CN105021549A - Nickel-alhumin chelate and preparation method and application thereof - Google Patents
Nickel-alhumin chelate and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nickel-alhumin chelate and its preparation method and application. The nickel-alhumin chelate is formed by chelating nickel ion and alhumin through a mercapto group or/and cysteine residues. According to the invention, a qualitative and quantitative detection method of the nickel-alhumin chelate is established so as to quantitatively detect an application of the nickel-alhumin chelate in evaluating the nickel pollution level of a region. By quantitative detection of the nickel-alhumin chelate in human serum of a region, nickel pollution condition of people in the region can be reflected indirectly so as to indirectly reflect the nickel pollution level of the region. Accuracy of the nickel-alhumin chelate quantitative detection method established in the invention is greatly raised, and detection repeatability is greatly enhanced.
Description
Technical field
The present invention relates to detection field, more particularly, relate to a kind of nickel-albumin chelate and its preparation method and application.
Background technology
Seralbumin (HSA) is the most rich in protein of content in mammalian plasma, and it can store and transport numerous endogenouss and exogenous material, and this and its specificity structure are closely related.Seralbumin is a kind of simple protein synthesized by liver, is only made up of amino acid, does not have modification group and other adjuncts.According to data information display, bovine serum albumin(BSA) (BSA) molecule is made up of 583 amino acid residues.And human serum albumins (HSA) 2 amino acid more than BSA, i.e. 585 amino acid residue compositions.Both difference is that BSA lacks the residue of HSA amino acid sequence 116 and 585.The most significant feature of albumin aminoacid ingredient is the tryptophane of low content, respectively containing 1 or 2 residue in HSA and BSA per molecule.Phenylalanine and isoleucine content are also lower.Halfcystine, leucine, glutamic acid and lysine content are all very high.A large amount of ionization residues provides the very high electric charge of albumin, and under the condition of PH7, per molecule albumin, with 185 ions, so just forms albumin ease of solubility.
The unique design feature of albumin is disulfide bond pattern.BSA sequence comprises 35 cysteine residues, forms 17 disulphide bridgeses, and residue nearest on adjacent halfcystine pair and chain links, and forms a pair ring of 10-47 residues in length.S-S key crossover adds rigidity and the stability of albumin structure.
Albuminous tertiary structure is considered to a kind of molecule of high degree of spiral, and learn that the residue of in HNI crystal about 67% participates in defining 28 α mono-conveyor screws by X-ray diffraction, 10% residue participates in forming β-bend, and 23% residue forms extended chain.
On physiological mechanism, albumin maintains 80% of human plasma colloid osmotic pressure.In addition, albumin is also the main carriers of various macromolecular substances in blood, plays an important role in metabolin transhipment, biological conversion.It is also the component regulating coagulation function and inflammatory reaction simultaneously.Human serum albumins is mainly used in research that is clinical, metabolic and genetic aspect.
Nickel common are noxious metals as one, is widely used among life, production, comprises and produces coin, jewelry, nickel alloy 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 disease (pneumonia, bronchitis, pulmonary fibrosis, asthma, pulmonary edema etc.), angiocardiopathy, kidney trouble, disease of skin 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 carcinogen (Group 1), using metallic nickel as 2B class carcinogen (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-albumin chelate and preparation method thereof, and set up the qualitative and quantitative analysis method of nickel-albumin chelate, quantitatively to detect the application of nickel-albumin chelate in the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by quantitatively detecting nickel-albumin chelate in regional crowd's serum, 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-albumin chelate, nickel ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned nickel-albumin chelate, comprises the following steps:
A) nickel and albuminous chelatropic reaction: add nickel ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction solution;
B) extraction of purifying nickel-albumin chelate: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary nickel ion in reaction solution, obtains nickel-albumin chelate.
Wherein, step B described in external synthetic method) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction solution that obtains nickel-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of nickel specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain nickel-albumin chelate.
Wherein, the preparation method of above-mentioned nickel-albumin chelate, also comprises step C): to the qualification of nickel-albumin chelate;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in nickel-albumin chelate 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-albumin chelate in preparation human body in the reagent of nickel-albumin chelate or kit.
The present invention also provides a kind of and at least comprises the kit of nickel described above-albumin chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching albuminous material or catching nickel.
The present invention also provides a kind of method of quantitative detection nickel-albumin chelate, using the above-mentioned nickel-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin chelate and enzyme linked immunological combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin chelate 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-albumin chelate and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis nickel-albumin chelate first;
2. the present invention proposes nickel-albumin chelate first and can be used for preparing application in the reagent or kit detecting nickel-albumin chelate in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of nickel-albumin chelate, quantitatively to detect the application of nickel-albumin chelate in the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by quantitatively detecting nickel-albumin chelate in regional crowd's serum, thus indirectly reflect this regional nickel contamination degree.Nickel-its accuracy of albumin chelate 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-albumin chelate;
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of nickel of the present invention-albumin chelate.
