaction, obtains reaction solution；
B the extraction of mercury-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed
And unnecessary mercury ion, produce mercury-IgG chelates.
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, answer mercury-IgG chelates
It is molten；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG
The filler that the opposite sex combines, 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, IgG is with filling out
Material specific binding；
(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；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(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；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(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-IgG chelates.
Wherein, the preparation method of above-mentioned mercury-IgG chelates, in addition to step C)：Identification to mercury-IgG chelates；
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-IgG chelates of extraction purification, add dilution buffer, and mix, so
After be 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 redissolved, so
Afterwards again using ICP-MS methods, AAS methods or ELISA method detection whether containing mercury and detect mercury content.
The present invention also provides a kind of mercury-IgG chelates described above and is preparing the examination of mercury-IgG chelates in detection human body
Application in agent or kit.
The present invention, which also provides, a kind of comprises at least kit of the mercury-IgG chelates described above as reference substance.
Preferably, coating buffer is also included in the kit, the coating buffer contains the albumen that can capture IgG or capture mercury metal
Material.
The present invention also provides a kind of method for quantitatively detecting mercury-IgG chelates, with the above-mentioned mercury-IgG chelas of known content
Compound is detected as reference substance using a pair of samples of following methods：ELISA, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are combined with inductivity coupled plasma mass spectrometry combined techniques, purification mercury-IgG chelates with enzyme linked immunological
Method, purification mercury-IgG chelates and atomic absorption spectrum combined techniques, purification mercury-IgG chelates and inductively coupled plasma matter
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement mercury-IgG chelates of the present invention and its preparation method and application, have the advantages that：
1. mercury-IgG chelates are extracted in present invention synthesis in vitro first from whole blood；
2. present invention firstly provides mercury-IgG chelates can be used for prepare detection blood sample in mercury-IgG chelates reagent or
Application in kit.
3. the present invention establishes the method for qualitative and quantitative detection of mercury-IgG chelates, so as to quantitative detection mercury-IgG chelates
Evaluating the application of a regional mercury pollution degree.Can be with by mercury-IgG chelates 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 present invention establishes
Mercury-IgG chelates quantitative detecting method its degree of accuracy greatly improve, and the repeatability of detection is greatly enhanced.
Brief description of the drawings
Fig. 1 is the non denatured electrophoretic band figure of mercury-IgG chelates of the present invention；
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of mercury-IgG chelates of the present invention；Wherein,
In Fig. 1, M Marker, 5 be mercury-IgG chelates；In Fig. 2, abscissa is protein band position, and ordinate is the protein band
Middle mercury metal energy.
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：
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 filler that can be specifically bound with IgG in the present invention, has the silica gel for the albumen that can capture IgG for surface
Or resin；
The albumen (anti-igg antibody) that can capture IgG in the present invention, including but not limited to rabbit Anti- IgGs H＆L,
Its brand is Abcam, model ab6715；
The filler that can be specifically bound with mercury in the present invention, have silica gel or the tree of the material that can capture mercury for surface
Fat；
The anti-Hg mAb of the material that can capture mercury in the present invention, including but not limited to mouse, buy from bar proud moral biotechnology
Company (BioWorld Inc), article No. AP7014；
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 KH2PO4 0.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl 8.0mg/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 phosphate 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 of the papain containing 1-2mg/ml 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, ddH2O7ML, sweet ammonia
Sour 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 also provides a kind of preparation method of mercury-IgG chelates, comprises the following steps：
A) mercury and IgG chelatropic reaction：Mercury ion is added in the IgG in people source or the IgG according to biological method restructuring
Chelatropic reaction is carried out, obtains reaction solution；
B the extraction of mercury-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed
And unnecessary mercury ion, mercury-IgG chelates are produced, are comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, answer mercury-IgG chelates
It is molten
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG
The filler that the opposite sex combines, 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, IgG is with filling out
Material specific binding；
(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；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(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；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(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-IgG chelates；
C) to the identification of mercury-IgG chelates, 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-IgG chelates of extraction purification, add dilution buffer, and mix, so
After be 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 redissolved, so
Afterwards again using ICP-MS methods, AAS methods or ELISA method detection whether containing mercury and detect mercury content.
The present invention, which also provides, a kind of comprises at least kit of the mercury-IgG chelates described above as reference substance.
