CN105044369B - Lead-chelated immune compound, and preparation method and application of compound - Google Patents

Lead-chelated immune compound, and preparation method and application of compound Download PDF

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Publication number
CN105044369B
CN105044369B CN201510413057.9A CN201510413057A CN105044369B CN 105044369 B CN105044369 B CN 105044369B CN 201510413057 A CN201510413057 A CN 201510413057A CN 105044369 B CN105044369 B CN 105044369B
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lead
immune complex
chelating type
solution
dilution buffer
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CN105044369A (en
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张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
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Guangdong Tenpai Medical Ltd By Share Ltd
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Shanghai Baihao Biotechnology Co Ltd
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    • 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
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Abstract

The invention provides a lead-chelated immune compound, and a preparation method and an application of the lead-chelated immune compound. The lead-chelated immune compound is one of the following compounds: a compound formed from an immune compound combined with lead ions, or a compound formed from carrier protein combined with lead and an antibody specifically binding to the carrier protein, or a compound formed from immune globulin combined with lead ions and carrier protein. The lead-chelated immune compound is an immune compound with relative specificity. Whether the lead-chelated immune compound is contained in serum of people in an area and the content of the compound is detected, so that the lead pollution degree in the area is indirectly reflected.

Description

A kind of lead chelating type immune complex and its preparation method and application
Technical field
The present invention relates to the detection of circulating immune complex is and in particular to a kind of lead chelating type immune complex.
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).The removing of immune complex depends greatly on cic's Size, antibody isotype, antigen-antibody relative concentration etc., only in limited instances immune complex precipitate cause tissue damage Wound, causes disease.The generation of multiple diseases is all closely bound up with the deposition of immune complex, such as systemic lupus erythematosus (sle), class wind Wet arthritis, nephrotic syndrome, cryoglobulinemia, vasculitiss, antibacterial, virus and parasitic infection, anaphylaxiss, itself Immunological diseases, dermatosiss etc..
With regard to the detection by quantitative of circulating immune complex, at present both at home and abroad through frequently with method mainly include Polyethylene Glycol (polyethylene glycol, peg) sedimentation method, ultracentrifugation, molecule ultrafiltration, gel filtration, double antibody sandwich method elisa Detection by quantitative, c1-q complement elisa detection by quantitative, but these methods can only detect non-specific circulating immune complex, many Number points out index only as Disease activity, and lacks disease specific and be worth.Although also there being scholar first to adopt the peg sedimentation method Purify out non-specific cic, recycle known antigens, combined enzyme-linked immunoadsorption principle, carry out specificity cic detection by quantitative, But also it is only applied to several known antigens disease such as hepatitis, hiv.Thus find a kind of quantitation of Specific Circulating Immune Complexes Detection method is most important.
Lead is that one kind common are noxious metals and environmental contaminants, and the main path of general population's contact or absorption lead has: Suck beverage or food, leaded medicine of absorption etc. that air, absorption lead container or the packaging of lead contamination contain.Lead is a kind of complete Body poisonous substance, can affect the histoorgans such as nerve, hemopoietic, endocrine, immunity, reproduction, liver, kidney, international tumor research Lead and its compound are also classified as 2b class people's carcinogen by mechanism (iarc).
Have lot of documents at present and report that lead can act on the albumen in each system of whole body, thus affecting body each group Knit the function of organ, the albumen of report includes: albumen in the albumen in renal tissue, the albumen in cerebral tissue, erythrocyte, Albumen in albumen in albumen in liver organization, intestinal tissue, lung tissue, metallothionein etc..
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.Circulating immune complex is a kind of Special Significance Protein, and lead may act on the protein of the multiple system of whole body, whether lead can act on circulating immune complex, and makes to follow The change of ring 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 lead chelating type immune complex, should Lead chelating type immune complex is a kind of immune complex of relative specificity, whether there is for detecting in regional crowd's serum Lead chelating type immune complex and its content, reflect this Pb pollution level indirectly.
For solving the above problems, the technical solution adopted in the present invention is as follows:
A kind of lead chelating type immune complex, this lead chelating type immune complex is one of following complex:
Lead ion is incorporated into the complex of immune complex formation, or
Lead ion be incorporated into carrier protein after with and the complex that formed of antibody that specifically binds of this carrier protein；Or
The complex that lead ion is combined to form with carrier protein after being incorporated into immunoglobulin.
Further, zinc fingerses, sulfydryl, half Guang ammonia are at least contained in the structure of described immune complex or carrier protein One of sour residue, lead ion and immune complex or carrier protein and zinc fingerses or sulfydryl or cysteine residues phase knot Close.
Further, described carrier protein be antibody protein, lipoprotein, hemoglobin, nuclear antigen, foreign sera antigen, One of virus, antibacterial, protozoon, anthelmintic or immunoglobulin.