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 Ago-Gel, polyacrylamide gel;
To catch albuminous material be the model that Abcam company produces is the Anti-mankind SerumAlbumin antibody of ab106349; Wherein, be of the present inventionly catch albuminous material with the material of albumin specific binding, anti-HSA antibody, anti-albumin antibodies;
The material purchase of catching nickel is the mouse-anti Ni mAb of RK14419 from the model of Ran Ke bio tech ltd, Guangzhou; 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 labeling such as horseradish peroxidase, alkaline phosphatase;
Substrate is methyl biphenyl amine (TMB) solution;
Cleansing solution 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(BSA) or skimmed milk power;
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;
Eluent be containing the PH of papain be 8.0 0.1mol/L Tris-HCL damping fluid;
Acidulant is nitric acid;
Sample-loading buffer is for containing 1M Tris-HCl (pH 6.8) 15.5mL, 1% bromophenol blue 2.5mL, ddH
2the Sample buffer (5X) of O7mL, glycocoll 25mL;
Electrophoretic buffer be containing Tris 3mg/ml, glycocoll 14.4mg/ml PH be the ddH of 6.8
2o solution.
The invention provides a kind of nickel-albumin chelate, nickel ion and albumin by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of nickel-albumin chelate, comprises the following steps:
A) nickel and albuminous chelatropic reaction: add nickel ion and carry out chelatropic reaction in the albumin in people source, obtain reaction solution;
B) extraction of purifying nickel-albumin chelate: adopt immune-affinity chromatography, remove unreacted albumin and unnecessary nickel ion in reaction solution, obtain nickel-albumin chelate, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction solution that obtains nickel-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column; Described can be the silica gel or the resin that contain anti-albumin antibodies with the filler of albumin specific binding;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of nickel specific binding, dress post after balance chromatographic 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 chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, with 2 liters of ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain nickel-albumin chelate;
C) to the qualification of nickel-albumin chelate, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in nickel-albumin chelate 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 kit of nickel described above-albumin chelate as standard items.
Preferably, also comprise coating buffer in this kit, this coating buffer contains the material of catching albuminous material or catching nickel.
In the present invention, the kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a kit for nickel in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, material, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught nickel.
Detect a kit for nickel in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, eluent, positive control, negative control etc.
Detect a kit for nickel in blood sample-albumin chelate, comprise containing the coating buffer of catching albuminous material, confining liquid, cleansing solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc.
Detect a kit for nickel in blood sample-albumin chelate, comprise as extracting reagent in purification whole blood needed for albumin, redissolve liquid, containing the coating buffer, confining liquid, cleansing solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching nickel.
Detecting a kit for nickel in blood sample-albumin chelate, comprising as extracting reagent, redissolution liquid, positive control, negative control etc. in purification whole blood needed for albumin.
Detecting a kit for nickel in blood sample-albumin chelate, comprising as extracting reagent, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc. in purification whole blood needed for albumin.
Detect a kit for nickel in blood sample-albumin chelate, comprise as extracting reagent, glue bed medium in purification whole blood needed for albumin, redissolve liquid needed for liquid, sample-loading buffer, protein band nickeliferous on dissolving glue bed, containing the coating buffer, confining liquid, cleansing solution, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material of catching nickel.
Detecting a kit for nickel in blood sample-albumin chelate, comprising as extracting liquid, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for albumin.
Detecting a kit for nickel in blood sample-albumin chelate, comprising as extracting liquid, acidulant, hydrogen peroxide, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for albumin.
In above-mentioned several kit, described positive control is standard items, is namely chelated with the albumin chelate of heavy metal nickel or is chelated with the BSA chelate of heavy metal nickel; Described negative control is the dilution buffer not containing standard items.
Mentioned reagent box is for detecting the albumin of chelating nickel, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides a kind of method of quantitative detection nickel-albumin chelate, using the above-mentioned nickel-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin chelate and enzyme linked immunological combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin chelate 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-albumin chelate can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned quantitative detection nickel-albumin chelate is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-albumin chelate, detects in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discards precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate 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 items with the nickel of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching nickel is added, and incubation: remove measuring samples, and wash with cleansing solution, after washing completes, add and be diluted to 5000-40000 anti-Ni Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the metallic nickel on anti-Ni Ab and albumin react and form antigen antibody complex;
6) enzyme conjugates incubation: remove anti-nickel antibody, and wash with cleansing solution, 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 cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) cessation 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 items respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
The method utilizes ELISA principle, specificity albumin in whole blood can be extracted, the albumin upper part extracted is chelated with heavy metal nickel, and the nickel on this part albumin catch by anti-nickel antibody, afterwards can again by horseradish peroxidase, the antibody of the enzyme labelings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the albumin 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 labelings 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 albumin.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect nickel-albumin chelate and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discards precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate 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 items with the nickel of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C.
