CN104987388A - Lead-albumin chelate as well as preparation method and application thereof - Google Patents

Lead-albumin chelate as well as preparation method and application thereof Download PDF

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Publication number
CN104987388A
CN104987388A CN201510411519.3A CN201510411519A CN104987388A CN 104987388 A CN104987388 A CN 104987388A CN 201510411519 A CN201510411519 A CN 201510411519A CN 104987388 A CN104987388 A CN 104987388A
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China
Prior art keywords
albumin
lead
inner complex
sample
chromatography column
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Inventor
张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
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Shanghai Baihao Biotechnology Co Ltd
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Shanghai Baihao Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • 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
    • 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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
    • G01N2333/765—Serum albumin, e.g. HSA

Abstract

The invention discloses a lead-albumin chelate as well as a preparation method and application thereof. The lead-albumin chelate is formed by chelating lead ions with albumin through sulfydryl or/and cysteine residues. According to the invention, a qualitative and quantitative detection method of the lead-albumin chelate is established for favorably quantitatively detecting the application of the lead-albumin chelate to the evaluation of the lead pollution degree of one region. By quantitatively detecting the lead-albumin chelates in serums of people in one region, the condition that people in the region are polluted by lead can be indirectly reflected, and further the lead pollution degree of the region is indirectly reflected. According to the quantitative detection method for the lead-albumin chelate, established by the invention, the accuracy is greatly improved, and the detection repeatability is greatly improved.

Description

A kind of lead-albumin inner complex and its preparation method and application
Technical field
The present invention relates to detection field, more particularly, relate to a kind of lead-albumin inner complex and its preparation method and application.
Background technology
Serum albumin (HSA) is the most rich in protein of content in mammalian plasma, and it can store and transport numerous endogenous and exogenous material, and this and its specificity structure are closely related.Serum albumin is a kind of simple protein synthesized by liver, is only made up of amino acid, does not have modification group and other appurtenants.According to data information display, bovine serum albumin (BSA) molecule is made up of 583 amino-acid residues.And human serum albumin (HSA) 2 amino acid more than BSA, i.e. 585 amino-acid residue compositions.Both difference is that BSA lacks the residue of HSA aminoacid sequence 116 and 585.The most significant feature of albumin aminoacid component is the tryptophane of low levels, respectively containing 1 or 2 residue in HSA and BSA per molecule.Phenylalanine and isoleucine content are also lower.Halfcystine, leucine, L-glutamic acid and lysine content are all very high.A large amount of ionization residues provides the very high electric charge of albumin, and under the condition of PH7, per molecule albumin, with 185 ions, so just forms albumin processable.
The unique constructional feature of albumin is disulfide linkage pattern.BSA sequence comprises 35 cysteine residues, forms 17 disulphide bridgeses, and residue nearest on adjacent halfcystine pair and chain links, and forms a pair ring of 10-47 residues in length.S-S key crossover adds rigidity and the stability of albumin structure.
Albuminous tertiary structure is considered to a kind of molecule of high degree of spiral, and learn that the residue of in HPB crystal about 67% participates in defining 28 α mono-spirochetes by X-ray diffraction, 10% residue participates in forming β-bend, and 23% residue forms extended chain.
On physiological mechanism, albumin maintains 80% of human plasma oncotic pressure.In addition, albumin is also the main carriers of various macromolecular substance in blood, plays an important role in metabolite transhipment, bio-transformation.It is also the component regulating coagulation function and Inflammatory response simultaneously.Human serum albumin is mainly used in research that is clinical, metabolic and genetic aspect.
Plumbous (Pb) is that one common are noxious metals and environmental pollutant, is widely used in industrial production environment, comprise smelt forging, lead battery is produced, the production of gasoline dope is filtered medium, lives closely bound up with people.For general population, mainly by sucking the air of Lead contamination, or drinking the wine with plumbous kettle or slicker solder kettle splendid attire, even using or abuse leaded pharmacological agent chronic disease and take in lead.Increasing research proves that lead can cause acute, chronic injury to body, even causes the generation of cancer.U.S.'s poisonous substance and disease are registered to affix one's name to and are announced research and show, lead is a kind of Systematic toxin, can affect the nerves, hematopoiesis, internal secretion, immunity, reproduction, liver, the histoorgan such as kidney, lead and compound thereof are also classified as 2B class people carcinogens by IARC (IARC).Visible lead is on the impact of body and important, very important.