Further, lead 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 the method preparing lead chelating type immune complex, comprise the following steps:
A) step synthesizing lead chelating type immune complex；
B) step purifying lead chelating type immune complex: using the prepared lead chela of immune-affinity chromatography removal step a) Carrier protein, specific antibody and the lead ion of reaction is had neither part nor lot in mould assembly immune complex.
Specifically, described step a) concretely comprises the following steps:
() prepares chelating agent solution: chelating agen is dissolved, is configured to chelating agent solution；
() formulation vehicle protein solution: carrier protein is added in borate buffer solution, prepared carrier protein is molten Liquid；
() 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；
() bag filter pretreatment: add edta-nahco in bag filter3Solution boils, and uses ddh after abandoning waste liquid2O rushes Wash, repeat this step 1-3 time；
V () is dialysed: the mixed liquor of step (iii) is loaded in the bag filter after processing through step (), uses ddh2O is saturating Analysis, changes water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid；
(vi) lead ion chelating: add hcl solution adjustment ph to 7.0 in the liquid in above-mentioned steps (v), be slowly added to Lead ion solution, shakes in Deca, after shaking table act on 2-30h, then repeat step (v) once, collect liquid, obtain Lead chelating type antigen；
(vii) combine: adding in above-mentioned lead chelating type antigen can be with the antibody of carrier protein specific binding, after reaction Obtain lead chelating type immune complex；
Described step b) specifically comprises the following steps that
I () redissolves the lead chelating type immune complex that above-mentioned a) step synthesizes in normal saline；
(ii) chromatographic column pretreatment: using dilution buffer flushing line, load energy and immune complex in chromatographic column 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, lead chelates Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, lead 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) dialyse: the eluent of step (v) is loaded bag filter ddh2O dialyses desalination, and after changing water 2-4 time, 4 DEG C saturating Analysis overnight, collects 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 lead specific binding again after dress post；
Described can be to be adsorbed with silica gel or the resin that can specifically bind material with lead with the filler of lead 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, lead chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined Thing can flow out with dilution buffer；
(ix) eluting: rinse pillar with dilution buffer after step (viii), to baseline balance, then using 0.5- 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 lead chelating type immune complex of purification.
Present invention also offers above-mentioned lead chelating type immune complex is in preparation detection lead chelating type circulating immune complex Reagent or enzyme linked immunological kit in application.
Present invention also offers a kind of enzyme linked immunological kit of above-mentioned detection lead chelating type circulating immune complex, this examination Agent box includes the standard substance of above-mentioned lead chelating type immune complex.
Further, mentioned reagent box also includes at least one following reagent: includes the egg of capture antigen antibody complex White be coated liquid, include that the antibody of capture metallic lead makees be coated liquid, enzyme labelled antibody.
A kind of method of detection by quantitative lead chelating type circulating immune complex, with the above-mentioned lead chelating type immunity of known content Complex is detected as standard substance, a pair of sample using following methods: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and Atomic 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 atomic absorption light Spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
Compared to existing technology, the beneficial effects of the present invention is:
1. synthesized lead chelating type immune complex first, and confirmed that lead may be present in immune system in body, and Demonstrate the presence of lead chelating type circulating immune complex；
2. present invention achieves the specific recognition of circulating immune complex, and achieve lead chelating type circulation immunity and be combined The detection by quantitative of thing, the method that the lead content level in immune angle testing machine body is provided, and the lead contamination for industrial area Level provides 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 line fluorescence analysiss figure of the electrophoretic band of immune complex；
Wherein, the m in Fig. 1 is marker, and ic is lead chelating type immune complex；Abscissa in Fig. 2 is protein band Position, vertical coordinate is lead metal energy in this protein band.
Specific embodiment
The present invention provides a kind of lead chelating type immune complex, and this lead chelating type immune complex is in following complex A kind of:
Lead ion is incorporated into the complex of immune complex formation, or
Lead ion be incorporated into carrier protein after with and the complex that formed of antibody that specifically binds of this carrier protein；Or
The complex that lead ion is combined to form with carrier protein after being incorporated into immunoglobulin.
Specifically, zinc fingerses, sulfydryl, cysteine are at least contained in the structure of described immune complex or carrier protein One of residue, lead ion and immune complex or carrier protein and zinc fingerses or sulfydryl or cysteine residues phase knot Close.
Further, described carrier protein be antibody protein, lipoprotein, hemoglobin, nuclear antigen, foreign sera antigen, One of virus, antibacterial, protozoon, anthelmintic or immunoglobulin.
Further, lead 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 the method preparing lead chelating type immune complex, comprise the following steps:
A) step synthesizing lead chelating type immune complex；
B) step purifying lead chelating type immune complex: using the prepared lead chela of immune-affinity chromatography removal step a) Carrier protein, specific antibody and the lead ion of reaction is had neither part nor lot in mould assembly immune complex.
