CN105137059A - 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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- CN105137059A CN105137059A CN201510411290.3A CN201510411290A CN105137059A CN 105137059 A CN105137059 A CN 105137059A CN 201510411290 A CN201510411290 A CN 201510411290A CN 105137059 A CN105137059 A CN 105137059A
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- mercury
- immune complex
- chelating type
- antibody
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- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 194
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 title claims abstract description 185
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000010668 complexation reaction Methods 0.000 title 1
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 47
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 47
- -1 mercury ions Chemical class 0.000 claims abstract description 7
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- 150000001875 compounds Chemical class 0.000 claims description 34
- 239000003480 eluent Substances 0.000 claims description 33
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims description 31
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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 and belong to medical domain, be specifically related to mercury chelating type immune complex and its preparation method and application.
Background technology
Immune complex (Immunecomplexes, IC) is the product that antigen-antibody combines, and according to the definition of national scientific and technical terminology validation board, refers to that antibody is combined with soluble antigen and the compound that formed.It is divided into two classes, and a kind of is immune complex fixing in tissue, and another kind is then the immune complex be free in blood, becomes CIC ELISA (circulatingimmunecomplexes, CIC).Immune complex is in removing and destroy in various antigen and play indispensable effect, and the result of organism immune response is exactly constantly produce immune complex.When body endoantigen (or antibody) quantity is slightly more than antibody (or antigen), the immune complex of medium molecule size will be formed, it was both not easily engulfed by phagocyte, did not discharge again by glomerulus filter opening, can be free on the long period in blood and other body fluid.When Vascular Permeability increases, this type of IC can with hypostasis at the capillary wall at some position or be entrenched in glomerular basement membrane, and activating complement causes immune complex deposit.These deposit or are embedded in the immune complex of vascular wall or tissue, are the initiating agent causing vascular basement membrane inflammation and tissue damage.Immune complex causes tissue damage by following several respects: 1) activating complement: the IC of deposition passes through activating complement, produce anaphylatoxin and the active fragment with chemotactic effect, chemotactic causes local vascular permeability increase to mast cell locally, basophilic granulocyte release active medium, causes and oozes out and local edema; 2) attract leukocyte infiltration and gather: neutrophil leucocyte discharges toxicity oxide and lysosomal enzyme when engulfing IC, damage adjacent tissue; 3) activated blood platelet: IC is by activated blood platelet release vasoactive amine material, and cause blood vessel dilatation, permeability increase, aggravation local is oozed out and oedema, and activates clotting mechanism, forms microthrombus, causes ischaemic, hemorrhage and necrosis.
The generation of various diseases is all closely bound up with the deposition of immune complex, as systemic loupus erythematosus, rheumatoid arthritis, nephrotic syndrome, cryoglobulinemia, vasculitis, bacterium, virus and parasitic infection, allergic reaction, autoimmune disease, skin disease etc.As far back as the nineties in last century, scholar is just had to find, in normal body; immune complex is constantly degraded; thus generally can't detect immune complex or only present low concentration, and in above-mentioned a series of disease, the immune complex in serum and CIC content significantly raise.Although CIC detects do not have disease specific, its detection can provide the information in immunopathogenesis, advancing of disease and prognosis.In certain micro-organisms infection, autoimmunity, detect the index that CIC ELISA can be used as Disease Activity, evaluates the function of body and detection curative effect.And in the last few years, scholars are devoted to study specific immune complex, study each side's relations of plane such as various specific immune complex and disease are caused a disease, disease progression, medical diagnosis on disease, and these researchs will impel us to have further understanding to disease.
Mercury (Mercury, Hg) is a kind of ubiquitous heavy metal, is widely used in industry, agricultural, pharmaceutical sector, is a kind of very important environmental contaminants.2011, U.S.'s poisonous substance and disease registration administration (UnitedStatesAgencyforToxicSubstancesandDiseaseRegistry, ATSDR) listed in (SubstancePriorityList) in material preferred list, and the World Health Organization (WHO) (WorldHealthOrganization, WHO) is also classified as one of chemicals of 10 large easily attractive health problems already.In the U.S., Hg is considered to one of four overall situation pollutants, drastically influence the healthy of people.Common mercury exposed population group comprises to be engaged in metal smelt, exploitation gold mine, to burn the crowd of coal, electric industry and wood producing, and for general population mainly through eat mercury pollution seafood, drink into mercury pollution water source, the mercuryvapour sucking fuel combustion or evaporation.In addition, the contact of mercury cosmetic, fill the ethyl mercury in mercury alloy dentistry filling material, contact vaccine antiseptic, the contact of mercurous pesticide is all common mercury exposure chamber, the life of mercury and people is closely related.2009, national health and nutrition find (NationalHealthandNutritionExaminationStudy in testing and investigating, NHANES), the mercury in U.S.'s Women of Childbearing Age body of 2% all exceeds standard, and visible mercury pollution is a problem demanding prompt solution.
American scholar A.Ramanaviciene in 2004 etc. find that in serum, CIC ELISA content pollutes relevant with local environment, find the increase along with the local pollution control order of severity, CIC content in local cow's serum significantly increases, difference has statistical significance, thus can as the index evaluating local pollution control.Actually but how environmental pollution stimulates CIC content in body significantly to raise, or be that a kind of multiple pollutant acting in conjunction causes it to raise, therefore whether the rising of CIC can cause body injury, and increase the neurological susceptibility of body for disease, these all require study.As everyone knows, CIC ELISA is a kind of protein of Special Significance, and mercury can act on the protein of the multiple system of whole body, and whether mercury can act on CIC ELISA, and the change of CIC ELISA content, activity change, function are changed, these still lack correlative study.
Summary of the invention
In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a kind of mercury chelating type immune complex, this mercury chelating type immune complex is a kind of immune complex of relative specificity, for detecting in regional crowd's serum whether there is mercury chelating type immune complex and content thereof, this regional mercury pollution degree of reflection indirectly.
For solving the problem, the technical solution adopted in the present invention is as follows:
A kind of mercury chelating type immune complex, this mercury chelating type immune complex is the one in following compound:
Mercury ion is incorporated into the compound that immune complex is formed, or
The compound that mercury ion is incorporated into carrier protein and can be formed with the antibody of this carrier protein specific binding; Or
The compound formed is combined with carrier protein after mercury ion is incorporated into immunoglobulin (Ig).
Further, described carrier protein or immune complex are at least containing a kind of structure in zinc fingers, sulfydryl, cysteine residues, and mercury ion is combined with carrier protein or immune complex by least one in zinc fingers, sulfydryl, cysteine residues.