In close relations between lead and multiple proteins, can be combined with multiple proteins, suppress the activity of multiple protein enzyme.Along with going deep into Toxicity of Lead research, people recognize that interaction that is plumbous and albumen plays important role in its intoxicating process gradually, be considered to the key point understanding plumbous toxicity mechanism, therefore, research that is plumbous and protein interaction just seemed ever more important.And the present mechanism about lead and protein effect is only tip of the iceberg, also has the relation between a lot of protein and lead not clear, thus strengthen for the relation between plumbous and protein most important.
Summary of the invention
For the problem that Lead contamination is serious, the object of the present invention is to provide a kind of lead-albumin inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of lead-albumin inner complex, so that detection by quantitative lead-albumin inner complex is in the application of an evaluation Pb pollution level.Indirectly can reflect the situation of this regional crowd by Lead contamination by the regional crowd's Lead in Serum-albumin inner complex of detection by quantitative one, thus indirectly reflect this Pb pollution level.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of lead-albumin inner complex, lead ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned lead-albumin inner complex, comprises the following steps:
A) plumbous with albuminous chelatropic reaction: to add lead ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying lead-albumin inner complex: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary lead ion in reaction soln, obtains plumbous-albumin inner complex.
Wherein, step B described in external synthesis method) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains lead-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of plumbous specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-albumin inner complex.
Wherein, the preparation method of above-mentioned lead-albumin inner complex, also comprises step C): to the qualification of lead-albumin inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in lead-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out leaded protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or the detection of AAS method whether containing plumbous and that detection is plumbous content.
The present invention also provides the application of a kind of lead described above-albumin inner complex in preparation human body in the reagent of lead-albumin inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of lead described above-albumin inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains catches albuminous material or catches plumbous material.
The present invention also provides the method for a kind of detection by quantitative lead-albumin inner complex, using the above-mentioned lead-albumin inner complex of known content as standard substance, 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 lead-albumin inner complex and enzyme linked immunological combined techniques, purification lead-albumin inner complex and atomic absorption spectrum combined techniques, purification lead-albumin inner 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.
Implement lead of the present invention-albumin inner complex and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis lead-albumin inner complex first;
2. the present invention proposes lead-albumin inner complex first and can be used for preparing application in the reagent or test kit detecting lead-albumin inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of lead-albumin inner complex, so that detection by quantitative lead-albumin inner complex is in the application of an evaluation Pb pollution level.Indirectly can reflect the situation of this regional crowd by Lead contamination by the regional crowd's Lead in Serum-albumin inner complex of detection by quantitative one, thus indirectly reflect this Pb pollution level.Lead-its accuracy of albumin inner complex quantitative detecting method that the present invention sets up greatly improves, and the repeatability of detection is greatly enhanced.
Accompanying drawing explanation
Fig. 1 is the electrophoretic band figure of lead of the present invention-albumin inner complex;
Fig. 2 is the fluorometric analysis figure of the electrophoresis strip of lead of the present invention-albumin inner complex.