Specifically, described step a) concretely comprises the following steps:
() prepares chelating agent solution: chelating agen is dissolved, is configured to chelating agent solution；
() formulation vehicle protein solution: carrier protein is added in borate buffer solution, prepared carrier protein is molten Liquid；
() 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；
() bag filter pretreatment: add edta-nahco in bag filter3Solution boils, and uses ddh after abandoning waste liquid2O rushes Wash, repeat this step 1-3 time；
V () is dialysed: the mixed liquor of step (iii) is loaded in the bag filter after processing through step (), uses ddh2O is saturating Analysis, changes water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid；
(vi) lead ion chelating: add hcl solution adjustment ph to 7.0 in the liquid in above-mentioned steps (v), be slowly added to Lead ion solution, shakes in Deca, after shaking table act on 2-30h, then repeat step (v) once, collect liquid, obtain Lead chelating type antigen；
(vii) combine: adding in above-mentioned lead chelating type antigen can be with the antibody of carrier protein specific binding, after reaction Obtain lead chelating type immune complex；
Described step b) specifically comprises the following steps that
I () redissolves the lead chelating type immune complex that above-mentioned a) step synthesizes in normal saline；
(ii) chromatographic column pretreatment: using dilution buffer flushing line, load energy and immune complex in chromatographic column 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, lead chelates Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, lead 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) dialyse: the eluent of step (v) loads bag filter ddh2O dialyses desalination, after changing water 2-4 time, dialyses for 4 DEG C 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 lead 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, lead chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined Thing can flow out with dilution buffer；
(ix) eluting: rinse pillar with dilution buffer after step (viii), to baseline balance, then using 0.5- 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 lead chelating type immune complex of purification.
Present invention also offers above-mentioned lead chelating type immune complex is in preparation detection lead chelating type circulating immune complex Reagent or enzyme linked immunological kit in application.
Present invention also offers a kind of enzyme linked immunological kit of above-mentioned detection lead chelating type circulating immune complex, this examination Agent box includes the standard substance of above-mentioned lead chelating type immune complex.
Further, mentioned reagent box also includes at least one following reagent: containing can capture antigen antibody complex Albumen be coated liquid, containing can capture metallic lead antibody 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 lead chelating type circulating immune complex, comprising: multiple containing antigen-antibody can be captured The albumen of compound be coated liquid, confining liquid, lavation buffer solution, anti-antibody lead, enzyme labelled antibody, substrate, terminate liquid, dilution buffer Liquid, positive control, negative control.
A kind of test kit for detecting lead chelating type circulating immune complex, comprising: multiple containing antigen-antibody can be captured The albumen of compound be coated liquid, confining liquid, lavation buffer solution, eluent, positive control, negative control.
A kind of test kit for detecting lead chelating type circulating immune complex, comprising: multiple containing antigen-antibody can be captured The albumen of compound be coated liquid, confining liquid, lavation buffer solution, eluent, nitric acid, hydrogen peroxide, positive control, negative control.
A kind of test kit for detecting lead chelating type circulating immune complex, including for purifying serum immune complex Required solution, redissolve liquid, containing can capture metallic lead antibody be coated liquid, confining liquid, lavation buffer solution, enzyme labelled antibody, bottom Thing, terminate liquid, dilution buffer, standard substance, negative control.
A kind of test kit for detecting lead chelating type circulating immune complex: include for purifying serum immune complex Required solution, redissolution liquid, positive control, negative control.
A kind of test kit for detecting lead chelating type circulating immune complex, including for purifying serum immune complex Required solution, redissolution liquid, nitric acid, hydrogen peroxide, positive control, negative control etc..
A kind of test kit for detecting lead chelating type circulating immune complex, including for purifying serum immune complex Required solution, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing metal liquid needed for protein band, containing can Capture metallic lead antibody be coated liquid, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, sun Property comparison, negative control etc..
A kind of test kit for detecting lead chelating type circulating immune complex, including for purifying serum immune complex On required solution, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed, liquid, the positive needed for protein band containing metal is right According to, negative control etc..
A kind of test kit for detecting lead chelating type circulating immune complex, including for purifying serum immune complex Required solution, glue bed medium, redissolve liquid, sample-loading buffer, on dissolving glue bed containing metal liquid needed for protein band, 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 complement c1q, cif Albumen, anti-c3 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 ap7019 " from Ba Ao get bio tech ltd article No. that the antibody of metallic lead includes but is not limited to buy Anti- pb 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 The tris-hcl buffer solution of papain；Described glue bed medium includes but is not limited to agarose gel, polyacrylamide coagulates Glue；Include but is not limited to peg solution, borate buffer solution for purifying solution needed for serum immune complex；Substrate include but It is not limited to tmb solution, abts solution；It is molten that described sample-loading buffer includes but is not limited to the buffering of the tris-hcl containing bromophenol blue Liquid；Enzyme labelled antibody is the antibody containing the enzyme labelling such as horseradish peroxidase, alkali phosphatase, such as hrp enzyme labelled antibody；Standard substance Including but not limited to the lead chelating type circulating immune complex of the present invention, other be chelated with the immune complex of lead metal；Positive Comparison include but is not limited to lead chelating type circulating immune complex of the present invention, other be chelated with the immune complex of lead metal, cloudy Property compares as dilution buffer.
