CN105137059B - Mercury chelating immune complex and preparation method and application thereof - Google Patents
Mercury chelating immune complex and preparation method and application thereof Download PDFInfo
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- CN105137059B CN105137059B CN201510411290.3A CN201510411290A CN105137059B CN 105137059 B CN105137059 B CN 105137059B CN 201510411290 A CN201510411290 A CN 201510411290A CN 105137059 B CN105137059 B CN 105137059B
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- hydrargyrum
- immune complex
- chelating
- chelating type
- solution
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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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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Abstract
The invention discloses a mercury chelating immune complex, a preparation method and application thereof, wherein the mercury chelating immune complex is one of the following complexes: the mercury ions are combined with the immune complex to form a complex, or the mercury ions are combined with the carrier protein and the antibody which can be specifically combined with the carrier protein to form a complex; or a complex formed by binding mercury ions to a carrier protein after binding to immunoglobulins. The mercury chelating immune complex is a relatively specific immune complex, is used for detecting whether the mercury chelating immune complex exists in serum of people in a region and the content of the mercury chelating immune complex, and indirectly reflects the mercury pollution degree of the region.
Description
Technical field
The present invention relates to belonging to medical domain and in particular to hydrargyrum chelating type immune complex and its preparation method and application.
Background technology
Immune complex (Immune complexes, IC) is the product that antigen-antibody combines, according to whole nation science and technology
The definition of noun validation board, refers to the complex that antibody is combined with soluble antigen and is formed.It is divided into two classes, Yi Zhongshi
Fixing immune complex in tissue, and another kind is then the immune complex being free in blood, becomes circulation immunity and is combined
Thing (circulating immune complexes, CIC).Immune complex plays in removing and the various antigens of destruction can not
The effect lacking, the result of organism immune response is exactly continuous generation immune complex.When body endoantigen (or antibody) quantity
When being slightly more than antibody (or antigen), the immune complex of medium molecule size will be formed, it is both difficult to be swallowed by phagocyte,
Can not be discharged by glomerule filter opening again, can be free on the long period in blood and other body fluid.When Vascular Permeability increases
When, such IC or can be entrenched in glomerular basement membrane in the capillary wall at some positions with hypostasis, and activating complement is led
Cause immune complex deposit.These deposit or are embedded in the immune complex of blood vessel wall or tissue, are to cause vascular basement membrane inflammation
Initiating agent with tissue injury.Immune complex can cause tissue injury by following several respects：1) activating complement：Deposition
IC passes through activating complement, produces anaphylatoxin and the active fragment with chemotactic effect, the mastocyte of chemotactic to local, basophilic
Property granulocyte release active medium cause local vascular permeability increase, cause and ooze out and local edema；2) attract leukocyte infiltration
With gather：Neutrophilic granulocyte release toxicity oxide and lysosomal enzyme when swallowing IC, damage adjacent tissue；3) activation blood is little
Plate：IC can discharge vasoactive amine material by activated blood platelet, leads to vasodilation, permeability to increase, aggravation local is oozed
Go out and edema, and activate clotting mechanism, form microthrombus, cause ischemia, bleeding and tissue necrosiss.
The generation of multiple diseases is all closely bound up with the deposition of immune complex, such as systemic lupus erythematosus (sle), rheumatoid
Arthritis, nephrotic syndrome, cryoglobulinemia, vasculitiss, antibacterial, virus and parasitic infection, anaphylaxiss, autoimmune
Disease, dermatosiss etc..Early in the nineties in last century, scholar is just had to find, in normal body, immune complex is constantly dropped
Solution, thus typically can't detect immune complex or only assume low concentration, and the immunity in a series of above-mentioned diseases, in serum
Complex is that CIC content significantly raises.Although CIC detection does not have disease specific, its detection is provided that with regard to immunological disease
The information of reason, advancing of disease and prognosis aspect.In certain micro-organisms infection, autoimmune, detect circulating immune complex
Can be used as the index of Disease Activity, the function evaluating body and detection curative effect.And in the last few years, it is special that scholars are devoted to research
The immune complex of the opposite sex, each side such as the various specific immune complex of research and disease are caused a disease, disease development, medical diagnosis on disease
Relation, and these researchs will promote us to have further understanding to disease.
Hydrargyrum (Mercury, Hg) is a kind of ubiquitous heavy metal, is widely used in industry, agricultural, pharmaceutical sector, is one
Plant very important environmental contaminants.2011, U.S.'s poisonous substance and disease registration administration (United States Agency for
Toxic Substances and Disease Registry, ATSDR) listed in material preferred list
(Substance Priority List), and World Health Organization (WHO) (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., Hg is considered as one of four overall situation pollutant,
Drastically influence the healthy of people.Common hydrargyrum exposed population group include being engaged in metal smelt, exploitation gold mine, burn coal,
Electric industry and the crowd of wood producing, and for general population mainly by eat mercury pollution seafood, drink into mercury pollution water source,
Suck the mercuryvapour of fuel combustion or evaporation.Additionally, the contact of mercury cosmetic, filling amalgam dentistry implant, contact epidemic disease
Ethyl hydrargyrum in Seedling preservative, the contact of mercurous insecticide are all common hydrargyrum exposure chambers, hydrargyrum close phase with the life of people
Close.2009, in national health and nutrition test investigation, find (National Health and Nutrition
Examination Study, NHANES), the hydrargyrum in 2% U.S.'s Women of Childbearing Age body is all exceeded it is seen that mercury pollution is one
Problem demanding prompt solution.
American scholar A.Ramanaviciene in 2004 etc. finds circulating immune complex content and local environment in serum
Pollution is relevant, finds the increase with the local pollution control order of severity, and the CIC content in local Ox blood serum dramatically increases, and difference has
Statistically significant, thus can be used as the index evaluating local pollution control.But environmental pollution is how to stimulate CIC in body actually
Content significantly raises, and is a kind of or multiple pollutant collective effect leads to its rising, and whether the rising of CIC therefore can lead to machine
Bulk damage, increases the susceptibility for disease for the body, these all require study.It is known that circulating immune complex is a kind of
The protein of Special Significance, and hydrargyrum may act on the protein of the multiple system of whole body, it is multiple whether hydrargyrum can act on circulation immunity
Compound, and make the change of circulating immune complex content, activity change, changing function, these still lack correlational study.
Content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of hydrargyrum chelating type immune complex, should
Hydrargyrum chelating type immune complex is a kind of immune complex of relative specificity, whether there is for detecting in regional crowd's serum
Hydrargyrum chelating type immune complex and its content, reflect this regional mercury pollution degree indirectly.
For solving the above problems, the technical solution adopted in the present invention is as follows：
A kind of hydrargyrum chelating type immune complex, this hydrargyrum chelating type immune complex is one of following complex：
Mercury ion is incorporated into the complex of immune complex formation, or
Mercury ion is incorporated into carrier protein and the complex that can be formed with the antibody of this carrier protein specific binding；Or
The complex that mercury ion is combined to form with carrier protein after being incorporated into immunoglobulin.
Further, described carrier protein or immune complex at least contain zinc fingerses, sulfydryl, in cysteine residues
A kind of structure, mercury ion passes through at least one of zinc fingerses, sulfydryl, cysteine residues and carrier protein or immune complex
In conjunction with.
Specifically, described carrier protein is antibody protein, lipoprotein, hemoglobin, nuclear antigen, foreign sera antigen, disease
One of poison, antibacterial, protozoon, anthelmintic.
Further, mercury ion is specifically bound with energy and carrier protein after chelating agen is combined with carrier protein again
Antibodies.
Further, described chelating agen be ITCBE, EDTA, polyphosphate, amino carboxylic acid, 1,3- diketone, in hydroxy carboxylic acid
One kind.
Present invention also offers two kinds of methods preparing hydrargyrum chelating type immune complex, obtain hydrargyrum chela by both approaches
Mould assembly immune complex.
Wherein, the first preparation method includes
A the step) synthesizing hydrargyrum chelating type immune complex：First chelating agen is combined with carrier protein, then with mercury ion network
Close；
B the step) purifying hydrargyrum chelating type immune complex：Using immune-affinity chromatography removal step A) the hydrargyrum chela that synthesizes
Carrier protein, specific antibody and the mercury ion of reaction is had neither part nor lot in mould assembly immune complex.
