CN105021549B - Nickel-albumin chelate as well as preparation method and application thereof - Google Patents
Nickel-albumin chelate as well as preparation method and application thereof Download PDFInfo
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- CN105021549B CN105021549B CN201510411356.9A CN201510411356A CN105021549B CN 105021549 B CN105021549 B CN 105021549B CN 201510411356 A CN201510411356 A CN 201510411356A CN 105021549 B CN105021549 B CN 105021549B
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Abstract
The invention discloses a nickel-albumin chelate and a preparation method and application thereof, wherein the nickel-albumin chelate is prepared by chelating nickel ions and albumin through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of a nickel-albumin chelate so as to quantitatively detect the application of the nickel-albumin chelate in evaluating the nickel pollution degree of one region. The condition of nickel pollution of people in a region can be indirectly reflected by quantitatively detecting the nickel-albumin chelate in the serum of people in the region, so that the nickel pollution degree of the region is indirectly reflected. The method for quantitatively detecting the nickel-albumin chelate is greatly improved in accuracy and repeatability.
Description
Technical field
The present invention relates to detection field, more specifically to a kind of nickel-albumin chelate and preparation method thereof and answers
With.
Background technology
Seralbumin (HSA) is the protein that content is most abundant in mammalian plasma, and it can store and transport crowd
More endogenous and exogenous material, this is closely related with its specificity structure.Seralbumin is the one kind synthesized by liver
Simple protein, only it is made up of amino acid, without modification group and other adjuncts.Shown according to data information, ox blood is pure
Albumen (BSA) molecule is made up of 583 amino acid residues.And human serum albumins (HSA) 2 amino acid more than BSA, i.e., 585
Individual amino acid residue composition.Both difference is the residue of BSA missing HSA amino acid sequences 116 and 585.Albumin
The most significant feature of aminoacid ingredient is the tryptophan of low content, contains 1 or 2 residue respectively in HSA and BSA per molecules.Benzene
Alanine and isoleucine content are also than relatively low.Cysteine, leucine, glutamic acid and lysine content are all very high.Largely
Ionization residue provide the very high electric charge of albumin, under conditions of PH7, per molecule albumin carries 185 ions,
Thus form albumin ease of solubility.
Albumin it is most unique be structurally characterized in that disulfide bond pattern.BSA sequences include 35 cysteine residues, form 17
Individual disulphide bridges, adjacent cysteine pair link with residue nearest on chain, form a pair of rings of 10-47 residues in length.S-S
Key is folded the rigidity and stability for adding albumin structure.
The tertiary structure of albumin is considered as a kind of molecule of high degree of spiral, is learnt by X-ray diffraction in HNI crystal
About 67% residue participates in foring 28 conveyor screws of α mono-, and 10% residue participates in forming β-bend, and 23% residue forms stretching, extension
Chain.
On physiological mechanism, albumin maintains the 80% of human plasma colloid osmotic pressure.In addition, albumin is also each in blood
The main carriers of kind macromolecular substances, played an important role in metabolin transhipment, bioconversion.It is simultaneously and regulation is solidifying
Blood function and the component of inflammatory reaction.Human serum albumins is mainly used in clinical, metabolism and the research of genetic aspect.
Nickel common are noxious metals as one kind, among being widely used in living, producing, including production coin, jewelry, nickel
Stainless steel alloy, manufacture Ni-Cd batteries etc., it is closely bound up with the life of people.With the development of science and technology people are using nickel as urging
Among production of the agent for producing carbon nano-particle, influence of the nickel for the mankind is further deepened.For general population,
Nickel mainly by eat, drink into, contact etc. mode take in nickel, certain smoking is also an important channel.Research is found, excessive
Nickel can cause body many places tissue and organ damage, cause the generation of a variety of diseases, including respiratory disease (pneumonia, branch gas
Guan Yan, pulmonary fibrosis, asthma, pulmonary edema etc.), angiocardiopathy, kidney trouble, disease of skin etc., or even also result in tumour
Generation.
Epidemiologic data shows that the worker of Long Term Contact nickel, it is suffered from the probability of tumor in respiratory system and greatly increased, and
It is also greatly increased because of the lethal probability of tumor in respiratory system.Animal model, external model experiment, it was also found that nickel can induce it is swollen
The generation of knurl.Nineteen ninety, international cancer research institution (International Agency for Research on Cancer,
IARC) using nickel compound as first kind carcinogen (Group 1), using metallic nickel as 2B class carcinogens (Group
2B).It can be seen that the health of nickel serious threat human body.
It is in close relations between nickel and multiple proteins, it can be combined with multiple proteins, suppress the activity of multiple protein enzyme.
With going deep into nickel toxicity research, people gradually recognize that the interaction of nickel and albumen is played the part of emphatically during its intoxicating
The role wanted, it is considered to be understand the key point of the toxicity mechanism of nickel, therefore, the research to nickel and protein interaction is just
Seem ever more important.And the mechanism now concerning nickel and protein effect is only tip of the iceberg, also many protein and nickel it
Between relation it is not clear, thus strengthen it is most important for the relation between nickel and protein.