Preferably, coating buffer is also included in the kit, the coating buffer contains the albumen that can capture IgG or can capture metal
The material of mercury.
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-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG,
Confining liquid, cleaning solution, the material for capturing mercury as secondary antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, loading are delayed
Fliud flushing, positive control, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG,
Confining liquid, cleaning solution, eluent, sample-loading buffer, positive control, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG,
Confining liquid, cleaning solution, eluent, sample-loading buffer, acidulant, hydrogen peroxide, standard items, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood
Agent, redissolve liquid, the coating buffer containing the material that can capture mercury, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution
Buffer solution, sample-loading buffer, positive control, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood
Agent, redissolve liquid, sample-loading buffer, positive control, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood
Agent, sample-loading buffer, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG,
Glue bed medium, liquid, sample-loading buffer are redissolved, liquid needed for protein band mercurous on glue bed is dissolved, contains the thing that can 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-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood
Agent, glue bed medium, redissolve liquid, sample-loading buffer, dissolve liquid, positive control, feminine gender needed for protein band mercurous on glue bed
Control etc..
The kit of mercury-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood
Agent, glue bed medium, redissolve liquid, sample-loading buffer, dissolve liquid, acidulant, peroxidating needed for protein band mercurous on glue bed
Hydrogen, positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with IgG chelates or the chela of heavy metal Hg
Closing has the BSA chelates of heavy metal Hg；The negative control is dilution buffer.
Mentioned reagent box is used for the IgG for detecting chelating mercury, to improve the accuracy of detection, repeatability, and is allowed in clinic
In be promoted.
The present invention also provides a kind of method for quantitatively detecting mercury-IgG chelates, with the above-mentioned mercury-IgG chelas of known content
Compound is detected as reference substance using a pair of samples of following methods：ELISA, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are combined with inductivity coupled plasma mass spectrometry combined techniques, purification mercury-IgG chelates with enzyme linked immunological
Method, purification mercury-IgG chelates and atomic absorption spectrum combined techniques, purification mercury-IgG chelates and inductively coupled plasma matter
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention,
Having of can listing of method with detection mercury chelating type immune complex is following several, but is not limited to following several.
Wherein, the reagent used in the above-mentioned method for quantitatively detecting mercury-IgG chelates is as follows：
Method one：ELISA (ELISA method) detects mercury-IgG chelates, detects in accordance with the following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with dilution buffer dilution
IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 are small
When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, 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：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；Corresponding multiple is diluted to dilution buffer dilution measuring samples, that is, dilutes 10-40 times, adds in micropore, 37 DEG C
Act on 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 dilution buffer dilutes and can capture the material of mercury or can react to form antigen antibody complex with mercury
Anti- Hg Ab, 37 DEG C of effect 1-2 hours, react the mercury metal on anti-Hg Ab and IgG；
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) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA read respectively measuring samples group and
The OD values of standard items, standard curve is drawn by standard items, the content for trying to achieve measuring samples (also can be without using ELIASA, directly
Qualitative detection is carried out by staining conditions).
This method utilizes ELISA principles, can extract the specific IgG in whole blood, the IgG tops extracted
Divide and be chelated with heavy metal Hg, and the mercury on the IgG of this part can be captured by the specific antibody of anti-mercury, afterwards can be peppery again
The antibody of the enzymes such as root peroxidase, alkaline phosphatase mark captures (the antibody nonrecognition coating protein), anti-in capture
Body can read OD values in the presence of developer and terminate liquid under instrument, and not contain the IgG 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 IgG.
Method two：Enzyme linked immunological is pressed with atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection mercury-IgG chelates
Detected according to following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with dilution buffer dilution
IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 are small
When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rpm centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Coating buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% cow's serums
As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, have been washed for albumin or skimmed milk power
Cheng Hou, elisa plate place 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；Corresponding multiple is diluted to dilution buffer dilution measuring samples, that is, dilutes 10-40 times, adds in micropore, 37 DEG C
Act on 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is small with elution 1-3
When.