Specifically, in the preparation method of described hydrargyrum chelating type immune complex,
(I) prepare chelating agent solution：Chelating agen is dissolved, is configured to chelating agent solution；
(II) formulation vehicle protein solution：Carrier protein is added in borate buffer solution, prepared carrier protein is molten
Liquid；
(III) it is stirred overnight：Step (I) chelating agent solution is added in the carrier protein solution of step (II), after stirring
Act on 2-30h in shaking table, obtain mixed liquor；
(IV) bag filter pretreatment：EDTA-NaHCO is added in bag filter3Solution boils, and uses ddH after abandoning waste liquid2O rushes
Wash, repeat this step 1-3 time；
(V) dialyse：The mixed liquor of step (III) is loaded in the bag filter after processing through step (IV), uses ddH2O is saturating
Analysis, changes water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid；
(VI) mercury ion chelating：Add HCl solution adjustment pH to 7.0 in the liquid in above-mentioned steps (V), be slowly added to
Mercury ion solution, shakes in Deca, after shaking table act on 2-30h, then repeat step (V) once, collect liquid, obtain
Hydrargyrum chelating type antigen；
(VII) combine：Adding in above-mentioned hydrargyrum chelating type antigen can be with the antibody of carrier protein specific binding, after reaction
Obtain hydrargyrum chelating type immune complex；
Described step B) comprise the following steps that：
I () is by above-mentioned A) the hydrargyrum chelating type immune complex of step synthesis redissolves in normal saline；
(ii) chromatographic column pretreatment：Using dilution buffer flushing line, chromatographic column loads energy and immune complex
The filler of specific binding, after dress post, continuation dilution buffer balances pillar；
The described filler that can specifically bind with immune complex can be with immune complex specificity junction mixture for being adsorbed with
The silica gel of matter or resin；
(ii) loading：After pillar balance, with dilution buffer dilution step (i) resulting solution, then upper prop, hydrargyrum chelates
Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer；
(iv) eluting：Rinse pillar using dilution buffer, to baseline balance, then using 0.05-0.10Mol/L's
Na2HPO4 solution carries out eluting；
V () collects：The eluent of collection step (iv), collects and makes protein renaturation immediately after finishing；
(vi) eluent collected loads bag filter ddH2O dialyses desalination, after changing water 2-4 time, 4 DEG C of dialysed overnight,
Collect sample；
(vii) chromatographic column pretreatment：Using new chromatographic column, use dilution buffer flushing line, fill in this chromatographic column
Enter and can balance pillar with dilution buffer with the filler of hydrargyrum specific binding again after dress post；
Described can be to be adsorbed with silica gel or the resin that can specifically bind material with hydrargyrum with the filler of hydrargyrum specific binding；
(viii) loading：After the pillar of step (vii) balances, dilute the sample of above-mentioned steps (vi) with dilution buffer
This, then by the sample upper prop after dilution, hydrargyrum chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined
Thing can flow out with dilution buffer；
(ix) eluting：Pillar is rinsed with dilution buffer, to baseline balance, then using 0.5- after step (viii)
The Na of 1.0Mol/L2HPO4Solution carries out eluting；
X () collects：The eluent of collection step (ix), collects and makes protein renaturation immediately after finishing；
(xi) bag filter ddH is loaded to the eluent of step (x)2O dialyses desalination, after changing water three times, dialyses for 4 DEG C
At night, collect sample, that is, obtain the hydrargyrum chelating type immune complex of purification.
Present invention also offers above-mentioned hydrargyrum chelating type immune complex is in the enzyme of preparation detection hydrargyrum chelating type immune complex
Application in linked immunoassay reagent kit.
Present invention also offers a kind of enzyme linked immunological kit of detection hydrargyrum chelating type immune complex, this test kit includes
The standard substance of hydrargyrum chelating type immune complex as claimed in claim 1.
Further, mentioned reagent box also includes at least one following reagent：Egg containing capture antigen antibody complex
White be coated liquid, the antibody containing capture metal Hg be coated liquid, enzyme labelled antibody.
A kind of method of detection by quantitative hydrargyrum chelating type immune complex is it is characterised in that hydrargyrum chelating type with known content
Immune complex is detected as standard substance, a pair of sample using following methods：Euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atom
Absorption spectrum combined techniqueses, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniqueses, purification immune complex and Atomic Absorption
Spectrum combined techniqueses, purification immune complex and inductivity coupled plasma mass spectrometry combined techniqueses, electrophoresis and enzyme linked immunological or atom are inhaled
Receive spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
Compared to existing technology, the beneficial effects of the present invention is：
1. synthesized hydrargyrum chelating type immune complex first, and confirmed that hydrargyrum may be present in immune system in body, and
Demonstrate the presence of hydrargyrum chelating type immune complex；
2. present invention achieves the specific recognition of immune complex, and achieve the quantitation of hydrargyrum chelating type immune complex
Detection, the method that the mercury content level in immune angle testing machine body is provided, and provide for the mercury pollution level of industrial area
Indirect indexes.
With reference to the accompanying drawings and detailed description the present invention is described in further detail.
Brief description
Fig. 1 is immune complex non denatured electrophoretic band figure；
Fig. 2 is the synchrotron radiation line x-ray fluorescence analysiss figure of the electrophoretic band of immune complex；
Wherein, the M in Fig. 1 is Marker, and IC is hydrargyrum chelating type immune complex；Abscissa in Fig. 2 is protein band
Position, vertical coordinate is hydrargyrum metal energy in this protein band.
Specific embodiment
A kind of hydrargyrum chelating type immune complex, this hydrargyrum chelating type immune complex is one of following complex：
Mercury ion is incorporated into the complex of immune complex formation, or
Mercury ion is incorporated into carrier protein and the complex that can be formed with the antibody of this carrier protein specific binding；Or
The complex that mercury ion is combined to form with carrier protein after being incorporated into immunoglobulin.
Specifically, described carrier protein or immune complex at least contain zinc fingerses, sulfydryl, one in cysteine residues
Plant structure, mercury ion passes through at least one of zinc fingerses, sulfydryl, cysteine residues and carrier protein or immune complex is tied
Close.
Specifically, described carrier protein is antibody protein, lipoprotein, hemoglobin, nuclear antigen, foreign sera antigen, disease
One of poison, antibacterial, protozoon, anthelmintic.
Further, mercury ion is specifically bound with energy and carrier protein after chelating agen is combined with carrier protein again
Antibodies.
Further, described chelating agen be ITCBE, EDTA, Glutathione (GSH), polyphosphate, amino carboxylic acid, 1,3-
One of diketone, hydroxy carboxylic acid.
Present invention also offers two kinds of methods preparing hydrargyrum chelating type immune complex, obtain hydrargyrum chela by both approaches
Mould assembly immune complex.
Wherein, the first preparation method includes
A the step) synthesizing hydrargyrum chelating type immune complex；
B the step) purifying hydrargyrum chelating type immune complex：Using immune-affinity chromatography removal step A) the hydrargyrum chela that synthesizes
Carrier protein, specific antibody and the mercury ion of reaction is had neither part nor lot in mould assembly immune complex.
Specifically, in the preparation method of described hydrargyrum chelating type immune complex,
(I) prepare chelating agent solution：Chelating agen is dissolved, is configured to chelating agent solution；
(II) formulation vehicle protein solution：Carrier protein is added in borate buffer solution, prepared carrier protein is molten
Liquid；
(III) it is stirred overnight：Step (I) chelating agent solution is added in the carrier protein solution of step (II), after stirring
Act on 2-30h in shaking table, obtain mixed liquor；
(IV) bag filter pretreatment：EDTA-NaHCO is added in bag filter3Solution boils, and uses ddH after abandoning waste liquid2O rushes
Wash, repeat this step 1-3 time；
(V) dialyse：The mixed liquor of step (III) is loaded in the bag filter after processing through step (IV), uses ddH2O is saturating
Analysis, changes water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid；
(VI) mercury ion chelating：Add HCl solution adjustment pH to 7.0 in the liquid in above-mentioned steps (V), be slowly added to
Mercury ion solution, shakes in Deca, after shaking table act on 2-30h, then repeat step (V) once, collect liquid, obtain
Hydrargyrum chelating type antigen；
(VII) combine：Adding in above-mentioned hydrargyrum chelating type antigen can be with the antibody of carrier protein specific binding, after reaction
Obtain hydrargyrum chelating type immune complex；
Described step B) comprise the following steps that：
I () is by above-mentioned A) the hydrargyrum chelating type immune complex of step synthesis redissolves in normal saline；
(ii) chromatographic column pretreatment：Using dilution buffer flushing line, chromatographic column loads energy and immune complex
The filler of specific binding, after dress post, continuation dilution buffer balances pillar；
(ii) loading：After pillar balance, with dilution buffer dilution step (i) resulting solution, then upper prop, hydrargyrum chelates
Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer；
(iv) eluting：Rinse pillar using dilution buffer, to baseline balance, then using 0.05-0.10Mol/L's
Na2HPO4 solution carries out eluting；
V () collects：The eluent of collection step (iv), collects and makes protein renaturation immediately after finishing；
(vi) eluent collected loads bag filter ddH2O dialyses desalination, after changing water 2-4 time, 4 DEG C of dialysed overnight,
Collect sample；
(vii) chromatographic column pretreatment：Using new chromatographic column, use dilution buffer flushing line, fill in this chromatographic column
Enter and can balance pillar with dilution buffer with the filler of hydrargyrum specific binding again after dress post；
(viii) loading：After the pillar of step (vii) balances, dilute the sample of above-mentioned steps (vi) with dilution buffer
This, then by the sample upper prop after dilution, hydrargyrum chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined
Thing can flow out with dilution buffer；
(ix) eluting：Pillar is rinsed with dilution buffer, to baseline balance, then using 0.5- after step (viii)
The Na of 1.0Mol/L2HPO4Solution carries out eluting；
X () collects：The eluent of collection step (ix), collects and makes protein renaturation immediately after finishing；
(xi) bag filter ddH is loaded to the eluent of step (x)2O dialyses desalination, after changing water three times, dialyses for 4 DEG C
At night, collect sample, that is, obtain the hydrargyrum chelating type immune complex of purification.