The content of the invention
For nickel contamination it is serious the problem of, it is an object of the invention to provide a kind of nickel-albumin chelate and its preparation
Method, and the qualitative and quantitative analysis method of nickel-albumin chelate is established, so that quantitative detection nickel-albumin chelate is being commented
The application of the regional nickel contamination degree of valency one.Can be with by nickel-albumin chelate in one regional crowd's serum of quantitative detection
Reflect situation of this regional crowd by nickel contamination indirectly, so as to reflect this regional nickel contamination degree indirectly.
The technical solution adopted for the present invention to solve the technical problems is：A kind of nickel-albumin chelate, nickel ion are provided
Formed with albumin by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned nickel-albumin chelate, comprises the following steps：
A) the chelatropic reaction of nickel and albumin：The albumin of human body is come from purification or according to the white of biological method restructuring
Nickel ion is added in albumen and carries out chelatropic reaction, obtains reaction solution；
B the extraction of nickel-albumin chelate) is purified：Using immune-affinity chromatography, remove unreacted in reaction solution
Albumin and unnecessary nickel ion, produce nickel-albumin chelate.
Wherein, step B described in external synthetic method) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution makes nickel-albumin chelate
Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column
The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin
Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus
Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of nickel specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid
Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces nickel-albumin chelate.
Wherein, the preparation method of above-mentioned nickel-albumin chelate, in addition to step C)：To the mirror of nickel-albumin chelate
It is fixed；
Wherein, step C) in it is specific as follows：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained nickel-albumin chelate of extraction purification, add dilution buffer, and mix
It is even, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：Nickeliferous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing nickel and detected the content of nickel.
The present invention also provides a kind of nickel-albumin chelate described above and is preparing nickel in detection human body-albumin chelating
Application in the reagent or kit of thing.
The present invention, which also provides, a kind of comprises at least nickel described above-kit of the albumin chelate as standard items.
Preferably, coating buffer is also included in the kit, the material or capture nickel that the coating buffer contains capture albumin
Material.
The present invention also provides a kind of method for quantitatively detecting nickel-albumin chelate, with the above-mentioned nickel of known content-white
Protein chelates thing is detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and atom
Absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin chelate with it is enzyme-linked
Immune combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin chelate and inductance
Coupled plasma mass spectrometry combined techniques, electrophoresis are combined with enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry
Method.
Implement nickel-albumin chelate of the present invention and its preparation method and application, have the advantages that：
1. the present invention synthesizes nickel-albumin chelate in vitro first；
2. present invention firstly provides nickel-albumin chelate can be used for preparing nickel-albumin chelate in detection blood sample
Application in reagent or kit.
3. the present invention establishes the method for qualitative and quantitative detection of nickel-albumin chelate, so as to quantitative detection nickel-albumin
Chelate is evaluating the application of a regional nickel contamination degree.Pass through nickel-albumin in one regional crowd's serum of quantitative detection
Chelate can reflect situation of this regional crowd by nickel contamination indirectly, so as to reflect this regional nickel contamination degree indirectly.
Its degree of accuracy of the nickel that the present invention establishes-albumin chelate quantitative detecting method greatly improves, and makes the repeated of detection
To greatly improving.
Brief description of the drawings
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, with reference to embodiment, the present invention is described further：
Unless otherwise indicated, the laboratory operating procedures being related to are the conventional step in this area to the present invention, agents useful for same, material
Material as following cited, do not include in the present invention come be reagent commonly used in the art or can be by mode purchased in market
Obtain：
Phosphate solution is 0.05-0.1mol/L Na2HPO4Solution or 0.5-1mol/L Na2HPO4Solution；
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
Glue bed medium is one kind in Ago-Gel, polyacrylamide gel；
The material for capturing albumin is the model ab106349 of Abcam companies production Anti- mankind Serum
Albumin antibody；Wherein, it is of the present invention equal with material, anti-HSA antibody, the anti-albumin antibodies of albumin specific binding
To capture the material of albumin；
The material purchase of nickel is captured from the model RK14419 of the Guangzhou Ran Ke bio tech ltd anti-Ni of mouse
mAb；Wherein, it is the thing for capturing nickel that the material of the present invention specifically bound with nickel, anti-Ni antibody, anti-nickel antibody, nickel are anti-
Matter；
Enzyme labelled antibody is one kind in the antibody containing the enzymes such as horseradish peroxidase, alkaline phosphatase mark；
Substrate is methyl biphenyl amine (TMB) solution；
Cleaning solution is to contain KH2PO40.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl 8.0mg/ml、KCl
0.2mg/ml, 0.5%Tween-20 pH are 7.4 0.15M PBS solutions；
Confining liquid is 1%-5% bovine serum albumin(BSA)s or skimmed milk power；
Dilution buffer is Na containing 1.5mg/mL2CO3、2.93mg/ml NaHCO3PH be 9.6 0.05M carbonate delay
Fliud flushing；
Enzyme labelled antibody is HRP enzyme labelled antibodies；
Terminate liquid is：By 21.7ml 2M H2SO4It is settled to 200ml ddH2In O；
Eluent is the 0.1mol/L Tris-HCL buffer solutions that the PH containing papain is 8.0；
Acidulant is nitric acid；
Sample-loading buffer is to contain 1M Tris-HCl (pH 6.8) 15.5mL, 1% bromophenol blue 2.5mL, ddH2It is O7mL, sweet
Propylhomoserin 25mL Sample buffer (5X)；
Electrophoretic buffer is the ddH that 3mg/ml containing Tris, glycine 14.4mg/ml PH are 6.82O solution.