Concrete, described carrier protein is the one in antibody protein, lipoprotein, haemoglobin, nuclear antigen, foreign sera antigen, virus, bacterium, protozoon, worm.
Further, mercury ion is combined with the antibody of energy and carrier protein specific binding after being combined with carrier protein by sequestrant again.
Further, described sequestrant is the one in ITCBE, EDTA, polyphosphate, amino carboxylic acid, 1,3-diketone, hydroxycarboxylic acid.
Present invention also offers the method that two kinds are prepared mercury chelating type immune complex, obtain mercury chelating type immune complex by these two kinds of methods.
Wherein, the first preparation method comprises
A) synthesize the step of mercury chelating type immune complex: be first combined with carrier protein by sequestrant, then with mercury ion complexing;
B) step of purification mercury chelating type immune complex: adopt immune-affinity chromatography removal step A) have neither part nor lot in the carrier protein of reaction, specific antibody and mercury ion in the mercury chelating type immune complex that synthesizes.
Particularly, in the preparation method of described mercury chelating type immune complex,
(I) chelating agent solution is prepared: dissolved by sequestrant, be mixed with chelating agent solution;
(II) formulation vehicle protein solution: carrier protein is joined in borate buffer solution, obtained carrier protein solution;
(III) stirring is spent the night: join in the carrier protein solution of step (II) by step (I) chelating agent solution, in shaking table effect 2-30h after stirring, obtain mixed liquor;
(IV) bag filter pre-service: add EDTA-NaHCO in bag filter
3solution boils, and uses ddH after abandoning waste liquid
2o rinses, and repeats this step 1-3 time;
(V) dialyse: the mixed liquor of step (III) is loaded in the bag filter after step (IV) process, use ddH
2o dialyses, and changes water 2-3 time, 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), slowly add mercury ion solution, dropping limit, limit is shaken, afterwards in shaking table effect 2-30h, then step (V) is repeated once, collect liquid, obtain mercury chelating type antigen;
(VII) combine: add in above-mentioned mercury chelating type antigen can with the antibody of carrier protein specific binding, obtain mercury chelating type immune complex after reaction;
Described step B) concrete steps are as follows:
I () is by above-mentioned A) step synthesis mercury chelating type immune complex redissolve in physiological saline;
(ii) chromatographic column pre-service: use dilution buffer flushing line, load in chromatographic column can with the filler of immune complex specific binding, after dress post, continuation dilution buffer balances pillar;
Described can with the filler of immune complex specific binding be adsorbed with can with the silica gel of immune complex specific binding material or resin;
(ii) loading: after pillar balance, with dilution buffer dilution step (i) gained solution, then upper prop, mercury chelating type immune complex is adsorbed on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer;
(iv) wash-out: use dilution buffer to rinse pillar, to baseline balance, then uses the Na2HPO4 solution of 0.05-0.10Mol/L to carry out wash-out;
V () collects: the eluent collecting step (iv), makes protein renaturation after collection immediately;
(vi) eluent collected loads bag filter ddH
2o dialyses desalination, and after changing water 2-4 time, 4 DEG C of dialysed overnight, collect sample;
(vii) chromatographic column pre-service: adopt new chromatographic column, uses dilution buffer flushing line, in this chromatographic column load can with the filler of mercury specific binding, dress post after balance pillar by dilution buffer again;
Described can with the filler of mercury specific binding be adsorbed with can with the silica gel of mercury specific binding material or resin;
(viii) loading: after the pillar balance of step (vii), the sample of above-mentioned steps (vi) is diluted by dilution buffer, then by the sample upper prop after dilution, mercury chelating type immune complex is adsorbed on filler, and unreacted antigen antibody complex can flow out with dilution buffer;
(ix) wash-out: rinse pillar by dilution buffer after step (viii), to baseline balance, then uses the Na of 0.5-1.0Mol/L
2hPO
4solution carries out wash-out;
X () collects: the eluent collecting step (ix), makes protein renaturation after collection immediately;
(xi) bag filter ddH is loaded to the eluent of step (x)
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, namely obtain the mercury chelating type immune complex of purifying.
Present invention also offers the application of above-mentioned mercury chelating type immune complex in the enzyme linked immunological kit of preparation detection mercury chelating type immune complex.
Present invention also offers a kind of enzyme linked immunological kit detecting mercury chelating type immune complex, this kit comprises the standard items of mercury chelating type immune complex as claimed in claim 1.
Further, mentioned reagent box also comprises the following reagent of at least one: the coating buffer of the albumen containing capture antigen antibody complex, containing coating buffer, the enzyme labelled antibody of antibody of catching metal Hg.
A kind of method of quantitative detection mercury chelating type immune complex, it is characterized in that, using the mercury chelating type immune complex of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification immune complex and atomic absorption spectrum combined techniques, purification immune complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Compared to existing technology, beneficial effect of the present invention is:
1. synthesize mercury chelating type immune complex first, and confirmed that mercury can be present in immune system in body, and demonstrate the existence of mercury chelating type immune complex;
2. present invention achieves the specific recognition of immune complex, and achieve the quantitative detection of mercury chelating type immune complex, the method detecting the mercury content level in body from immune angle is provided, and provide indirect indexes for the mercury pollution level of industrial area.
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
Accompanying drawing explanation
Fig. 1 is immune complex non denatured electrophoretic band figure;
Fig. 2 is the synchrotron radiation line x-ray fluorescence analysis figure of the electrophoretic band of immune complex;
Wherein, the M in Fig. 1 is Marker, IC is mercury chelating type immune complex; Horizontal ordinate in Fig. 2 is protein band position, and ordinate is mercury metal energy in this protein band.
Embodiment
A kind of mercury chelating type immune complex, this mercury chelating type immune complex is the one in following compound:
Mercury ion is incorporated into the compound that immune complex is formed, or
The compound that mercury ion is incorporated into carrier protein and can be formed with the antibody of this carrier protein specific binding; Or
The compound formed is combined with carrier protein after mercury ion is incorporated into immunoglobulin (Ig).
Concrete, described carrier protein or immune complex are at least containing a kind of structure in zinc fingers, sulfydryl, cysteine residues, and mercury ion is combined with carrier protein or immune complex by least one in zinc fingers, sulfydryl, cysteine residues.
Concrete, described carrier protein is the one in antibody protein, lipoprotein, haemoglobin, nuclear antigen, foreign sera antigen, virus, bacterium, protozoon, worm.