Present invention also offers above-mentioned hydrargyrum chelating type immune complex is in the enzyme of preparation detection hydrargyrum chelating type immune complex
Application in linked immunoassay reagent kit.
Present invention also offers a kind of enzyme linked immunological kit of detection hydrargyrum chelating type immune complex, this test kit includes
The standard substance of hydrargyrum chelating type immune complex as claimed in claim 1.
Further, mentioned reagent box also includes at least one following reagent：Egg containing capture antigen antibody complex
White be coated liquid, the antibody containing capture metal Hg be coated liquid, enzyme labelled antibody.
In the present invention, the test kit enabling the object of the invention can be listed following several, but is not limited to this.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including：It is combined containing antigen-antibody can be captured
The albumen of thing be coated liquid, confining liquid, lavation buffer solution, anti-hydrargyrum antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer,
Positive control, negative control.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including：It is combined containing antigen-antibody can be captured
The albumen of thing be coated liquid, confining liquid, lavation buffer solution, eluent, positive control, negative control.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including：It is combined containing antigen-antibody can be captured
The albumen of thing be coated liquid, confining liquid, lavation buffer solution, eluent, nitric acid, hydrogen peroxide, positive control, negative control.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including for purifying serum immune complex institute
Need solution, redissolve liquid, containing can capture mercury metal antibody be coated liquid, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate,
Terminate liquid, dilution buffer, standard substance, negative control.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex：Including for purifying serum immune complex institute
Need solution, redissolve liquid, positive control, negative control.
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including for purifying serum immune complex institute
Need solution, redissolve liquid, nitric acid, hydrogen peroxide, positive control, negative control etc..
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including for purifying serum immune complex institute
Need solution, glue bed medium, redissolve liquid, sample-loading buffer, dissolve the liquid needed for protein band containing antigen antibody complex on glue bed
Body, containing can capture mercury metal antibody be coated liquid, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, dilution
Buffer, positive control, negative control etc..
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including for purifying serum immune complex institute
Need solution, glue bed medium, redissolve liquid, sample-loading buffer, dissolve the liquid needed for protein band containing antigen antibody complex on glue bed
Body, positive control, negative control etc..
A kind of test kit for detecting hydrargyrum chelating circulating immune complex, including for purifying serum immune complex institute
Need solution, glue bed medium, redissolve liquid, sample-loading buffer, dissolve the liquid needed for protein band containing antigen antibody complex on glue bed
Body, nitric acid, hydrogen peroxide, positive control, negative control etc..
In above-mentioned several test kit, the albumen of described capture circulating immune complex, including but not limited to C1Q., CIF
Albumen, anti-C_3 antibody, such as article No. are the C1q Recombinant Protein of " NOVUS H00000712-p01 "；Described capture
The Mus that it is " bar is proud to obtain AP7014 " from Ba Ao get bio tech ltd article No. that the antibody of mercury metal includes but is not limited to buy
Anti- Hg mAb；Confining liquid is the bovine serum albumin of 1%-5% or defatted milk powder for mass concentration；Eluent includes but is not limited to wood
The Tris-HCl buffer solution of melon protease；Described glue bed medium includes but is not limited to agarose gel, polyacrylamide gel；
Include but is not limited to PEG solution, borate buffer solution for purifying solution needed for serum immune complex；Substrate includes but does not limit
In TMB solution, ABTS solution；Described sample-loading buffer includes but is not limited to the Tris-HCl buffer solution containing bromophenol blue；Enzyme
Labeling antibody is the antibody containing the enzyme labelling such as horseradish peroxidase, alkali phosphatase, such as HRP enzyme labelled antibody；Standard substance include
But be not limited to the present invention hydrargyrum chelating type circulating immune complex, other be chelated with the immune complex of hydrargyrum metal；Positive control
Including but not limited to hydrargyrum chelating type circulating immune complex of the present invention, other be chelated with the immune complex of hydrargyrum metal, negative right
According to for dilution buffer.
Present invention also offers a kind of method of detection by quantitative hydrargyrum chelating type immune complex, with the hydrargyrum chelating of known content
Type immune complex is detected as standard substance, a pair of sample using following methods：Euzymelinked immunosorbent assay (ELISA), enzyme linked immunological with former
Sub- absorption spectrum combined techniqueses, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniqueses, purification immune complex and atom are inhaled
Receive spectrum combined techniqueses, purification immune complex and inductivity coupled plasma mass spectrometry combined techniqueses, electrophoresis and enzyme linked immunological or atom
Absorption spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
In the present invention, having that the method with detecting hydrargyrum chelating type immune complex can be listed is following several, but not
It is limited to following several.
Unless otherwise indicated, the laboratory operating procedures being related to are the conventional step in this area to the present invention, and reagent, material are such as
Following cited, that does not enumerate out in the present invention is commonly used in the art or can be obtained by commercial mode,
The following is the test that the present invention uses as follows with reagent：
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example：Take the Na of 1.5g2CO3With
2.93g NaHCO3Dissolving plus ddH2O is settled to 1000mL；
Lavation buffer solution is the 0.15M PBS of pH7.4, compound method example：Take the KH of 0.2g2PO4, 2.90g
Na2HPO4·12H2KCl, 0.5mL Tween-20 of NaCl, 0.2g of O, 8.0g, dissolving plus ddH2O is settled to 1000mL；
Confining liquid is bovine serum albumen solution, compound method example：Take 0.1g bovine serum albumin, add lavation buffer solution dilute
Release and be settled to 100mL；
Terminate liquid is 2M H2SO4, compound method example：Take the ddH of 178.3mL2O, to ddH2Dropwise add dense in O along wall
H2SO4, stirring while adding, it is settled to 200mL；
The pH of substrate buffer solution is 5.0, Na2HPO4Molar concentration be 0.2M, citric acid molar concentration be 0.1M, often
The preparation method of the substrate buffer solution of 50mL is as follows：Take 1.42gNa2HPO4, 0.96g citric acid, be subsequently adding ddH2O to 50mL,
Obtain final product；
Substrate is methyl biphenyl amine (abbreviation TMB) solution, and this methyl biphenyl amine (TMB) solution is by the group according to following ratio
Assignment system forms：TMB：Substrate buffer solution：0.75%H2O2=0.5mL：10mL：32 μ L, wherein TMB are the methyl biphenyl of 2g/L
Amine ethanol solution；
The albumen that immune complex can be captured is can be with the albumen of immune complex specific binding, including but not limited to
As C1Q., CIF albumen, anti-C_3 antibody；In following examples, can capture immune complex albumen specifically used be
Article No. is the C1q Recombinant Protein of " NOVUS H00000712-p01 "；
The material of capture hydrargyrum is to buy the Mus being " bar is proud to obtain AP7014 " from Ba Ao get bio tech ltd article No. to resist
HgmAb；Wherein, the material specifically binding with hydrargyrum of the present invention, anti-Hg antibody, anti-hydrargyrum antibody are the material capturing hydrargyrum；
Described can be rabbit anti-human serum albumin antibodies with the antibody of human serum albumin's specific binding, be commercially available；
Described below dilution multiple proportions is w/v.