The present invention provides a kind of nickel-albumin chelate, and nickel ion passes through sulfydryl or/and cysteine residues with albumin
Chelating forms.
The present invention also provides a kind of preparation method of nickel-albumin chelate, comprises the following steps：
A) the chelatropic reaction of nickel and albumin：Nickel ion is added in the albumin in people source and carries out chelatropic reaction, is obtained anti-
Answer solution；
B the extraction of nickel-albumin chelate) is purified：Using immune-affinity chromatography, remove unreacted in reaction solution
Albumin and unnecessary nickel ion, nickel-albumin chelate is produced, is comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution makes nickel-albumin chelate
Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column
The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；It is described to be specifically bound with albumin
Filler be silica gel or resin containing anti-albumin antibodies；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin
Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus
Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of nickel specific binding, chromatographic column is balanced with dilution buffer again after filling post；It is described to be filled out with what nickel was specifically bound
Expect for silica gel or resin containing anti-nickel antibody；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid
Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water
After three times, 4 DEG C of dialysed overnights, sample is collected, produces nickel-albumin chelate；
C) to the identification of nickel-albumin chelate, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained nickel-albumin chelate of extraction purification, add dilution buffer, and mix
It is even, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：Nickeliferous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing nickel and detected the content of nickel.
The present invention, which also provides, a kind of comprises at least nickel described above-kit of the albumin chelate as standard items.
Preferably, coating buffer is also included in the kit, the material or capture nickel that the coating buffer contains capture albumin
Material.
In the present invention, can realize the kit of the object of the invention can list following several, but be not limited to this.
The kit of nickel-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin
Liquid, confining liquid, cleaning solution, the material for capturing nickel, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, sun as secondary antibody
Property control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin
Liquid, confining liquid, cleaning solution, eluent, positive control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, include the coating of the material containing capture albumin
Liquid, confining liquid, cleaning solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Reagent is taken, liquid is redissolved, is the coating buffer of material containing capture nickel, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilute
Release buffer solution, positive control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Take reagent, redissolve liquid, positive control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Take reagent, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Reagent, glue bed medium are taken, liquid, sample-loading buffer is redissolved, dissolves liquid needed for protein band nickeliferous on glue bed, containing capture nickel
The coating buffer of material, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative right
According to etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Take reagent, glue bed medium, redissolve liquid, sample-loading buffer, liquid needed for nickeliferous protein band on dissolving glue bed, positive control,
Negative control etc..
The kit of nickel-albumin chelate in a kind of detection blood sample, including carried as needed for albumin in purification whole blood
Reagent, glue bed medium are taken, liquid, sample-loading buffer is redissolved, dissolves liquid, acidulant, mistake needed for protein band nickeliferous on glue bed
Hydrogen oxide, positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with the albumin chelate of heavy metal nickel
Or it is chelated with the BSA chelates of heavy metal nickel；The negative control is the dilution buffer for not containing standard items.
Mentioned reagent box is used for the albumin for detecting chelating nickel, to improve the accuracy of detection, repeatability, and is allowed to facing
It is promoted in bed.
The present invention also provides a kind of method for quantitatively detecting nickel-albumin chelate, with the above-mentioned nickel of known content-white
Protein chelates thing is detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and atom
Absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin chelate with it is enzyme-linked
Immune combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin chelate and inductance
Coupled plasma mass spectrometry combined techniques, electrophoresis are combined with enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry
Method.In the present invention, the having of can listing of method with detection nickel-albumin chelate is following several, but is not limited to following
It is several.
Wherein, the reagent used in the above-mentioned method for quantitatively detecting nickel-albumin chelate is as follows：
Method one：ELISA (ELISA method) detects nickel-albumin chelate, detects in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With
Anti- HSA Ab are diluted to 1000-8000 times by dilution buffer, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37
DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp
5-8 minutes are centrifuged, centrifugation discards precipitation, 1000rmp centrifugation 5-8 minutes, and centrifugation discards precipitation, and 1000rmp centrifuges 5-8 minutes,
Centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen
As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power
After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) material of nickel can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed
After the completion of washing, addition is diluted to 5000-40000 times of anti-Ni Ab with dilution buffer, 37 DEG C of effect 1-2 hours, makes anti-Ni
Ab forms antigen antibody complex with the metal nickel reactant on albumin；
6) enzyme conjugates incubates：Anti- nickel antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute
Release the enzyme labelled antibody of buffer solution dilution, the concentration for making the enzyme labelled antibody of dilution is 2 μ g/mL, 37 DEG C of effect 1-2 hours, make its with
Enzyme labelled antibody reacts；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C
Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore；
9) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively
With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA
Colour developing situation carries out qualitative detection).