6) detect：Sample, chelated in Atomic Absorption Spectrometer detection in the mercury on IgG, and draw mark from ELISA micropores
Directrix curve, read respective value；
The embodiment combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principles, utilizes atomic absorption light
Spectrometer detection is chelated in the mercury on IgG, due to only containing IgG in solution, and it is (negative right without any heavy metal in agents useful for same
It is feminine gender according to group result), result will not be interfered, thus when the result read is shown as the positive, you can prove inspection
Measure the mercury metal chelated on IgG.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection mercury-
IgG chelates detect in accordance with the following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with dilution buffer dilution
IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 10-40 hours, or 37 DEG C of water-bath 1-3 are small
When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, 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：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；Corresponding multiple is diluted to dilution buffer dilution measuring samples, that is, dilutes 20-80 times, adds in micropore, 37 DEG C
Act on 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is small with elution 1-3
When.
6) it is acidified：Add acidulant in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified；
7) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled in the solution eluted from ELISA agent plates, in
Detection chelates the mercury in IgG under icp mses, and draws standard curve, 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 IgG, due to only containing IgG in solution, and is free of in agents useful for same
Any heavy metal (negative control group result is feminine gender), will not interfere to result, thus works as read result and be shown as
When positive, you can prove to detect the mercury metal chelated on IgG.
Method four：Mercury-IgG chelates are purified to chelate with enzyme linked immunological combined techniques (method of purification+ELISA method) detection mercury-IgG
Thing, detect in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method is redissolved the IgG of the purification of blood out using whole blood extraction method, obtains IgG redissolution liquid；
2) anti-Hg Ab are coated on solid phase carrier：With anti-Hg Ab to 2500-20000 times of dilution buffer dilution, add
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；
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 from the IgG of extraction redissolution liquid, make measuring samples；With known content
Mercury-IgG chelates make standard items；Corresponding multiple is diluted with dilution buffer, that is, dilutes 10-40 times, is added in micropore, 37 DEG C
Act on 1-2 hours；
5) enzyme conjugates incubates：IgG redissolution liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, adds and uses
The enzyme labelled antibody of dilution buffer dilution, the concentration for making the enzyme labelled antibody of dilution is 2 μ g/ml, 37 DEG C of effect 1-2 hours, makes it
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) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA read respectively measuring samples group and
The OD values of standard items, (also can directly it be passed through without using ELIASA by the content for compared with standard item group, trying to achieve measuring samples
Staining conditions carry out qualitative detection).
Method five：Mercury-IgG chelates are purified to detect in blood sample with atomic absorption spectrum combined techniques (method of purification+AAS methods)
Mercury-IgG chelates, are detected in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) detect：Sample in redissolution liquid from step 1), chelated in Atomic Absorption Spectrometer detection in the mercury on IgG,
And standard curve is drawn, read respective value.
Method six：Purify mercury-IgG chelates and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS methods)
Mercury-IgG chelates are detected, are detected in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) it is acidified：Sampled in redissolution liquid from step 1), add acidulant (such as nitric acid) in the solution and solution is carried out
Acidifying, sealing overnight, are thoroughly acidified；
3) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled from solution, in inductively coupled plasma matter
Detection is chelated in the mercury on IgG under spectrometer, and draws standard curve, reads respective value.
Method seven：Electrophoresis and ELISA (electrophoresis-ELISA method) detection mercury-IgG chelates, in accordance with the following steps
Detection：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
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 prepared；
3) it is loaded：Take the μ L of redissolution liquid 8 in step 1) to 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, after treatment by the albumen one
Band is dissolved among liquid, then recycles the detection of ELISA principles to be dissolved in the mercury content in liquid.Further, it is also possible to utilize this side
IgG isoelectric point, molecular weight and content of method detection chelating mercury etc..
Method eight：Electrophoresis and atomic absorption spectrography (AAS) (electrophoresis-AAS methods) detection mercury-IgG chelates, according to following step
Rapid detection：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
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 prepared；
3) it is loaded：Sampled in redissolution liquid from step 1), add sample-loading buffer, and mixed, be then loaded onto sample
In groove；
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, after treatment by the albumen one
Band is dissolved among liquid, then recycles the detection of AAS principles to be dissolved in the mercury content in liquid.Further, it is also possible to utilize the method
IgG isoelectric point, molecular weight and content of detection chelating mercury etc..
Method nine：Inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ICP-MS methods) detects mercury-IgG chelates, presses
Detected according to following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
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 prepared；
3) it is loaded：Sampled in redissolution liquid from step 1), add sample-loading buffer, and mixed, be then loaded onto sample
In groove；
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, after treatment by the albumen one
Band is dissolved among liquid, then recycles the detection of ICP-MS principles to be dissolved in the mercury content in liquid.Further, it is also possible to utilize this side
IgG isoelectric point, molecular weight and content of method detection chelating mercury etc..