Further, mercury ion is combined with the antibody of energy and carrier protein specific binding after being combined with carrier protein by sequestrant again.
Further, described sequestrant is the one in ITCBE, EDTA, glutathione (GSH), polyphosphate, amino carboxylic acid, 1,3-diketone, hydroxycarboxylic acid.
Present invention also offers the method that two kinds are prepared mercury chelating type immune complex, obtain mercury chelating type immune complex by these two kinds of methods.
Wherein, the first preparation method comprises
A) step of mercury chelating type immune complex is synthesized;
B) step of purification mercury chelating type immune complex: adopt immune-affinity chromatography removal step A) have neither part nor lot in the carrier protein of reaction, specific antibody and mercury ion in the mercury chelating type immune complex that synthesizes.
Particularly, in the preparation method of described mercury chelating type immune complex,
(I) chelating agent solution is prepared: dissolved by sequestrant, be mixed with chelating agent solution;
(II) formulation vehicle protein solution: carrier protein is joined in borate buffer solution, obtained carrier protein solution;
(III) stirring is spent the night: join in the carrier protein solution of step (II) by step (I) chelating agent solution, in shaking table effect 2-30h after stirring, obtain mixed liquor;
(IV) bag filter pre-service: add EDTA-NaHCO in bag filter
3solution boils, and uses ddH after abandoning waste liquid
2o rinses, and repeats this step 1-3 time;
(V) dialyse: the mixed liquor of step (III) is loaded in the bag filter after step (IV) process, use ddH
2o dialyses, and changes water 2-3 time, 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), slowly add mercury ion solution, dropping limit, limit is shaken, afterwards in shaking table effect 2-30h, then step (V) is repeated once, collect liquid, obtain mercury chelating type antigen;
(VII) combine: add in above-mentioned mercury chelating type antigen can with the antibody of carrier protein specific binding, obtain mercury chelating type immune complex after reaction;
Described step B) concrete steps are as follows:
I () is by above-mentioned A) step synthesis mercury chelating type immune complex redissolve in physiological saline;
(ii) chromatographic column pre-service: use dilution buffer flushing line, load in chromatographic column can with the filler of immune complex specific binding, after dress post, continuation dilution buffer balances pillar;
(ii) loading: after pillar balance, with dilution buffer dilution step (i) gained solution, then upper prop, mercury chelating type immune complex is adsorbed on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer;
(iv) wash-out: use dilution buffer to rinse pillar, to baseline balance, then uses the Na2HPO4 solution of 0.05-0.10Mol/L to carry out wash-out;
V () collects: the eluent collecting step (iv), makes protein renaturation after collection immediately;
(vi) eluent collected loads bag filter ddH
2o dialyses desalination, and after changing water 2-4 time, 4 DEG C of dialysed overnight, collect sample;
(vii) chromatographic column pre-service: adopt new chromatographic column, uses dilution buffer flushing line, in this chromatographic column load can with the filler of mercury specific binding, dress post after balance pillar by dilution buffer again;
(viii) loading: after the pillar balance of step (vii), the sample of above-mentioned steps (vi) is diluted by dilution buffer, then by the sample upper prop after dilution, mercury chelating type immune complex is adsorbed on filler, and unreacted antigen antibody complex can flow out with dilution buffer;
(ix) wash-out: rinse pillar by dilution buffer after step (viii), to baseline balance, then uses the Na of 0.5-1.0Mol/L
2hPO
4solution carries out wash-out;
X () collects: the eluent collecting step (ix), makes protein renaturation after collection immediately;
(xi) bag filter ddH is loaded to the eluent of step (x)
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, namely obtain the mercury chelating type immune complex of purifying.
Present invention also offers the application of above-mentioned mercury chelating type immune complex in the enzyme linked immunological kit of preparation detection mercury chelating type immune complex.
Present invention also offers a kind of enzyme linked immunological kit detecting mercury chelating type immune complex, this kit comprises the standard items of mercury chelating type immune complex as claimed in claim 1.
Further, mentioned reagent box also comprises the following reagent of at least one: the coating buffer of the albumen containing capture antigen antibody complex, containing coating buffer, the enzyme labelled antibody of antibody of catching metal Hg.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
For detecting a kit for mercury chelating CIC ELISA, comprising: containing can coating buffer, confining liquid, lavation buffer solution, anti-mercury antibody, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, the negative control of albumen of capture antigen antibody complex.
For detecting a kit for mercury chelating CIC ELISA, comprising: containing can coating buffer, confining liquid, lavation buffer solution, eluent, positive control, the negative control of albumen of capture antigen antibody complex.
For detecting a kit for mercury chelating CIC ELISA, comprising: containing can coating buffer, confining liquid, lavation buffer solution, eluent, nitric acid, hydrogen peroxide, positive control, the negative control of albumen of capture antigen antibody complex.
For detecting a kit for mercury chelating CIC ELISA, comprise for solution needed for serum immune complex of purifying, redissolve liquid, containing coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, standard items, the negative control of antibody can catching mercury metal.
A kind of kit for detecting mercury chelating CIC ELISA: comprise for solution needed for serum immune complex of purifying, redissolution liquid, positive control, negative control.
For detecting a kit for mercury chelating CIC ELISA, comprise for solution needed for serum immune complex of purifying, redissolution liquid, nitric acid, hydrogen peroxide, positive control, negative control etc.
For detecting a kit for mercury chelating CIC ELISA, comprise for solution needed for serum immune complex of purifying, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid needed for the protein band of antigen antibody complex, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of antibody can catching mercury metal.
For detecting a kit for mercury chelating CIC ELISA, comprise for solution needed for serum immune complex of purifying, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, positive control, negative control etc. needed for the protein band of antigen antibody complex.
For detecting a kit for mercury chelating CIC ELISA, comprise for solution needed for serum immune complex of purifying, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, nitric acid, hydrogen peroxide, positive control, negative control etc. needed for the protein band of antigen antibody complex.
