CN104987386A - Lead-fibrinogen chelate as well as preparation method and application thereof - Google Patents

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

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CN104987386A
CN104987386A CN201510413238.1A CN201510413238A CN104987386A CN 104987386 A CN104987386 A CN 104987386A CN 201510413238 A CN201510413238 A CN 201510413238A CN 104987386 A CN104987386 A CN 104987386A
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fibrinogen
lead
inner complex
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chromatography column
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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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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
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen

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Abstract

The invention discloses a lead-fibrinogen chelate as well as a preparation method and application thereof. The lead-fibrinogen chelate is formed by chelating lead ions with fibrinogen through sulfydryl or/and cysteine residues, and can be used for preparing a reagent for detecting the lead-fibrinogen chelate of a human body. According to the invention, the lead ions are proved to directly act on fibrinogens for the first time; a qualitative and quantitative detection method of the lead-fibrinogen chelate is established for detecting the contents of the lead-fibrinogens in bodies of people in one region, so that the lead pollution degree of the region and the influence on the heath of people are indirectly reflected. According to the quantitative detection method for the lead-fibrinogen chelate, established by the invention, the accuracy is high, and the repeatability is good.

Description

A kind of lead-Fibrinogen inner complex and its preparation method and application
Technical field
The present invention relates to the immunology detection of heavy metal ion, be specifically related to a kind of lead-Fibrinogen inner complex and its preparation method and application.
Background technology
Its physiological function of Fibrinogen (fibrinogen, Fg) mainly participates in coagulation process directly as factor I.In blood coagulation common pathway, the first cracking Fibrinogen of zymoplasm two A α chain aminoterminal Arg16-Gly17 discharge a pair Fibrinogen peptide A, form fibrinogen monomer I; A pair Fibrinogen peptide B is discharged at cracking Fibrinogen two B β chain aminoterminal Arg14-Gly15, form fibrinogen monomer II, the polymerization position of exposed fibers proteinogen monomer, by Non-covalent binding, defines the unstable former monomer of soluble fibrin.At factor XIII and the Ca of activation 2+effect under, fibrinogen monomer is cross-linked mutually, generates stable soluble fibrin former, and holds wherein by the formed elements of blood, forms firmly thrombus.
Except participation blood coagulation, Fibrinogen also has other several functions, and as being combined with platelet membrane glycoprotein Ⅱb/III a, mediate platelet aggregation reacts, and participates in atherosclerosis and tumour hematogenous metastasis etc.; Fibrinogen level also affects blood viscosity, it is found that plasma fibrinogen level rising is the important risk factor of cardiovascular and cerebrovascular, thrombotic diseases especially in recent years.Plasma fibrinogen is also acute phase protein, much stress under situation, as infection, severe trauma etc. also can raise in the short period of time.
Plumbous content in vivo exceedes certain level will cause to health the infringement being difficult to recover, it can lead fume, lead dust and various oxide form are taken in body by human body through respiratory tract and digestive tube, by the Competition with calcium ion, oxidative damage, the mechanism such as apoptosis, cause based on nerve, digestion, the general of hemopoietic system obstacle, gradual, persistence non-reversibility disease, and its harm also may be genetic to the next generation.Anaemia is one of saturnine early symptom, and lead can suppress the activity of many enzymes in protoheme building-up process.Lead poisoning can cause vasospasm, angina abdominis, retinal arterioles spasm and hypertension, often causes tiny arteriosclerosis.Plumbous renal impairment often shows as the pathology such as interstitial nephritis or atrophic ephritis, and reabsorption function reduction is early stage symptom.Effects of Lead Exposure also may affect reproductive function, and the probability of the plumbous women's generation Infertility of contact, miscarriage and stillborn foetus increases, and by with it placental metastasis to fetus.Per os lead poisoning person liver is one of main damaged organ, hepatomegaly can be caused to present jaundice, even liver cirrhosis or hepatic necrosis, and hepatic injury may be that in liver, arteriolospasm causes caused by local asphyxia.Lead causes Porphyrin Metabolism obstacle, suppresses containing sulfydryl enzyme, interference vegetative nerve.
The plumbous albumen that can act in each system of whole body of existing lot of documents report at present, thus affecting the function of Body organs, the albumen reported comprises: albumen, metallothionein(MT) etc. in the albumen in the albumen in renal tissue, the albumen in cerebral tissue, red corpuscle, the albumen in liver organization, the albumen in intestinal tissue, lung tissue.
Research finds to send out plumbous, and error at measurment is large, and tooth is plumbous, bone is plumbous and organize lead to draw materials not easily, and lead in urine mainly reflects plumbous excretion situation.China's " children's Lead-poisoning and lead poisoning prevention guide " regulation in 2006, the examination of children blood lead levels can adopt peripheral blood or venous blood, but diagnosis must adopt venous blood.But the content affecting the protein binding lead of immunity of organism sphaeroprotein has no precedent detection.