IgG in method seven to nine can come out (such as supercentrifugation, high pressure liquid chromatography (HPLC) with a variety of Methods For Purifications
Method, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), IgG out will be purified and redissolved, take a certain amount of IgG,
Using electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, (different Jie can be used as needed in gel slab
Matter) 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
Protein redissolved with corresponding double solvents in solution, you can to detect related IgG content at a particular wavelength, can also profit
Detect to chelate in the mercury content on IgG with principles such as ELISA, AAS, ICP-MS, due to only containing IgG in solution, and it is used
Without any heavy metal (negative control group result is feminine gender) in reagent, result will not be interfered, thus work as what is read
When being as a result shown as the positive, you can prove to detect the mercury metal chelated on IgG.
Embodiment 1：The preparation method of mercury-IgG chelates, comprises the following steps：
A) mercury and IgG chelatropic reaction：Mercury ion is added in the IgG in people source and carries out chelatropic reaction, obtains reaction 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) IgG solution：Weigh 4.0mg IgG to be dissolved in 4.0mL 0.01M pH9.0 borate buffer solutions, fully vibrate molten
Solution, it is configured to 1.0mg/mL protein solution；
3)5mmol/L EDTA+200mmol/LNaHCO3Solution：Weigh EDTA2H2O 1.86g、NaHCO316.8g is molten
In 900mL ultra-pure waters, 1000ml, autoclaving, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
4) ITCBE (buying from Japanese colleague's chemistry institute, article No. M030)
5) bag filter (molecular cut off 14000) (Bioshop Inc)
The preparation process of standard items：
A. the preparation of standard items
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) weigh 4.0mgIgG to be dissolved in 4.0mL 0.01M pH9.0 borate buffer solutions, fully vibration dissolving, be configured to
1.0mg/mL protein solution；
4) liquid for slowly preparing step 2 is added in IgG 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 ITCBE not combined with IgG；
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 dialysed is preserved in -20 DEG C of packing.
B the extraction of mercury-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed
And unnecessary mercury ion, mercury-IgG chelates are produced, are comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, answer mercury-IgG chelates
It is molten；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG
The filler that the opposite sex combines, 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, IgG is with filling out
Material specific binding；
(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；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(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；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(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-IgG chelates
C) to the identification of mercury-IgG chelates, 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-IgG chelates of 8 μ L extraction purifications, add 2 μ L sample-loading buffers, and
Mix, be then 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 degree；Stop electrophoresis when moving to glue bottom to bromophenol blue；
(4) detect：Mercurous protein band is found out on glue bed, the protein band is taken out, protein band is redissolved, so
The content of mercury is detected whether containing mercury and detected again afterwards using AAS methods.
D) testing result
(1) electrophoresis result：
Wherein, Fig. 1 is the non denatured electrophoretic band figure of mercury-IgG chelates of the present invention.
(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) 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 (1mmx are excited with 11.5keV monochromatic synchrotron radiation light
3mm) position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always, counts knot
Hot spot moves on to the band other end during beam.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings, and it is used for
The Ar signal peaks for coming from air and content constant carry out normalization to other element peaks, to offset beam intensity change to signal
Influenceed caused by strong and weak.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way under the same conditions.
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of mercury-IgG chelates of the present invention, horizontal in figure
Coordinate is protein band position, and ordinate is mercury metal energy (content) value in the protein band.
(3) use in the mercury-IgG chelates that graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination the present embodiment obtains
Mercury content, its content is 146.944 μ g/L.