6) detect: sample from the micropore of elisa plate, detect the nickel of chelating on albumin in Atomic Absorption Spectrometer, 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 Spectrometer is utilized to detect the nickel of chelating on albumin, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable metallic nickel detecting chelating on albumin.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect nickel-albumin chelate and detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 1000-8000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discards precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate 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 items with the nickel of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add eluent, wash-out 1-3 hour at 37 DEG C.
6) acidifying: in step 5) in solution in add acidulant acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) elisa plate solution get 0.5ml liquid, under icp ms, detect chelating in albuminous nickel, 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 albumin is detected with icp ms, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable metallic nickel detecting chelating on albumin.
method four:purifying nickel-albumin chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detect nickel-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) anti-Ni Ab is coated on solid phase carrier: by dilution buffer, anti-Ni Ab is diluted to 5000-40000 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 cleansing solution, after washing completes, add using 1%-5% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: from step 1) solution in sample, make measuring samples; Standard items are made with the nickel of known content-albumin chelate; Be diluted to 10-40 doubly with dilution buffer dilution measuring samples, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove step 4) in measuring samples, and to wash with cleansing solution, 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 cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) cessation 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 items 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-albumin chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detect nickel in blood sample-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) detect: from step 1) sample the solution that obtains, detect the nickel of chelating on albumin in Atomic Absorption Spectrometer, and drawing standard curve, readout value.
method six:purifying nickel-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect nickel-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) acidifying: from step 1) sample the solution that obtains, add acidulant (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 albumin, and drawing standard curve, readout value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ELISA method/AAS method/ICP-MS method) detect nickel-albumin chelate, detect in accordance with the following steps:
1) from whole blood, albumin is extracted: adopt the polyglycol PEG precipitation method or ultracentrifugation or the method such as molecule ultrafiltration or gel filtration from the albumin whole blood extraction purification blood, and redissolve in solution by albumin, obtain albumin solution;
2) glue bed is prepared: select Ago-Gel or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare glue bed;
3) application of sample: get step 1) the solution 8 μ L that obtains adds 2 μ L sample-loading buffers, mixing, of short duration centrifugal; (noticing that step can not be boiled herein)
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: 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 albuminous isoelectric point of the method detection chelating nickel, molecular weight and content etc. can also be utilized.
Albumin in method seven can with multiple Methods For Purification out (such as supercentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the albumin of purifying out is redissolved in solution, get a certain amount of albumin, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different medium can be adopted as required), the difference such as isoelectric point runs out of different bands, find out the respective strap being rich in nickel, protein in gel is redissolved in solution, namely relevant albuminous content can be detected at a particular wavelength, also the principle such as ELISA or AAS or ICP-MS can be utilized to detect the nickel content of chelating on albumin, owing to only containing albumin in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, the i.e. provable metallic nickel detecting chelating on albumin.
embodiment 1: the preparation method of nickel-albumin chelate, comprises the following steps:
A) nickel and albuminous chelatropic reaction: add nickel ion and carry out chelatropic reaction in the albumin in people source, obtain reaction solution;
Preparation of reagents:
1) borate buffer solution (0.01M): take 0.31g boric acid and be dissolved in 400ml ultrapure water, regulates pH to 9.0 with the NaOH of 0.1M, is settled to 500mL.
2) albumin solution: take 4.0mg albumin and be dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the protein solution of 1.0mg/mL;
3) EDTA-NaHCO3 mixed solution: 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) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of nickel-albumin chelate:
1) process of bag filter: EDTA-NaHCO bag filter being put into 500ml
3in mixed solution, 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 bag filter 4 DEG C and spend the night.During use, put on one's gloves, take out bag filter, with its surfaces externally and internally of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg albumin is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the albumin solution prepared, dropping limit, limit is shaken, and being placed in temperature is 25 DEG C, and rotating speed is react 24h in the shaking table of 100r/min, then to dialyse 24h with bag filter, remove the ITCBE be not combined with albumin;
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 albuminous degeneration 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 bag filter;
7) by step 6) dialysis after liquid preserve in-20 DEG C of packing, obtain reaction solution.