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-Fibrinogen inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of lead-Fibrinogen inner complex, so that detection by quantitative lead-Fibrinogen inner complex is in the application of an evaluation Pb pollution level.By the regional crowd's Pb in Blood-Fibrinogen inner complex of detection by quantitative one, indirectly can reflect the situation of this regional crowd by Lead contamination, 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-Fibrinogen inner complex, this lead-Fibrinogen inner complex be lead ion and Fibrinogen by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned lead-Fibrinogen inner complex, i.e. external synthesis method, comprises the following steps:
The synthesis of A) lead-Fibrinogen huge legendary turtle compound: add lead ion and carry out chelatropic reaction purifying in the Fibrinogen that comes from human body or the Fibrinogen according to biological method restructuring, obtain reaction soln;
B) lead-Fibrinogen inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted Fibrinogen, specific antibody and lead ion in reaction soln, obtain plumbous-Fibrinogen inner complex.
As preferably, described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) lead-Fibrinogen inner complex be dissolved in physiological saline, obtain plumbous-Fibrinogen chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of Fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatography column;
Described can with the filler of Fibrinogen specific binding be adsorbed with can with the silica gel of Fibrinogen specific binding material or resin;
(3) loading: after chromatography column balance, with dilution buffer dilution step (1) described solution, then upper prop, make Fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use 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 with the filler of plumbous specific binding be adsorbed with can with the silica gel of plumbous specific binding material or resin;
(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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-Fibrinogen inner complex.
As preferably, the preparation method of above-mentioned lead-Fibrinogen inner complex, also comprises the following authentication step to lead-Fibrinogen inner complex, specifically comprises the following steps:
(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-Fibrinogen inner complex of obtaining of extraction purification, add sample-loading 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 the protein band containing lead, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS detection whether containing plumbous and that detection is plumbous content.
The present invention also provides the application of a kind of lead described above-Fibrinogen inner complex in preparation human body in the reagent of lead-Fibrinogen inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of lead described above-Fibrinogen inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching fibrinogenic material or catching metallic lead.
The present invention also provides the method for a kind of detection by quantitative lead-Fibrinogen inner complex, using the above-mentioned lead-Fibrinogen 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-Fibrinogen inner complex and enzyme linked immunological combined techniques, purification lead-Fibrinogen inner complex and atomic absorption spectrum combined techniques, purification lead-Fibrinogen 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.
Compared to existing technology, beneficial effect of the present invention is:
1. the present invention synthesizes lead-Fibrinogen inner complex first;
2. the present invention proposes lead-Fibrinogen inner complex first and can be used for preparing application in the reagent or test kit detecting lead-Fibrinogen inner complex in blood sample;
3. present invention achieves specific recognition and the detection by quantitative of lead-Fibrinogen inner complex, so that the content of detection by quantitative crowd Pb in Blood-Fibrinogen inner complex, to evaluate the application of a Pb pollution level, for the Lead contamination level of industrial area provides indirect indexes.The accuracy of lead-Fibrinogen inner complex quantitative detecting method that the present invention sets up is high, reproducible.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of lead of the present invention-Fibrinogen inner complex;
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of lead of the present invention-Fibrinogen 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; Agents useful for same, material, as non-specified otherwise, are all considered as to buy from commercial mode:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Catch fibrinogenic material be can with the material of Fibrinogen specific binding, by commercially available acquisition, as the human fibrinogen antibody A nti-human Fibrinogen that article No. is " NovusA-0577 ", in following embodiment, described material with Fibrinogen specific binding, antifibrin original antibody, anti-FG antibody are and catch fibrinogenic material;
Enzyme labelled antibody is the one in the antibody containing the enzyme labelling such as horseradish peroxidase, alkaline phosphatase;
Dilution buffer is the 0.05M carbonate buffer solution of pH 9.6, compound method example: the Na getting 1.5g 2cO 3with the NaHCO of 2.93g 3dissolving adds ddH 2o is settled to 1000mL;
Lavation buffer solution is the 0.15M PBS solution of pH7.4, compound method example: the KH getting 0.2g 2pO 4, 2.90g Na 2hPO 412H 2kCl, 0.5mLTween-20 of NaCl, 0.2g of O, 8.0g, dissolve and add ddH 2o is settled to 1000mL;
Confining liquid is bovine serum albumin solution, compound method example: get 0.1g bovine serum albumin, adds lavation buffer solution dilution and is settled to 100mL;
Stop buffer is 2M H 2sO 4, compound method example: the ddH getting 178.3mL 2o, enriching H 2sO 4be settled to 200mL;
Substrate is methyl diphenyl amine (TMB) solution, compound method example: get the methyl diphenyl amine ethanolic soln that 0.5mL concentration is 2g/L, add substrate dilution and be diluted to 10mL;
Substrate buffer solution pH is 5.0, wherein Na 2hPO 4volumetric molar concentration be 0.2M, the volumetric molar concentration of citric acid is 0.1M, compound method example: get 1.42gNa 2hPO 4, 0.96g citric acid, then add ddH 2o to 50mL, to obtain final product;