This method utilizes ELISA principles, can extract the specific albumin in whole blood, the white egg extracted
White upper part is chelated with heavy metal nickel, and the nickel on this part albumin is captured by anti-nickel antibody, afterwards can be again by horseradish
The antibody of the enzymes such as peroxidase, alkaline phosphatase mark captures (the antibody nonrecognition coating protein), the antibody in capture
In the presence of developer and terminate liquid, OD values can be read under instrument, and do not contain the albumin of chelated mineral nickel, then not
It can be captured by the specific antibody of anti-nickel, the antibody institute that will not be also marked with enzymes such as horseradish peroxidase, alkaline phosphatases
Capture, and metallic nickel (negative control group result is feminine gender) is not contained in agents useful for same yet, thus work as read OD value results
When being shown as the positive, you can prove to detect the metallic nickel chelated on albumin.
Method two：Enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection nickel-albumin chelate
Detect in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With
Anti- HSA Ab are diluted to 1000-8000 times by dilution buffer, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37
DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp
5-8 minutes are centrifuged, centrifugation discards precipitation, 1000rmp centrifugation 5-8 minutes, and centrifugation discards precipitation, and 1000rmp centrifuges 5-8 minutes,
Centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen
As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power
After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C
Lower elution 1-3 hours.
6) detect：Sample from the micropore of elisa plate, chelated in Atomic Absorption Spectrometer detection in the nickel on albumin,
And standard curve is drawn, readout value；
This method combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principles, utilizes atomic absorption spectrum
Instrument detection is chelated in the nickel on albumin, due to only containing albumin in solution, and it is (cloudy without any heavy metal in agents useful for same
Property control group result be feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can card
The bright metallic nickel for detecting to chelate on albumin.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection nickel-
Albumin chelate detects in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With
Anti- HSA Ab are diluted to 1000-8000 times by dilution buffer, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37
DEG C water-bath 1-3 hours, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp
5-8 minutes are centrifuged, centrifugation discards precipitation, 1000rmp centrifugation 5-8 minutes, and centrifugation discards precipitation, and 1000rmp centrifuges 5-8 minutes,
Centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen
As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power
After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C
Lower elution 1-3 hours.
6) it is acidified：Acidulant is added in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified,
Hydrogen peroxide is added, and heats and catches up with acid；
7) detect：0.5ml liquid is taken from the solution of the elisa plate of step 6), in icp mses
Lower detection chelates the nickel in albumin, and draws standard curve, readout value.
This method is on the basis of using ELISA principles, with reference to sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption
Sense couple plasma mass spectrometer detection is chelated in the nickel on albumin, due to only containing albumin, and agents useful for same in solution
In without any heavy metal (negative control group result for feminine gender), result will not be interfered, thus work as read result
When being shown as the positive, you can prove to detect the metallic nickel chelated on albumin.
Method four：Purifying nickel-albumin chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detection nickel-white egg
White chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel
The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) anti-Ni Ab are coated on solid phase carrier：Anti- Ni Ab are diluted to 5000-40000 times with dilution buffer,
Add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen
As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power
After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Sampled in solution from step 1), make measuring samples；With known content
Nickel-albumin chelate makees standard items；10-40 times is diluted to dilution buffer dilution measuring samples, is added in micropore, 37 DEG C
Act on 1-2 hours；
5) enzyme conjugates incubates：The measuring samples in step 4) are removed, and are washed with cleaning solution, treat that washing is completed
Afterwards, the enzyme labelled antibody that addition is diluted with dilution buffer, the concentration for making the enzyme labelled antibody of dilution are 2 μ g/mL, 37 DEG C of effect 1-2
Hour, it is reacted with enzyme labelled antibody；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C
Lucifuge acts on 30 minutes；
7) terminating reaction：Terminate liquid is added dropwise to each micropore；
8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively
With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA
Colour developing situation carries out qualitative detection).
Method five：Purifying nickel-albumin chelate and atomic absorption spectrum combined techniques (method of purification+AAS methods) detection blood sample
Middle nickel-albumin chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel
The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) detect：Sample in the solution obtained from step 1), chelated in Atomic Absorption Spectrometer detection on albumin
Nickel, and standard curve is drawn, readout value.
Method six：Purifying nickel-albumin chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS
Method) detection nickel-albumin chelate, is detected in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel
The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) it is acidified：Sampled in the solution obtained from step 1), add acidulant (such as nitric acid) in the solution and solution is carried out
Acidifying, sealing overnight, thoroughly acidifying, add hydrogen peroxide, and heat and catch up with acid；
3) detect：0.5ml liquid is taken in the solution obtained from step 2), is detected under icp mses
Chelate in the nickel on albumin, and draw standard curve, readout value.
Method seven：Electrophoresis and ELISA or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques
(electrophoresis-ELISA method/AAS methods/ICP-MS methods) detects nickel-albumin chelate, detects in accordance with the following steps：
1) albumin is extracted from whole blood：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel
The methods of filtering, redissolves albumin in solution from the albumin in whole blood extraction purification blood, obtains albumin solution；
2) glue bed is prepared：Selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gels as needed
Deng medium is used as, glue bed is conventionally prepared；
3) it is loaded：The μ L of solution 8 for taking step 1) to obtain add 2 μ L sample-loading buffers, mix, of short duration centrifugation；(pay attention to herein
Step can not boil)
4) electrophoresis：Electrophoresis plate is connected, is separated by electrophoresis；
5) detect：The protein band containing nickel is found out on glue bed, the band is taken out, the protein band is dissolved in liquid
Among, ELISA or ICP-MS are then utilized respectively again or AAS detects the nickel content being dissolved in liquid.Further, it is also possible to utilize this
Isoelectric point, molecular weight and content of albumin of method detection chelating nickel etc..
