CN104987403A - Nickel-hemoglobin chelate as well as preparation method and application thereof - Google Patents

Nickel-hemoglobin chelate as well as preparation method and application thereof Download PDF

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CN104987403A
CN104987403A CN201510413332.7A CN201510413332A CN104987403A CN 104987403 A CN104987403 A CN 104987403A CN 201510413332 A CN201510413332 A CN 201510413332A CN 104987403 A CN104987403 A CN 104987403A
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nickel
oxyphorase
inner complex
sample
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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    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins

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Abstract

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

Description

A kind of nickel-oxyphorase inner complex and its preparation method and application
Technical field
The present invention relates to detection field, more particularly, relate to a kind of nickel-oxyphorase inner complex and its preparation method and application.
Background technology
Oxyphorase (hemoglobin; HB; HGB) be a kind of protein being responsible for delivery oxygen in higher organisms.The albumen that blood is taken on a red color.Three kinds of Hb A hemoglobin adults and Hb-A, A is had in human erythrocyte 2and F.Hb-A is the main component of oxyphorase in adult normal's red corpuscle, Hb-A 2that submember accounts for 2%, and Hb-F accounts for below 2-3% in the red corpuscle of adult normal or children, Hb-F starts embryo the main component being produced as fetal erythrocyte October, in the red corpuscle of FTNB, Hb-F accounts for 70-80%, all the other are Hb-A, account for 50% after two months, be less than 10% after four months, after six months, large portion disappears.Oxyphorase, except transports oxygen carbonic acid gas, can also cause vasospasm and vasoconstriction.The oxyphorase of the three kinds of forms existed in vivo, Oxyhemoglobins, reduced form oxyphorase and methemoglobin, wherein Oxyhemoglobins contracting vessel patency is the strongest, and methemoglobin does not almost have vasoconstriction active.
Nickel (Nickel; Ni) closely bound up with the health of human body; first it is that human body maintains healthy necessary trace element; it is present in multiple hydrogenase; the redox reaction of catalysis hydrogen, participates in the synthesis of multiple zymoprotein and the metabolism of cytohormone and pigment, have promote the absorption of iron and erythrocytic growth, activating enzyme to form coenzyme, strengthen Regular Insulin, hypoglycemic, protect the effects such as cardiovascular; if therefore human body lacks nickel, the generation of disease can be caused.But true in life, due to environmental pollution, seldom lack nickel in human body, the content of contrary nickel is usually excessive, usually causes the generation of disease, even threat to life.
Nickel common are noxious metals as one, is widely used among life, production, comprises and produces coin, jewelry, nickelalloy stainless steel, manufacture Ni-Cd battery etc., closely bound up with the life of people.Along with the progress of science and technology, people using nickel as catalyzer for the production of among the production of carbon nano-particle, deepened again the impact of nickel for the mankind further.For general population, nickel mainly takes in nickel by eating, drinking into modes such as, contacts, and certain smoking is also an important channel.Research finds, excessive nickel can cause body many places tissue and organ damage, cause the generation of various diseases, comprise respiratory system disease (pneumonia, bronchitis, pulmonary fibrosis, asthma, pulmonary edema etc.), cardiovascular disorder, kidney disease, dermatosis etc., even also can lead oncogenic generation.
Epidemiologic data shows, the workman of Long Term Contact nickel, and it suffers from probability of tumor in respiratory system increases greatly, and it also increases greatly because of the probability that tumor in respiratory system is lethal.The test of animal model, external model also finds, nickel can the generation of induced tumor.Nineteen ninety, international cancer research institution (International Agency forResearch on Cancer, IARC) using nickel compound as first kind carcinogenic substance (Group 1), using metallic nickel as 2B class carcinogenic substance (Group 2B).Visible, the health of nickel serious threat human body.
In close relations between nickel and multiple proteins, can be combined with multiple proteins, suppress the activity of multiple protein enzyme.Along with going deep into nickel toxicity research, people recognize that the interaction of nickel and albumen plays important role in its intoxicating process gradually, be considered to the key point of toxicity mechanism understanding nickel, therefore, the research of nickel and protein interaction just seemed ever more important.And the present mechanism about nickel and protein effect is only tip of the iceberg, also has the relation between a lot of protein and nickel not clear, thus strengthen for the relation between nickel and protein most important.
Summary of the invention