The compound method example of elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/mL, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min, obtain elutriant;
Sample-loading buffer can be formulated by the component of following ratio: Tris-HCl:1% tetrabromophenol sulfonphthalein: ddH 2o: glycine=15.5:2.5:7:25, wherein the pH of Tris-HCl is 6.8, volumetric molar concentration is 1M;
The compound method of electrophoretic buffer is as follows: get 3.0g Tris, 14.4g glycine, be dissolved in 800mLddH 2in O, after adjusting pH to 8.3, be settled to 1L;
The mouse-anti Pb mAb that to catch plumbous material be article No. is " cling to proud AP7019 ", resists with the material of plumbous specific binding, anti-Pb antibody, two anti-, anti-antibody lead, Pb the material being and catching plumbous described in following embodiment;
The invention provides a kind of lead-Fibrinogen inner complex, lead ion and Fibrinogen by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of lead-Fibrinogen inner complex, comprises the following steps:
The synthesis of A) lead-Fibrinogen huge legendary turtle compound: add lead ion and carry out chelatropic reaction purifying in the Fibrinogen that comes from human body or the Fibrinogen according to biological method restructuring, obtain reaction soln;
B) lead-Fibrinogen inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted Fibrinogen, specific antibody and lead ion in reaction soln, obtain plumbous-Fibrinogen inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) lead-Fibrinogen inner complex be dissolved in physiological saline, obtain plumbous-Fibrinogen chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of Fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatography column;
Described can with the filler of Fibrinogen specific binding be adsorbed with can with the silica gel of Fibrinogen specific binding material or resin;
(3) loading: after chromatography column balance, with dilution buffer dilution step (1) described solution, then upper prop, make Fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use 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 with the filler of plumbous specific binding be adsorbed with can with the silica gel of plumbous specific binding material or resin;
(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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-Fibrinogen inner complex.
C): to the qualification of lead-Fibrinogen inner complex, specifically comprise the following steps:
(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-Fibrinogen inner complex of obtaining of extraction purification, add sample-loading 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 the protein band containing lead, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS detection 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-Fibrinogen inner complex as standard substance.
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-Fibrinogen inner complex, comprising: containing can be used for catching the coating buffer of fibrinogenic material, confining liquid, lavation buffer solution, plumbous material can being caught as two anti-, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative controls etc.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprising: containing the coating buffer, confining liquid, lavation buffer solution, elutriant, positive control, negative control etc. that can be used for catching fibrinogenic material.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprising: containing the coating buffer, confining liquid, lavation buffer solution, elutriant, souring agent, hydrogen peroxide, standard substance, negative control etc. that can be used for catching fibrinogenic material.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise to extract reagent needed for Fibrinogen for extracting, redissolve liquid, comprise coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, standard substance, negative control etc. containing can be used for catching plumbous material.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise and extract reagent, redissolution liquid, positive control, negative control etc. for extracting needed for Fibrinogen.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise and extract reagent, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc. for extracting needed for Fibrinogen.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise and to extract reagent, glue bed medium needed for Fibrinogen for extracting, to redissolve on liquid, sample loading buffer, dissolving glue bed containing liquid needed for plumbous protein band, containing the coating buffer, confining liquid, lavation buffer solution, enzyme labelled antibody, substrate, stop buffer, positive control, negative control etc. that can be used for catching plumbous material.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise and to extract reagent, glue bed medium needed for Fibrinogen for extracting, redissolving on liquid, sample loading buffer, dissolving glue bed containing liquid, positive control, negative control etc. needed for plumbous protein band.
Detect a test kit for lead in blood sample-Fibrinogen inner complex, comprise and to extract reagent, glue bed medium needed for Fibrinogen for extracting, redissolving on liquid, sample loading buffer, dissolving glue bed containing liquid, nitric acid, hydrogen peroxide, positive control, negative control etc. needed for plumbous protein band.
In above-mentioned several test kit, described positive control is standard substance, the Fibrinogen inner complex being namely chelated with heavy metal lead or the BSA inner complex being chelated with heavy metal lead (as BSA-ITCBE-Pb mixture); Described negative control is dilution buffer.
Mentioned reagent box, for detecting lead-Fibrinogen inner complex, to improve accuracy and the repeatability of detection, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative lead-Fibrinogen inner complex, using the above-mentioned lead-Fibrinogen 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-Fibrinogen inner complex and enzyme linked immunological combined techniques, purification lead-Fibrinogen inner complex and atomic absorption spectrum combined techniques, purification lead-Fibrinogen 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-Fibrinogen inner complex can list, but be not limited to following several.
The following stated dilution multiple proportions is weightmeasurement ratio.