Albumin in method seven can come out (such as supercentrifugation or HPLC with a variety of Methods For Purifications
Or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), albumin out will be purified and redissolved in solution, taken
A certain amount of albumin, using electric charge shifting principle, electrophoresis (electrophoresis, EP) is carried out, (can basis in gel slab
Need to use different medium) on can run out of different bands according to molecular weight, isoelectric point etc. are different, search out rich in the corresponding of nickel
Band, the protein in gel is redissolved in solution, you can to detect the content of related albumin at a particular wavelength, also may be used
It is white due to only containing in solution to detect to chelate in the nickel content on albumin using principles such as ELISA or AAS or ICP-MS
Albumen, and result will not be interfered without any heavy metal (negative control group result is feminine gender) in agents useful for same, thus
When the result read is shown as the positive, you can prove to detect the metallic nickel chelated on albumin.
Embodiment 1：The preparation method of nickel-albumin chelate, comprises the following steps：
A) the chelatropic reaction of nickel and albumin：Nickel ion is added in the albumin in people source and carries out chelatropic reaction, is obtained anti-
Answer solution；
Preparation of reagents：
1) borate buffer solution (0.01M)：Weigh 0.31g boric acid to be dissolved in 400ml ultra-pure waters, adjusted with 0.1M NaOH
PH to 9.0, is settled to 500mL.
2) albumin solution：Weigh 4.0mg albumin to be dissolved in 4.0mL 0.01M pH9.0 borate buffer solutions, fully
Vibration dissolving, it is configured to 1.0mg/mL protein solution；
3) EDTA-NaHCO3 mixed solutions：Weigh EDTA2H2O 1.86g、NaHCO316.8g is dissolved in 900mL ultra-pure waters
In, it is settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH adjustment pH to 8.0；
4) ITCBE is bought from Japanese colleague's chemistry institute, article No. M030；
5) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of nickel-albumin chelate：
1) processing of bag filter：Bag filter is put into 500ml EDTA-NaHCO3In mixed solution, 10min is boiled；Incline
Abandon EDTA/NaHCO3Liquid, gently rinsed with ultra-pure water, then 10min is boiled with 500ml 5mmol/L EDTA solution；Discard and boil
Liquid is boiled, is thoroughly cleaned with ultra-pure water, adds 4 DEG C of ultra-pure water immersion bag filter overnight.In use, putting on one's gloves, bag filter is taken out,
With its surfaces externally and internally of ultra-pure water cleaning down；
2) 2.0mg ITCBE are taken to be dissolved in 2ml DMSO；
3) 4.0mg albumin is taken to be dissolved in 4.0ml borate buffer solutions；
4) liquid for slowly preparing step 2) is added in the albumin solution that step 3) is prepared, and is shaken, is put when being added dropwise
In temperature be 25 DEG C, rotating speed be 100r/min shaking table in react 24h, then with bag filter dialyse 24h, remove not with albumin
With reference to ITCBE；
5) liquid obtained by step 4) is adjusted into pH value to 7.0 with 1mol/L HCl, is then slowly gradually added dropwise 80 μ l's
1mmol/L nickel ion solution, vibrated when being added dropwise, in case nickel ion precipitates albuminous degeneration；
6) solution obtained by step 5) is placed in temperature as 25 DEG C, rotating speed is to react 2h in 100r/min shaking table, with saturating
Analysis bag carries out dialysis 24h；
7) liquid after step 6) is dialysed preserves in -20 DEG C of packing, obtains reaction solution.
B the extraction of nickel-albumin chelate) is purified：Using immune-affinity chromatography, reaction solution (i.e. step 7) is removed
Obtained reaction solution) in unreacted albumin and unnecessary nickel ion, produce nickel-albumin chelate, specific steps
It is as follows：
(1) sample dissolution：Physiological saline is added into the reaction solution obtained by step 7) makes nickel-albumin chelate
Redissolve；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, energy and albumin are loaded in chromatographic column
The filler of specific binding, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, albumin
Specifically bound with filler；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus
Acid salt solution is eluted, and obtains eluent I；
(5) collect：Collect eluent I；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times
Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column
With the filler of nickel specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid
Salting liquid is eluted, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water
After three times, 4 DEG C of dialysed overnights, sample is collected, produces nickel-albumin chelate；
C) to the identification of nickel-albumin chelate, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using Ago-Gel as medium；
(2) it is loaded：Take step B) in the obtained nickel-albumin chelate of 8 μ L extraction purifications, add 2 μ L sample-loading buffers,
And mix, then it is loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, electrophoretic buffer is added and carries out electrophoresis；In electrophoresis process, electric current is 22mA constant currents,
Environment temperature is 4 DEG C；Electrophoresis to bromophenol blue stops electrophoresis when moving to glue bottom；
(4) detect：Nickeliferous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing nickel and detected the content of nickel.