In above-mentioned several kit, described in catch the albumen of CIC ELISA, include but not limited to C1Q, CIF albumen, anti-C_3 antibody, if article No. is the C1qRecombinantProtein of " NOVUSH00000712-p01 "; It is the mouse-anti HgmAb of " cling to proud must AP7014 " that described antibody of catching mercury metal includes but not limited to buy from Ba Ao get bio tech ltd article No.; Bovine serum albumin or the skimmed milk power of confining liquid to be mass concentration be 1%-5%; Eluent includes but not limited to the Tris-HCl buffer solution of papain; Described glue bed medium includes but not limited to Ago-Gel, polyacrylamide gel; For purifying, solution needed for serum immune complex includes but not limited to PEG solution, borate buffer solution; Substrate includes but not limited to TMB solution, ABTS solution; Described sample-loading buffer includes but not limited to the Tris-HCl buffer solution containing bromophenol blue; Enzyme labelled antibody is the antibody containing the enzyme labeling such as horseradish peroxidase, alkaline phosphatase, as HRP enzyme labelled antibody; Standard items include but not limited to mercury chelating type CIC ELISA of the present invention, other are chelated with the immune complex of mercury metal; Positive control includes but not limited to mercury chelating type CIC ELISA of the present invention, other are chelated with the immune complex of mercury metal, and negative control is dilution buffer.
Present invention also offers a kind of method of quantitative detection mercury chelating type immune complex, using the mercury chelating type immune complex of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification immune complex and atomic absorption spectrum combined techniques, purification immune complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
In the present invention, following several with having of can listing of the method detecting mercury chelating type immune complex, but be not limited to following several.
The present invention unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine, reagent, material are as cited by following, that does not enumerate out in the present invention is that this area is commonly used or can be obtained by commercial mode, is below that the test reagent that the present invention uses is as follows:
Dilution buffer is the 0.05M carbonate buffer solution of pH9.6, compound method example: the Na getting 1.5g
2cO
3with the NaHCO of 2.93g
3dissolving adds ddH
2o is settled to 1000mL;
Lavation buffer solution is the 0.15MPBS damping fluid of pH7.4, compound method example: the KH getting 0.2g
2pO
4, 2.90g Na
2hPO
412H
2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH
2o is settled to 1000mL;
Confining liquid is bovine serum albumen solution, compound method example: get 0.1g bovine serum albumin, adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2MH
2sO
4, compound method example: the ddH getting 178.3mL
2o, to ddH
2dropwise dense H is added along wall in O
2sO
4, limit edged stirs, and is settled to 200mL;
The pH of substrate buffer solution is 5.0, Na
2hPO
4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, the preparation method of the substrate buffer solution of every 50mL is as follows: get 1.42gNa
2hPO
4, 0.96g citric acid, then add ddH
2o to 50mL, to obtain final product;
Substrate is methyl biphenyl amine (being called for short TMB) solution, and this methyl biphenyl amine (TMB) solution is by formulated according to the component of following ratio: TMB: substrate buffer solution: 0.75%H
2o
2=0.5mL:10mL:32 μ L, wherein TMB is the methyl biphenyl amine ethanolic solution of 2g/L;
The albumen can catching immune complex be can with the albumen of immune complex specific binding, include but not limited to as C1Q, CIF albumen, anti-C_3 antibody; In following examples, the albumen can catching immune complex specifically uses article No. to be the C1qRecombinantProtein of " NOVUSH00000712-p01 ";
The material of catching mercury is that to buy from Ba Ao get bio tech ltd article No. be the mouse-anti HgmAb of " cling to proud must AP7014 "; Wherein, of the present inventionly the material of catching mercury is with the material of mercury specific binding, anti-Hg antibody, anti-mercury antibody;
Described can be rabbit anti-human serum albumin antibodies with the antibody of human serum albumins specific binding, for commercially available;
The following stated dilution multiple proportions is w/v.
Method one: ELISA method detects mercury chelating type immune complex, and concrete steps are as follows:
1) bag quilt: the albumen can catching immune complex is coated on solid phase carrier: with dilution buffer dilution coating protein to 2500-20000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, washed rear elisa plate and placed 1 hour in 37 DEG C;
3) measuring samples is added, and incubation: blood sampling, make measuring samples; Standard items are made with the mercury chelating type immune complex of known content; With dilution buffer dilution 10-40 times, add in micropore, 37 DEG C of effect 1-2 hour;
4) material of catching mercury is added, and incubation: remove measuring samples, and wash with cleansing solution, after washing completes, add with dilution buffer dilution 2500-20000 anti-mercury antibody doubly, 37 DEG C of effect 1-2 hour, make the mercury metal on itself and immune complex react;
5) enzyme conjugates incubation: remove anti-Hg antibody, and wash with cleansing solution, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and anti-Hg antibody response;
6) substrate incubation: remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: drip stop buffer to each micropore;
8) getting wavelength is 405nm, after adding stop buffer, the OD value of testing sample group and standard items is read respectively in stipulated time inherent microplate reader, by comparing with standard items group, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of testing sample.
The method utilizes enzyme linked immunosorbent assay (ELISA) (ELISA) principle, nospecific immunity compound in serum can be extracted, the immune complex upper part extracted is chelated with heavy metal Hg, and mercury on this part immune complex can by with the material of mercury specific binding or can with mercury react the specific antibody of the anti-mercury forming antigen antibody complex catch, afterwards can again by horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value is read under instrument, and the immune complex not containing chelated mineral mercury, then can not catch by the specific antibody of anti-mercury, also can not with horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase caught, and also not containing mercury metal (negative control group result is negative) in agents useful for same, thus when read OD value result is shown as the positive, the i.e. provable mercury metal detecting chelating on CIC ELISA.
Method two: ELISA method+AAS method detects mercury chelating type immune complex, and concrete steps are as follows:
1) bag quilt: the albumen can catching immune complex is coated on solid phase carrier, is diluted to 2500-20000 doubly by dilution buffer, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
2) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C.After having washed, elisa plate is placed 1 hour in 36.5-37.5 DEG C;
3) measuring samples is added, and incubation: from circulation system sampling, make measuring samples; Standard items are made with the mercury chelating type immune complex of known content; With dilution buffer dilution 10-40 times, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, adding extension rate is 5-80 eluent doubly, at 37-57 DEG C, act on 0-2 hour; Eluent;
5) detect: sample from ELISA micropore, detect the mercury of chelating on immune complex in Atomic Absorption Spectrometer, and drawing standard curve, read respective value.
The method further on the basis of enzyme linked immunological principle in conjunction with atomic absorption spectrum (AAS) principle, Atomic Absorption Spectrometer is utilized to detect the mercury of chelating on CIC ELISA, owing to only containing immune complex in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable mercury metal detecting chelating on CIC ELISA.