Embodiment
Below, in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention, and agents useful for same, material are as cited by following, and the reagent being this area conventional not enumerating out in the present invention maybe can be obtained by commercial mode:
Phosphate solution is 0.05-0.1mol/L Na 2hPO 4solution or 0.5-1mol/L Na 2hPO 4solution;
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Glue bed medium is the one in sepharose, polyacrylamide gel;
To catch albuminous material be the model that Abcam company produces is the Anti-mankind SerumAlbumin antibody of ab106349; Wherein, be of the present inventionly catch albuminous material with the material of albumin specific binding, anti-HSA antibody, anti-albumin antibodies;
Catch the mouse-anti PbmAb that the plumbous material model bought from Ba Ao get bio tech ltd is AP7019; Wherein, of the present invention and the material of plumbous specific binding, anti-Pb antibody, anti-antibody lead, plumbous anti-being catch plumbous material;
Enzyme labelled antibody is the one in the antibody containing the enzyme labelling such as horseradish peroxidase, alkaline phosphatase;
Substrate is methyl diphenyl amine (TMB) solution;
Washings is for containing KH 2pO 40.2mg/ml, Na 2hPO 412H 2the pH of O 2.90mg/ml, NaCl8.0mg/ml, KCl 0.2mg/ml, 0.5%Tween-20 is the 0.15M PBS solution of 7.4;
Confining liquid is 1%-5% bovine serum albumin or skim-milk;
Dilution buffer is for containing 1.5mg/mL Na 2cO 3, 2.93mg/ml NaHCO 3pH be 9.6 0.05M carbonate buffer solution;
Enzyme labelled antibody is HRP enzyme labelled antibody;
Stop buffer is: by the 2M H of 21.7ml 2sO 4be settled to the ddH of 200ml 2in O;
Elutriant be containing the PH of papoid be 8.0 0.1mol/L Tris-HCL damping fluid;
Souring agent is nitric acid;
Sample-loading buffer is for containing 1M Tris-HCl (pH 6.8) 15.5mL, 1% tetrabromophenol sulfonphthalein 2.5mL, ddH 2the Sample buffer (5X) of O7mL, glycine 25mL;
Electrophoretic buffer be containing Tris 3mg/ml, glycine 14.4mg/ml PH be the ddH of 6.8 2o solution.
The invention provides a kind of lead-albumin inner complex, lead ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of lead-albumin inner complex, comprises the following steps:
A) plumbous and albuminous chelatropic reaction: add lead ion and carry out chelatropic reaction in the albumin in people source, obtain reaction soln;
B) extraction of purifying lead-albumin inner complex: adopt immune-affinity chromatography, remove unreacted albumin and unnecessary lead ion in reaction soln, obtain plumbous-albumin inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains lead-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column; Described can be the silica gel or the resin that contain anti-albumin antibodies with the filler of albumin specific binding;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of plumbous specific binding, dress post after balance chromatography column by dilution buffer again; Described can be the silica gel or the resin that contain anti-antibody lead with the filler of plumbous specific binding;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-albumin inner complex;
C) to the qualification of lead-albumin inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in lead-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out leaded protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or the detection of AAS method whether containing plumbous and that detection is plumbous content.
The present invention also provides a kind of and at least comprises the test kit of lead described above-albumin inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains catches albuminous material or catches plumbous material.
In the present invention, the test kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a test kit for lead in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, material, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught lead.
Detect a test kit for lead in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, elutriant, positive control, negative control etc.
Detect a test kit for lead in blood sample-albumin inner complex, comprise containing the coating buffer of catching albuminous material, confining liquid, washings, elutriant, souring agent, hydrogen peroxide, positive control, negative control etc.
Detect a test kit for lead in blood sample-albumin inner complex, comprise as extracting reagent in purification whole blood needed for albumin, redissolve liquid, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of catching plumbous material.
Detecting a test kit for lead in blood sample-albumin inner complex, comprising as extracting reagent, redissolution liquid, positive control, negative control etc. in purification whole blood needed for albumin.
Detecting a test kit for lead in blood sample-albumin inner complex, comprising as extracting reagent, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc. in purification whole blood needed for albumin.
Detect a test kit for lead in blood sample-albumin inner complex, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid needed for liquid, sample-loading buffer, protein band leaded on dissolving glue bed, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of catching plumbous material.
Detecting a test kit for lead in blood sample-albumin inner complex, comprising as extracting liquid, positive control, negative control etc. needed for protein band leaded on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for albumin.