Method one：ELISA method detects hydrargyrum chelating type immune complex, comprises the following steps that：
1) it is coated：The albumen that immune complex can be captured is coated on solid phase carrier：Dilute bag with dilution buffer
By albumen to 2500-20000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, storage
Deposit refrigerator；
2) close：Remove dilution buffer, and washed with cleaning mixture, after the completion of waiting to wash, addition confining liquid, 37 DEG C
Place 1 hour, remove confining liquid, and washed with cleaning mixture, after the completion of washing, elisa plate is placed 1 hour in 37 DEG C；
3) add measuring samples, and incubate：Blood sampling, makees measuring samples；Compound with the hydrargyrum chelating type immunity of known content
Thing makees standard substance；Dilute 10-40 times with dilution buffer, add in micropore, 37 DEG C of effect 1-2 hours；
4) add the material of capture hydrargyrum, and incubate：Remove measuring samples, and washed with cleaning mixture, wait to have washed
Cheng Hou, addition dilution buffer dilutes 2500-20000 times of anti-hydrargyrum antibody, and 37 DEG C of effect 1-2 hours are so as to answer with immunity
Mercury metal reaction on compound；
5) enzyme conjugates incubate：Remove anti-Hg antibody, and washed with cleaning mixture, after the completion of waiting to wash, add with dilute
Release buffer dilution HRP enzyme labelled antibody, 37 DEG C effect 1-2 hours so as to anti-Hg antibody response；
6) substrate incubates：Remove enzyme labelled antibody, and washed with cleaning mixture, after the completion of waiting to wash, addition substrate, 37 DEG C
Lucifuge acts on 30 minutes；
7) terminating reaction：Deca terminate liquid is to each micropore；
8) take wavelength to be 405nm, after adding terminate liquid, testing sample group was read within the stipulated time on microplate reader respectively
With the OD value of standard substance, by comparing with standard item group, the content trying to achieve testing sample (also can not use microplate reader, directly lead to
Cross colour developing situation and carry out qualitative detection).
The method utilizes enzyme-linked immunosorbent assay (ELISA) principle, can be combined the nonspecific immunity in serum
Thing extracts, and the immune complex extracting partly is chelated with heavy metal Hg, and the hydrargyrum on this partial immunity complex
Can be by the material specifically binding with hydrargyrum or the specific antibody institute that the anti-hydrargyrum forming antigen antibody complex can be reacted with hydrargyrum
Capture, can be captured (this antibody nonrecognition bag by the antibody of the enzyme labelling such as horseradish peroxidase, alkali phosphatase afterwards again
By albumen), the antibody in capture, in the presence of developer and terminate liquid, reads OD value under instrument, and does not contain chelating gold
Belong to the immune complex of hydrargyrum, then will not be captured by the specific antibody of anti-hydrargyrum, also will not be with horseradish peroxidase, alkaline phosphorus
The antibody of the enzyme labelling such as sour enzyme is captured, and does not also contain mercury metal (negative control group result is feminine gender) in agents useful for same, because
And when the OD value result being read is shown as the positive, you can prove to detect the mercury metal of chelating in circulating immune complex.
Method two：ELISA method+AAS method detects hydrargyrum chelating type immune complex, comprises the following steps that：
1) it is coated：The albumen that immune complex can be captured is coated on solid phase carrier, is diluted to dilution buffer
2500-20000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
2) close：Remove dilution buffer, and washed with cleaning mixture, after the completion of waiting to wash, addition confining liquid, 37 DEG C
Place 1 hour, remove confining liquid, and washed with cleaning mixture.After the completion of washing, elisa plate places 1 in 36.5-37.5 DEG C
Hour；
3) add measuring samples, and incubate：From blood circulation sampling, make measuring samples；Hydrargyrum chelating type with known content
Immune complex makees standard substance；Dilute 10-40 times with dilution buffer, add in micropore, 37 DEG C of effect 1-2 hours；
4) eluting：Remove measuring samples, and washed with cleaning mixture, after the completion of waiting to wash, addition extension rate is 5-
80 times of eluent, acts on 0-2 hour at 37-57 DEG C；Eluent；
5) detect：Sample from ELISA micropore, chelate the hydrargyrum on immune complex in Atomic Absorption Spectrometer detection,
And draw standard curve, read respective value.
The method further combines atomic absorption spectrum (AAS) principle on the basis of enzyme linked immunological principle, using former
Sub- absorption spectrometer detection chelates the hydrargyrum in circulating immune complex, contains only immune complex due in solution, and used
Do not contain any heavy metal (negative control group result be feminine gender) in reagent, result will not be interfered, thus work as and read
When result is shown as the positive, you can prove to detect the mercury metal of chelating in circulating immune complex.
Method three：ELISA method+ICP-MS method detects hydrargyrum chelating type immune complex, comprises the following steps that：
1) it is coated：The albumen that immune complex can be captured is coated on solid phase carrier：Dilute bag with dilution buffer
By 00-20000 times of protein 25, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, storage
Refrigerator；
2) close：Remove dilution buffer, and washed with cleaning mixture, after the completion of waiting to wash, addition confining liquid, 37 DEG C
Place 1 hour, remove confining liquid, and washed with cleaning mixture.After the completion of washing, elisa plate places 1 in 36.5-37.5 DEG C
Hour；
3) add measuring samples, and incubate：From blood circulation sampling, make measuring samples；Hydrargyrum chelating type with known content
Immune complex makees standard substance；Dilute 10-40 times with dilution buffer, add in micropore, 37 DEG C of effect 1-2 hours；
4) eluting：Remove measuring samples, and washed with cleaning mixture, after the completion of waiting to wash, add eluent, in 37 DEG C
Lower effect 0-2 hour；
5) it is acidified：Add acidulant that solution is acidified in the solution, sealing overnight, is thoroughly acidified；
6) detect：Add hydrogen peroxide, and heat and catch up with acid, sampling, detects chela under icp mses
Together in the hydrargyrum on immune complex, read respective value.
The method further combines inductively coupled plasma mass spectrometry (ICP- on the basis of enzyme linked immunological principle
MS) principle, chelates the hydrargyrum in circulating immune complex with ICP-MS detection, contains only immune complex due in solution, and
Do not contain any heavy metal (negative control group result is feminine gender) in agents useful for same, result will not be interfered, thus work as and read
When the result taking is shown as the positive, you can prove to detect the mercury metal of chelating in circulating immune complex.
Method four：Purification CIC method+ELISA method detection hydrargyrum chelating type immune complex, comprises the following steps that：
1) extract nonspecific immunity complex：Using the Polyethylene Glycol PEG sedimentation method, ultracentrifugation, molecule ultrafiltration, gel
The methods such as filtration are extracted nonspecific immunity complex and are redissolved the immune complex double solvents purifying out in solution；
2) use can capture the material of hydrargyrum and be coated on solid phase carrier：With dilution buffer dilution coated antibody to 2500-
20000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
3) close：Remove dilution buffer, and washed with cleaning mixture, after the completion of waiting to wash, plus 1%-5% Ox blood serum
Albumin or defatted milk powder, as confining liquid, are placed 1 hour for 37 DEG C, are removed confining liquid, and washed with cleaning mixture, washed
Cheng Hou, elisa plate is placed 1 hour in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Sample from the redissolution liquid of the nonspecific immunity complex extracting, make to be checked
Sample；Standard substance are made with the hydrargyrum chelating type immune complex of known content；Dilute 10-40 times with dilution buffer, add to micropore
In, 37 DEG C of effect 1-2 hours；
5) enzyme conjugates incubate：Remove the redissolution liquid of nonspecific immunity complex, and washed with cleaning mixture, to be washed
After the completion of washing, the HRP enzyme labelled antibody that addition is diluted with dilution buffer, 37 DEG C of effect 1-2 hours are so as to anti-with anti-Hg antibody
Should；
6) substrate incubates：Remove HRP enzyme labelled antibody, and washed with cleaning mixture, after the completion of waiting to wash, add substrate,
37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction：Deca terminate liquid is to each micropore.
8) take wavelength to be 405nm, after adding terminate liquid, testing sample group was read within the stipulated time on microplate reader respectively
With the OD value of standard substance, by comparing with standard item group, the content trying to achieve testing sample (also can not use microplate reader, directly lead to
Cross colour developing situation and carry out qualitative detection).
Method five：Purification CIC method+AAS method detection hydrargyrum chelating type immune complex, comprises the following steps that：
1) extract nonspecific immunity complex：Using the Polyethylene Glycol PEG sedimentation method, ultracentrifugation, molecule ultrafiltration, gel
The methods such as filtration are extracted nonspecific immunity complex and are redissolved the immune complex purifying out；
2) detect：From step 1) sample solution, chelate the hydrargyrum on immune complex in Atomic Absorption Spectrometer detection,
Read respective value.