Method three: ELISA method+ICP-MS method detects mercury chelating type immune complex, and concrete steps are as follows:
1) bag quilt: the albumen can catching immune complex is coated on solid phase carrier: with dilution buffer dilution coating protein 2500-20000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C.After having washed, elisa plate is placed 1 hour in 36.5-37.5 DEG C;
3) measuring samples is added, and incubation: from circulation system sampling, make measuring samples; Standard items are made with the mercury chelating type immune complex of known content; With dilution buffer dilution 10-40 times, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add eluent, at 37 DEG C, act on 0-2 hour;
5) acidifying: add acidulant in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and acid is caught up with in heating, sampling, detects the mercury of chelating on immune complex under icp ms, reads respective value.
The method further on the basis of enzyme linked immunological principle in conjunction with inductively coupled plasma mass spectrometry (ICP-MS) principle, the mercury of chelating on CIC ELISA is detected with ICP-MS, owing to only containing immune complex in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable mercury metal detecting chelating on CIC ELISA.
Method four: purification CIC method+ELISA method detects mercury chelating type immune complex, and concrete steps are as follows:
1) nospecific immunity compound is extracted: utilize the method such as the polyglycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration to extract nospecific immunity compound and redissolve in solution by the immune complex double solvents of purifying out;
2) be coated on solid phase carrier with the material can catching mercury: with dilution buffer dilution coated antibody to 2500-20000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate is placed 1 hour in 36.5-37.5 DEG C;
4) measuring samples is added, and incubation: sample from the redissolution liquid of the nospecific immunity compound extracted, make measuring samples; Standard items are made with the mercury chelating type immune complex of known content; With dilution buffer dilution 10-40 times, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: the redissolution liquid removing nospecific immunity compound, and wash with cleansing solution, after washing completes, add the HRP enzyme labelled antibody with dilution buffer dilution, 37 DEG C of effect 1-2 hour, make itself and anti-Hg antibody response;
6) substrate incubation: remove HRP enzyme labelled antibody, and wash with cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation reaction: drip stop buffer to each micropore.
8) getting wavelength is 405nm, after adding stop buffer, the OD value of testing sample group and standard items is read respectively in stipulated time inherent microplate reader, by comparing with standard items group, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of testing sample.
Method five: purification CIC method+AAS method detects mercury chelating type immune complex, and concrete steps are as follows:
1) nospecific immunity compound is extracted: utilize the method such as the polyglycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration to extract nospecific immunity compound and redissolved by the immune complex of purifying out;
2) detect: from step 1) sample solution, detect the mercury of chelating on immune complex in Atomic Absorption Spectrometer, read respective value.
Method six: purification CIC method+ICP-MS method detects mercury chelating type immune complex, and concrete steps are as follows:
1) nospecific immunity compound is extracted: utilize the method such as the polyglycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration to extract nospecific immunity compound and redissolved by the immune complex of purifying out;
2) acidifying: from step 1) sample solution, add respective acids agent in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
3) detect: add hydrogen peroxide, and heating catch up with acid, and then get heating catch up with acid after solution 0.5mL under icp ms, detect the mercury of chelating on immune complex, read respective value.
Method four, method five and method six are all first isolate immune complex, then adopt method for detecting specificity, measure the content of mercury on mercury chelating type immune complex in immune complex; Namely first physical separation means are adopted, as supercentrifugation, HPLC, gel-filtration chromatography etc., separated from test plasma sample by immune complex and redissolve in physiological saline, recycling ELISA principle, atomic absorption spectrum detect or carry out detecting the mercury content on inductively coupled plasma mass spectrometry detection mercury chelating type immune complex.
Method seven: electrophoresis+ELISA/AAS/ICP-MS method detects mercury chelating type immune complex, and concrete steps are as follows:
1) nospecific immunity compound is extracted: utilize the method such as the polyglycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration to extract nospecific immunity compound and redissolved by the immune complex of purifying out, obtain the solution of immune complex;
2) glue bed is prepared: select suitable medium (as Ago-Gel, polyacrylamide gel etc.) as required, prepare glue bed as requested;
3) application of sample: from step 1) solution sample 8 μ L, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
Sample-loading buffer (Samplebuffer) can be formulated by the component of following ratio: Tris-HCl:1% bromophenol blue: ddH
2o: glycocoll=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M.
4) electrophoresis: connect electrophoresis plate, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of isoelectric point;
Described electrophoretic buffer obtains by following method: get Tris3.0g, glycocoll 14.4g, be dissolved in 800mLddH
2in O, regulate pH to 8.3, then add ddH
2o to 1000mL, to obtain final product;
5) detect: on glue bed, find out the protein band containing immune complex, this band is taken out, after treatment band is dissolved, and then utilize the principles such as ELISA, ICP-MS, AAS to detect mercury content respectively; In addition, the isoelectric point of the immune complex of the method detection chelating mercury, molecular weight and content etc. can also be utilized.
Method seven is on the basis of method four, method five, method six, mercury chelating type immune complex is extracted from whole blood, gel electrophoresis is adopted to be separated extracted mercury chelating type immune complex again, and then the protein band found out from electrophoretic 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 solution, its compound method example: take 0.31g boric acid and be dissolved in 400mLddH
2in O, regulate pH to 9.0 by the NaOH aqueous solution of 0.1Mol/L, be settled to 500mL, obtain final product;
Following EDTA-NaHCO
3the compound method example of solution: get 1.86gEDTA2H
2o and 16.8gNaHCO
3, be dissolved in 900mLddH
2in O, adjust pH to 8.0 with 1.0MNaOH and be settled to 1000mL, autoclaving, to obtain final product, 25 ± 2 DEG C of preservations;
The molecular cut off of following bag filter is 14000, buys from BioshopInc; Bag filter is through following process: EDTA-NaHCO bag filter being put into 500mL volume
3in solution, boil 10min; Tipping EDTA-NaHCO
3solution, uses ddH
2o rinsing, then boil 10min with 500mL5mmol/LEDTA; Discard boiling liquid, thoroughly use ddH
2o cleans, and adds a large amount of ddH
2o soaks bag filter 4 DEG C and spends the night; During use, put on one's gloves, take out bag filter, with a large amount of ddH
2its surfaces externally and internally of O cleaning down.