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects lead-Fibrinogen inner complex, detects in accordance with the following steps:
1) bag quilt: can catch fibrinogenic material to 1000-8000 times with dilution buffer dilution, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid, and wash with lavation buffer solution for 37 DEG C, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: from recycle system sampling, make sample to be tested; Standard substance are made with the lead of known content-Fibrinogen inner complex; By dilution buffer, sample to be tested and standard substance are all diluted to 20-80 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
4) material can catching lead is added, and incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, add with dilution buffer dilution 50000-400000 anti-antibody lead doubly, 37 DEG C of effect 1-2 hour, make the metallic lead on anti-antibody lead and Fibrinogen react;
5) enzyme conjugates incubation: remove anti-antibody lead, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and HRP enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: add stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, elisa plate is placed in OD value microplate reader reading respectively sample to be tested and standard substance, drawing standard curve, tries to achieve the content of sample to be tested.
In present method, step 8) also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
The method utilizes ELISA principle, specificity Fibrinogen in whole blood can be extracted, the Fibrinogen upper part extracted is chelated with heavy metal lead, and lead on this part Fibrinogen can catch by the specific antibody of anti-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 Fibrinogen 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 on Fibrinogen.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect lead-Fibrinogen inner complex and detect in accordance with the following steps:
1) bag quilt: fibrinogenic material can be caught, as antifibrin original antibody is coated on solid phase carrier: with dilution buffer dilution antifibrin original antibody to 1000-8000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: sample from the recycle system, make sample to be tested; Standard substance are made with the lead of known content-Fibrinogen inner complex; Be diluted to 20-80 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, washing, adds elutriant, wash-out 1-3 hour at 37 DEG C;
5) detect: sample from ELISA micropore, detect the lead of chelating on Fibrinogen in Atomic Absorption Spectroscopy AAS, read respective value;
This embodiment utilizes ELISA principle to catch Fibrinogen, and detects the lead of chelating on Fibrinogen in conjunction with atomic absorption spectrum (AAS) instrument; Owing to only containing Fibrinogen 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 on Fibrinogen.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect lead-Fibrinogen inner complex and detect in accordance with the following steps:
1) bag quilt: fibrinogenic material can be caught, as antifibrin original antibody is coated on solid phase carrier, with dilution buffer dilution antifibrin original antibody to 1000-8000 times, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
3) sample to be tested is added, and incubation: from recycle system sampling, make sample to be tested; Standard substance are made with the lead of known content-Fibrinogen inner complex; Be diluted to 20-80 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove sample to be tested, and wash with corresponding lavation buffer solution, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C;
5) acidifying: in step 4) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, thorough acidifying;
6) detect: add hydrogen peroxide, and heating catch up with acid, and from ELISA agent plate wash-out solution in get 0.5mL liquid, under icp ms, detect chelating in fibrinogenic lead, read respective value.
The method, on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, detects the lead of chelating on Fibrinogen with icp ms; Namely first adopt ELISA principle to be extracted by lead-Fibrinogen inner complex, then adopt sense couple plasma mass spectrometer to carry out detection by quantitative to the lead of chelating on Fibrinogen; Owing to only containing Fibrinogen 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 on Fibrinogen.
method four:purification lead-Fibrinogen inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect lead-Fibrinogen inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific Fibrinogen is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, Fibrinogen is extracted from whole blood, and the Fibrinogen extracted is redissolved, obtain fibrinogenic solution;
2) bag quilt: be coated on solid phase carrier by anti-antibody lead, dilutes anti-antibody lead to 50000-400000 times by dilution buffer, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, stores refrigerator;
3) close: remove dilution buffer, and wash with lavation buffer solution, after washing completes, add confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with corresponding lavation buffer solution, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add sample to be tested, and incubation: from step 1) solution sample, make sample to be tested; Standard substance are made with the lead of known content-Fibrinogen inner complex; Be diluted to 20-80 doubly by dilution buffer, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove sample to be tested, and wash with lavation buffer solution, after washing completes, adds the enzyme labelled antibody with dilution buffer dilution, and 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and washing with corresponding lavation buffer solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: add stop buffer to each micropore;
8) get wavelength 450nm, after adding stop buffer, microplate reader reads the OD value of sample to be tested and standard substance respectively, drawing standard curve, try to achieve the content of sample to be tested.
In present method, also can not use microplate reader, directly carry out qualitative detection by colour developing situation.
method five:purification lead-Fibrinogen inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect lead in blood sample-Fibrinogen inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific Fibrinogen is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, Fibrinogen is extracted from whole blood, and the Fibrinogen extracted is redissolved, obtain fibrinogen solution;
2) detect: from step 1) solution sample, detect the lead of chelating on Fibrinogen in Atomic Absorption Spectroscopy AAS, read respective value.
method six:purification lead-Fibrinogen inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect lead-Fibrinogen inner complex, detect in accordance with the following steps:
1) from whole blood, non-specific Fibrinogen is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, Fibrinogen is extracted from whole blood, and the Fibrinogen extracted is redissolved, obtain fibrinogenic solution;
2) acidifying: from step 1) solution sample, add nitric acid in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
3) detect: add hydrogen peroxide, and heating gets 0.5mL solution after catching up with acid, detects the lead of chelating on Fibrinogen under icp ms, read respective value.