A further object of the present invention is to provide a kind of method of detection by quantitative lead chelating type circulating immune complex, with As standard substance, a pair of sample using following methods is detected the above-mentioned lead chelating type immune complex knowing content: enzyme connection Immunization, enzyme linked immunological and atomic 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, electricity Swimming and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
In the present invention, having that the method with detecting lead 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 buffer 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 albumin solution, compound method example: takes 0.1g bovine serum albumin, adds washing buffer Liquid dilution is 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: takes 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 complement c1q, cif albumen, anti-c3 antibody；In following examples, can capture immune complex albumen specifically used be Article No. is the c1q recombinant protein of " novus h00000712-p01 "；
The Mus anti-pb mab that material with lead specific binding is " bar is proud to obtain ap7019 " for article No.；In implementation below Described material with lead specific binding, anti-pb antibody, anti-antibody lead be capture lead material；
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 lead chelating type immune complex, specifically comprises the following steps that
1) it is coated: the albumen that can capture immune complex is coated on solid phase carrier, is diluted with 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 lavation buffer solution, after the completion of waiting to wash, plus confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with lavation buffer solution, after the completion of washing, elisa plate is placed in 37 DEG C 1 little When；
3) add testing sample, and incubate: from blood circulation sampling, make testing sample；Lead 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) add the material of capture lead, and incubate: remove testing sample, and washed with lavation buffer solution, to be washed After the completion of washing, addition dilution buffer dilutes 50000-400000 times of anti-antibody lead, 37 DEG C act on 1-2 hours so as to Metallic lead reaction on immune complex；
5) enzyme conjugates incubate: remove anti-antibody lead, and are washed with lavation buffer solution, after the completion of waiting to wash, add With the hrp enzyme labelled antibody of dilution buffer dilution, 37 DEG C of effect 1-2 hours are so as to react with anti-antibody lead；
6) substrate incubates: remove enzyme labelled antibody, and washed with lavation buffer solution, after the completion of waiting to wash, add substrate, 37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction: Deca terminate liquid to each micropore；
8) take wavelength to be 405nm, elisa plate is placed in the od that testing sample group and standard substance are read respectively on microplate reader Value, and draw standard curve, try to achieve the content of testing sample.
In this method, step 8) detection when, also can not use microplate reader, directly qualitative inspection be carried out by colour developing situation.
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 lead, and the lead on this partial immunity complex Can be by the material specifically binding with lead or the specific antibody institute that the anti-lead forming antigen antibody complex can be reacted with lead 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 lead, then will not be captured by the specific antibody of anti-lead, 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 metallic lead (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 metallic lead of chelating in circulating immune complex.
Method two: elisa method+aas method detects lead chelating type immune complex, specifically comprises the following steps that
1) it is coated: the albumen that can capture immune complex 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 lavation buffer solution, after the completion of waiting to wash, plus confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with lavation buffer solution, after the completion of washing, elisa plate is placed in 37 DEG C 1 little When；
3) add testing sample, and incubate: from blood circulation sampling, make testing sample；Lead 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 testing sample, and washed with lavation buffer solution, after the completion of waiting to wash, add eluent, in 1-2 hour is acted at 37 DEG C；
5) detect: sample from elisa micropore, chelate the lead on immune complex in Atomic Absorption Spectrometer detection, 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 lead 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 metallic lead of chelating in circulating immune complex.
Method three: elisa method+icp-ms method detects lead chelating type immune complex, specifically comprises the following steps that
1) it is coated: the albumen that can capture immune complex 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 lavation buffer solution, after the completion of waiting to wash, plus confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with lavation buffer solution, after the completion of washing, elisa plate is placed in 37 DEG C 1 little When；
3) add testing sample, and incubate: from blood circulation sampling, make testing sample；Lead 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 testing sample, and washed with lavation buffer solution, after the completion of waiting to wash, add eluent, in 1-3 hour is acted at 37 DEG C；
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 lead 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 lead 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 metallic lead of chelating in circulating immune complex.