Method six：Purification CIC method+ICP-MS method detection hydrargyrum chelating type immune complex, comprises the following steps that：
1) extract nonspecific immunity complex：Using the Polyethylene Glycol PEG sedimentation method, ultracentrifugation, molecule ultrafiltration, gel
The methods such as filtration are extracted nonspecific immunity complex and are redissolved the immune complex purifying out；
2) it is acidified：From step 1) sample solution, add corresponding acidulant that solution is acidified in the solution, sealed
At night, thoroughly it is acidified；
3) detect：Add hydrogen peroxide, and heat and catch up with acid, then take heating to catch up with the solution 0.5mL after acid in inductance coupling again
Close detection under plasma mass spectrograph and chelate the hydrargyrum on immune complex, read respective value.
Method four, method five and method six are all first to isolate immune complex, then adopt method for detecting specificity, measure
The content of hydrargyrum on hydrargyrum chelating type immune complex in immune complex；First adopt physical separation means, such as supercentrifugation, height
Pressure liquid chromatography, gel-filtration chromatography etc., immune complex are separated from test plasma sample and redissolve in life
In reason saline, recycle ELISA principle, atomic absorption spectrum detection or carry out detecting inductively coupled plasma mass spectrometry detection
Mercury content on hydrargyrum chelating type immune complex.
Method seven：Electrophoresis method+ELISA/AAS/ICP-MS method detects hydrargyrum chelating type immune complex, comprises the following steps that：
1) extract nonspecific immunity complex：Using the Polyethylene Glycol PEG sedimentation method, ultracentrifugation, molecule ultrafiltration, gel
The methods such as filtration are extracted nonspecific immunity complex and are redissolved the immune complex purifying out, obtain the molten of immune complex
Liquid；
2) prepare glue bed：Select suitable medium (as agarose gel, polyacrylamide gel etc.) as needed, according to
Requirement prepares glue bed；
3) it is loaded：From step 1) solution sample 8 μ L, add 2 μ L sample-loading buffers, and mix, be then loaded onto sample
In product groove；
Sample-loading buffer (Sample buffer) can be formulated by the component of following ratio：Tris-HCl：1% bromine phenol
Blue：ddH2O：Glycine=15.5：2.5：7：25, the wherein pH of Tris-HCl are 6.8, molar concentration is 1M.
4) electrophoresis：Connect electrophoresis plate, carry out electrophoresis, and according to demand that albumen is isoparametric according to molecular weight, isoelectric point, IP
Difference carries out separating；
Described electrophoretic buffer can be obtained by the following method：Take Tris3.0g, glycine 14.4g, be dissolved in
800mLddH2In O, adjust pH to 8.3, be subsequently adding ddH2O to 1000mL, obtains final product；
5) detect：Protein band containing immune complex is found out on glue bed, this band is taken out, after treatment will
Band dissolves, and is then utilized respectively the principles such as ELISA, ICP-MS, AAS detection mercury content again；Further, it is also possible to utilize the method
The isoelectric point, IP of immune complex of detection chelating hydrargyrum, molecular weight and content etc..
Method seven is on the basis of method four, method five, method six, and hydrargyrum chelating type immune complex is carried from whole blood
Take out, then the hydrargyrum chelating type immune complex being extracted is carried out separate using gel electrophoresis, and then from electrophoretic band
Find out the protein band containing immune complex, carry out assay.
Embodiment 1
In the present embodiment, ITCBE buys from Japanese colleague's chemistry institute, article No. M030；
Following borate buffer solutions, its compound method example：Weigh 0.31g boric acid and be dissolved in 400mLddH2In O, use
The NaOH aqueous solution of 0.1Mol/L adjusts pH to 9.0, is settled to 500mL, obtains final product；
Following EDTA-NaHCO3The compound method example of solution：Take 1.86g EDTA 2H2O and 16.8g NaHCO3, molten
In 900mLddH2In O, adjust pH to 8.0 with 1.0M NaOH and be settled to 1000mL, autoclaving, obtain final product, 25 ± 2 DEG C of preservations；
The molecular cut off of following bag filters is 14000, buys from Bioshop Inc；Bag filter is through following process：Will
Bag filter puts into the EDTA-NaHCO of 500mL volume3In solution, boil 10min；Tipping EDTA-NaHCO3Solution, uses ddH2O floats
Wash, then boil 10min with 500mL 5mmol/L EDTA；Discard boiling liquid, thoroughly use ddH2O cleans, and adds substantial amounts of ddH2O
Soak 4 DEG C of bag filter overnight；During use, put on one's gloves, take out bag filter, use substantial amounts of ddH2Its surfaces externally and internally of O cleaning down.
The present embodiment provides a kind of hydrargyrum chelating type immune complex, comprises the following steps：
A) synthesize hydrargyrum chelating type immune complex, concrete operation step is as follows：
(I) prepare chelating agent solution：2.0mg ITCBE is dissolved in 2mL DMSO solvent, is configured to chelating agent solution；
(II) prepare human serum albumin solution：The human serum albumin of 4.0mg is added to the pH=9.0's of 4mL
In 0.01M borate buffer solution, prepared human serum albumin solution；
(III) it is stirred overnight：Step (I) chelating agent solution is slowly added into the human serum albumin solution of step (II)
In, stir in Deca, in 25 DEG C, act on 24h in the shaking table of 100r/min, then dialysed 24h with bag filter, remove not with people
The ITCBE that serum albumin combines, obtains mixed liquor；
(IV) bag filter pretreatment：EDTA-NaHCO is added in bag filter3Solution boils, and uses ddH after abandoning waste liquid2O rushes
Wash；Repeat this step 2 time；
(V) dialyse：The mixed liquor of step (III) is loaded in the bag filter after processing through step (IV), uses ddH2O is saturating
Analysis, changes ddH2O 3 times, after 4 DEG C of dialysed overnight, collects liquid；
(VI) mercury ion chelating：Add HCl solution adjustment pH to 7.0 in the liquid in above-mentioned steps (V), be slowly added to
The 1mmol/L mercury ion solution of 80 μ L, shakes in Deca, after shaking table act on 2-30h, then repeat step (V) 1 time,
Collect liquid, obtain hydrargyrum chelating type antigen；
(VII) combine：Add in hydrargyrum chelating type antigen prepared by step (VI) and can tie with human serum albumin's specificity
Hydrargyrum chelating type immune complex is obtained after the antibody response closing；
B) purify hydrargyrum chelating type immune complex, concrete operation step is as follows：
I () is by above-mentioned A) the hydrargyrum chelating type immune complex of step synthesis redissolves in normal saline；
(ii) chromatographic column pretreatment：Using dilution buffer flushing line, chromatographic column loads energy and immune complex
The filler of specific binding, after dress post, continuation dilution buffer balances pillar；
(ii) loading：After pillar balance, with dilution buffer dilution step (i) resulting solution, then upper prop, hydrargyrum chelates
Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer；
(iv) eluting：Rinse pillar using dilution buffer, to baseline balance, then using 0.05-0.10Mol/L's
Na2HPO4Solution carries out eluting；
V () collects：The eluent of collection step (iv), collects and makes protein renaturation immediately after finishing；
(vi) eluent collected loads bag filter ddH2O dialyses desalination, after changing water 2-4 time, 4 DEG C of dialysed overnight,
Collect sample；
(vii) chromatographic column pretreatment：Using new chromatographic column, use dilution buffer flushing line, fill in this chromatographic column
Enter and can balance pillar with dilution buffer with the filler of hydrargyrum specific binding again after dress post；
(viii) loading：After the pillar of step (vii) balances, dilute the sample of above-mentioned steps (vi) with dilution buffer
This, then by the sample upper prop after dilution, hydrargyrum chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined
Thing can flow out with dilution buffer；
(ix) eluting：Pillar is rinsed with dilution buffer, to baseline balance, then using 0.5- after step (viii)
The Na of 1.0Mol/L2HPO4Solution carries out eluting；
X () collects：The eluent of collection step (ix), collects and makes protein renaturation immediately after finishing；
(xi) bag filter ddH is loaded to the eluent of step (x)2O dialyses desalination, after changing water three times, dialyses for 4 DEG C
At night, collect sample, that is, obtain the hydrargyrum chelating type immune complex of purification.