The present embodiment provides a kind of mercury chelating type immune complex, comprises the following steps:
A) synthesize mercury chelating type immune complex, concrete operation step is as follows:
(I) chelating agent solution is prepared: be dissolved in by 2.0mgITCBE in 2mLDMSO solvent, be mixed with chelating agent solution;
(II) human serum albumin solution is prepared: joined by the human serum albumins of 4.0mg in the 0.01M borate buffer solution of the pH=9.0 of 4mL, obtained human serum albumin solution;
(III) stirring is spent the night: slowly join in the human serum albumin solution of step (II) by step (I) chelating agent solution, dropping limit, limit is stirred, in 25 DEG C, 24h is acted in the shaking table of 100r/min, then to dialyse 24h with bag filter, remove the ITCBE be not combined with human serum albumins, obtain mixed liquor;
(IV) bag filter pre-service: add EDTA-NaHCO in bag filter
3solution boils, and uses ddH after abandoning waste liquid
2o rinses; Repeat this step 2 time;
(V) dialyse: the mixed liquor of step (III) is loaded in the bag filter after step (IV) process, use ddH
2o dialyses, and changes ddH
2o3 time, after 4 DEG C of dialysed overnight, collect liquid;
(VI) mercury ion chelating: add HCl solution adjustment pH to 7.0 in the liquid in above-mentioned steps (V), slowly add the 1mmol/L mercury ion solution of 80 μ L, dropping limit, limit is shaken, afterwards in shaking table effect 2-30h, then step (V) 1 time is repeated, collect liquid, obtain mercury chelating type antigen;
(VII) combine: add in mercury chelating type antigen prepared by step (VI) can with the antibody response of human serum albumins specific binding after obtain mercury chelating type immune complex;
B) purification mercury chelating type immune complex, concrete operation step is as follows:
I () is by above-mentioned A) step synthesis mercury chelating type immune complex redissolve in physiological saline;
(ii) chromatographic column pre-service: use dilution buffer flushing line, load in chromatographic column can with the filler of immune complex specific binding, after dress post, continuation dilution buffer balances pillar;
(ii) loading: after pillar balance, with dilution buffer dilution step (i) gained solution, then upper prop, mercury chelating type immune complex is adsorbed on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer;
(iv) wash-out: use dilution buffer to rinse pillar, to baseline balance, then uses the Na of 0.05-0.10Mol/L
2hPO
4solution carries out wash-out;
V () collects: the eluent collecting step (iv), makes protein renaturation after collection immediately;
(vi) eluent collected loads bag filter ddH
2o dialyses desalination, and after changing water 2-4 time, 4 DEG C of dialysed overnight, collect sample;
(vii) chromatographic column pre-service: adopt new chromatographic column, uses dilution buffer flushing line, in this chromatographic column load can with the filler of mercury specific binding, dress post after balance pillar by dilution buffer again;
(viii) loading: after the pillar balance of step (vii), the sample of above-mentioned steps (vi) is diluted by dilution buffer, then by the sample upper prop after dilution, mercury chelating type immune complex is adsorbed on filler, and unreacted antigen antibody complex can flow out with dilution buffer;
(ix) wash-out: rinse pillar by dilution buffer after step (viii), to baseline balance, then uses the Na of 0.5-1.0Mol/L
2hPO
4solution carries out wash-out;
X () collects: the eluent collecting step (ix), makes protein renaturation after collection immediately;
(xi) bag filter ddH is loaded to the eluent of step (x)
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, namely obtain the mercury chelating type immune complex of purifying.
Mercury chelating type immune complex prepared by the present embodiment, is separated further by gel electrophoresis, and carries out detection Qualitative Identification by inductivity coupled plasma mass spectrometry or atomic absorption spectrum, and concrete authentication step is as follows:
1) glue bed is prepared: select non-denaturing polyacrylamide gel as medium as required, prepare glue bed;
2) application of sample: get the mercury chelating type immune complex solution that 8 μ L purify out, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
Described sample-loading buffer (Samplebuffer) can be formulated by the component of following ratio: Tris-HCl:1% bromophenol blue: ddH
2o: glycocoll=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer, in electrophoresis process, electric current is 22mA constant current, and environment temperature is 4 degree; Stop electrophoresis when moving to bottom glue to bromophenol blue, non denatured electrophoretic band figure is shown in Fig. 1;
Described electrophoretic buffer obtains by following method: get Tris3.0g, glycocoll 14.4g, be dissolved in 800mLddH
2in O, regulate pH to 8.3, then add ddH
2o to 1000mL, to obtain final product;
4) detect: on glue bed, find out the protein band containing antigen antibody complex, this band is taken out, after treatment band is dissolved, and then utilize inductivity coupled plasma mass spectrometry or atomic absorption spectrum detection whether containing mercury and mercury content.
By the SRXRF of micronutrient levels in protein band analysis, at the 4W1 of BEPC (BEPC), " synchrotron radiation bunch completes, and in storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGTInc.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AXIL software data processing, and with deriving from air and the Ar signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
The synchrotron radiation line x-ray fluorescence analysis figure of the electrophoretic band of plumbous chelating type immune complex prepared by the method for the present embodiment refers to Fig. 2, and the horizontal ordinate in Fig. 2 is protein band position, and ordinate is mercury metal energy in this protein band.
Mercury chelating type immune complex in the present embodiment, can be used for preparing the reagent detecting mercury chelating 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
Adopt mercury chelating type immune complex provided by the invention as standard items, adopt Checkerboard titration method determination C1Q, the best effort concentration of anti-Hg antibody and the optimum diluting multiple of blood plasma, Checkerboard titration method concrete steps are as follows:
1) immobilized: C1Q to be coated on solid phase carrier, press the doubling dilution of 1:2500,1:5000,1:10000,1:20000 by dilution buffer, add in elisa plate micropore, at 4 DEG C, deposit 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) incubation: add test plasma sample and mercury chelating type immune complex standard items respectively, press the doubling dilution of 1:10,1:20,1:40 by dilution buffer, add in elisa plate micropore, 37 DEG C of incubations 2 hours;
4) catch: remove test plasma, washing, adds with the anti-Hg antibody of dilution buffer by the doubling dilution of 1:2500,1:5000,1:10000,1:20000, and 37 DEG C act on 2 hours, the mercury metal on itself and CIC ELISA is reacted;
5) enzyme conjugates incubation: remove anti-mercury antibody, with lavation buffer solution washing, adds the enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C of incubation 1-2 hour, makes itself and anti-Hg antibody response;
6) substrate incubation: remove enzyme labelled antibody, after washing, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer to elisa plate micropore;
7) detect: the OD value of testing each hole sample under 405nm wavelength.