Method four, method five and method six are all isolate Fibrinogen by whole blood extraction method, then adopt method for detecting specificity, measure content plumbous on lead-Fibrinogen inner complex in Fibrinogen; Namely first physical sepn means are adopted, as ultracentrifugation, HPLC, gel-filtration chromatography etc., separated from test plasma sample by Fibrinogen and redissolve in physiological saline, recycling ELISA principle, atomic absorption spectrum detect or carry out detecting the lead content on inductively coupled plasma mass spectrometry detection lead-Fibrinogen inner complex.
Method seven: electrophoretic method+ELISA/AAS/ICP-MS method detects lead-Fibrinogen inner complex, specific as follows:
1) from whole blood, non-specific Fibrinogen is extracted: adopt the methods such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, Fibrinogen is extracted from whole blood, the Fibrinogen extracted is redissolved in physiological saline, obtains fibrinogenic solution;
2) glue bed is prepared: select suitable medium (as sepharose, polyacrylamide gel etc.) as required, prepare corresponding glue bed according to corresponding requirements;
3) application of sample: from step 1) solution get 8 μ L solution, make standard substance with the lead of known content-Fibrinogen inner complex, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, with electrophoretic buffer, carry out electrophoresis, and according to demand albumen is separated according to molecular weight, the isoparametric difference of iso-electric point;
5) detect: find out on glue bed containing plumbous protein band, this band is taken out, this protein band is dissolved, and then the lead content utilizing the principle such as ELISA or ICP-MS or AAS to detect respectively.
In addition, the iso-electric point of this method detection lead-Fibrinogen inner complex, molecular weight and content etc. can also be utilized.
In method seven, Fibrinogen is extracted from whole blood, then adopt gel electrophoresis to be separated extracted Fibrinogen, then find out the respective strap being rich in lead, then detect the content of associated fiber proteinogen, namely Fibrinogen can by multiple Methods For Purification out (such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the Fibrinogen of purifying out is redissolved in solution, get a certain amount of Fibrinogen, 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 the content of associated fiber proteinogen can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the lead content of chelating on Fibrinogen, owing to only containing Fibrinogen 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 on Fibrinogen.
embodiment 1:synthesis method synthesis lead-Fibrinogen huge legendary turtle compound, comprises the following steps:
Lead prepared by the present embodiment-Fibrinogen huge legendary turtle compound, is separated further by gel electrophoresis, and carries out detection Qualitative Identification by inductivity coupled plasma mass spectrometry or atomic absorption spectrum.
The compound method example of the reagent that the present embodiment uses is as follows:
1) borate buffer solution, its volumetric molar concentration is 0.01M, and its preparation method example is as follows: take 0.31g boric acid and be dissolved in 400mLddH 2in O, regulate pH to 9.0 with the NaOH of 0.1mol/L, be settled to 500mL.
2) EDTA-NaHCO 3solution, its preparation method is as follows: get 1.86g EDTA2H 2o and 16.8g NaHCO 3, be dissolved in 900mLddH 2in O, adjust pH to 8.0 with 1.0M NaOH and be settled to 1000mL, autoclaving, room temperature preservation;
3) ITCBE buys from Japanese colleague's chemistry institute, article No. M030;
4) fibrinogen solution: take 4.0mg Fibrinogen and be dissolved in 4.0mL0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the fibrinogen solution of 1.0mg/mL;
5) molecular weight cut-off 14000 of dialysis tubing, buys from Bioshop Inc;
The pre-treatment of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500mL 3in solution, boil 10min; Tipping EDTA-NaHCO 3solution, uses ddH 2o is rinsing gently, then boils 10min with 500mL5mmol/L EDTA; Discard boiling liquid, thoroughly use ddH 2o cleans, and adds a large amount of ddH 2o soaks dialysis tubing 4 DEG C and spends the night.During use, put on one's gloves, take out dialysis tubing, with a large amount of ddH 2its surfaces externally and internally of O cleaning down;
Prepare the method for lead-Fibrinogen inner complex, comprise the following steps:
The synthesis of A) lead-Fibrinogen huge legendary turtle compound: add lead ion and carry out chelatropic reaction in the Fibrinogen in people source, obtain reaction soln;
1) getting 2.0mg ITCBE is dissolved in 2mLDMSO;
2) liquid slowly step 1 prepared adds in fibrinogen solution, and dropping limit, limit is shaken, and in 25 DEG C, acts on 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, removes the ITCBE be not combined with Fibrinogen;
3) by the liquid 1mol/L HCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l 1mmol/L lead ion solution gradually, dropping limit, limit vibrates, in order to avoid lead ion makes protein denaturation precipitate;
4) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the dialysis tubing handled well;
5) liquid of having dialysed is preserved in-20 DEG C of packing, obtain the reaction soln of lead-Fibrinogen inner complex.