Hydrargyrum chelating type immune complex prepared by the present embodiment, is separated further by gel electrophoresiss, and passes through inductance
Coupled plasma mass spectrometry or atomic absorption spectrum carry out detecting Qualitative Identification, concrete authentication step is as follows：
1) prepare glue bed：Select non-denaturing polyacrylamide gel as medium as needed, prepare glue bed；
2) it is loaded：Take the hydrargyrum chelating type immune complex solution that 8 μ L purify out, add 2 μ L sample-loading buffers, and mix
Even, then it is loaded onto in sample cell；
Described sample-loading buffer (Sample buffer) can be formulated by the component of following ratio：Tris-HCl：1%
Bromophenol blue：ddH2O：Glycine=15.5：2.5：7：25, the wherein pH of Tris-HCl are 6.8, molar concentration is 1M；
3) electrophoresis：Connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer, in electrophoresis process, electric current is 22mA constant current, environment
Temperature is 4 degree；Move to stopping electrophoresis during glue bottom to bromophenol blue, non denatured electrophoretic band figure is shown in Fig. 1；
Described electrophoretic buffer can be obtained by the following method：Take Tris3.0g, glycine 14.4g, be dissolved in
800mLddH2In O, adjust pH to 8.3, be subsequently adding ddH2O to 1000mL, obtains final product；
4) detect：Protein band containing antigen antibody complex is found out on glue bed, this band is taken out, after treatment
Band is dissolved, then recycles inductivity coupled plasma mass spectrometry or atomic absorption spectrum to detect whether to contain containing hydrargyrum and hydrargyrum
Amount.
Will be " same in the 4W1 of BEPC (BEPC) for the SRXRF analysis of micronutrient levelss in protein band
Complete on step radiation bunch, in storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample mobile station (TSA200
Type, stand upright Han Guang company in Beijing) can the stepper motor of computer controls drive lower edge X, Y two-dimensional square move up with change into
Penetrate facula position, moving step length is 0.0025mm.The X-ray going out from electromagnetic radiation is by Si (Li) detector (PGT Inc.LS
30143-DS) detect, probe with incident SR line copline and is mutually perpendicular to, and away from sample irradiation point 20mm, signal is divided with PGT multiple tracks
Analyzer (MCA 4000) obtains output.Excite sample with the monochromatic synchrotron radiation light of 11.5keV, adjust launching spot (1mmx
3mm) position is allowed to be in band one end, and in the minute of 300s, hot spot uniformly slowly moves along band always, counts knot
During bundle, hot spot moves on to this band other end.Take a spectrum along the every 1mm in electrophoresis direction.Using AX IL software data processing, and it is used for
Come from air and the Ar signal peak of content constant carries out normalization to other element peaks, changed to signal with offsetting beam intensity
The strong and weak impact producing.Measure the fluorescence Spectra of quantitative criterion dry glue film at identical conditions in the same way.
The synchrotron radiation line x-ray fluorescence of the electrophoretic band of lead chelating type immune complex of method preparation of the present embodiment divides
Analysis figure refers to Fig. 2, and the abscissa in Fig. 2 is protein band position, and vertical coordinate is hydrargyrum metal energy in this protein band.
Hydrargyrum chelating type immune complex in the present embodiment, can be used for preparing the reagent that detection hydrargyrum chelates immune complex,
Or can be used for preparing in enzyme linked immunological kit.
The determination of testing conditions
1. the determination of the optimum diluting multiple of complement protein best effort concentration and blood plasma
The hydrargyrum chelating type immune complex being provided using the present invention, as standard substance, determines complement using Checkerboard titration method
The optimum diluting multiple of C1q, the best effort concentration of anti-Hg antibody and blood plasma, Checkerboard titration method comprises the following steps that：
1) immobilized：C1Q. is coated on solid phase carrier, presses 1 with dilution buffer:2500、1:5000、1:10000、
1:20000 doubling dilution, adds in elisa plate micropore, deposits 18 hours at 4 DEG C；
2) close：Remove dilution buffer, washing, plus confining liquid, place 1 hour for 37 DEG C, remove confining liquid, washing；
3) incubate：Respectively plus test plasma sample and hydrargyrum chelating type immune complex standard substance, press 1 with dilution buffer:
10、1:20、1:40 doubling dilution, adds in elisa plate micropore, and 37 DEG C incubate 2 hours；
4) capture：Remove test plasma, washing, addition presses 1 with dilution buffer:2500、1:5000、1:10000、1:
The anti-Hg antibody of 20000 doubling dilution, 37 DEG C act on 2 hours so as to react with the mercury metal in circulating immune complex；
5) enzyme conjugates incubate：Remove anti-hydrargyrum antibody, washed with lavation buffer solution, add the enzyme that antibody concentration is 2 μ g/mL
Labeling antibody, 37 DEG C incubate 1-2 hours so as to anti-Hg antibody response；
6) substrate incubates：Remove enzyme labelled antibody, after washing, add substrate, 37 DEG C of lucifuges act on 30 minutes, to elisa plate
Micropore adds terminate liquid；
7) detect：The OD value of each hole sample is tested under 405nm wavelength.
In the present embodiment, using the present invention provide hydrargyrum chelated complexes standard substance as positive control, respectively to be not added with
Detection sample controlled trial group as negative control 1, that is, sequentially added C1q albumen, confining liquid, anti-hydrargyrum antibody, enzyme mark and
Substrate；
To be not added with the controlled trial group of anti-hydrargyrum antibody as negative control 2, that is, sequentially add C1q albumen, confining liquid, treated
Survey blood plasma, enzyme mark and substrate；
Using not enzyme-added target controlled trial group as negative control 3, C1q albumen, confining liquid, blood to be measured are sequentially added
Slurry, anti-hydrargyrum antibody and substrate；
To be not added with the controlled trial group detecting sample and anti-hydrargyrum antibody as negative control 4 simultaneously, that is, sequentially add C1q
Albumen, confining liquid, enzyme mark and substrate；
To be not added with the controlled trial group of immune complex trapping agent C1q albumen as blank 1, that is, add closing
Liquid, test plasma, anti-hydrargyrum antibody, enzyme mark and substrate；
And only to add the controlled trial group of substrate for blank 2, only to add PBS for blank 3.
In euzymelinked immunosorbent assay (ELISA), first with have with immune complex the C1Q. albumen of affinity or CIF albumen or
Anti-C3 albumen adsorbs immune complex as trapping agent, and the nonspecific immunity complex in serum is extracted, and extracts
Partly be chelated with heavy metal Hg on immune complex out, and the hydrargyrum on this partial immunity complex can by with hydrargyrum specificity
In conjunction with material or can react with hydrargyrum form the specific antibody of anti-hydrargyrum of antigen antibody complex and captured, afterwards can again by
The enzyme labelled antibody of the enzyme labelling such as horseradish peroxidase, alkali phosphatase is captured, and in the presence of developer and terminate liquid,
OD value can be read under instrument, you can prove to detect the mercury metal of chelating in circulating immune complex.
In euzymelinked immunosorbent assay (ELISA) test, the different diluted concentration of C1Q. albumen, the diluted concentration of anti-hydrargyrum antibody and
As shown in table 1, in table, each numerical value is meansigma methodss to the parameter optimization of the diluted concentration of test plasma；
Table 1：C1q and Hg antibody best effort concentration and the determination of diluted plasma multiple
We can see that from table 1 when complement protein C1q dilution multiple proportions be 1:2500th, the dilution of dilution rate of blood plasma
Multiple proportions is 1:20th, the dilution multiple proportions of anti-Hg antibody is 1:When 2500, OD value is maximum, under this best effort concentration conditions, ELISA
Positive control and negative control ELISA testing result are as shown in table 2,
Table 2 ELISA positive control and negative control ELISA testing result
As shown in table 2, the OD value of negative control group and blank control group is respectively less than 0.1, so this working concentration is as
Good working concentration.
2nd, ELISA eluent best effort concentration and elution time determine
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-hydrargyrum antibody with enzyme labelled antibody incubation, with not
Eluent with concentration carries out eluting, then detects OD value by microplate reader, during determining optimal Elisa wash-out concentration and eluting
Between, comprise the following steps that：
(1) it is coated：Anti- Hg antibody is pressed 1 with dilution buffer:2500 doubling dilution, adds in elisa plate micropore, and 4
DEG C overnight 18h；
(2) close：Remove dilution buffer, after washing, plus confining liquid, place 1 hour for 37 DEG C, remove confining liquid, and wash
Wash；
(3) enzyme-added labeling antibody：Remove confining liquid, after washing, it is 2 μ g/ that addition dilution buffer is diluted to antibody concentration
The HRP enzyme labelled antibody of mL, 37 DEG C effect 2 hours so as to anti-Hg antibody response；
(4) eluting：Remove enzyme labelled antibody, with dilution buffer, eluent is diluted, make Papain in eluent
The concentration of enzyme：Concentration=1 of antibody in enzyme labelled antibody:80、1:40、1:20、1:10、1:5, make at a temperature of being respectively placed in 37 DEG C
With 1h, 2h, 3h；Remove eluent, washing, after the completion of waiting to wash, add substrate, 37 DEG C of lucifuges act on 30 minutes；
The compound method example of described eluent：By papain with pH be 8.0, molar concentration 0.1mol/L
Tris-HCI buffer becomes 2mg/mL, adds dithiothreitol, DTT (DTT), and the concentration being configured to dithiothreitol, DTT is
1mmol/L, 37 DEG C of incubation 30min；
(5) Deca terminate liquid is to each micropore；
(6) every group of OD value is read respectively under the Detection wavelength of 405nm on microplate reader, concrete outcome referring to table 3,
Table 3：ELISA eluent best effort densitometer elution time
By comparing OD value, with judge on ELISA hole wall combine hydrargyrum anti-enzyme mark complex eluting degree, when OD value
When low, anti-hydrargyrum antibody-enzyme mark complex eluting degree reaches maximum.