A kind of determination of the testing conditions for the method for quantitatively detecting mercury-IgG chelates of the present invention：
1. the determination of the optimum diluting multiple of complement protein best effort concentration and blood plasma
Step is as follows：
(1) IgG Ab dilution buffers are diluted (1 according to following proportioning:500000、1:1000000、1:2000000、
1:4000000), add in elisa plate micropore, anti-igg Ab is coated on solid phase carrier, three rows of each concentration coating, 4 DEG C
18 hours overnight；
(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 press following concentration dilution (1:10、1:20、1:40), add in micropore, according to above-mentioned coated anti-
IgG Ab concentration, the anti-igg Ab's of same concentration is separately added into different dilution factor blood plasma, and 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
Following concentration dilution (i.e. 1:2500、1:5000、1:10000、1:20000), according to each identical anti-igg Ab, serum-dilution
Concentration, the anti-Hg Ab of various concentrations respectively add 2 holes, and 37 DEG C act on 1 hour, react the mercury metal on anti-Hg Ab and IgG；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2ng/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-
IgG chelates react；
(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) with adding substrate solution same speed and order that terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and reads each hole OD values respectively.Root
According to each hole OD value numerical value, anti-igg Ab, anti-Hg-Ab best effort concentration and the optimum diluting multiple of blood plasma are selected.
In experiment simultaneously using made reference substance as positive control, select IgG antibody+closing+Hg resist+enzyme mark+substrate (i.e.
It is not added with detecting sample) it is right as feminine gender as negative control 1, IgG antibody+closing+blood plasma+enzyme mark+substrate (being not added with Hg to resist)
According to 2, IgG antibody+closing+enzyme mark+substrate (be not added with detecting sample and Hg resists) as negative control 3, IgG antibody+closing+blood
Slurry+Hg is anti-+ and substrate (i.e. not enzyme-added mark) is used as negative control 4, and closing+blood plasma+Hg resists+enzyme mark+substrate (being not added with IgG antibody)
As blank control 1, only add substrate and PBS as blank control 2；Testing result is shown in Table 1-2.
Table 1：The determination of anti-igg Ab and Hg antibody best effort concentration and diluted plasma multiple
Table 2：ELISA positive controls and negative control ELISA testing results
Shown by table 1-2 data, we can see that when the dilution factor of human IgG antibody is 1:1000000th, whole blood dilution factor
For 1:20th, the anti-dilution factors of Hg are 1:When 5000, OD values are maximum, although OD values are less than 0.8, the negative control group corresponding to it
OD values are all less than 0.1, and the positive controls corresponding to it, and OD values are more than 0.8, so selecting the concentration corresponding to this value to make
For best effort concentration, (i.e. human IgG antibody's concentration is 1:1000000, whole blood dilution factor is 1:20, Hg anti-diluted concentrations are 1:
5000)。
2.ELISA eluent best effort concentration and time determine
Step is as follows：(1) human IgG antibody is diluted to 1000000 times (mass volume ratios) with dilution buffer, added
In elisa plate micropore, 37 DEG C of water-baths 3 hours, refrigerator is stored；
(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) confining liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, addition is diluted with dilution buffer
HRP enzyme labelled antibodies, 37 DEG C act on 2 hours, it is reacted with human IgG antibody；
(4) eluent is prepared：By papain with pH 8.0,0.1mol/L Tris-HCI buffers into 1-
2mg/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 the papain in eluent dense
Degree：Enzyme labelled antibody concentration ratio=1:80、1:40、1:20、1:10、1:5, wherein, each concentration makees 3 multiple holes, is respectively placed in
1h, 2h, 3h are eluted at a temperature of 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) with adding substrate solution same speed and order that terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed on ELIASA and reads every group of OD value respectively, is led to
The optimum concentration and elution time for compared with PBS group, comparing eluent are crossed, concrete outcome is referring to table 3.
Table 3：ELISA eluent best effort concentration and elution time determine
| 1:5 | 1:10 | 1:20 | 1:40 | 1:80 | |
| 1h | 0.281 | 0.168 | 0.081 | 0.114 | 0.469 |
| 2h | 0.250 | 0.115 | 0.050 | 0.183 | 0.438 |
| 3h | 0.225 | 0.106 | 0.100 | 0.196 | 0.441 |
From table 4 we it can be found that papain in eluent concentration and enzyme labelled antibody the ratio between concentration=
1:When 20, i.e. during the concentration 100ng/ml of papain, each group OD values are below other several groups, illustrate that the concentration eluent is washed
De- effect is optimal (to reach maximum, thus OD values by the human IgG antibody combined on ELISA hole walls-enzyme mark compound elution degree
It is minimum)；And action time either 1h, 2h, 3h, each group OD value changes are little, it is seen that with the extension of time, enzyme activity by
Decrescence weak, in the case where enzyme concentration is constant, digestibility can not be improved by extending digestion time, so eluent in this experiment
Action time is that 1-3h all may be used.