Method four: purification cic method+elisa method detection lead chelating type immune complex, specifically comprise the following steps that
1) extract nonspecific immunity complex: using the side such as the peg sedimentation method, ultracentrifugation, molecule ultrafiltration, gel filtration Method extracts nonspecific immunity complex, purification immune complex out is redissolved, obtains the solution of immune complex；
2) it is coated: the material that use can capture lead is coated on solid phase carrier, with dilution buffer dilution coating protein extremely 50000-400000 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 lavation buffer solution, after the completion of waiting to wash, plus confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with lavation buffer solution, after the completion of washing, elisa plate is placed in 37 DEG C 1 little When；
4) sample solution plus testing sample, and incubate: from step 1), make testing sample；Lead with known content Chelating type immune complex makees standard substance；Dilute 10-40 times with dilution buffer, add in micropore, 37 DEG C of effect 1-2 hours；
5) enzyme conjugates incubate: testing sample, and being washed with lavation buffer solution, after the completion of waiting to wash, add with dilute Release the hrp enzyme labelled antibody of buffer dilution, 37 DEG C of effect 1-2 hours are so as to react with anti-antibody lead；
6) substrate incubates: removes hrp enzyme labelled antibody, and is washed with lavation buffer solution, after the completion of waiting to wash, adds bottom Thing, 37 DEG C of lucifuges act on 30 minutes；
7) terminating reaction: Deca terminate liquid to each micropore.
8) take wavelength to be 405nm, after adding terminate liquid, elisa plate is placed in microplate reader and reads testing sample group respectively With the od value of standard substance, draw standard curve, try to achieve the content of testing sample；
In this method, step 8) in, also can not use microplate reader, directly qualitative detection be carried out by colour developing situation.
Method five: purification cic method+aas method detection lead chelating type immune complex, specifically comprise the following steps that
1) extract nonspecific immunity complex: using the side such as the peg sedimentation method, ultracentrifugation, molecule ultrafiltration, gel filtration Method extracts nonspecific immunity complex, purification immune complex out is redissolved, obtains the solution of immune complex；
2) detect: from step 1) solution sample, in Atomic Absorption Spectrometer detection chelate on immune complex Lead, reads respective value.
Method six: purification cic method+icp-ms method detection lead chelating type immune complex, specifically comprise the following steps that
1) extract nonspecific immunity complex: using the side such as the peg sedimentation method, ultracentrifugation, molecule ultrafiltration, gel filtration Method extracts nonspecific immunity complex, purification immune complex out is redissolved, obtains the solution of immune complex；
2) be acidified: from step 1) solution sample, in the solution add acidulant solution is acidified, sealed At night, thoroughly it is acidified；
3) detect: add hydrogen peroxide, and heat and catch up with acid, sampling, detects chela under icp mses Together in the lead 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 lead on lead 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 Lead content on lead chelating type immune complex.
Method seven: electrophoresis method+elisa/aas/icp-ms method detection lead chelating type immune complex, specifically comprise the following steps that
1) extract nonspecific immunity complex: using the side such as the peg sedimentation method, ultracentrifugation, molecule ultrafiltration, gel filtration Method extracts nonspecific immunity complex, purification immune complex out is redissolved, obtains the solution of immune complex；
2) prepare glue bed: select suitable medium (as agarose gel, polyacrylamide gel etc.) as needed, according to Requirement prepares glue bed；
3) be 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；
Described sample-loading buffer (sample buffer) can be formulated by the component of following ratio: tris-hcl:1% Bromophenol blue: ddh2O: the ph of glycine=15.5:2.5:7:25, wherein tris-hcl is 6.8, molar concentration is 1m；
4) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer, and according to demand by albumen according to molecular weight, etc. Electricity is put isoparametric difference and is carried 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: the protein band containing immune complex is found out on glue bed, this band is taken out, band is dissolved, Then it is utilized respectively the principles such as elisa, icp-ms, aas detection lead content again；Further, it is also possible to using the method detection chelating The isoelectric point, IP of the immune complex of lead, molecular weight and content etc..
Method seven is on the basis of method four, method five, method six, and lead chelating type immune complex is extracted, Again using gel electrophoresis the lead chelating type immune complex being extracted is carried out separate, and then find out from electrophoretic band containing The protein band of immune complex, carries 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 lead chelating type immune complex, comprises the following steps:
A) synthesize lead chelating type immune complex, concrete operation step is as follows:
I () prepares 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: add edta-nahco in bag filter3Solution boils, and uses ddh after abandoning waste liquid2O rushes Wash；Repeat this step 2 time；
V () is dialysed: 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) lead 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 lead 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 lead chelating type antigen；
(vii) combine: add in lead chelating type antigen prepared by step (vi) and can tie with human serum albumin's specificity Lead chelating type immune complex is obtained after the antibody response closing；
B) purify lead chelating type immune complex, concrete operation step is as follows:
I () redissolves the lead chelating type immune complex that above-mentioned a) step synthesizes in normal saline；
(ii) chromatographic column pretreatment: using dilution buffer flushing line, load energy and immune complex in chromatographic column 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, lead chelates Type immune complex adsorbs on filler, and unreacted carrier protein, antibody, lead 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) dialyse: the eluent of step (v) loads bag filter ddh2O dialyses desalination, after changing water 2-4 time, dialyses for 4 DEG C 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 lead 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, lead chelating type immune complex adsorbs on filler, and unreacted antigen-antibody is combined Thing can flow out with dilution buffer；
(ix) eluting: rinse pillar with dilution buffer after step (viii), to baseline balance, then using 0.5- 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 lead chelating type immune complex of purification.
