CN104987401A - Cadmium-hemoglobin chelate as well as preparation method and application thereof - Google Patents

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

Info

Publication number
CN104987401A
CN104987401A CN201510413021.0A CN201510413021A CN104987401A CN 104987401 A CN104987401 A CN 104987401A CN 201510413021 A CN201510413021 A CN 201510413021A CN 104987401 A CN104987401 A CN 104987401A
Authority
CN
China
Prior art keywords
cadmium
oxyphorase
inner complex
sample
chromatography column
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510413021.0A
Other languages
Chinese (zh)
Inventor
张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Baihao Biotechnology Co Ltd
Original Assignee
Shanghai Baihao Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Baihao Biotechnology Co Ltd filed Critical Shanghai Baihao Biotechnology Co Ltd
Priority to CN201510413021.0A priority Critical patent/CN104987401A/en
Publication of CN104987401A publication Critical patent/CN104987401A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/795—Porphyrin- or corrin-ring-containing peptides
    • G01N2333/805—Haemoglobins; Myoglobins

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention discloses a cadmium-hemoglobin chelate as well as a preparation method and an application thereof. The cadmium-hemoglobin chelate is formed by chelating cadmium ions and hemoglobin by virtue of sulfydryl or/and cysteine residues. The invention establishes a quantitative and qualitative detection method of the cadmium-hemoglobin chelate, so as to perform a quantitative detection on the application of the cadmium-hemoglobin chelate in evaluating the cadmium pollution level in an area. By performing the quantitative detection on the cadmium-hemoglobin chelate in sera of a group of people in the area, the condition of cadmium-polluted people in the area can be indirectly reflected, so as to indirectly reflect the cadmium pollution level of the area. The cadmium-hemoglobin chelate quantitative detection method established by the invention has the advantages that the accuracy is greatly improved, and the repeatability of the detection is greatly improved.

Description

A kind of cadmium-oxyphorase inner complex and its preparation method and application
Technical field
The present invention relates to detection field, more particularly, relate to a kind of cadmium-oxyphorase inner complex and its preparation method and application.
Background technology
Oxyphorase (hemoglobin; HB; HGB) be a kind of protein being responsible for delivery oxygen in higher organisms.The albumen that blood is taken on a red color.Three kinds of Hb A hemoglobin adults and Hb-A, A is had in human erythrocyte 2and F.Hb-A is the main component of oxyphorase in adult normal's red corpuscle, Hb-A 2that submember accounts for 2%, and Hb-F accounts for below 2-3% in the red corpuscle of adult normal or children, Hb-F starts embryo the main component being produced as fetal erythrocyte October, in the red corpuscle of FTNB, Hb-F accounts for 70-80%, all the other are Hb-A, account for 50% after two months, be less than 10% after four months, after six months, large portion disappears.Oxyphorase, except transports oxygen carbonic acid gas, can also cause vasospasm and vasoconstriction.The oxyphorase of the three kinds of forms existed in vivo, Oxyhemoglobins, reduced form oxyphorase and methemoglobin, wherein Oxyhemoglobins contracting vessel patency is the strongest, and methemoglobin does not almost have vasoconstriction active.
Cadmium (Cd) is that one common are noxious metals and environmental pollutant, is widely used in industrial production environment, comprises plating, process industry pigment, stabilizer for plastics, Ni-Cr battery etc., lives closely bound up with people.For general population, mainly by sucking the air of cadmium pollution, and the edible modes such as the farm crop (as containing cadmium rice) of cadmium rural sewage disposal farm production that contain take in cadmium.Certainly, smoking is also another important sources that Chronic Cadmium sucks.Along with the develop rapidly of economy, cadmium pollution is increasing for the impact of the mankind.
In close relations between cadmium and multiple proteins, can be combined with multiple proteins, suppress the activity of multiple protein enzyme.Along with going deep into of studying cadmium toxicity, people recognize that the interaction of cadmium and albumen plays important role in its intoxicating process gradually, be considered to the key point of toxicity mechanism understanding cadmium, therefore, the research of cadmium and protein interaction just seemed ever more important.And the present mechanism about cadmium and protein effect is only tip of the iceberg, also have the relation between a lot of protein and cadmium not clear, thus strengthening research is most important for the relation between cadmium and protein.