B) lead-Fibrinogen inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted Fibrinogen, specific antibody and lead ion in reaction soln, obtain plumbous-Fibrinogen inner complex, specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) lead-Fibrinogen inner complex be dissolved in physiological saline, obtain plumbous-Fibrinogen chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of Fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with dilution buffer dilution step (1) described solution, then upper prop, make Fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use 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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-Fibrinogen inner complex.
C) to the qualification of lead-Fibrinogen huge legendary turtle compound, 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-Fibrinogen inner complex of obtaining of extraction purification, redissolve in physiological saline, take out 8 μ L, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis with electrophoretic buffer; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 DEG C; Stop electrophoresis when moving to bottom glue to tetrabromophenol sulfonphthalein, Fig. 1 is the non denatured electrophoretic band figure of lead of the present invention-Fibrinogen huge legendary turtle compound;
(4) detect: on glue bed, find out the protein band containing lead, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS detection whether containing plumbous and that detection is plumbous content.
D) qualification result
1) AAS detected result
Get step C) isolated protein band solution, with the content of heavy metal lead in graphite furnace atomic absorption spectrometry (AAS) rough determination Fibrinogen, as shown in the table, with (NH 4) 2sO 4solution is blank;
Content plumbous in table 1 Fibrinogen
Sample name Pb(μg/L)
Sample to be tested 430.731
(NH 4) 2SO 4 0
2) 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.LS30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA 4000) 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-Fibrinogen 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 testing conditions
1. the determination of the optimum diluting multiple of antifibrin original antibody, anti-Pb antibody best effort concentration and blood plasma.
Euzymelinked immunosorbent assay (ELISA) detects the method for lead-Fibrinogen inner complex, specifically comprises the following steps:
1) bag quilt: antifibrin original antibody albumen is coated on solid phase carrier, respectively by dilution buffer by coating protein with the doubling dilution of 1:1000,1:2000,1:4000 and 1:8000, add in elisa plate micropore, each concentration bag, by three rows, is preserved 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing;
3) sample to be tested is added: by dilution buffer by the doubling dilution of test plasma sample by 1:20,1:40,1:80, add in micropore, make standard substance with the lead of known content-Fibrinogen inner complex, negative control and blank are set, add in micropore, 37 DEG C act on 1 hour;
4) anti-Pb antibody is added: remove test plasma sample, washing, add by dilution buffer by 1:50000,1:100000,1,200000 and the anti-Pb antibody of doubling dilution of 1:400000,37 DEG C of effects 1 hour, make the metallic lead on itself and Fibrinogen react;
5) enzyme-added mark: remove anti-Pb antibody, washing, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, make itself and anti-Pb antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer;
7) detect: the OD value reading standard substance, test plasma, positive control, negative control and blank sample under 450nm wavelength in microplate reader respectively.
In the present embodiment, adopt lead provided by the invention-Fibrinogen inner complex standard substance as positive control, respectively not add test plasma as negative control 1, namely add antifibrin original antibody, confining liquid, anti-Pb antibody, enzyme labelled antibody and substrate successively;
Not add the controlled trial group of anti-Pb antibody as negative control 2, namely add antifibrin original antibody, confining liquid, test plasma, enzyme labelled antibody and substrate successively;
Not add the controlled trial group of enzyme labelled antibody as negative control 3, namely add antifibrin original antibody, confining liquid, test plasma, anti-Pb antibody and substrate successively;
Not add the controlled trial group of sample to be tested and anti-Pb antibody as negative control 4 simultaneously, namely add antifibrin original antibody, confining liquid, enzyme labelled antibody and substrate successively;
Not add the controlled trial group of antifibrin original antibody work for blank 1, namely add confining liquid, test plasma, anti-Pb antibody, enzyme labelled antibody and substrate;
And with the controlled trial group only adding substrate for blank 2, only to add the controlled trial group of PBS for blank 3.
Table 2 is the sample OD Value Data of different antifibrin original antibody dilution multiple proportions, diluted plasma multiple proportions, anti-Pb antibody dilution multiple proportions,
Detected result under table 2 different antifibrin original antibody, anti-Pb antibody and diluted plasma multiple proportions
As known from Table 2, when the dilution multiple proportions of antifibrin original antibody is 1:2000, sample OD value is greater than the dilution multiple proportions of other antifibrin original antibody under parallel condition; In this group sample, when diluted plasma multiple proportions is 1:40, when anti-Pb antibody dilution multiple proportions is 1:50000, OD value is maximum, is 0.731.