For the problem that nickel contamination is serious, the object of the present invention is to provide a kind of nickel-oxyphorase inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of nickel-oxyphorase inner complex, so that detection by quantitative nickel-oxyphorase inner complex is in the application of the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by nickel-oxyphorase inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional nickel contamination degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of nickel-oxyphorase inner complex, nickel ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned nickel-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of nickel and oxyphorase: add nickel ion in the oxyphorase coming from human body or the oxyphorase of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying nickel-oxyphorase inner complex: adopt immune-affinity chromatography, removes unreacted oxyphorase and unnecessary nickel ion in reaction soln, obtains nickel-oxyphorase 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 nickel-oxyphorase 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 oxyphorase 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), oxyphorase 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 nickel 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 nickel-oxyphorase inner complex.
Wherein, the preparation method of above-mentioned nickel-oxyphorase inner complex, also comprises step C): to the qualification of nickel-oxyphorase 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 nickel-oxyphorase 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 nickeliferous protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing nickel and detection nickel.
The present invention also provides the application of a kind of nickel described above-oxyphorase inner complex in preparation human body in the reagent of nickel-oxyphorase inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of nickel described above-oxyphorase inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching oxyphorase or the material of catching nickel.
The present invention also provides the method for a kind of detection by quantitative nickel-oxyphorase inner complex, using the above-mentioned nickel-oxyphorase 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, purifying nickel-oxyphorase inner complex and enzyme linked immunological combined techniques, purifying nickel-oxyphorase inner complex and atomic absorption spectrum combined techniques, purifying nickel-oxyphorase 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 nickel of the present invention-oxyphorase inner complex and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis nickel-oxyphorase inner complex first;
2. the present invention proposes nickel-oxyphorase inner complex first and can be used for preparing application in the reagent or test kit detecting nickel-oxyphorase inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of nickel-oxyphorase inner complex, so that detection by quantitative nickel-oxyphorase inner complex is in the application of the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by nickel-oxyphorase inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional nickel contamination degree.Nickel-its accuracy of oxyphorase 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 non denatured electrophoretic band figure of nickel of the present invention-oxyphorase inner complex;
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of nickel of the present invention-oxyphorase 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;
The anti-hemoglobin antibodies of the material of catching oxyphorase to be model that Jiang Lai bio tech ltd, Shanghai produces be KAY0043; Wherein, of the present inventionly the material of catching oxyphorase is with the material of oxyphorase specific binding, anti-HB antibody, anti-hemoglobin antibodies;
The mouse-anti Ni mAb of the material of catching nickel to be model that Ran Ke bio tech ltd, Guangzhou produces be RK14419; Wherein, of the present inventionly resist with the material of nickel specific binding, anti-Ni antibody, anti-nickel antibody, nickel the material being and catching nickel;
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;
The formula of erythrocyte cracked liquid is as follows: 139.6mmol/L NH 4cl, 16.96mmol/L Tris, adjusts pH to 7.2 with 1mol/L HCl;
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 nickel-oxyphorase inner complex, nickel ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of nickel-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of nickel and oxyphorase: add nickel ion and carry out chelatropic reaction in the oxyphorase in people source, obtain reaction soln;
B) extraction of purifying nickel-oxyphorase inner complex: adopt immune-affinity chromatography, remove unreacted oxyphorase and unnecessary nickel ion in reaction soln, obtain nickel-oxyphorase inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains nickel-oxyphorase 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 oxyphorase 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-hemoglobin antibodies with the filler of oxyphorase specific binding;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), oxyphorase 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 nickel specific binding, dress post after balance chromatography column by dilution buffer again; Described can be the silica gel or the resin that contain anti-nickel antibody with the filler of nickel 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 nickel-oxyphorase inner complex;
C) to the qualification of nickel-oxyphorase 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 nickel-oxyphorase 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 nickeliferous protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing nickel and detection nickel.
The present invention also provides a kind of and at least comprises the test kit of nickel described above-oxyphorase inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching oxyphorase or the material of catching nickel.