Summary of the invention
For the problem that cadmium pollution is serious, the object of the present invention is to provide a kind of cadmium-oxyphorase inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of cadmium-oxyphorase inner complex, so that detection by quantitative cadmium-oxyphorase inner complex is in the application of the regional cadmium pollution degree of evaluation one.Indirectly can reflect the situation of this regional crowd by cadmium pollution by cadmium-oxyphorase inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional cadmium pollution degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of cadmium-oxyphorase inner complex, cadmium ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned cadmium-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of cadmium and oxyphorase: add cadmium ion in the oxyphorase coming from human body or the oxyphorase of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying cadmium-oxyphorase inner complex: adopt immune-affinity chromatography, removes unreacted oxyphorase and unnecessary cadmium ion in reaction soln, obtains cadmium-oxyphorase inner complex.
Wherein, step B described in external synthesis method) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains cadmium-oxyphorase inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of oxyphorase specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), oxyphorase and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant I of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of cadmium specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant II of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample, obtains cadmium-oxyphorase inner complex.
Wherein, the preparation method of above-mentioned cadmium-oxyphorase inner complex, also comprises step C): to the qualification of cadmium-oxyphorase inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in cadmium-oxyphorase inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing cadmium, 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 cadmium and detection cadmium.
The present invention also provides the application of a kind of cadmium described above-oxyphorase inner complex in preparation human body in the reagent of cadmium-oxyphorase inner complex or test kit.
The present invention also provides a kind of and at least comprises the test kit of cadmium described above-oxyphorase inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching oxyphorase or the material of catching cadmium.
The present invention also provides the method for a kind of detection by quantitative cadmium-oxyphorase inner complex, using the above-mentioned cadmium-oxyphorase inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification cadmium-oxyphorase inner complex and enzyme linked immunological combined techniques, purification cadmium-oxyphorase inner complex and atomic absorption spectrum combined techniques, purification cadmium-oxyphorase inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement cadmium of the present invention-oxyphorase inner complex and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis cadmium-oxyphorase inner complex first;
2. the present invention proposes cadmium-oxyphorase inner complex first and can be used for preparing application in the reagent or test kit detecting cadmium-oxyphorase inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of cadmium-oxyphorase inner complex, so that detection by quantitative cadmium-oxyphorase inner complex is in the application of the regional cadmium pollution degree of evaluation one.Indirectly can reflect the situation of this regional crowd by cadmium pollution by cadmium-oxyphorase inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional cadmium pollution degree.Cadmium-its accuracy of oxyphorase inner complex quantitative detecting method that the present invention sets up greatly improves, and the repeatability of detection is greatly enhanced.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of cadmium of the present invention-oxyphorase inner complex;
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of cadmium of the present invention-oxyphorase inner complex.