As known from Table 3, when the concentration of papain in eluent：Concentration=1 of antibody in enzyme labelled antibody:When 20, respectively
Group OD value is below other groups, illustrates to reach optimum in this concentration elution effect；No matter and action time be 1h, 2h,
3h, all less it is seen that prolongation over time, enzyme activity gradually weakens each group OD value changes, in the case that enzyme concentration is constant,
Extend digestion time can not improve digestibility, so in this experiment the action time of eluent all may be used for 1-3h.
Application Example
Hydrargyrum chelating type immune complex in 1.ELISA method detection blood plasma
100 plasma samples are detected, concrete operations condition is as follows by the ELISA method being provided using method one：
1) immobilized：C1Q. is coated on polypropylene solid phase carrier, presses 1 with dilution buffer：2500 multiple proportions is dilute
Release, add in elisa plate micropore, at 4 DEG C, deposit 16 hours；
2) close：Remove dilution buffer, washing, plus confining liquid, place 1 hour for 37 DEG C, remove confining liquid, washing；
3) it is loaded：Respectively plus test plasma sample and standard substance, sample is pressed 1 with dilution buffer：20 doubling dilution,
Add in elisa plate micropore, 37 DEG C incubate 1.5 hours；
4) capture：Remove test plasma, washing, addition presses 1 with dilution buffer:The anti-hydrargyrum of 2500 doubling dilution resists
Body, 37 DEG C incubate 1.5 hours；
5) enzyme conjugates incubate：Remove anti-hydrargyrum antibody, washing, addition antibody concentration is the enzyme labelled antibody of 2 μ g/mL, 37 DEG C
Incubate 1.5 hours；
6) substrate incubates：Remove enzyme labelled antibody, washing, after the completion of waiting to wash, add substrate, 37 DEG C of lucifuges act on 30 points
Clock, adds terminate liquid to elisa plate micropore；
7) detect：Test plasma sample and the immunity of above-mentioned hydrargyrum chelating type are read respectively under 405nm wavelength on microplate reader
The OD value of complex standard substance, result is as shown in table 4.
Table 4 adopts the measured result of a pair 100 parts of specimen blood plasma of method
2nd, chromium chelating type immune complex in ELISA+AAS method detection blood plasma
100 plasma samples are entered by the method being combined with AAS method using the Elisa method that the inventive method two provides
Row detection, concrete operations condition is as follows：
1) immobilized：Complement CIF albumen is coated on polystyrene solid phase carrier, presses 1 with dilution buffer:2500 times
Ratio dilution, adds in elisa plate micropore, deposits 16 hours at 4 DEG C；
2) close：Remove dilution buffer, washing, plus confining liquid, place 1 hour for 37 DEG C, remove confining liquid, washing；
3) incubate：Respectively plus standard substance and test plasma sample, sample is pressed 1 with dilution buffer:20 doubling dilution,
Add in elisa plate micropore, 37 DEG C incubate 2 hours；
4) eluting：Remove testing sample, washing, add the eluent that Papain enzyme concentration is 100 μ g/mL, at 37 DEG C
Effect 1 hour；
5) detect：It is combined in circulation immunity using chelating in Atomic Absorption Spectrometer examination criteria product and test plasma sample
Lead on thing, testing result is as shown in table 5.
Table 5 adopts the measured result to 100 parts of specimen blood plasma for the method two
Application Example 3：
The method being combined with ICP-MS using the Elisa that the inventive method three provides, the hydrargyrum chelating type being provided with embodiment 1
100 plasma samples are detected, concrete operations condition is as follows by immune complex standard substance：
1) immobilized：Anti-C_3 antibody is coated on polystyrene solid phase carrier, presses 1 with dilution buffer:2500 multiple proportions
Dilution, adds in elisa plate micropore, and 37 DEG C of water-baths were stored in refrigerator after 1 hour；
2) close：Remove dilution buffer, washing, plus confining liquid, place 1 hour for 37 DEG C, remove confining liquid, washing；
3) incubate：Respectively plus standard substance and test plasma sample, sample is pressed 1 with dilution buffer:20 doubling dilution,
Add in elisa plate micropore, 37 DEG C incubate 2 hours；
4) eluting：Remove testing sample, washing, add the eluent that Papain enzyme concentration is 100 μ g/mL, at 37 DEG C
Effect 1 hour；
5) it is acidified：Add nitric acid that solution is acidified, sealing overnight, is thoroughly acidified, and adds hydrogen peroxide, and heat and catch up with
Acid；
6) detect：Sample the hydrargyrum that detection chelates in circulating immune complex under icp mses,
Read respective value and calculate content, result is as shown in table 6.
Table 6 adopts the measured result to 100 parts of specimen blood plasma for the method three
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μ | 0.118 | 0.104 | 0.069 | 0.001 | 0.085 | 0.031 | 0.004 | 0.096 | 0.077 | 0.122 |
| g/L | ||||||||||
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 0.017 | 0.06 | 0.078 | 0.01 | 0.006 | 0.097 | 0.126 | 0.07 | 0.048 | 0.082 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 0.117 | 0.108 | 0.144 | 0.048 | 0.04 | 0.029 | 0.018 | 0.113 | 0.031 | 0.136 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 0.155 | 0.099 | 0.137 | 0.091 | 0.125 | 0.032 | 0.103 | 0.058 | 0.132 | 0.16 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 0.069 | 0.112 | 0.02 | 0.125 | 0.149 | 0.052 | 0.015 | 0.102 | 0.11 | 0.129 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 0.117 | 0.142 | 0.026 | 0.15 | 0.115 | 0.114 | 0.041 | 0.055 | 0.103 | 0.072 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 0.109 | 0.112 | 0.013 | 0.159 | 0.039 | 0.046 | 0.021 | 0.016 | 0.148 | 0.083 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 0.159 | 0.074 | 0.039 | 0.062 | 0.105 | 0.15 | 0.12 | 0.004 | 0.042 | 0.019 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 0.043 | 0.017 | 0.142 | 0.005 | 0.032 | 0.121 | 0.132 | 0.055 | 0.07 | 0.114 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 0.09 | 0.158 | 0.153 | 0.049 | 0.032 | 0.154 | 0.05 | 0.145 | 0.032 | 0.004 |
Above-mentioned embodiment is only the preferred embodiment of the present invention it is impossible to limit the scope of protection of the invention with this,
The change of any unsubstantiality that those skilled in the art is done on the basis of the present invention and replacement belong to institute of the present invention
Claimed scope.