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 nickel in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, material, enzyme labelled antibody, erythrocyte cracked liquid, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught nickel.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, elutriant, erythrocyte cracked liquid, positive control, negative control etc.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, elutriant, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise to extract reagent needed for full blood hemoglobin as purifying, redissolve liquid, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, erythrocyte cracked liquid, positive control, negative control etc. of material of catching nickel.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise and extract reagent, redissolution liquid, erythrocyte cracked liquid, positive control, negative control etc. needed for the full blood hemoglobin of purification.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise and extract reagent, redissolution liquid, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc. needed for the full blood hemoglobin of purification.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise as the extraction reagent of full blood hemoglobin of purifying, glue bed medium, redissolve liquid needed for protein band nickeliferous on liquid, sample-loading buffer, dissolving glue bed, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, erythrocyte cracked liquid, positive control, negative control etc. of material of catching nickel.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise and extract liquid, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed as purifying needed for full blood hemoglobin.
Detect a test kit for nickel in blood sample-oxyphorase inner complex, comprise and extract liquid, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed as purifying needed for full blood hemoglobin.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the oxyphorase inner complex of heavy metal nickel or is chelated with the BSA inner complex of heavy metal nickel; Described negative control is the dilution buffer not containing standard substance.
Mentioned reagent box is for detecting the oxyphorase of chelating nickel, 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 nickel-oxyphorase inner complex, using the above-mentioned nickel-oxyphorase 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, purifying nickel-oxyphorase inner complex and enzyme linked immunological combined techniques, purifying nickel-oxyphorase inner complex and atomic absorption spectrum combined techniques, purifying nickel-oxyphorase 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 nickel-oxyphorase inner complex can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative nickel-oxyphorase inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-oxyphorase inner complex, detects in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 500-4000 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, add deionized water by whole blood, then add erythrocyte cracked liquid, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20-80 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching nickel is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and be diluted to 50000-400000 anti-Ni Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the metallic nickel on anti-Ni Ab and oxyphorase react and form immune complex;
6) enzyme conjugates incubation: remove anti-nickel antibody, 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 oxyphorase in whole blood can be extracted, the oxyphorase upper part extracted is chelated with heavy metal nickel, and the nickel on this part oxyphorase catch by anti-nickel antibody, 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 oxyphorase not containing chelated mineral nickel, then can not catch by the specific antibody of anti-nickel, also can not with horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase caught, and also not containing metallic nickel (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 nickel detecting chelating on oxyphorase.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect nickel-oxyphorase inner complex and detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 500-4000 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, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rpm, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20-80 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 nickel of chelating on oxyphorase 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 nickel of chelating on oxyphorase, owing to only containing oxyphorase 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 nickel detecting chelating on oxyphorase.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect nickel-oxyphorase inner complex and detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 500-4000 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, add deionized water by whole blood, then add erythrocyte cracked liquid, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20-80 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 the nickel of oxyphorase, 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 nickel of chelating on oxyphorase is detected with icp ms, owing to only containing oxyphorase 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 nickel detecting chelating on oxyphorase.
method four:purifying nickel-oxyphorase inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect nickel-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) anti-Ni Ab is coated on solid phase carrier: by dilution buffer, anti-Ni Ab is diluted to 50000-400000 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20-80 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:purifying nickel-oxyphorase inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect nickel in blood sample-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) detect: from step 1) sample the solution that obtains, detect the nickel of chelating on oxyphorase in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value.