In the present embodiment, adopt mercury chelated complexes standard items provided by the invention as positive control, respectively not add the control test group of detection sample as negative control 1, namely add C1q albumen, confining liquid, anti-mercury antibody, enzyme mark and substrate successively;
Not add the control test group of anti-mercury antibody as negative control 2, namely add C1q albumen, confining liquid, test plasma, enzyme mark and substrate successively;
C1q albumen, confining liquid, test plasma, anti-mercury antibody and substrate is added successively using not enzyme-added target control test group as negative control 3, namely;
Not add the control test group of detection sample and anti-mercury antibody as negative control 4 simultaneously, namely add C1q albumen, confining liquid, enzyme mark and substrate successively;
Not add the control test group of immune complex trapping agent C1q albumen as blank 1, namely add confining liquid, test plasma, anti-mercury antibody, enzyme mark and substrate;
And with the control test group only adding substrate for blank 2, only to add PBS damping fluid for blank 3.
In euzymelinked immunosorbent assay (ELISA), first adsorb immune complex there is the C1Q albumen of compatibility or CIF albumen or anti-C3 albumen with immune complex as trapping agent, nospecific immunity compound in serum is extracted, the immune complex upper part extracted is chelated with heavy metal Hg, and mercury on this part immune complex can by with the material of mercury specific binding or can with mercury react the specific antibody of the anti-mercury forming antigen antibody complex catch, afterwards can again by horseradish peroxidase, the enzyme labelled antibody of the enzyme labelings such as alkaline phosphatase caught, and under the effect of developer and stop buffer, OD value can be read under instrument, the i.e. provable mercury metal detecting chelating on CIC ELISA.
In euzymelinked immunosorbent assay (ELISA) test, the parameter optimization of the dilute concentration of different C1Q albumen, the anti-dilute concentration of mercury antibody and the dilute concentration of test plasma is as shown in table 1, and in table, each numerical value is mean value;
The determination of table 1:C1q and Hg antibody best effort concentration and diluted plasma multiple
From table 1 we can find out when complement protein C1q dilution multiple proportions be 1:2500, the dilution multiple proportions of dilution rate of blood plasma is 1:20, the dilution multiple proportions of anti-Hg antibody is 1:2500 time, OD value is maximum, under this best effort concentration conditions, ELISA positive control and negative control ELISA testing result as shown in table 2
Table 2ELISA positive control and negative control ELISA testing result
As shown in table 2, the OD value of negative control group and blank group is all less than 0.1, so this working concentration is as best effort concentration.
2, ELISA eluent best effort concentration and elution time are determined
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-mercury antibody and enzyme labelled antibody incubation, carry out wash-out with the eluent of variable concentrations, then detect OD value by microplate reader, to determine best Elisa wash-out concentration and elution time, concrete steps are as follows:
(1) bag quilt: the doubling dilution anti-Hg antibody dilution buffer being pressed 1:2500, adds in elisa plate micropore, 4 DEG C of 18h that spend the night;
(2) close: remove dilution buffer, after washing, add confining liquid, place 1 hour, remove confining liquid, and wash for 37 DEG C;
(3) add enzyme labelled antibody: remove confining liquid, after washing, add and be diluted to by dilution buffer the HRP enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C act on 2 hours, make itself and anti-Hg antibody response;
(4) wash-out: remove enzyme labelled antibody, by dilution buffer, eluent is diluted, make the concentration of papain in eluent: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove eluent, washing, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
The compound method example of described eluent: be 8.0 by papain pH, the Tris-HCI buffer of volumetric molar concentration 0.1mol/L becomes 2mg/mL, add dithiothreitol (DTT) (DTT) again, the concentration being mixed with dithiothreitol (DTT) is 1mmol/L, hatches 30min for 37 DEG C;
(5) stop buffer is dripped to each micropore;
(6) read respectively in microplate reader under the determined wavelength of 405nm and often organize OD value, concrete outcome see table 3,
Table 3:ELISA eluent best effort densimeter elution time
By comparing OD value, to judge the mercury anti-enzyme mark compound wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-mercury antibody-enzyme mark compound wash-out degree reaches maximum.
As known from Table 3, the concentration when papain in eluent: in enzyme labelled antibody during the concentration=1:20 of antibody, each group OD value, all lower than other groups, illustrates and reaches optimum in this concentration elution effect; And no matter action time is 1h, 2h, 3h, the change of each group OD value is all little, as seen along with the prolongation of time, enzyme activity weakens gradually, when enzyme concentration is constant, extend digestion time can not improve digestibility, so in this experiment the action time of eluent be that 1-3h all can.
Application Example
1.ELISA method detects mercury chelating type immune complex in blood plasma
The ELISA method that employing method one provides, detect 100 plasma samples, concrete operations condition is as follows:
1) immobilized: C1Q to be coated on polypropylene solid phase carrier, to press the doubling dilution of 1:2500 by dilution buffer, add in elisa plate micropore, at 4 DEG C, deposit 16 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) application of sample: add test plasma sample and standard items respectively, presses the doubling dilution of 1:20 by dilution buffer by sample, add in elisa plate micropore, 37 DEG C of incubations 1.5 hours;
4) catch: remove test plasma, washing, adds with the anti-mercury antibody of dilution buffer by the doubling dilution of 1:2500,37 DEG C of incubations 1.5 hours;
5) enzyme conjugates incubation: remove anti-mercury antibody, washing, adds the enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C of incubations 1.5 hours;
6) substrate incubation: remove enzyme labelled antibody, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer to elisa plate micropore;
7) detect: the OD value reading test plasma sample and above-mentioned mercury chelating type immune complex standard items under 405nm wavelength in microplate reader respectively, result is as shown in table 4.
Table 4 adopts the measured result of method a pair 100 parts of sample blood plasma
2, ELISA+AAS method detects chromium chelating type immune complex in blood plasma
The Elisa method adopting the inventive method two to provide and the method for AAS methods combining, detect 100 plasma samples, concrete operations condition is as follows:
1) immobilized: complement CIF albumen to be coated on polystyrene solid phase carrier, to press the doubling dilution of 1:2500 by dilution buffer, add in elisa plate micropore, at 4 DEG C, deposit 16 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) incubation: add standard items and test plasma sample respectively, presses the doubling dilution of 1:20 by dilution buffer by sample, add in elisa plate micropore, 37 DEG C of incubations 2 hours;
4) wash-out: remove testing sample, washing, adds the eluent that Papain enzyme concentration is 100 μ g/mL, acts on 1 hour at 37 DEG C;
5) detect: adopt the lead of chelating on CIC ELISA in Atomic Absorption Spectrometer examination criteria product and test plasma sample, testing result is as shown in table 5.