Detecting a test kit for lead in blood sample-albumin inner complex, comprising as extracting liquid, souring agent, hydrogen peroxide, positive control, negative control etc. needed for protein band leaded on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for albumin.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the albumin inner complex of heavy metal lead or is chelated with the BSA inner complex of heavy metal lead; Described negative control is the dilution buffer not containing standard substance.
Mentioned reagent box is for detecting the albumin of chelating lead, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative lead-albumin inner complex, using the above-mentioned lead-albumin inner complex of known content as standard substance, 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 lead-albumin inner complex and enzyme linked immunological combined techniques, purification lead-albumin inner complex and atomic absorption spectrum combined techniques, purification lead-albumin inner 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 detecting that the method for lead-albumin inner complex can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative lead-albumin inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects lead-albumin inner complex, detects in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching lead is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and be diluted to 50000-400000 anti-Pb Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the metallic lead in anti-Pb Ab and albumin react and form immune complex;
6) enzyme conjugates incubation: remove anti-antibody lead, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
The method utilizes ELISA principle, specificity albumin in whole blood can be extracted, the albumin upper part extracted is chelated with heavy metal lead, and the lead in this part albumin catch by anti-antibody lead, afterwards can again by horseradish peroxidase, the antibody of the enzyme labellings 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 can be read under instrument, and the albumin not containing chelated mineral lead, then can not catch by the specific antibody of anti-lead, also can not with horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase caught, and also not containing metallic lead (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 metallic lead detecting chelating in albumin.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect lead-albumin inner complex and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) detect: sample from the micropore of elisa plate, detect the lead of chelating in albumin in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value;
In conjunction with atomic absorption spectrum (AAS) principle on the basis that the method utilizes ELISA principle, Atomic Absorption Spectroscopy AAS is utilized to detect the lead of chelating in albumin, owing to only containing albumin 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 metallic lead detecting chelating in albumin.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect lead-albumin inner complex and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, 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) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) acidifying: in step 5) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) elisa plate solution get 0.5ml liquid, under icp ms, detect chelating in albuminous lead, and drawing standard curve, readout value.
The method is on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, the lead of chelating in albumin is detected with icp ms, owing to only containing albumin 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 metallic lead detecting chelating in albumin.
method four:purification lead-albumin inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect lead-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) anti-Pb Ab is coated on solid phase carrier: by dilution buffer, anti-Pb Ab is diluted to 1000-8000 doubly, 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, store refrigerator;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: from step 1) solution sample, make measuring samples; Standard substance are made with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove step 4) in measuring samples, and to wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: stop buffer is dropped to each micropore;
8) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
method five:purification lead-albumin inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect lead in blood sample-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) detect: from step 1) sample the solution that obtains, detect the lead of chelating in albumin in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value.
method six:purification lead-albumin inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect lead-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) acidifying: from step 1) sample the solution that obtains, add souring agent (as nitric acid) in the solution 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;
3) detect: from step 2) get 0.5ml liquid the solution that obtains, under icp ms, detect the lead of chelating in albumin, and drawing standard curve, readout value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) or atomic absorption spectrometry or inductivity coupled plasma mass spectrometry combined techniques (electrophoretic method-ELISA method/AAS method/ICP-MS method) detect lead-albumin inner complex, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare glue bed;
3) application of sample: get step 1) the solution 8 μ L that obtains adds 2 μ L sample-loading buffers, mixing, of short duration centrifugal; (noticing that step can not be boiled herein)
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: find out on glue bed containing plumbous protein band, this band is taken out, this protein band is dissolved among liquid, and then utilize ELISA or ICP-MS or AAS to detect the lead content be dissolved in liquid respectively.In addition, the albuminous iso-electric point of this method detection chelating lead, molecular weight and content etc. can also be utilized.