D) testing result
(1) content of beary metal in graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination HSA：It the results are shown in Table 1
Table 1 is content of beary metal (μ g/L) in albumin
| Sample name | Ni(μg/L) |
| HSA | 162.811 |
| (NH4)2SO4 | 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 for active electrophoresis be be added without SDS and dredge
Under conditions of the denaturants such as base ethanol, polyacrylamide gel electrophoresis is carried out to the protein for keeping activity, is usually used in the mirror of enzyme
Fixed, Isozyme Analysis and purification.Not plus SDS native polyacrylamide gel electrophoresis can make large biological molecule in electrophoresis process
Middle its natural shape of holding and electric charge, their separation are made according to the difference of its electrophoretic mobility and the molecular sieve of gel
With, thus higher resolution ratio can be obtained, especially remain to keep the large biological molecule such as protein and enzyme after electrophoretic separation
Bioactivity, therefore the right swimming lane M is denaturation Marker, is served only for detecting, does not mark effect, and swimming lane 1 is albumin
Band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis：
4W1 " of the SRXRF analyses of micronutrient levels in BEPC (BEPC) is synchronous in protein band
Completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200
Type, Beijing are stood upright Han Guang companies) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into
Penetrate facula position, moving step length 0.0025mm.From the X ray that electromagnetic radiation goes out by Si (Li) detector (PGT Inc.LS
30143-DS) detect, probe is with incident SR lines copline and being mutually perpendicular to, and away from sample irradiation point 20mm, signal is divided with PGT multiple tracks
Analyzer (MCA4000) obtains output.Sample, regulation launching spot (1mmx3mm) are excited with 11.5keV monochromatic synchrotron radiation light
Position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always, at the end of counting
Hot spot moves on to the band other end.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings, and with deriving from
Air and the Ar signal peaks of content constant carry out normalization to other element peaks, to offset beam intensity change to signal strength
Caused influence.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way under the same conditions.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of nickel of the present invention-albumin chelate, is schemed
Middle abscissa is protein band position, and ordinate is each metal energy of the band (content) value.
A kind of determination of the testing conditions for the method for quantitatively detecting 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) it is 1 according to anti-HSA Ab and the mass volume ratio of dilution buffer by anti-HSA Ab dilution buffers:
1000、1:2000、1:4000、1:8000 are diluted, and add in elisa plate micropore, anti-HSA Ab are coated in into solid phase carrier
On, each concentration is coated with three rows, and 4 DEG C are stayed overnight 18 hours；
(2) dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is made with 2% bovine serum albumin(BSA)
For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) measuring samples are 1 by measuring samples and the mass volume ratio of dilution buffer:10、1:20、1:40 progress are dilute
Release, add in micropore, according to above-mentioned coated anti-HSA Ab concentration, being separately added into for the anti-HSA Ab of same concentration is different dilute
Degree of releasing blood plasma, 37 DEG C act on 1 hour；
(4) measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, add anti-Ni Ab, anti-Ni Ab are pressed
Anti- Ni Ab and the mass volume ratio of dilution buffer are 1:5000、1:10000、1:20000、1:40000 are diluted, according to
Each identical anti-HSA Ab, serum-dilution concentration, the anti-Ni Ab of various concentrations respectively add 2 holes, and 37 DEG C act on 1 hour, make anti-Ni
Metal nickel reactant on Ab and HSA；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2 μ g/mL, removes anti-Ni antibody, and is washed with cleaning solution,
Wait after the completion of washing, addition dilution buffer dilutes HRP enzyme labelled antibodies, and 37 DEG C act on 1 hour, make HRP enzyme labelled antibodies and nickel-
Albumin chelate reacts；
(6) enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects
30 minutes；
(7) terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads each hole OD respectively
Value.According to each hole OD value numerical value, anti-HSA Ab, anti-Ni Ab best effort concentration and blood plasma optimum diluting multiple are selected.
Simultaneously using made standard items as positive control in experiment, anti-HSA Ab+ closings+anti-Ni Ab+ enzymes mark+bottom is selected
Thing (being not added with measuring samples) is used as negative control 1；Anti- HSA Ab+ closings+blood plasma+enzyme mark+substrate (being not added with anti-Ni Ab) is made
For negative control 2；Anti- HSA Ab+ closings+enzyme mark+substrate (being not added with measuring samples and anti-Ni Ab) are used as negative control 3；It is anti-
HSA Ab+ closings+blood plasma+anti-Ni Ab+ substrates (i.e. not enzyme-added mark) are used as negative control 4；Closing+blood plasma+anti-Ni Ab+ enzyme marks
+ substrate (being not added with anti-HSA Ab captures HSA) is used as blank control 1；Only add substrate as blank control 2 and PBS as blank
Control 3.Testing result is shown in Table 2-3.
Table 2：Checkerboard titration method determines anti-HSA Ab, anti-Ni Ab best effort concentration and blood plasma optimal dilution times
Several data (each data are all average value)
Table 3：ELISA positive controls and negative control ELISA testing results
Shown by table 2-3 data, we can see that when anti-HSA Ab dilution factor is 1:2000th, whole blood dilution factor is 1:
20th, the anti-dilution factors of Ni are 1:When 10000, OD values are maximum, so selecting the concentration corresponding to this value as best effort concentration.