Claims (9)
1. a kind of hydrargyrum chelating type immune complex is it is characterised in that this hydrargyrum chelating type immune complex is incorporated into load for mercury ion
Body protein and the complex that can be formed with the antibody of this carrier protein specific binding；
Described hydrargyrum chelating type immune complex is obtained by following preparation method：
A）The step of synthesis hydrargyrum chelating type immune complex；
（Ⅰ）Prepare chelating agent solution：Chelating agen is dissolved, is configured to chelating agent solution；
（Ⅱ）Formulation vehicle protein solution：Carrier protein is added in borate buffer solution, prepared carrier protein solution；
（Ⅲ）It is stirred overnight：By step（I）Chelating agent solution is added to step（II）Carrier protein solution in, stirring is after shaking
Bed effect 2-30h, obtains mixed liquor；
（Ⅳ）Bag filter pretreatment：EDTA-NaHCO is added in bag filter3Solution boils, and uses ddH after abandoning waste liquid2O rinses, weight
This step 1-3 time again；
（V）Dialysis：By step（III）Mixed liquor load through step（Ⅳ）In bag filter after process, use ddH2O dialyses, and changes water
2-3 time, after 4 DEG C of dialysed overnight, collect liquid；
（VI）Mercury ion chelates：In above-mentioned steps（V）In liquid in add HCl solution adjustment pH to 7.0, be slowly added to hydrargyrum from
Sub- solution, shakes in Deca, after shaking table act on 2-30h, then by the liquid obtaining load through step（Ⅳ）After process
Bag filter in, with ddH2O dialysis, change water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid, obtain hydrargyrum chelating type antigen；
（VII）In conjunction with：Above-mentioned hydrargyrum chelating type antigen adds and can obtain after reaction with the antibody of carrier protein specific binding
Hydrargyrum chelating type immune complex；
B）The step of purification hydrargyrum chelating type immune complex：Using immune-affinity chromatography removal step A）The hydrargyrum chelating type of synthesis
The carrier protein of reaction and the antibody of this carrier protein specific binding and mercury ion is had neither part nor lot in immune complex；
（i）By above-mentioned A）The hydrargyrum chelating type immune complex of step synthesis redissolves in normal saline；
（ii）Chromatographic column pretreatment：Using dilution buffer flushing line, loading in chromatographic column can be special with immune complex
Property combine filler, dress post after continuation dilution buffer balance pillar；
（ii）Loading：After pillar balance, use dilution buffer dilution step（i）Resulting solution, then upper prop, hydrargyrum chelating type is exempted from
Epidemic disease complex adsorbs on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer；
（iv）Eluting：Rinse pillar using dilution buffer, to baseline balance, then using the Na of 0.05-0.10Mol/L2HPO4
Solution carries out eluting；
（v）Collect：Collection step（iv）Eluent, collect finish after make protein renaturation immediately；
（vi）The eluent collected loads bag filter ddH2O dialyses desalination, after changing water 2-4 time, 4 DEG C of dialysed overnight, and collection sample
This；
（vii）Chromatographic column pretreatment：Using new chromatographic column, use dilution buffer flushing line, this chromatographic column loads energy
With the filler of hydrargyrum specific binding, after dress post, balance pillar with dilution buffer again；
（viii）Loading：Treat step（vii）Pillar balance after, dilute above-mentioned steps with dilution buffer（vi）Sample, so
Afterwards by the sample upper prop after dilution, hydrargyrum chelating type immune complex adsorbs on filler, unreacted antigen antibody complex meeting
Flow out with dilution buffer；
（ix）Eluting：Step（viii）Rinse pillar with dilution buffer afterwards, to baseline balance, then using 0.5-
The Na of 1.0Mol/L2HPO4Solution carries out eluting；
（x）Collect：Collection step（ix）Eluent, collect finish after make protein renaturation immediately；
（xi）By step（x）Eluent load bag filter ddH2O dialyses desalination, after changing water three times, 4 DEG C of dialysed overnight, and collect
Sample, that is, obtain the hydrargyrum chelating type immune complex of purification.
2. hydrargyrum chelating type immune complex according to claim 1 is it is characterised in that described carrier protein at least contains zinc
Refer to structure, sulfydryl, a kind of structure in cysteine residues, mercury ion pass through zinc fingerses, sulfydryl, in cysteine residues at least
One kind is combined with carrier protein.
3. hydrargyrum chelating type immune complex according to claim 2 is it is characterised in that described carrier protein is antibody egg
In vain, one of lipoprotein, hemoglobin, nuclear antigen, foreign sera antigen, virus, antibacterial, protozoon, anthelmintic.
4. it is characterised in that wherein, mercury ion passes through chelating agen to hydrargyrum chelating type immune complex according to claim 1
The antibodies specifically binding with energy and carrier protein again after being combined with carrier protein.
5. a kind of preparation method of hydrargyrum chelating type immune complex as claimed in claim 1 is it is characterised in that this preparation method
Including：
A）The step of synthesis hydrargyrum chelating type immune complex；
（Ⅰ）Prepare chelating agent solution：Chelating agen is dissolved, is configured to chelating agent solution；
（Ⅱ）Formulation vehicle protein solution：Carrier protein is added in borate buffer solution, prepared carrier protein solution；
（Ⅲ）It is stirred overnight：By step（I）Chelating agent solution is added to step（II）Carrier protein solution in, stirring is after shaking
Bed effect 2-30h, obtains mixed liquor；
（Ⅳ）Bag filter pretreatment：EDTA-NaHCO is added in bag filter3Solution boils, and uses ddH after abandoning waste liquid2O rinses, weight
This step 1-3 time again；
（V）Dialysis：By step（III）Mixed liquor load through step（Ⅳ）In bag filter after process, use ddH2O dialyses, and changes water
2-3 time, after 4 DEG C of dialysed overnight, collect liquid；
（VI）Mercury ion chelates：In above-mentioned steps（V）In liquid in add HCl solution adjustment pH to 7.0, be slowly added to hydrargyrum from
Sub- solution, shakes in Deca, after shaking table act on 2-30h, then by the liquid obtaining load through step（Ⅳ）After process
Bag filter in, with ddH2O dialysis, change water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid, obtain hydrargyrum chelating type antigen；
（VII）In conjunction with：Above-mentioned hydrargyrum chelating type antigen adds and can obtain after reaction with the antibody of carrier protein specific binding
Hydrargyrum chelating type immune complex；
B）The step of purification hydrargyrum chelating type immune complex：Using immune-affinity chromatography removal step A）The hydrargyrum chelating type of synthesis
The carrier protein of reaction and the antibody of this carrier protein specific binding and mercury ion is had neither part nor lot in immune complex；
（i）By above-mentioned A）The hydrargyrum chelating type immune complex of step synthesis redissolves in normal saline；
（ii）Chromatographic column pretreatment：Using dilution buffer flushing line, loading in chromatographic column can be special with immune complex
Property combine filler, dress post after continuation dilution buffer balance pillar；
（ii）Loading：After pillar balance, use dilution buffer dilution step（i）Resulting solution, then upper prop, hydrargyrum chelating type is exempted from
Epidemic disease complex adsorbs on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer；
（iv）Eluting：Rinse pillar using dilution buffer, to baseline balance, then using the Na of 0.05-0.10Mol/L2HPO4
Solution carries out eluting；
（v）Collect：Collection step（iv）Eluent, collect finish after make protein renaturation immediately；
（vi）The eluent collected loads bag filter ddH2O dialyses desalination, after changing water 2-4 time, 4 DEG C of dialysed overnight, and collection sample
This；
（vii）Chromatographic column pretreatment：Using new chromatographic column, use dilution buffer flushing line, this chromatographic column loads energy
With the filler of hydrargyrum specific binding, after dress post, balance pillar with dilution buffer again；
（viii）Loading：Treat step（vii）Pillar balance after, dilute above-mentioned steps with dilution buffer（vi）Sample, so
Afterwards by the sample upper prop after dilution, hydrargyrum chelating type immune complex adsorbs on filler, unreacted antigen antibody complex meeting
Flow out with dilution buffer；
（ix）Eluting：Step（viii）Rinse pillar with dilution buffer afterwards, to baseline balance, then using 0.5-
The Na of 1.0Mol/L2HPO4Solution carries out eluting；
（x）Collect：Collection step（ix）Eluent, collect finish after make protein renaturation immediately；
（xi）By step（x）Eluent load bag filter ddH2O dialyses desalination, after changing water three times, 4 DEG C of dialysed overnight, and collect
Sample, that is, obtain the hydrargyrum chelating type immune complex of purification.
6. hydrargyrum chelating type immune complex as claimed in claim 1 is in the enzyme linked immunological of preparation detection chelating hydrargyrum immune complex
Application in test kit.
7. a kind of enzyme linked immunological kit of detection chelating hydrargyrum immune complex is it is characterised in that this test kit includes standard substance；
Described standard substance are hydrargyrum chelating type immune complex as claimed in claim 1.
8. enzyme linked immunological kit according to claim 7 is it is characterised in that this test kit also includes below at least one
Reagent：Containing capture antigen antibody complex albumen be coated liquid, containing capture metal Hg antibody be coated liquid, enzyme mark resist
Body.
9. a kind of method of detection by quantitative hydrargyrum chelating type immune complex is it is characterised in that claim 1 institute with known content
The hydrargyrum chelating type immune complex stated is detected as standard substance, a pair of sample using following methods：Euzymelinked immunosorbent assay (ELISA), enzyme
Connection immunity is compound with atomic absorption spectrum combined techniqueses, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniqueses, purification immunity
Thing and atomic absorption spectrum combined techniqueses, purification immune complex and inductivity coupled plasma mass spectrometry combined techniqueses, electrophoresis and enzyme connection
Immunity or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
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| CN101139398A (en) * | 2007-09-27 | 2008-03-12 | 南京农业大学 | Preparation method of heavy metal mercury monoclonal antibody |
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