Embodiment
Below, in conjunction with embodiment, the present invention is described further:
Unless otherwise indicated, the laboratory operating procedures related to is the step of this area routine in the present invention, and agents useful for same, material are as cited by following, and the reagent being this area conventional not enumerating out in the present invention maybe can be obtained by commercial mode:
Phosphate solution is 0.05-0.1mol/L Na 2hPO 4solution or 0.5-1mol/L Na 2hPO 4solution;
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Glue bed medium is the one in sepharose, polyacrylamide gel;
The anti-hemoglobin antibodies of the material of catching oxyphorase to be model that Jiang Lai bio tech ltd, Shanghai produces be KAY0043; Wherein, of the present inventionly the material of catching oxyphorase is with the material of oxyphorase specific binding, anti-HB antibody, anti-hemoglobin antibodies;
The Cadmium resistance antibody of the material of catching cadmium to be model that Ba Ao get bio tech ltd produces be AP7015; Wherein, of the present inventionly resist with the material of cadmium specific binding, anti-Cd antibody, Cadmium resistance antibody, cadmium the material being and catching cadmium;
Enzyme labelled antibody is the one in the antibody containing the enzyme labelling such as horseradish peroxidase, alkaline phosphatase;
Substrate is methyl diphenyl amine (TMB) solution;
The formula of erythrocyte cracked liquid is as follows: 139.6mmol/L NH 4cl, 16.96mmol/L Tris, adjusts pH to 7.2 with 1mol/L HCl;
Washings is for containing KH 2pO 40.2mg/ml, Na 2hPO 412H 2the pH of O 2.90mg/ml, NaCl8.0mg/ml, KCl 0.2mg/ml, 0.5%Tween-20 is the 0.15M PBS solution of 7.4;
Confining liquid is 1%-5% bovine serum albumin or skim-milk;
Dilution buffer is for containing 1.5mg/mL Na 2cO 3, 2.93mg/ml NaHCO 3pH be 9.6 0.05M carbonate buffer solution;
Enzyme labelled antibody is HRP enzyme labelled antibody;
Stop buffer is: by the 2M H of 21.7ml 2sO 4be settled to the ddH of 200ml 2in O;
Elutriant be containing the PH of papoid be 8.0 0.1mol/L Tris-HCL damping fluid;
Souring agent is nitric acid;
Sample-loading buffer is for containing 1M Tris-HCl (pH 6.8) 15.5mL, 1% tetrabromophenol sulfonphthalein 2.5mL, ddH 2the Sample buffer (5X) of O7mL, glycine 25mL;
Electrophoretic buffer be containing Tris 3mg/ml, glycine 14.4mg/ml PH be the ddH of 6.8 2o solution.
The invention provides a kind of cadmium-oxyphorase inner complex, cadmium ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides the preparation method of a kind of cadmium-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of cadmium and oxyphorase: add cadmium ion and carry out chelatropic reaction in the oxyphorase in people source, obtain reaction soln;
B) extraction of purifying cadmium-oxyphorase inner complex: adopt immune-affinity chromatography, remove unreacted oxyphorase and unnecessary cadmium ion in reaction soln, obtain cadmium-oxyphorase inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains cadmium-oxyphorase inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of oxyphorase specific binding, after dress post, continue to use dilution buffer balance chromatography column; Described can be the silica gel or the resin that contain anti-hemoglobin antibodies with the filler of oxyphorase specific binding;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), oxyphorase and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I (6) and dialyse: by the elutriant I of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of cadmium specific binding, dress post after balance chromatography column by dilution buffer again; Described can be the silica gel or the resin that contain Cadmium resistance antibody with the filler of cadmium specific binding;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant II of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain cadmium-oxyphorase inner complex;
C) to the qualification of cadmium-oxyphorase inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in cadmium-oxyphorase inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing cadmium, 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 cadmium and detection cadmium.
The present invention also provides a kind of and at least comprises the test kit of cadmium described above-oxyphorase inner complex as standard substance.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material of catching oxyphorase or the material of catching cadmium.
In the present invention, the test kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, material, enzyme labelled antibody, erythrocyte cracked liquid, substrate, stop buffer, dilution buffer, positive control, negative control etc. as two anti-caught cadmiums.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, elutriant, erythrocyte cracked liquid, positive control, negative control etc.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise the coating buffer containing the material of catching oxyphorase, confining liquid, washings, elutriant, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise to extract reagent needed for full blood hemoglobin as purifying, redissolve liquid, containing catch cadmium material coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, erythrocyte cracked liquid, positive control, negative control etc.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise and extract reagent, redissolution liquid, erythrocyte cracked liquid, positive control, negative control etc. needed for the full blood hemoglobin of purification.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise and extract reagent, redissolution liquid, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc. needed for the full blood hemoglobin of purification.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise and to extract reagent, glue bed medium as purifying needed for full blood hemoglobin, to redissolve on liquid, sample-loading buffer, dissolving glue bed containing liquid needed for the protein band of cadmium, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, erythrocyte cracked liquid, positive control, negative control etc. of material of catching cadmium.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise and to extract reagent, glue bed medium as purifying needed for full blood hemoglobin, redissolving on liquid, sample-loading buffer, dissolving glue bed containing liquid, positive control, negative control etc. needed for the protein band of cadmium.