B) extraction of purifying nickel-albumin chelate: adopt immune-affinity chromatography, remove reaction solution (the i.e. step 7) reaction solution that obtains) in unreacted albumin and unnecessary nickel ion, obtain nickel-albumin chelate, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction solution that obtains nickel-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of nickel specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, with 2 liters of ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain nickel-albumin chelate;
C) to the qualification of nickel-albumin chelate, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using Ago-Gel as medium;
(2) application of sample: get step B) in nickel-albumin chelate 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 environment temperature is 4 DEG C; Electrophoresis is stopped when electrophoresis to bromophenol blue 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) testing result
(1) content of beary metal in GFAAS (graphite furnace atomic absorption spectrometry) (AAS) Preliminary Determination HSA: the results are shown in Table 1
Table 1 is content of beary metal in albumin (μ g/L)
| Sample name | Ni(μg/L) |
| HSA | 162.811 |
| (NH 4) 2SO 4 | 0.249 |
| 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-albumin chelate.
Native polyacrylamide gel electrophoresis (Native-PAGE) or be called that active electrophoresis is under the condition not adding the denaturant 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 resolution can be obtained, especially after electrophoretic separation, still can keep the biologically active of the biomacromolecule such as protein and enzyme, therefore the right swimming lane M is sex change Marker, only for detecting, do not mark effect, swimming lane 1 is albuminous band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of BEPC (BEPC).In storage rings, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X ray gone out from electromagnetic radiation is detected by Si (Li) detector (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 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 signal peak of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of nickel of the present invention-albumin chelate, and in figure, horizontal ordinate is protein band position, and ordinate is each metal energy of this band (content) value.
the determination of the testing conditions of the method for a kind of quantitative detection nickel-albumin chelate of the present invention:
1. the determination of the optimum diluting multiple of anti-albumin antibodies, anti-nickel antibody best effort concentration and blood plasma
Step is as follows:
(1) be that 1:1000,1:2000,1:4000,1:8000 dilute by anti-HSA Ab dilution buffer according to the mass volume ratio of anti-HSA Ab and dilution buffer, add in elisa plate micropore, anti-HSA Ab is coated on solid phase carrier, each concentration bag is by three rows, and 4 DEG C are spent the night 18 hours;
(2) remove dilution buffer, and wash with cleansing solution, after washing completes, with 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C;
(3) measuring samples is that 1:10,1:20,1:40 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HSA Ab concentration of above-mentioned bag quilt, the anti-HSAAb of same concentration adds different dilutability blood plasma respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with cleansing solution, after washing completes, add anti-Ni Ab, anti-Ni Ab is that 1:5000,1:10000,1:20000,1:40000 dilute by the mass volume ratio of anti-Ni Ab and dilution buffer, and according to each identical anti-HSA Ab, serum-dilution concentration, the anti-Ni Ab of variable concentrations respectively adds 2 holes, 37 DEG C act on 1 hour, the metallic nickel on anti-Ni Ab and HSA are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-Ni antibody, and wash with cleansing solution, 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-albumin chelate are reacted;
(6) remove enzyme labelled antibody, and wash with cleansing solution, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and detects, read each hole OD value respectively.According to each hole OD value numerical value, select anti-HSA Ab, the best effort concentration of anti-Ni Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made standard items as positive control in test, select anti-HSA Ab+ close+anti-Ni Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HSA Ab+ closes+and blood plasma+enzyme mark+substrate (namely not adding anti-Ni Ab) is as negative control 2; Anti-HSA Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-Ni Ab) is as negative control 3; Anti-HSA Ab+ closes+and blood plasma+anti-Ni Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+blood plasma+anti-Ni Ab+ enzyme mark+substrate (namely do not add anti-HSA Ab and catch HSA) is as blank 1; Only add substrate as blank 2 and PBS as blank 3.Testing result is in Table 2-3.
Table 2: Checkerboard titration method determines the data (each data are all mean value) of anti-HSA Ab, the best effort concentration of anti-Ni Ab and the optimum diluting multiple of blood plasma
Table 3:ELISA positive control and negative control ELISA testing result
Shown by table 2-3 data, we can find out, and the dilutability as anti-HSA Ab is 1:2000, whole blood dilutability is when be the anti-dilutability of 1:20, Ni being 1:10000, OD value is maximum, so select concentration corresponding to this value as best effort concentration.