Application Example 1
Take using the mercury-IgG chelates in 100 parts of sample blood plasma of ELISA method detection, follow the steps below detection：
ELISA (ELISA method) detects mercury-IgG chelates, detects in accordance with the following steps：
1) anti-igg Ab is coated on solid phase carrier：Anti-igg Ab to 1000000 times is diluted with dilution buffer, is 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, add toluene, dissolve cell membrane；
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：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；Corresponding multiple is diluted to dilution buffer dilution measuring samples, that is, dilutes 20 times, adds in micropore, 37 DEG C of works
With 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 5000 times, and 37 DEG C act on 1 hour, make anti-HgAb and the mercury metal on IgG anti-
Should；
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) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA and reads measuring samples and mark respectively
The OD values (also directly can carry out qualitative detection by staining conditions without using ELIASA) of quasi- product, testing result is as shown in table 4.
Table 4：The ELISA testing results of mercury-IgG chelates 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-IgG chelates, are detected in accordance with the following steps：
1) IgG material will can be captured, as anti-igg antibody (anti-igg Ab) is coated on solid phase carrier：It is slow with dilution
Fliud flushing dilutes anti-igg Ab to 1000000 times, adds 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, add toluene, dissolve cell membrane；
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：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；20 times are diluted to dilution buffer dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, waits after the completion of washing, adds with papain
Concentration be 100ng/ml eluent, elute 1 hour；
6) detect：Sample, chelated in Atomic Absorption Spectrometer detection in the mercury on IgG, detected value is such as from ELISA micropores
Shown in table 5.
Table 5：The AAS testing results of mercury-IgG chelates in 100 parts of blood samples
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μg/L | 0.015 | 0.111 | 0.072 | 0.018 | 0.074 | 0.11 | 0.087 | 0.031 | 0.1 | 0.089 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 0.019 | 0.07 | 0.107 | 0.117 | 0.037 | 0.045 | 0.014 | 0.079 | 0.114 | 0.11 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 0.023 | 0.003 | 0.034 | 0.073 | 0.101 | 0.1 | 0.03 | 0.108 | 0.077 | 0.004 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 0.024 | 0.08 | 0.033 | 0.032 | 0.031 | 0.035 | 0.112 | 0.071 | 0.115 | 0.052 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 0.086 | 0.046 | 0.084 | 0.015 | 0.057 | 0.064 | 0.052 | 0.061 | 0.006 | 0.108 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 0.023 | 0.003 | 0.025 | 0.049 | 0.063 | 0.119 | 0.06 | 0.044 | 0.054 | 0.02 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 0.064 | 0.112 | 0.12 | 0.088 | 0.117 | 0.031 | 0.055 | 0.118 | 0.041 | 0.077 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 0.119 | 0.001 | 0.048 | 0.02 | 0.102 | 0.111 | 0.072 | 0.009 | 0.066 | 0.087 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 0.046 | 0.022 | 0.109 | 0.049 | 0.06 | 0.093 | 0.12 | 0.017 | 0.015 | 0.068 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 0.101 | 0.003 | 0.042 | 0.061 | 0.107 | 0.08 | 0.049 | 0.048 | 0.022 | 0.06 |
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-IgG chelate contents in this blood sample, are detected in accordance with the following steps：
1) IgG material will can be captured, as anti-igg antibody (anti-igg Ab) is coated on solid phase carrier：It is slow with dilution
Fliud flushing dilutes anti-igg Ab to 1000000 times, adds in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) 100 parts of standard blood samples are taken to add toluene as measuring samples, dissolve cell membrane；
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：Measuring samples, the mercury-IgG chelates with known content added in step 2)
Make standard items；20 times are diluted to dilution buffer dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, waits after the completion of washing, adds with papain
Concentration be 100ng/ml eluent, elute 1 hour；
6) it is acidified：Add nitric acid in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified；
7) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled from solution, in inductively coupled plasma matter
Detection chelates the mercury in IgG under spectrometer, and detected value is as shown in table 6.