The OD detected value of corresponding positive control, negative control and blank when the dilution multiple proportions that table 3 is antifibrin original antibody is 1:2000, diluted plasma multiple proportions is 1:40, anti-Pb antibody dilution multiple proportions is 1:50000,
The detected result of table 3 positive control, negative control and blank
As known from Table 3, negative control group OD detected value is less than 0.1, and under this optimal conditions is described, the systematic error of present method is little, meets analytical procedure requirement, so select concentration corresponding to this value as best effort concentration.
2.ELISA elutriant best effort concentration and elution time are determined
For seeking optimum elution requirement, by euzymelinked immunosorbent assay (ELISA) after anti-Pb antibody and enzyme labelled antibody incubation, carry out wash-out with the elutriant of different concns, then detect OD value by microplate reader, concrete steps are as follows:
(1) bag quilt: be coated on solid phase carrier by anti-Pb antibody, press the doubling dilution of 1:50000 by dilution buffer, adds in elisa plate micropore, preserves 16 hours for 4 DEG C;
(2) close: remove dilution buffer, after washing, add confining liquid, place 1 hour, remove confining liquid, and wash for 37 DEG C;
(3) add enzyme labelled antibody: remove confining liquid, after washing, add and be diluted to by dilution buffer the HRP enzyme labelled antibody that antibody concentration is 2 μ g/mL, 37 DEG C act on 2 hours, make itself and anti-Pb antibody response;
(4) wash-out: remove enzyme labelled antibody, by dilution buffer, elutriant is diluted, make the concentration of papoid in elutriant: the concentration=1:80,1:40,1:20,1:10,1:5 of antibody in enzyme labelled antibody, acts on 1h, 2h, 3h under being positioned over 37 DEG C of temperature respectively; Remove elutriant, washing, after washing completes, adds substrate, and 37 DEG C of lucifuge effects 30 minutes, add stop buffer termination reaction;
(5) under the determined wavelength of 450nm, in microplate reader, read the OD value of each micropore respectively, concrete outcome see table 4,
Detected result under the different elutriant extension rate of table 4
By comparing OD value, to judge anti-Pb antibody-hrp-antibody complex wash-out degree that ELISA hole wall combines, when OD value is minimum, anti-Pb antibody-hrp-antibody complex wash-out degree reaches maximum.As shown in table 4, the concentration when papoid in elutriant: in enzyme labelled antibody during the concentration=1:20 of antibody; And action time is when being 1h, 2h, 3h, each group of OD value change is little, the visible prolongation along with the time, enzyme activity weakens gradually, when enzyme concn is constant, extends digestion time and can not improve digestibility, so in this experiment the action time of elutriant be that 1-3h all can, in sum, we select elutriant 1:20 as the suitableeest working concentration, and 1-3h is as the suitableeest elution time.
Application Example
application Example 1
Employing euzymelinked immunosorbent assay (ELISA) (ELISA method) detects the lead-Fibrinogen inner complex in 100 parts of sample blood plasma, and the method namely adopting specific embodiment method one to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by antifibrin original antibody, is diluted to 2000 times by dilution buffer, adds in elisa plate micropore, preserves 4 DEG C of storages in refrigerator after 1 hour for 37 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, and place 1 hour, remove confining liquid for 37 DEG C, washing, elisa plate 37 DEG C places 4 DEG C of storages in refrigerator after 1 hour;
3) application of sample: using sample blood plasma as sample to be tested, makes standard substance with the lead of known content-Fibrinogen inner complex, by dilution buffer, sample to be tested and standard substance is all diluted to 40 times, adds in micropore, and 37 DEG C act on 1 hour;
4) add anti-Pb antibody: remove sample, washing, add the anti-Pb antibody being diluted to 50000 times by dilution buffer, 37 DEG C of effects 1 hour, make the metallic lead on itself and Fibrinogen react;
5) enzyme-added mark: remove anti-Pb antibody, washing, adds the enzyme labelled antibody that antibody concentration is 2 μ g/mL, and 37 DEG C act on 1 hour, make itself and anti-Pb antibody response;
6) substrate incubation: remove enzyme labelled antibody, washing, add substrate, 37 DEG C of lucifuge effect 30min, add stop buffer to each micropore;
7) detect: the OD value reading sample to be tested and standard substance under 450nm wavelength in microplate reader respectively, result is as shown in table 5.
The measured result of table 5 method a pair 100 parts of sample blood plasma
In this application embodiment 1, step 7) in, microplate reader also can not be used to detect, but directly carry out qualitative detection by colour developing.
application Example 2
Adopt enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) to detect lead-Fibrinogen inner complex in 100 parts of sample blood plasma, the method namely adopting specific embodiment method two to record detects, and concrete operation step is as follows:
1) bag quilt: antifibrin original antibody is coated on solid phase carrier, with dilution buffer dilution coating protein to 2000 times, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, preserve at elisa plate 4 DEG C;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the lead of known content-Fibrinogen inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 40 times, add in micropore, 37 DEG C of effect 1-2 hour;
4) wash-out: remove test plasma sample, washing, adds the Na of 0.8mol/L 2hPO 4solution, 37 DEG C act on 2 hours;
5) detect: sample from ELISA micropore, detect the lead of chelating on Fibrinogen in Atomic Absorption Spectroscopy AAS, result is as shown in table 6.