Table 5 adopts method two to the measured result of 100 parts of sample blood plasma
application Example 3:
The method that Elisa and the ICP-MS adopting the inventive method three to provide combines, the mercury chelating type immune complex standard items provided with embodiment 1, detect 100 plasma samples, concrete operations condition is as follows:
1) immobilized: to be coated in by anti-C_3 antibody on polystyrene solid phase carrier, to press the doubling dilution of 1:2500 by dilution buffer, add in elisa plate micropore, 37 DEG C of water-baths were stored in refrigerator after 1 hour;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) incubation: add standard items and test plasma sample respectively, presses the doubling dilution of 1:20 by dilution buffer by sample, add in elisa plate micropore, 37 DEG C of incubations 2 hours;
4) wash-out: remove testing sample, washing, adds the eluent that Papain enzyme concentration is 100 μ g/mL, acts on 1 hour at 37 DEG C;
5) acidifying: add nitric acid and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
6) detect: sample and detect the mercury of chelating on CIC ELISA under icp ms, read respective value and calculate content, result is as shown in table 6.
Table 6 adopts method three to the measured result of 100 parts of sample blood plasma
| 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; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.
Claims (10)
1. a mercury chelating type immune complex, is characterized in that, this mercury chelating type immune complex is the one in following compound:
Mercury ion is incorporated into the compound that immune complex is formed, or
The compound that mercury ion is incorporated into carrier protein and can be formed with the antibody of this carrier protein specific binding; Or
The compound formed is combined with carrier protein after mercury ion is incorporated into immunoglobulin (Ig).
2. mercury chelating type immune complex according to claim 1, it is characterized in that, described carrier protein or immune complex are at least containing a kind of structure in zinc fingers, sulfydryl, cysteine residues, and mercury ion is combined with carrier protein or immune complex by least one in zinc fingers, sulfydryl, cysteine residues.
3. mercury chelating type immune complex according to claim 2, is characterized in that, described carrier protein is the one in antibody protein, lipoprotein, haemoglobin, nuclear antigen, foreign sera antigen, virus, bacterium, protozoon, worm.
4. mercury chelating type immune complex according to claim 1, is characterized in that, wherein, mercury ion is combined with the antibody of energy and carrier protein specific binding after being combined with carrier protein by sequestrant again.
5. a preparation method for mercury chelating type immune complex as claimed in claim 1, it is characterized in that, this preparation method comprises:
A) step of mercury chelating type immune complex is synthesized;
B) step of purification mercury chelating type immune complex: adopt immune-affinity chromatography removal step A) have neither part nor lot in the carrier protein of reaction, specific antibody and mercury ion in the mercury chelating type immune complex that synthesizes.
6. preparation method according to claim 5, is characterized in that,
Described steps A) concrete steps are:
(I) chelating agent solution is prepared: dissolved by sequestrant, be mixed with chelating agent solution;
(II) formulation vehicle protein solution: carrier protein is joined in borate buffer solution, obtained carrier protein solution;
(III) stirring is spent the night: join in the carrier protein solution of step (II) by step (I) chelating agent solution, in shaking table effect 2-30h after stirring, obtain mixed liquor;
(IV) bag filter pre-service: add EDTA-NaHCO in bag filter
3solution boils, and uses ddH after abandoning waste liquid
2o rinses, and repeats this step 1-3 time;
(V) dialyse: the mixed liquor of step (III) is loaded in the bag filter after step (IV) process, use ddH
2o dialyses, and changes water 2-3 time, 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), slowly add mercury ion solution, dropping limit, limit is shaken, afterwards in shaking table effect 2-30h, then step (V) is repeated once, collect liquid, obtain mercury chelating type antigen;
(VII) combine: add in above-mentioned mercury chelating type antigen can with the antibody of carrier protein specific binding, obtain mercury chelating type immune complex after reaction;
Described step B) concrete steps are as follows:
I () is by above-mentioned A) step synthesis mercury chelating type immune complex redissolve in physiological saline;
(ii) chromatographic column pre-service: use dilution buffer flushing line, load in chromatographic column can with the filler of immune complex specific binding, after dress post, continuation dilution buffer balances pillar;
(ii) loading: after pillar balance, with dilution buffer dilution step (i) gained solution, then upper prop, mercury chelating type immune complex is adsorbed on filler, and unreacted carrier protein, antibody, mercury ion flow out with dilution buffer;
(iv) wash-out: use dilution buffer to rinse pillar, to baseline balance, then uses the Na of 0.05-0.10Mol/L
2hPO
4solution carries out wash-out;
V () collects: the eluent collecting step (iv), makes protein renaturation after collection immediately;
(vi) eluent collected loads bag filter ddH
2o dialyses desalination, and after changing water 2-4 time, 4 DEG C of dialysed overnight, collect sample;
(vii) chromatographic column pre-service: adopt new chromatographic column, uses dilution buffer flushing line, in this chromatographic column load can with the filler of mercury specific binding, dress post after balance pillar by dilution buffer again;
(viii) loading: after the pillar balance of step (vii), the sample of above-mentioned steps (vi) is diluted by dilution buffer, then by the sample upper prop after dilution, mercury chelating type immune complex is adsorbed on filler, and unreacted antigen antibody complex can flow out with dilution buffer;
(ix) wash-out: rinse pillar by dilution buffer after step (viii), to baseline balance, then uses the Na of 0.5-1.0Mol/L
2hPO
4solution carries out wash-out;
X () collects: the eluent collecting step (ix), makes protein renaturation after collection immediately;
(xi) bag filter ddH is loaded to the eluent of step (x)
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, namely obtain the mercury chelating type immune complex of purifying.
7. mercury chelating type immune complex as claimed in claim 1 detects the application in the enzyme linked immunological kit of mercury chelating type immune complex in preparation.
8. detect an enzyme linked immunological kit for mercury chelating type immune complex, it is characterized in that, this kit comprises the standard items of mercury chelating type immune complex as claimed in claim 1.
9. enzyme linked immunological kit according to claim 8, is characterized in that, this kit also comprises the following reagent of at least one: the coating buffer of the albumen containing capture antigen antibody complex, containing coating buffer, the enzyme labelled antibody of antibody of catching metal Hg.
10. one kind is quantitatively detected the method for mercury chelating type immune complex, it is characterized in that, using the mercury chelating type immune complex according to claim 1 of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification immune complex and atomic absorption spectrum combined techniques, purification immune complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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| CN108982864A (en) * | 2018-06-13 | 2018-12-11 | 广东腾湃医疗股份有限公司 | A kind of novel purification mercury chelating type immune complex method |
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