Albumin in method seven can with multiple Methods For Purification out (such as ultracentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the albumin of purifying out is redissolved in solution, get a certain amount of albumin, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different media can be adopted as required), the difference such as iso-electric point runs out of different bands, find out and be rich in plumbous respective strap, protein in gel is redissolved in solution, namely relevant albuminous content can be detected at a particular wavelength, also the principle such as ELISA or AAS or ICP-MS can be utilized to detect the lead content of chelating in albumin, owing to only containing albumin 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, the i.e. provable metallic lead detecting chelating in albumin.
embodiment 1: the preparation method of lead-albumin inner complex, comprises the following steps:
A) plumbous and albuminous chelatropic reaction: add lead ion and carry out chelatropic reaction in the albumin in people source, obtain reaction soln;
Preparation of reagents:
1) borate buffer solution (0.01M): take 0.31g boric acid and be dissolved in 400ml ultrapure water, regulates pH to 9.0 with the NaOH of 0.1M, is settled to 500mL.
2) albumin solution: take 4.0mg albumin and be dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the protein solution of 1.0mg/mL;
3) EDTA-NaHCO3 mixing solutions: take EDTA2H 2o 1.86g, NaHCO 316.8g is dissolved in 900mL ultrapure water, adjusts pH to 8.0 be settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH;
4) ITCBE buys from Japanese colleague's chemistry institute, and article No. is M030;
5) dialysis tubing is purchased from Bioshop Inc, molecular weight cut-off 14000.
The chelation step of lead-albumin inner complex:
1) process of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500ml 3in mixing solutions, boil 10min; Tipping EDTA/NaHCO 3liquid, with ultrapure water rinsing gently, then boils 10min with the 5mmol/LEDTA solution of 500ml; Discard boiling liquid, thoroughly clean with ultrapure water, add ultrapure water immersion dialysis tubing 4 DEG C and spend the night.During use, put on one's gloves, take out dialysis tubing, with its surfaces externally and internally of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg albumin is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the albumin solution prepared, dropping limit, limit is shaken, and being placed in temperature is 25 DEG C, and rotating speed is react 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, removing not with the ITCBE of albumin bound;
5) by step 4) the liquid 1mol/L HCl adjust ph to 7.0 of gained, then slowly drip the 1mmol/L lead ion solution of 80 μ l gradually, dropping limit, limit vibrates, in order to avoid lead ion makes protein denaturation precipitate;
6) by step 5) to be placed in temperature be 25 DEG C for the solution of gained, rotating speed is react 2h in the shaking table of 100r/min, carries out dialysis 24h with dialysis tubing;
7) by step 6) dialysis after liquid preserve in-20 DEG C of packing, obtain reaction soln.
B) extraction of purifying lead-albumin inner complex: adopt immune-affinity chromatography, remove reaction soln (the i.e. step 7) reaction soln that obtains) in unreacted albumin and unnecessary lead ion, obtain plumbous-albumin inner complex, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction soln that obtains lead-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of plumbous specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-albumin inner complex;
C) to the qualification of lead-albumin inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using sepharose as medium;
(2) application of sample: get step B) in lead-albumin inner complex of obtaining of 8 μ L extraction purification, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, add electrophoretic buffer and carry out electrophoresis; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 DEG C; Electrophoresis is stopped when electrophoresis to tetrabromophenol sulfonphthalein moves to bottom glue;
(4) detect: on glue bed, find out leaded protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or the detection of AAS method whether containing plumbous and that detection is plumbous content.
D) detected result
(1) heavy metal content in graphite furnace atomic absorption spectrometry (AAS) rough determination HSA: the results are shown in Table 1
Table 1 is heavy metal content in albumin (μ g/L)
Sample name Pb(μg/L)
HSA 587.408
(NH 4) 2SO 4 0
N.S 0
N.S 0
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of lead of the present invention-albumin inner complex.