Detect a test kit for cadmium in blood sample-oxyphorase inner complex, comprise and to extract reagent, glue bed medium as purifying needed for full blood hemoglobin, redissolving on liquid, sample-loading buffer, dissolving glue bed containing liquid, souring agent, hydrogen peroxide, erythrocyte cracked liquid, positive control, negative control etc. needed for the protein band of cadmium.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the oxyphorase inner complex of heavy metal cadmium or is chelated with the BSA inner complex of heavy metal cadmium; Described negative control is the dilution buffer not containing standard substance.
Mentioned reagent box is for detecting the oxyphorase of chelating cadmium, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides the method for a kind of detection by quantitative cadmium-oxyphorase inner complex, using the above-mentioned cadmium-oxyphorase inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification cadmium-oxyphorase inner complex and enzyme linked immunological combined techniques, purification cadmium-oxyphorase inner complex and atomic absorption spectrum combined techniques, purification cadmium-oxyphorase inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention, following several with having of detecting that the method for cadmium-oxyphorase inner complex can list, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative cadmium-oxyphorase inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects cadmium-oxyphorase inner complex, detects in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB A b is diluted to 500-4000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching cadmium is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and be diluted to 12500-100000 anti-Cd Ab doubly by dilution buffer, 37 DEG C of effect 1-2 hour, make the cadmium metal on anti-Cd Ab and oxyphorase react and form immune complex;
6) enzyme conjugates incubation: remove Cadmium resistance antibody, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
The method utilizes ELISA principle, specificity oxyphorase in whole blood can be extracted, the oxyphorase upper part extracted is chelated with heavy metal cadmium, and the cadmium on this part oxyphorase catch by Cadmium resistance antibody, afterwards can again by horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase is caught (this antibody nonrecognition coating protein), the antibody of catching is under the effect of developer and stop buffer, OD value can be read under instrument, and the oxyphorase not containing chelated mineral cadmium, then can not catch by the specific antibody of Cadmium resistance, also can not with horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase caught, and also not containing cadmium metal (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 cadmium metal detecting chelating on oxyphorase.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect cadmium-oxyphorase inner complex and detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 500-4000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rpm, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) detect: sample from the micropore of elisa plate, detect the cadmium of chelating on oxyphorase in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value;
In conjunction with atomic absorption spectrum (AAS) principle on the basis that the method utilizes ELISA principle, Atomic Absorption Spectroscopy AAS is utilized to detect the cadmium of chelating on oxyphorase, owing to only containing oxyphorase in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable cadmium metal detecting chelating on oxyphorase.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect cadmium-oxyphorase inner complex and detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 500-4000 doubly, add in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add elutriant, wash-out 1-3 hour at 37 DEG C.
6) acidifying: in step 5) in solution in add souring agent acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) elisa plate solution get 0.5ml liquid, under icp ms, detect chelating in the cadmium of oxyphorase, and drawing standard curve, readout value.
The method is on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, the cadmium of chelating on oxyphorase is detected with icp ms, owing to only containing oxyphorase in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, i.e. the provable cadmium metal detecting chelating on oxyphorase.
method four:purification cadmium-oxyphorase inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect cadmium-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) anti-Cd Ab is coated on solid phase carrier: by dilution buffer, anti-Cd Ab is diluted to 12500-100000 doubly, adds in elisa plate micropore, 4 DEG C of 16-18 hour that spend the night, or 37 DEG C of water-bath 1-3 hour, store refrigerator;
3) close: remove dilution buffer, and wash with washings, after washing completes, add using 1%-5% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C, after having washed, elisa plate places 1-2 hour in 36.5-37.5 DEG C;
4) add measuring samples, and incubation: from step 1) solution sample, make measuring samples; Standard substance are made with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 10-40 doubly, adds in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: remove step 4) in measuring samples, and to wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/mL, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
6) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
7) termination reaction: stop buffer is dropped to each micropore;
8) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in microplate reader and detects, read the OD value of measuring samples and standard substance respectively, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by colour developing situation) of measuring samples.