Lead 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 lead 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: the ph of glycine=15.5:2.5:7:25, wherein tris-hcl is 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: the protein band containing metal is found out on glue bed, this band is taken out, band is dissolved, then sharp again Detected whether containing lead and lead content with inductivity coupled plasma mass spectrometry or atomic absorption spectrum.
In protein band, the srxrf analysis of micronutrient levelss is " synchronous in the 4w1 of BEPC (bepc) Complete on 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.ls30143-ds) detect, probe with incident sr line copline and is mutually perpendicular to, away from sample irradiation point 20mm, signal pgt MCA (mca 4000) obtains output.Excite sample with the monochromatic synchrotron radiation light of 11.5kev, adjust launching spot (1mmx3mm) position is allowed to be in band one end, and in the minute of 300s, hot spot uniformly slowly moves along band always, At the end of counting, 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 with from air and the ar signal peak of content constant carries out normalization to other element peaks, to offset beam intensity change The impact that signal strength is produced.Measure the fluorescence Spectra of quantitative criterion dry glue film at identical conditions in the same way.
Using GFAAS (aas) Preliminary Determination lead manufactured in the present embodiment chelating type immune complex In lead content, its content be 60.518 μ g/l, blank borate buffer solution detected value be 0.613 μ g/l.
The synchrotron radiation line x line 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 lead metal energy in this protein band.
The present invention provides lead chelating type immune complex, can be used for preparing the examination of detection lead chelating type circulating immune complex Agent, 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 lead 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-pb antibody and blood plasma, Checkerboard titration method specifically comprises the following steps that
1) immobilized: complement c1q is coated on solid phase carrier, with dilution buffer press 1:2500,1:5000,1:10000, The doubling dilution of 1:20000, 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 lead chelating type immune complex standard substance, press 1 with dilution buffer: 10th, the doubling dilution of 1:20,1:40, adds in elisa plate micropore, and 37 DEG C incubate 2 hours；
4) capture: remove test plasma, washing, addition dilution buffer press 1:50000,1:100000,1:200000, The anti-pb antibody of the doubling dilution of 1:400000,37 DEG C of effects 2 hours are so as to anti-with the metallic lead in circulating immune complex Should；
5) enzyme conjugates incubate: remove anti-antibody lead, are 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-pb antibody response；
6) substrate incubates: removes enzyme labelled antibody, after washing, adds substrate, 37 DEG C of lucifuges act on 30 minutes, to elisa plate Micropore adds terminate liquid；
7) detect: test the od value of each hole sample under 405nm wavelength.
In the present embodiment, using the present invention provide lead 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-antibody lead, enzyme mark and Substrate；
To be not added with the controlled trial group of anti-antibody lead 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-antibody lead and substrate；
To be not added with the controlled trial group detecting sample and anti-antibody lead 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-antibody lead, enzyme mark and substrate；
And only to add the controlled trial group of substrate for blank 2, only to add pbs buffer for blank 3.
In euzymelinked immunosorbent assay (ELISA), first with have with immune complex the complement 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 lead on immune complex out, and the lead on this partial immunity complex can by with lead specificity In conjunction with material or can react with lead form the specific antibody of anti-lead 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 metallic lead of chelating in circulating immune complex.
In euzymelinked immunosorbent assay (ELISA) test, the different diluted concentration of complement c1q albumen, the diluted concentration of anti-antibody lead 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；
Testing result under the different complement c1q of table 1, anti-antibody lead and diluted plasma multiple
As known from Table 1, when complement protein c1q dilution multiple proportions be 1:5000, dilution rate of blood plasma dilution multiple proportions be 1:20, When the dilution multiple proportions of anti-antibody lead is 1:50000, od value is maximum, is 0.862, under this best effort concentration conditions, elisa sun Property comparison and negative control elisa testing result as shown in table 2,
The testing result of table 2 positive control, negative control and blank
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.