method six:purifying nickel-oxyphorase inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect nickel-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
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 nickel of chelating on oxyphorase, 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 nickel-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
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) obtain-solution 8 μ L 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: on glue bed, find out the protein band containing nickel, this band is taken out, this protein band is dissolved among liquid, and then utilize ELISA or ICP-MS or AAS to detect the nickel content be dissolved in liquid respectively.In addition, the iso-electric point of the oxyphorase of this method detection chelating nickel, molecular weight and content etc. can also be utilized.
After oxyphorase in method seven discharges from red corpuscle can with multiple Methods For Purification out (such as ultracentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the oxyphorase of purifying out is redissolved in solution, get a certain amount of oxyphorase, 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 the respective strap being rich in nickel, protein in gel is redissolved in solution, namely the content of relevant oxyphorase can be detected at a particular wavelength, also the principle such as ELISA or AAS or ICP-MS can be utilized to detect the nickel content of chelating on oxyphorase, owing to only containing oxyphorase 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 nickel detecting chelating on oxyphorase.
Below in conjunction with embodiment, the present invention is further described.
embodiment 1: the preparation method of nickel-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of nickel and oxyphorase: add nickel ion and carry out chelatropic reaction in the oxyphorase 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) hemoglobin solutions: take 4.0mg oxyphorase 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 nickel-oxyphorase 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 oxyphorase is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the hemoglobin solutions 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, remove the ITCBE be not combined with oxyphorase;
5) by step 4) the liquid 1mol/L HCl adjust ph to 7.0 of gained, then slowly drip the 1mmol/L nickel ion solution of 80 μ l gradually, dropping limit, limit vibrates, in order to avoid nickel 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 nickel-oxyphorase inner complex: adopt immune-affinity chromatography, remove reaction soln (the i.e. step 7) reaction soln that obtains) in unreacted oxyphorase and unnecessary nickel ion, obtain nickel-oxyphorase inner complex, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction soln that obtains nickel-oxyphorase 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 oxyphorase 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), oxyphorase 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 nickel 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 nickel-oxyphorase inner complex;
C) to the qualification of nickel-oxyphorase 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 nickel-oxyphorase 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 nickeliferous protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing nickel and detection nickel.
D) detected result
(1) heavy metal content in graphite furnace atomic absorption spectrometry (AAS) rough determination HB: the results are shown in Table 1
Table 1 is heavy metal content in oxyphorase (μ g/L)
Sample name Ni(μg/L)
HB 247.22
(NH 4) 2SO 4 0.56
N.S 0.002
N.S 0
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of nickel of the present invention-oxyphorase 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 left lane M is sex change Marker, only for detecting, do not mark effect, swimming lane Hb is hemoglobin electrophoresis 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 nickel of the present invention-oxyphorase 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 nickel-oxyphorase inner complex of the present invention:
1. the determination of the optimum diluting multiple of anti-hemoglobin antibodies, anti-nickel antibody best effort concentration and whole blood
Step is as follows:
(1) be that 1:500,1:1000,1:2000,1:4000 dilute by anti-HB Ab dilution buffer according to the mass volume ratio of anti-HB Ab and dilution buffer, add in elisa plate micropore, anti-HB 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 1:20,1:40,1:80 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HB Ab concentration of above-mentioned bag quilt, the anti-HB Ab of same concentration adds different extent of dilution whole blood respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with washings, after washing completes, add anti-Ni Ab, anti-Ni Ab is that 1:50000,1:100000,1:200000,1:400000 dilute by the mass volume ratio of anti-Ni Ab and dilution buffer, and according to each identical anti-HB Ab, whole blood weaker concn, the anti-Ni Ab of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the metallic nickel on anti-Ni Ab and HB are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-Ni 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 nickel-oxyphorase 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-HB Ab, the best effort concentration of anti-Ni Ab and the optimum diluting multiple of whole blood.