method five:purification cadmium-oxyphorase inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect cadmium in blood sample-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) detect: from step 1) sample the solution that obtains, detect the cadmium of chelating on oxyphorase in Atomic Absorption Spectroscopy AAS, and drawing standard curve, readout value.
method six:purification cadmium-oxyphorase inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect cadmium-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) acidifying: from step 1) sample the solution that obtains, add souring agent (as nitric acid) in the solution and carry out acidifying to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
3) detect: from step 2) get 0.5ml liquid the solution that obtains, under icp ms, detect the cadmium of chelating on oxyphorase, and drawing standard curve, readout value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) or atomic absorption spectrometry or inductivity coupled plasma mass spectrometry combined techniques (electrophoretic method-ELISA method/AAS method/ICP-MS method) detect cadmium-oxyphorase inner complex, detect in accordance with the following steps:
1) from whole blood, oxyphorase is extracted: adopt the polyoxyethylene glycol PEG precipitator method or ultracentrifugation or the method such as molecule ultrafiltration or gel-filtration from the oxyphorase whole blood extraction purification blood, and redissolve in solution by oxyphorase, obtain hemoglobin solutions;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare glue bed;
3) application of sample: get step 1) 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 cadmium, this band is taken out, this protein band is dissolved among liquid, and then utilize ELISA or ICP-MS or AAS to detect the cadmium content be dissolved in liquid respectively.In addition, the iso-electric point of the oxyphorase of this method detection chelating cadmium, molecular weight and content etc. can also be utilized.
After oxyphorase in method seven discharges from red corpuscle can with multiple Methods For Purification out (such as ultracentrifugation or HPLC or gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the oxyphorase of purifying out is redissolved in solution, get a certain amount of oxyphorase, utilize electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), can according to molecular weight on gel slab (different media can be adopted as required), the difference such as iso-electric point runs out of different bands, find out the respective strap being rich in cadmium, protein in gel is redissolved in solution, namely the content of relevant oxyphorase can be detected at a particular wavelength, also the principle such as ELISA or AAS or ICP-MS can be utilized to detect the cadmium content of chelating on oxyphorase, owing to only containing oxyphorase in solution, and not containing any heavy metal (negative control group result is negative) in agents useful for same, interference can not be caused to result, thus when read result is shown as the positive, the i.e. provable cadmium metal detecting chelating on oxyphorase.
Below in conjunction with embodiment, the present invention is further described.
embodiment 1: the preparation method of cadmium-oxyphorase inner complex, comprises the following steps:
A) chelatropic reaction of cadmium and oxyphorase: add cadmium ion and carry out chelatropic reaction in the oxyphorase in people source, obtain reaction soln;
Preparation of reagents:
1) borate buffer solution (0.01M): take 0.31g boric acid and be dissolved in 400ml ultrapure water, regulates pH to 9.0 with the NaOH of 0.1M, is settled to 500mL.
2) hemoglobin solutions: take 4.0mg oxyphorase and be dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, dissolving of fully vibrating, is mixed with the protein solution of 1.0mg/mL;
3) EDTA-NaHCO3 mixing solutions: take EDTA2H 2o 1.86g, NaHCO 316.8g is dissolved in 900mL ultrapure water, adjusts pH to 8.0 be settled to 1000ml, autoclaving, room temperature preservation with 1.0M NaOH;
4) ITCBE buys from Japanese colleague's chemistry institute, and article No. is M030;
5) dialysis tubing is purchased from Bioshop Inc, molecular weight cut-off 14000.