2. quantitatively detect the determination of eluent best effort concentration and elution time in method two, three in the method for nickel-albumin chelate
Step is as follows: (1) bag quilt: anti-NiAb dilution buffer be diluted to 10000 times (mass volume ratios), adds in elisa plate micropore, 37 DEG C of water-baths 3 hours, stores refrigerator;
(2) close: remove dilution buffer, and wash with cleansing solution, after washing completes, with 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid, and wash with cleansing solution for 37 DEG C;
(3) remove confining liquid, and wash with cleansing solution, 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 eluent: papain 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, eluent is diluted, make the concentration of the papain in eluent 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 eluent, and wash with cleansing solution, 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 optimum concentration and the elution time of eluent, concrete outcome is see table 4.
Table 4:ELISA eluent best effort concentration and elution time are determined
From table 4, we can find, as the ratio=1:20 of the concentration of the papain in eluent 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 compound 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 concentration is constant, extends digestion time and can not improve digestibility, so the elution time of eluent is that 1-3h all can in this test.
application Example 1
Get and adopt ELISA method to detect nickel-albumin chelate in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-albumin chelate, detect in accordance with the following steps:
1) anti-HSA Ab is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 37 DEG C of water-baths 1 hour, store refrigerator;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discards precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, 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 items with the nickel of known content-albumin chelate; With dilution buffer dilution, measuring samples is diluted to 20 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 cleansing solution, after washing completes, add and dilute 10000 times by dilution buffer, 37 DEG C act on 1 hour, the metallic nickel on anti-Ni Ab and albumin are reacted;
6) enzyme conjugates incubation: remove anti-nickel antibody, and wash with cleansing solution, 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 cleansing solution, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) cessation 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 testing result of nickel-albumin chelate 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-albumin chelate in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the circulation system, make measuring samples, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discards precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) or skimmed milk power as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, 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 items with the nickel of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add the eluent that Papain enzyme concentration is 100ng/ml, wash-out 1-3 hour;
6) detect: sample from the micropore of elisa plate, detect the nickel of chelating on albumin in Atomic Absorption Spectrometer, detected value is as shown in table 6.
The AAS testing result of nickel-albumin chelate 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-albumin chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) albuminous material can be caught, as anti-albumin antibodies (anti-HSA Ab) is coated on solid phase carrier: by dilution buffer, anti-HSA Ab is diluted to 2000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) 100 parts of standard blood samples are got as measuring samples;
3) close: remove dilution buffer, and wash with cleansing solution, after washing completes, add 2% bovine serum albumin(BSA) as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with cleansing solution, 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 items with the nickel of known content-albumin chelate; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with cleansing solution, after washing completes, add the eluent that Papain enzyme concentration is 100ng/ml, wash-out 1-3 hour;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) sample the solution that obtains, under icp ms, detect chelating in albuminous nickel, detected value is as shown in table 7.
The ICP-MS testing result of nickel-albumin chelate 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-albumin chelate, is characterized in that, nickel ion and albumin by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for nickel as claimed in claim 1-albumin chelate, is characterized in that, comprise the following steps:
A) nickel and albuminous chelatropic reaction: add nickel ion in the albumin coming from human body or the albumin of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction solution;
B) extraction of purifying nickel-albumin chelate: adopt immune-affinity chromatography, removes unreacted albumin and unnecessary nickel ion in reaction solution, obtains nickel-albumin chelate.
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 solution that obtains nickel-albumin chelate is redissolved;
(2) balance chromatographic column: use dilution buffer to rinse the pipeline of chromatographic column, load in chromatographic column can with the filler of albumin specific binding, after dress post, continue to use dilution buffer balance chromatographic column;
(3) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (1), albumin and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain eluent I;
(5) collect: collect eluent I;
(6) dialyse: by the eluent of the collection in step (5), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatographic column: adopt new chromatographic column, use dilution buffer flushing line, in this chromatographic column load can with the filler of nickel specific binding, dress post after balance chromatographic column by dilution buffer again;
(8) loading: after chromatographic column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatographic column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain eluent II;
(10) collect: collect eluent II;
(11) dialyse: by the eluent of the collection in step (10), dress bag filter, uses ddH
2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain nickel-albumin chelate.
4. the preparation method of nickel according to claim 2-albumin chelate, is characterized in that, also comprise step C): to the qualification of nickel-albumin chelate;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in Ago-Gel, polyacrylamide gel as medium;
(2) application of sample: get step B) in nickel-albumin chelate 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-albumin chelate in the reagent or kit preparing to detect nickel-albumin chelate in blood sample.
6. one kind at least comprises the kit of nickel as claimed in claim 1-albumin chelate as standard items.
7. kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching albuminous material or catching nickel.
8. one kind is quantitatively detected the method for nickel-albumin chelate, it is characterized in that, using the nickel according to claim 1-albumin chelate of known content as standard items, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin chelate and enzyme linked immunological combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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