2. quantitatively detect eluent best effort concentration and elution in method two, three in the method for nickel-albumin chelate
The determination of time
Step is as follows：(1) it is coated with：Anti- NiAb is diluted to 10000 times (mass volume ratios) with dilution buffer, added
In elisa plate micropore, 37 DEG C of water-baths 3 hours, refrigerator is stored；
(2) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure with 2% ox blood
As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and washed with cleaning solution；
(3) confining liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, addition is diluted with dilution buffer
Enzyme labelled antibody, enzyme labelled antibody are diluted to concentration as 2 μ g/mL, and 37 DEG C act on 2 hours, it is reacted with anti-Ni Ab；
(4) eluent is prepared：By papain with pH 8.0,0.1mol/L Tris-HCI buffers into
100ng/ml, add 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT)s (DTT)；
(5) enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes papain in eluent
The ratio between concentration and the concentration of enzyme labelled antibody are 1:80、1:40、1:20、1:10、1:5 (wherein each concentration makees 3 multiple holes), respectively
1h, 2h, 3h are eluted at a temperature of being positioned over 37 DEG C；
(6) eluent is removed, and is washed with cleaning solution, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects 30
Minute；
(7) terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads every group of OD respectively
Value, by compared with PBS group, comparing the optimum concentration and elution time of eluent, concrete outcome is referring to table 4.
Table 4：ELISA eluent best effort concentration and elution time determine
We are it can be found that when the concentration of the papain in eluent and the ratio between the concentration of enzyme labelled antibody from table 4
=1:When 20, each group OD values are below other several groups, illustrate that the concentration elution effect is optimal (i.e. by ELISA hole walls
With reference to anti-Ni Ab- enzyme marks compound elution degree reach maximum, thus OD values are minimum)；And elution time either 1h, 2h,
3h, each group OD value changes are little, it is seen that with the extension of time, enzyme activity gradually weakens, in the case where enzyme concentration is constant,
Digestibility can not be improved by extending digestion time, so the elution time of eluent all may be used for 1-3h in this experiment.
Application Example 1
Take using nickel-albumin chelate in 100 parts of sample blood plasma of ELISA method detection, follow the steps below inspection
Survey：ELISA (ELISA method) detects nickel-albumin chelate, detects in accordance with the following steps：
1) anti-HSA Ab are coated on solid phase carrier：Anti- HSA Ab are diluted to 2000 times with dilution buffer, added
In elisa plate micropore, 37 DEG C of water-baths 1 hour, refrigerator is stored；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp
5-8 minutes are centrifuged, centrifugation discards precipitation, 1000rmp centrifugation 5-8 minutes, and centrifugation discards precipitation, and 1000rmp centrifuges 5-8 minutes,
Centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, adds 2% bovine serum albumin
It is white be used as confining liquid, 37 DEG C of placements 1 hour, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate in
37 DEG C are placed 1 hour；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer dilution, added in micropore, 37 DEG C act on 1 hour；
5) material of nickel can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed
After the completion of washing, addition dilution buffer dilutes 10000 times, and 37 DEG C act on 1 hour, make anti-Ni Ab and the metal on albumin
Nickel reactant；
6) enzyme conjugates incubates：Anti- nickel antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute
The HRP enzyme labelled antibodies of buffer solution dilution are released, 37 DEG C act on 1 hour, it is reacted with enzyme labelled antibody；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C
Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore；
9) it is 405nm to take wavelength, after adding terminate liquid, elisa plate is placed on ELIASA and detected, and reads treat sample respectively
Product OD values (also directly can carry out qualitative detection) without using ELIASA by the situation that develops the color, and specific detected value is as shown in table 5.
Table 5：The ELISA testing results of nickel-albumin chelate in 100 parts of blood samples
Application Example 2
Using in enzyme linked immunological and minute mark this blood sample of atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection 100
Nickel-albumin chelate, is detected in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With
Anti- HSA Ab are diluted to 2000 times by dilution buffer, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation, 1000rmp
5-8 minutes are centrifuged, centrifugation discards precipitation, 1000rmp centrifugation 5-8 minutes, and centrifugation discards precipitation, and 1000rmp centrifuges 5-8 minutes,
Centrifugation discards precipitation；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood
Albumen or skimmed milk power are as confining liquid, and 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, and washing is completed
Afterwards, elisa plate is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain
The eluent for 100ng/ml is spent, elutes 1-3 hours；
6) detect：Sample from the micropore of elisa plate, chelated in Atomic Absorption Spectrometer detection in the nickel on albumin,
Detected value is as shown in table 6.
Table 6：The AAS testing results of nickel-albumin chelate in 100 parts of blood samples
Application Example 3
100 minute marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods)
Nickel-albumin chelate content in this blood sample, is detected in accordance with the following steps：
1) material of albumin will can be captured, as anti-albumin antibodies (anti-HSA Ab) are coated on solid phase carrier：With
Anti- HSA Ab are diluted to 2000 times by dilution buffer, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) 100 parts of standard blood samples are taken as measuring samples；
3) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood
As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and are washed with cleaning solution, after the completion of washing, elisa plate
Placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Add step 2) in measuring samples, with the nickel of known content-albumin chela
Compound makees standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain
The eluent for 100ng/ml is spent, elutes 1-3 hours；
6) it is acidified：Add nitric acid in solution in step 5) to be acidified solution, overnight, thoroughly acidifying, adds for sealing
Enter hydrogen peroxide, and heat and catch up with acid；
7) detect：Sampled in the solution obtained from step 6), under icp mses detection chelate in
The nickel of albumin, detected value are as shown in table 7.