The ICP-MS testing results of 6 100 parts of sample blood sample mercury-IgG chelates of table
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μg/L | 0.034 | 0.095 | 0.049 | 0 | 0.067 | 0.036 | 0.002 | 0.077 | 0.011 | 0.01 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 0.015 | 0.12 | 0.034 | 0.009 | 0.049 | 0.067 | 0.066 | 0.067 | 0.064 | 0.035 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 0.095 | 0.05 | 0.105 | 0.082 | 0.114 | 0.093 | 0.059 | 0.116 | 0.035 | 0.037 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 0.088 | 0.024 | 0.037 | 0.091 | 0.019 | 0.04 | 0.087 | 0.08 | 0.083 | 0.051 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 0.067 | 0.014 | 0.031 | 0.02 | 0.072 | 0.104 | 0.117 | 0.054 | 0.045 | 0.049 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 0.116 | 0.057 | 0.006 | 0.063 | 0.052 | 0.011 | 0.014 | 0.016 | 0.103 | 0.016 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 0.09 | 0.058 | 0.067 | 0.116 | 0.093 | 0.016 | 0.046 | 0.12 | 0.054 | 0.051 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 0.079 | 0.059 | 0.028 | 0.092 | 0.038 | 0.005 | 0.115 | 0.105 | 0.086 | 0.104 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 0.116 | 0.064 | 0.018 | 0.082 | 0.007 | 0.034 | 0.085 | 0.117 | 0.098 | 0.12 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 0.069 | 0.028 | 0.023 | 0.083 | 0.059 | 0.041 | 0.119 | 0.012 | 0.08 | 0.009 |
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 (6)
1. a kind of mercury-IgG chelates, mercury ion is formed with IgG by sulfydryl or/and cysteine residues chelating；
The preparation method of the mercury-IgG chelates, comprises the following steps：
A）The chelatropic reaction of mercury and IgG：Mercury ion is added in the IgG in people source or according to the IgG of biological method restructuring to carry out
Chelatropic reaction, obtain reaction solution；
B）The extraction of mercury-IgG chelates：Using immune-affinity chromatography, unreacted IgG and unnecessary is removed in reaction solution
Mercury ion, produce mercury-IgG chelates；
The step B）It is specific as follows：
（1）Sample dissolution：To by step A）Physiological saline is added in obtained reaction solution, redissolves mercury-IgG chelates；
（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with IgG specificity
With reference to 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, IgG are special with filler
The opposite sex combines；
（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate
Solution is eluted；
（5）Collect：Step is passed through in collection（4）Eluent after elution, makes protein renaturation immediately after collection；
（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；
（10）Collect：Step is passed through in collection（9）Eluent after elution, makes protein renaturation immediately after collection；
（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-IgG chelates.
2. mercury-IgG chelates according to claim 1, it is characterised in that its preparation methods steps B）Also include step afterwards
Rapid C）：Identification to mercury-IgG chelates；
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）It is middle to purify obtained mercury-IgG chelates, dilution buffer is added, and mix, 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 redissolved, Ran Houzai
Using ICP-MS methods, AAS methods or ELISA method detection whether containing mercury and detect mercury content.
3. a kind of mercury-IgG chelates as claimed in claim 1 are preparing the reagent for detecting mercury-IgG chelates in blood sample or examination
Application in agent box.
4. a kind of comprise at least kit of the mercury-IgG chelates as claimed in claim 1 as standard items.
5. kit according to claim 4, it is characterised in that also including coating buffer, the coating buffer, which contains, can capture IgG
Albumen or the material of mercury metal can be captured.
A kind of 6. method for quantitatively detecting mercury-IgG chelates, it is characterised in that with described in the claim 1 of known content
Mercury-IgG chelates are detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and original
Sub- absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification mercury-IgG chelates with it is enzyme-linked
Immune combined techniques, purification mercury-IgG chelates and atomic absorption spectrum combined techniques, purification mercury-IgG chelates and inductive etc.
Gas ions mass spectrum combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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| CN101139396A (en) * | 2007-08-08 | 2008-03-12 | 华南理工大学 | A kind of cadmium chelate monoclonal antibody and its preparation method and application |
| 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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|---|---|---|---|---|
| JPS5045687A (en) * | 1973-08-24 | 1975-04-23 | ||
| US5612016A (en) * | 1988-04-01 | 1997-03-18 | Immunomedics, Inc. | Conjugates of antibodies and bifunctional ligands |
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| CN101139396A (en) * | 2007-08-08 | 2008-03-12 | 华南理工大学 | A kind of cadmium chelate monoclonal antibody and its preparation method and application |
| 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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