Simultaneously using made standard substance as positive control in test, select anti-HB Ab+ close+anti-Ni Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HB Ab+ closes+and whole blood+enzyme mark+substrate (namely not adding anti-Ni Ab) is as negative control 2; Anti-HB Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-Ni Ab) is as negative control 3; Anti-HB Ab+ closes+and whole blood+anti-Ni Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+whole blood+anti-Ni Ab+ enzyme mark+substrate (namely do not add anti-HB Ab and catch HB) 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-HB Ab, anti-Ni Ab
The data (each data are all mean value) of the optimum diluting multiple of best effort concentration and whole blood
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-HB Ab is 1:1000, whole blood extent of dilution be the anti-extent of dilution of 1:80, Ni 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 nickel-oxyphorase inner complex
Step is as follows: (1) bag quilt: anti-Ni antibody 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-Ni 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-Ni 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 nickel-oxyphorase inner complex in 100 parts of sample whole bloods, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-oxyphorase inner complex, detect in accordance with the following steps:
1) anti-HB Ab is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 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, add deionized water by whole blood, then add erythrocyte cracked liquid, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 80 times, adds in micropore, 37 DEG C act on 1 hour;
5) material can catching nickel 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 nickel on anti-Ni Ab and oxyphorase are reacted;
6) enzyme conjugates incubation: remove anti-nickel antibody, 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 nickel-oxyphorase 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 nickel-oxyphorase inner complex in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 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, add deionized water by whole blood, then add erythrocyte cracked liquid, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 80 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 nickel of chelating on oxyphorase in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 6.
The AAS detected result of nickel-oxyphorase 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 nickel-oxyphorase chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get 100 parts of standard blood samples as measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, 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 nickel of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 80 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 the nickel of oxyphorase, detected value is as shown in table 7.
The ICP-MS detected result of nickel-oxyphorase 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. nickel-oxyphorase inner complex, is characterized in that, nickel ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for nickel as claimed in claim 1-oxyphorase inner complex, is characterized in that, comprise the following steps:
A) chelatropic reaction of nickel and oxyphorase: add nickel ion in the oxyphorase coming from human body or the oxyphorase of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying nickel-oxyphorase inner complex: adopt immune-affinity chromatography, removes unreacted oxyphorase and unnecessary nickel ion in reaction soln, obtains nickel-oxyphorase 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 nickel-oxyphorase 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 oxyphorase 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), oxyphorase 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 nickel 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 nickel-oxyphorase inner complex.
4. the preparation method of nickel according to claim 2-oxyphorase inner complex, is characterized in that, also comprise step C): to the qualification of nickel-oxyphorase 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 nickel-oxyphorase 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 nickeliferous protein band, this protein band is taken out and dissolves, and then adopt ELISA method or ICP-MS method or AAS method to detect the content whether containing nickel and detection nickel.
5. the application of nickel as claimed in claim 1-oxyphorase inner complex in the reagent or test kit preparing to detect nickel-oxyphorase inner complex in blood sample.
6. one kind at least comprises the test kit of nickel as claimed in claim 1-oxyphorase 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 oxyphorase or the material of catching nickel.
8. the method for detection by quantitative nickel-oxyphorase inner complex, it is characterized in that, using the nickel according to claim 1-oxyphorase 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, purifying nickel-oxyphorase inner complex and enzyme linked immunological combined techniques, purifying nickel-oxyphorase inner complex and atomic absorption spectrum combined techniques, purifying nickel-oxyphorase 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.
CN201510413332.7A 2015-07-14 2015-07-14 Nickel-hemoglobin chelate as well as preparation method and application thereof Pending CN104987403A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123343A2 (en) * 2005-05-18 2006-11-23 Ramot At Tel Aviv University Ltd. Biologically active metal-coated proteins

Non-Patent Citations (2)

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Title
刘冬等: "《生物分离技术》", 31 December 2007 *
吴士良等: "《生物化学与分子生物学实验教程》", 28 February 2009 *

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