Table 7：The ICP-MS testing results of nickel-albumin chelate in 100 parts of blood samples
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (5)
1. the preparation method of a kind of nickel-albumin chelate, it is characterised in that the nickel-albumin chelate is by nickel ion and in vain
Albumen is formed by sulfydryl or/and cysteine residues chelating；Specifically include following steps：
A）The chelatropic reaction of nickel and albumin：In the albumin that purification comes from the albumin of human body or recombinated according to biological method
Middle addition nickel ion carries out chelatropic reaction, obtains reaction solution；Step A）Specifically include as follows：
1）The processing of bag filter：Bag filter is put into 500ml EDTA-NaHCO3In mixed solution, 10min is boiled；Tipping
EDTA/NaHCO3Liquid, gently rinsed with ultra-pure water, then 10min is boiled with 500ml 5mmol/L EDTA solution；Discard and boil
Liquid, thoroughly cleaned with ultra-pure water, add 4 DEG C of ultra-pure water immersion bag filter overnight；In use, putting on one's gloves, bag filter is taken out, is used
Its surfaces externally and internally of ultra-pure water cleaning down；
2）2.0mg ITCBE are taken to be dissolved in 2ml DMSO；
3）4.0mg albumin is taken to be dissolved in 4.0ml borate buffer solutions；
4）Slowly by step 2）The liquid of preparation adds step 3）In the albumin solution of preparation, shaken when being added dropwise, be placed in temperature
Spend for 25 DEG C, rotating speed is to react 24h in 100r/min shaking table, is then dialysed 24h with bag filter, remove not with albumin combination
ITCBE；
5）By step 4）The liquid of gained adjusts pH value to 7.0 with 1mol/L HCl, is then slowly gradually added dropwise 80 μ l's
1mmol/L nickel ion solution, vibrated when being added dropwise, in case nickel ion precipitates albuminous degeneration；
6）By step 5）The solution of gained is placed in temperature as 25 DEG C, and rotating speed is to react 2h in 100r/min shaking table, uses bag filter
Carry out dialysis 24h；
7）By step 6）Liquid after dialysis preserves in -20 DEG C of packing, obtains reaction solution；
B）Purify the extraction of nickel-albumin chelate：Using immune-affinity chromatography, unreacted white egg in reaction solution is removed
White and unnecessary nickel ion, produce nickel-albumin chelate；
The step B）It is specific as follows：
（1）Sample dissolution：To by step A）Physiological saline is added in obtained reaction solution answers nickel-albumin chelate
It is molten；
（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with albumin
Property combine filler, fill post after, be continuing with dilution buffer balance chromatographic column；
（3）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（1）Middle sample, then upper prop, albumin is with filling out
Material specific binding；
（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate
Solution is eluted, and obtains eluent I；
（5）Collect：Collect eluent I；
（6）Dialysis：By step（5）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C
Dialysed overnight, collect sample；
（7）Balance chromatographic column：Using new chromatographic column, with dilution buffer flushing line, energy and nickel are loaded in the chromatographic column
The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；
（8）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（6）Middle sample, then upper prop；
（9）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, it is then molten using 0.5-1.0mol/L phosphate
Liquid is eluted, and obtains eluent II；
（10）Collect：Collect eluent II；
（11）Dialysis：By step（10）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4
DEG C dialysed overnight, sample is collected, produces nickel-albumin chelate；
C）Identification to nickel-albumin chelate；Wherein, step C）In it is specific as follows：
（1）Prepare glue bed：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
（2）Sample-adding：Take step B）The nickel that middle extraction purification obtains-albumin chelate, dilution buffer is added, and mixed, so
After be loaded onto in sample cell；
（3）Electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
（4）Detection：Nickeliferous protein band is found out on glue bed, the protein band is taken out and dissolved, then uses ELISA again
Method or ICP-MS methods or AAS methods detect whether containing nickel and detected the content of nickel.
A kind of 2. reagent of the nickel-albumin chelate in detection blood sample is prepared of nickel-albumin chelate as claimed in claim 1
Or the application in kit.
3. a kind of comprise at least nickel as claimed in claim 1-kit of the albumin chelate as standard items.
4. kit according to claim 3, it is characterised in that also including coating buffer, the coating buffer contains the white egg of capture
White material or the material of capture nickel.
A kind of 5. method for quantitatively detecting nickel-albumin chelate, it is characterised in that with described in the claim 1 of known content
Nickel-albumin chelate as standard items, detected using a pair of samples of following methods：ELISA, enzyme-linked exempt from
Epidemic disease chelates with atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purifying nickel-albumin
Thing chelates with enzyme linked immunological combined techniques, purifying nickel-albumin chelate and atomic absorption spectrum combined techniques, purifying nickel-albumin
Thing and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductively coupled plasma
Mass spectrum combined techniques.
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