Table 6 method two is to the measured result of 100 parts of sample blood plasma
application Example 3:
Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) is adopted to detect lead-Fibrinogen inner complex in 100 parts of sample blood plasma, namely the method adopting specific embodiment method three to record detects, and concrete operation step is as follows:
1) bag quilt: be coated on solid phase carrier by antifibrin original antibody, with dilution buffer dilution coating protein to 2000 times, adds in elisa plate micropore, preserves 18 hours for 4 DEG C;
2) close: remove dilution buffer, washing, adds confining liquid, places 1 hour, removes confining liquid for 37 DEG C, washing, elisa plate 4 DEG C preservation;
3) application of sample: make sample to be tested with sample blood plasma, makes standard substance with the lead of known content-Fibrinogen inner complex, by dilution buffer, sample to be tested and standard substance is diluted to 40 times, add in micropore, and 37 DEG C act on 2 hours;
4) wash-out: remove sample to be tested and standard substance, washing, adds and is diluted to by dilution buffer the elutriant that Papain enzyme concn is 100ng/mL, 37 DEG C of wash-outs 2 hours;
5) acidifying: add 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;
6) detect: sampling, under icp ms, detect the lead of chelating on Fibrinogen, result is as shown in table 7.
Table 7 method three is to the measured result of 100 parts of sample blood plasma
Above-mentioned embodiment is only the preferred embodiment of the present invention; can not limit the scope of protection of the invention with this, change and the replacement of any unsubstantiality that those skilled in the art does on basis of the present invention all belong to the present invention's scope required for protection.

Claims (8)

1. lead-Fibrinogen inner complex, is characterized in that, this lead-Fibrinogen inner complex be lead ion and Fibrinogen by sulfydryl or/and cysteine residues chelating forms.
2. the preparation method of lead-Fibrinogen inner complex as claimed in claim 1, comprises the following steps:
The synthesis of A) lead-Fibrinogen huge legendary turtle compound: add lead ion and carry out chelatropic reaction purifying in the Fibrinogen that comes from human body or the Fibrinogen according to biological method restructuring, obtain reaction soln;
B) lead-Fibrinogen inner complex purifying: adopt immune-affinity chromatography, removal step A) unreacted Fibrinogen, specific antibody and lead ion in reaction soln, obtain plumbous-Fibrinogen inner complex.
3. method as claimed in claim 2, is characterized in that,
Described step B) in specifically comprise the following steps:
(1) sample dissolution: by above-mentioned steps A) lead-Fibrinogen inner complex be dissolved in physiological saline, obtain plumbous-Fibrinogen chelate solution;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of Fibrinogen specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with dilution buffer dilution step (1) described solution, then upper prop, make Fibrinogen and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the Na of 0.05-0.10mol/L 2hPO 4solution carries out wash-out;
(5) collect: the elutriant collecting step (4), makes albumen restore immediately after collection;
(6) dialyse: by the elutriant of the collection in step (5) dress dialysis tubing, use 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 Na of 0.5-1.0mol/L 2hPO 4solution carries out wash-out;
(10) collect: the elutriant collecting step (9), makes albumen restore immediately after collection;
(11) dialyse: by the elutriant of step (10) dress dialysis tubing, use ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain plumbous-Fibrinogen inner complex.
4. the preparation method of lead according to claim 2-Fibrinogen inner complex, is characterized in that, step B) after also comprise step C): to the qualification of lead-Fibrinogen inner complex;
Wherein, step C) in specifically comprise the following steps:
(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-Fibrinogen inner complex of obtaining of extraction purification, add sample-loading 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 the protein band containing lead, this protein band is taken out, protein band is dissolved, and then adopt ICP-MS or AAS detection whether containing plumbous and that detection is plumbous content.
5. the application of lead as claimed in claim 1-Fibrinogen inner complex in the reagent or test kit preparing to detect lead-Fibrinogen inner complex in blood sample.
6. one kind at least comprises the test kit of lead as claimed in claim 1-Fibrinogen 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 the material of catching fibrinogenic material or catching metallic lead.
8. the method for detection by quantitative lead-Fibrinogen inner complex, it is characterized in that, using the lead according to claim 1-Fibrinogen 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-Fibrinogen inner complex and enzyme linked immunological combined techniques, purification lead-Fibrinogen inner complex and atomic absorption spectrum combined techniques, purification lead-Fibrinogen 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.
CN201510413238.1A 2015-07-14 2015-07-14 Lead-fibrinogen chelate as well as preparation method and application thereof Pending CN104987386A (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands

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WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands

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何春燕、喻红: "《医学生物化学实验指导》", 30 September 2010 *
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Application publication date: 20151021