The chelation step of cadmium-oxyphorase inner complex:
1) process of dialysis tubing: EDTA-NaHCO dialysis tubing being put into 500ml 3in mixing solutions, boil 10min; Tipping EDTA/NaHCO 3liquid, with ultrapure water rinsing gently, then boils 10min with the 5mmol/LEDTA solution of 500ml; Discard boiling liquid, thoroughly clean with ultrapure water, add ultrapure water immersion dialysis tubing 4 DEG C and spend the night.During use, put on one's gloves, take out dialysis tubing, with its surfaces externally and internally of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg oxyphorase is dissolved in 4.0ml borate buffer solution;
4) slowly by step 2) liquid prepared adds step 3) in the hemoglobin solutions prepared, dropping limit, limit is shaken, and being placed in temperature is 25 DEG C, and rotating speed is react 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, remove the ITCBE be not combined with oxyphorase;
5) by step 4) the liquid 1mol/L HCl adjust ph to 7.0 of gained, then slowly drip the 1mmol/L cadmium-ion solution of 80 μ l gradually, dropping limit, limit vibrates, in order to avoid cadmium ion makes protein denaturation precipitate;
6) by step 5) to be placed in temperature be 25 DEG C for the solution of gained, rotating speed is react 2h in the shaking table of 100r/min, carries out dialysis 24h with dialysis tubing;
7) by step 6) dialysis after liquid preserve in-20 DEG C of packing, obtain reaction soln.
B) extraction of purifying cadmium-oxyphorase inner complex: adopt immune-affinity chromatography, remove reaction soln (the i.e. step 7) reaction soln that obtains) in unreacted oxyphorase and unnecessary cadmium ion, obtain cadmium-oxyphorase inner complex, concrete steps are as follows:
(1) sample dissolution: to through step 7) add physiological saline in the reaction soln that obtains cadmium-oxyphorase inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of oxyphorase specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), oxyphorase and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I;
(6) dialyse: by the elutriant I of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of cadmium specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant II of the collection in step (10), dress dialysis tubing, with 2 liters of ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain cadmium-oxyphorase inner complex;
C) to the qualification of cadmium-oxyphorase inner complex, concrete steps are as follows:
(1) glue bed is prepared: prepare glue bed using sepharose as medium;
(2) application of sample: get step B) in cadmium-oxyphorase inner complex of obtaining of 8 μ L extraction purification, add 2 μ L sample-loading buffers, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, add electrophoretic buffer and carry out electrophoresis; In electrophoresis process, electric current is 22mA constant current, and envrionment temperature is 4 DEG C; Electrophoresis stops electrophoresis to when moving to bottom glue to tetrabromophenol sulfonphthalein;
(4) detect: on glue bed, find out the protein band containing cadmium, 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 cadmium and detection cadmium.
D) detected result
(1) heavy metal content in graphite furnace atomic absorption spectrometry (AAS) rough determination HB: the results are shown in Table 1
Table 1 is heavy metal content in oxyphorase (μ g/L)
Sample name Cd(μg/L)
HB 167.78
(NH 4) 2SO 4 0.05
N.S 0.04
N.S 0
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of cadmium of the present invention-oxyphorase inner complex.
Native polyacrylamide gel electrophoresis (Native-PAGE) or be called that active electrophoresis is under the condition not adding the denaturing agent such as SDS and thin base ethanol, to keeping active protein to carry out polyacrylamide gel electrophoresis, be usually used in the qualification of enzyme, Isozyme Analysis and purification.The native polyacrylamide gel electrophoresis not adding SDS can make biomacromolecule in electrophoresis process, keep its natural shape and electric charge, their separation is the molecular sieve effect of difference according to its electrophoretic mobility and gel, thus higher resolving power can be obtained, especially after electrophoretic separation, still can keep the biological activity of the biomacromolecule such as protein and enzyme, therefore left lane M is sex change Marker, only for detecting, do not mark effect, swimming lane Hb is hemoglobin electrophoresis band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of Beijing electron positron collider (BEPC).In storage ring, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X-ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.LS 30143-DS), probe is mutually vertical with incident SR line copline, apart from sample irradiation point 20mm, signal obtains with PGT MCA (MCA4000) and exports.By the monochromatic synchrotron radiation light excited sample of 11.5keV, launching spot (1mmx3mm) position is regulated to make it to be in band one end, in the minute of 300s, hot spot is always along band evenly slowly movement, and at the end of counting, hot spot moves on to this band the other end.A spectrum is got along the every 1mm in electrophoresis direction.Adopt AX IL software data processing, and with deriving from air and the Ar fignal center of content constant carries out normalization to other element peak, to offset the impact that beam intensity change produces signal power.Measure the fluorescence Spectra of the dry glued membrane of quantitative criterion at identical conditions in the same way.