Native polyacrylamide gel electrophoresis (Native-PAGE) or be called that active electrophoresis is under the condition not adding the denaturing agent such as SDS and thin base ethanol, to keeping active protein to carry out polyacrylamide gel electrophoresis, be usually used in the qualification of enzyme, Isozyme Analysis and purification.The native polyacrylamide gel electrophoresis not adding SDS can make biomacromolecule in electrophoresis process, keep its natural shape and electric charge, their separation is the molecular sieve effect of difference according to its electrophoretic mobility and gel, thus higher resolving power can be obtained, especially after electrophoretic separation, still can keep the biological activity of the biomacromolecule such as protein and enzyme, therefore the right swimming lane M is sex change Marker, only for detecting, do not mark effect, swimming lane 1 is albuminous band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of Beijing electron positron collider (BEPC).In storage ring, 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 (PGT Inc.LS 30143-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 the other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar fignal center 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.
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of lead of the present invention-albumin inner complex, and in figure, X-coordinate is protein band position, and ordinate zou is each metal energy of this band (content) value.
the determination of the testing conditions of the method for a kind of detection by quantitative lead-albumin inner complex of the present invention:
1. the determination of the optimum diluting multiple of anti-albumin antibodies, anti-antibody lead best effort concentration and blood plasma
Step is as follows:
(1) be that 1:1000,1:2000,1:4000,1:8000 dilute by anti-HSA Ab dilution buffer according to the mass volume ratio of anti-HSA Ab and dilution buffer, add in elisa plate micropore, anti-HSA Ab is coated on solid phase carrier, each concentration bag is by three rows, and 4 DEG C are spent the night 18 hours;
(2) remove dilution buffer, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) measuring samples is that 1:10,1:20,1:40 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HSA Ab concentration of above-mentioned bag quilt, the anti-HSAAb of same concentration adds different extent of dilution blood plasma respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with washings, after washing completes, add anti-Pb Ab, anti-Pb Ab is that 1:50000,1:100000,1:200000,1:400000 dilute by the mass volume ratio of anti-Cr Ab and dilution buffer, and according to each identical anti-HSA Ab, serum-dilution concentration, the anti-PbAb of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the metallic lead on anti-Pb Ab and HSA are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-Pb antibody, and wash with washings, after washing completes, add with dilution buffer dilution HRP enzyme labelled antibody, 37 DEG C act on 1 hour, HRP enzyme labelled antibody and lead-albumin inner complex are reacted;
(6) remove enzyme labelled antibody, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and detects, read each hole OD value respectively.According to each hole OD value numerical value, select anti-HSA Ab, the best effort concentration of anti-Pb Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made standard substance as positive control in test, select anti-HSA Ab+ close+anti-Pb Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HSA Ab+ closes+and blood plasma+enzyme mark+substrate (namely not adding anti-Pb Ab) is as negative control 2; Anti-HSA Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-Pb Ab) is as negative control 3; Anti-HSA Ab+ closes+and blood plasma+anti-Pb Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+blood plasma+anti-Pb Ab+ enzyme mark+substrate (namely do not add anti-HSA Ab and catch HSA) is as blank 1; Only add substrate as blank 2 and PBS as blank 3.Detected result is in Table 2-3.
Table 2: Checkerboard titration method determines anti-HSA Ab, anti-Pb Ab
The data (each data are all mean value) of the optimum diluting multiple of best effort concentration and blood plasma
Table 3:ELISA positive control and negative control ELISA detected result
By table 2-3 data presentation, when we can find out, and the extent of dilution as anti-HSA Ab is 1:2000, whole blood extent of dilution be the anti-extent of dilution of 1:20, Pb is 1:50000, OD value is maximum, so select concentration corresponding to this value as best effort concentration.
2. the determination of elutriant best effort concentration and elution time in method two, three in the method for detection by quantitative lead-albumin inner complex
Step is as follows: (1) bag quilt: anti-Pb Ab dilution buffer be diluted to 50000 times (mass volume ratios), add in elisa plate micropore, 37 DEG C of water-baths 3 hours, stores refrigerator;
(2) close: remove dilution buffer, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) remove confining liquid, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, it is 2 μ g/mL that enzyme labelled antibody is diluted to concentration, and 37 DEG C act on 2 hours, itself and anti-Pb Ab are reacted;
(4) prepare elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 100ng/ml, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min;
(5) enzyme labelled antibody is removed, by dilution buffer, elutriant is diluted, make the concentration of the papoid in elutriant and the ratio of the concentration of enzyme labelled antibody be 1:80,1:40,1:20,1:10,1:5 (wherein each concentration does 3 multiple holes), be positioned over wash-out 1h, 2h, 3h at 37 DEG C of temperature respectively;
(6) remove elutriant, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get 405nm wavelength, after adding stop buffer, be placed in by elisa plate in microplate reader and detect, read respectively and often organize OD value, by comparing with PBS damping fluid group, compare optimal concentration and the elution time of elutriant, concrete outcome is see table 4.