2.elisa eluent best effort concentration and elution time determine
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-antibody lead 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, specifically comprise the following steps that
(1) it is coated: by anti-pb antibody dilution buffer by the doubling dilution of 1:50000, add in elisa plate micropore, 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-pb antibody response；
(4) eluting: remove enzyme labelled antibody, with dilution buffer, eluent is diluted, make Papain in eluent The concentration of enzyme: the concentration of antibody=1:80,1:40,1:20,1:10,1:5 in enzyme labelled antibody, makees 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) under the Detection wavelength of 405nm, every group of od value is read on microplate reader respectively, concrete outcome referring to table 3,
Testing result under the different eluent extension rate of table 3
By comparing od value, with judge on elisa hole wall combine lead anti-enzyme mark complex eluting degree, when od value When low, anti-antibody lead-enzyme mark complex eluting degree reaches maximum.
As known from Table 3, when the concentration of papain in eluent: in enzyme labelled antibody during the concentration=1:20 of antibody, 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
Lead 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: complement c1q is coated in pressed with dilution buffer on polypropylene solid phase carrier 1:5000 multiple proportions 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, press the doubling dilution of 1:20 with dilution buffer, add to In elisa plate micropore, 37 DEG C incubate 1.5 hours；
4) capture: remove test plasma, washing, the anti-lead that addition dilution buffer presses the doubling dilution of 1:50000 resists Body, 37 DEG C incubate 1.5 hours；
5) enzyme conjugates incubate: remove anti-antibody lead, washing, and 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, and 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 lead 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, lead 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, with dilution buffer press 1:5000 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 the doubling dilution of 1:20 with dilution buffer, 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 100ng/ml, at 37 DEG C Effect 1 hour；
5) detect: be 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 supplies, is exempted from the lead chelating type that embodiment 1 provides 100 plasma samples are detected, concrete operations condition is as follows by epidemic disease complex standard substance:
1) immobilized: anti-c3 antibody is coated in the multiple proportions pressing 1:5000 on polystyrene solid phase carrier with dilution buffer 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 the doubling dilution of 1:20 with dilution buffer, 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 100ng/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: sampling, detection under icp mses chelates the lead in circulating immune complex, 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
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 (10)

1. a kind of lead chelating type immune complex is it is characterised in that this lead chelating type immune complex is incorporated into load for lead ion After body protein with and the complex that formed of antibody that specifically binds of this carrier protein；
Described lead chelating type immune complex is prepared from by following steps:
A) step synthesizing lead chelating type immune complex, concrete operation step is as follows:
() prepares chelating agent solution: chelating agen is dissolved, is configured to chelating agent solution；
() formulation vehicle protein solution: carrier protein is added in borate buffer solution, prepared carrier protein solution；
() is stirred overnight: step (i) chelating agent solution is added in the carrier protein solution of step (ii), stirring is after shaking Bed effect 2-30h, obtains mixed liquor；
() bag filter pretreatment: add edta-nahco in bag filter3Solution boils, and uses ddh after abandoning waste liquid2O rinses, weight This step 1-3 time again；
V () is dialysed: the mixed liquor of step (iii) is loaded in the bag filter after processing through step (), uses ddh2O dialyses, and changes water 2-3 time, after 4 DEG C of dialysed overnight, collect liquid；
(vi) lead ion chelating: in the liquid in above-mentioned steps (v) add hcl solution adjustment ph to 7.0, be slowly added to lead from Sub- solution, shakes in Deca, after shaking table act on 2-30h, then by the liquid obtaining load through step () process after Bag filter in, with ddh2o dialysis, change water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid, obtain lead chelating type antigen；
(vii) combine: add in above-mentioned lead chelating type antigen and can obtain after reaction with the antibody of carrier protein specific binding Lead chelating type immune complex；
B) step purifying lead chelating type immune complex: using the prepared lead chelating type of immune-affinity chromatography removal step a) The carrier protein of reaction and the antibody of this carrier protein specific binding and lead ion, concrete behaviour is had neither part nor lot in immune complex Make step as follows:
I () redissolves the lead chelating type immune complex that above-mentioned a) step synthesizes 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；
(iii) loading: after pillar balance, with dilution buffer dilution step (i) resulting solution, then upper prop, lead chelating type Immune complex adsorbs on filler, and unreacted carrier protein, antibody, lead 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 () 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, and collection sample This；
(vii) chromatographic column pretreatment: using new chromatographic column, use dilution buffer flushing line, load energy in this chromatographic column With the filler of lead specific binding, after dress post, balance pillar with dilution buffer again；
(viii) loading: after the pillar of step (vii) balances, dilute the sample of above-mentioned steps (vi) with dilution buffer, so Afterwards by the sample upper prop after dilution, lead chelating type immune complex adsorbs on filler, unreacted antigen antibody complex meeting Flow out with dilution buffer；
(ix) eluting: rinse pillar with dilution buffer after step (viii), to baseline balance, then using 0.5- 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) eluent of step (x) is loaded bag filter ddh2O dialyses desalination, after changing water three times, 4 DEG C of dialysed overnight, and collect Sample, that is, obtain the lead chelating type immune complex of purification.
2. lead chelating type immune complex as claimed in claim 1 is it is characterised in that in the structure of described carrier protein at least Containing one of zinc fingerses, sulfydryl, cysteine residues, the zinc fingerses of lead ion and carrier protein or sulfydryl or half Guang Histidine residue combines.
3. lead chelating type immune complex as claimed in claim 2 is it is characterised in that described carrier protein is lipoprotein, blood One of Lactoferrin, nuclear antigen, foreign sera antigen, virus, antibacterial, protozoon, anthelmintic or immunoglobulin.
4. the method preparing lead chelating type immune complex as claimed in claim 1 is it is characterised in that comprise the following steps:
A) step synthesizing lead chelating type immune complex；
B) step purifying lead chelating type immune complex: using the prepared lead chelating type of immune-affinity chromatography removal step a) The carrier protein of reaction and the antibody of this carrier protein specific binding and lead ion is had neither part nor lot in immune complex.
5. method as claimed in claim 4 it is characterised in that
Described step a) concretely comprises the following steps:
() prepares chelating agent solution: chelating agen is dissolved, is configured to chelating agent solution；
() formulation vehicle protein solution: carrier protein is added in borate buffer solution, prepared carrier protein solution；
() is stirred overnight: step (i) chelating agent solution is added in the carrier protein solution of step (ii), stirring is after shaking Bed effect 2-30h, obtains mixed liquor；
() bag filter pretreatment: add edta-nahco in bag filter3Solution boils, and uses ddh after abandoning waste liquid2O rinses, weight This step 1-3 time again；
V () is dialysed: the mixed liquor of step (iii) is loaded in the bag filter after processing through step (), uses ddh2O dialyses, and changes water 2-3 time, after 4 DEG C of dialysed overnight, collect liquid；
(vi) lead ion chelating: in the liquid in above-mentioned steps (v) add hcl solution adjustment ph to 7.0, be slowly added to lead from Sub- solution, shakes in Deca, after shaking table act on 2-30h, then by the liquid obtaining load through step () process after Bag filter in, with ddh2o dialysis, change water 2-3 time, after 4 DEG C of dialysed overnight, collection liquid, obtain lead chelating type antigen；
(vii) combine: add in above-mentioned lead chelating type antigen and can obtain after reaction with the antibody of carrier protein specific binding Lead chelating type immune complex；
Described step b) specifically comprises the following steps that
I () redissolves the lead chelating type immune complex that above-mentioned a) step synthesizes 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；
(iii) loading: after pillar balance, with dilution buffer dilution step (i) resulting solution, then upper prop, lead chelating type Immune complex adsorbs on filler, and unreacted carrier protein, antibody, lead 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 () 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, and collection sample This；
(vii) chromatographic column pretreatment: using new chromatographic column, use dilution buffer flushing line, load energy in this chromatographic column With the filler of lead specific binding, after dress post, balance pillar with dilution buffer again；
(viii) loading: after the pillar of step (vii) balances, dilute the sample of above-mentioned steps (vi) with dilution buffer, so Afterwards by the sample upper prop after dilution, lead chelating type immune complex adsorbs on filler, unreacted antigen antibody complex meeting Flow out with dilution buffer；
(ix) eluting: rinse pillar with dilution buffer after step (viii), to baseline balance, then using 0.5- 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) eluent of step (x) is loaded bag filter ddh2O dialyses desalination, after changing water three times, 4 DEG C of dialysed overnight, and collect Sample, that is, obtain the lead chelating type immune complex of purification.
6. lead chelating type immune complex as claimed in claim 1 chelates the reagent of circulating immune complex in preparation detection lead In application.
7. lead chelating type immune complex as claimed in claim 1 chelates the enzyme connection of circulating immune complex in preparation detection lead Application in immune reagent kit.
8. a kind of detection lead chelates the enzyme linked immunological kit of circulating immune complex it is characterised in that this test kit includes marking Quasi- product, described standard substance are lead chelating type immune complex as claimed in claim 1.
9. enzyme linked immunological kit as claimed in claim 8 is it is characterised in that this test kit also includes the examination of below at least one Agent: containing capture antigen antibody complex albumen be coated liquid, containing capture metallic lead antibody be coated liquid, enzyme mark resist Body.
10. a kind of method of detection by quantitative lead chelating type circulating immune complex is it is characterised in that wanted with the right of known content Ask the lead chelating type immune complex described in 1 as standard substance, a pair of sample using following methods is detected: enzyme linked immunological Method, enzyme linked immunological and atomic absorption spectrum combined techniqueses, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniqueses, purification immunity Complex and atomic absorption spectrum combined techniqueses, purification immune complex and inductivity coupled plasma mass spectrometry combined techniqueses, electrophoresis and Enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniqueses.
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