Table 4:ELISA elutriant best effort concentration and elution time are determined
From table 4, we can find, as the ratio=1:20 of the concentration of the papoid in elutriant with the concentration of enzyme labelled antibody, each group of OD value is all lower than other several groups, this concentration elution effect optimum (reach maximum by the anti-Pb Ab-enzyme mark mixture wash-out degree that ELISA hole wall combines, thus OD value is minimum) is described; And no matter elution 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 concn is constant, extends digestion time and can not improve digestibility, so the elution time of elutriant is that 1-3h all can in this test.
application Example 1
Get and adopt ELISA method to detect lead-albumin inner complex in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects lead-albumin inner complex, detect in accordance with the following steps:
1) anti-HSA Ab is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 37 DEG C of water-baths 1 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) material can catching lead is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and dilute 50000 times by dilution buffer, 37 DEG C act on 1 hour, the metallic lead in anti-Pb Ab and albumin are reacted;
6) enzyme conjugates incubation: remove anti-antibody lead, and wash with washings, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, itself and enzyme labelled antibody are reacted;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) getting wavelength is 405nm, after adding stop buffer, is placed in by elisa plate in microplate reader and detects, and read measuring samples OD value (also can not use microplate reader, directly carry out qualitative detection by colour developing situation) respectively, concrete detected value is as shown in table 5.
The ELISA detected result of lead-albumin inner complex in table 5:100 part blood sample
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the lead-albumin inner complex in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the recycle system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; With dilution buffer dilution, measuring samples is diluted to 40 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) detect: sample from the micropore of elisa plate, detect the lead of chelating in albumin in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 6.
The AAS detected result of lead-albumin inner complex in table 6:100 part blood sample
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the lead-albumin chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) 100 parts of standard blood samples are got as measuring samples;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the lead of known content-albumin inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) sample the solution that obtains, under icp ms, detect chelating in albuminous lead, detected value is as shown in table 7.
The ICP-MS detected result of lead-albumin inner complex in table 7:100 part blood sample
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.

Claims (8)

1. lead-albumin inner complex, is characterized in that, lead ion and albumin by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for lead as claimed in claim 1-albumin inner complex, is characterized in that, comprise the following steps:
A) plumbous with albuminous chelatropic reaction: to add lead ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying lead-albumin inner complex: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary lead ion in reaction soln, obtains plumbous-albumin inner complex.
3. preparation method according to claim 2, is characterized in that,
Described step B) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains lead-albumin inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of plumbous specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-albumin inner complex.
4. the preparation method of lead according to claim 2-albumin inner complex, is characterized in that, also comprise step C): to the qualification of lead-albumin inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in lead-albumin inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out leaded protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or the detection of AAS method whether containing plumbous and that detection is plumbous content.
5. the application of lead as claimed in claim 1-albumin inner complex in the reagent or test kit preparing to detect lead-albumin inner complex in blood sample.
6. one kind at least comprises the test kit of lead as claimed in claim 1-albumin inner complex as standard substance.
7. test kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains catches albuminous material or catch plumbous material.
8. the method for detection by quantitative lead-albumin inner complex, it is characterized in that, using the lead according to claim 1-albumin inner complex of known content as standard substance, 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 lead-albumin inner complex and enzyme linked immunological combined techniques, purification lead-albumin inner complex and atomic absorption spectrum combined techniques, purification lead-albumin inner 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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Application publication date: 20151021