Fig. 2 is the synchrotron radiation X line fluorometric analysis figure of the electrophoretic band of cadmium of the present invention-oxyphorase inner complex, and in figure, X-coordinate is protein band position, and ordinate zou is each metal energy of this band (content) value.
the determination of the testing conditions of the method for a kind of detection by quantitative cadmium-oxyphorase inner complex of the present invention:
1. the determination of anti-hemoglobin antibodies, the anti-optimum diluting multiple every antibody best effort concentration and whole blood
Step is as follows:
(1) be that 1:500,1:1000,1:2000,1:4000 dilute by anti-HB Ab dilution buffer according to the mass volume ratio of anti-HB Ab and dilution buffer, add in elisa plate micropore, anti-HB Ab is coated on solid phase carrier, each concentration bag is by three rows, and 4 DEG C are spent the night 18 hours;
(2) remove dilution buffer, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) measuring samples is that 1:10,1:20,1:40 dilute by the mass volume ratio of measuring samples and dilution buffer, add in micropore, according to the anti-HB Ab concentration of above-mentioned bag quilt, the anti-HB Ab of same concentration adds different extent of dilution whole blood respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with washings, after washing completes, add anti-Cd Ab, anti-Cd Ab is that 1:12500,1:25000,1:50000,1:100000 dilute by the mass volume ratio of anti-Cd Ab and dilution buffer, and according to each identical anti-HB Ab, whole blood weaker concn, the anti-Cd Ab of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the cadmium metal on anti-Cd Ab and HB are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2 μ g/mL, removes anti-Cd antibody, and wash with washings, after washing completes, add with dilution buffer dilution HRP enzyme labelled antibody, 37 DEG C act on 1 hour, HRP enzyme labelled antibody and cadmium-oxyphorase inner complex are reacted;
(6) remove enzyme labelled antibody, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and detects, read each hole OD value respectively.According to each hole OD value numerical value, select anti-HB Ab, the best effort concentration of anti-Cd Ab and the optimum diluting multiple of whole blood.
Simultaneously using made standard substance as positive control in test, select anti-HB Ab+ close+anti-Cd Ab+ enzyme mark+substrate (namely not adding measuring samples) is as negative control 1; Anti-HB Ab+ closes+and whole blood+enzyme mark+substrate (namely not adding anti-Cd Ab) is as negative control 2; Anti-HB Ab+ closes+and enzyme mark+substrate (namely not adding measuring samples and anti-Cd Ab) is as negative control 3; Anti-HB Ab+ closes+and whole blood+anti-Cd Ab+ substrate (i.e. not enzyme-added mark) is as negative control 4; Closed+whole blood+anti-Cd Ab+ enzyme mark+substrate (namely do not add anti-HB Ab and catch HB) is as blank 1; Only add substrate as blank 2 and PBS as blank 3.Detected result is in Table 2-3.
Table 2: Checkerboard titration method determines the data (each data are all mean value) of anti-HB Ab, the best effort concentration of anti-Cd Ab and the optimum diluting multiple of whole blood
Table 3:ELISA positive control and negative control ELISA detected result
By table 2-3 data presentation, when we can find out, and the extent of dilution as anti-HB Ab is 1:1000, whole blood extent of dilution be the anti-extent of dilution of 1:20, Cd is 1:12500, OD value is maximum, so select concentration corresponding to this value as best effort concentration.
2. the determination of elutriant best effort concentration and elution time in method two, three in the method for detection by quantitative cadmium-oxyphorase inner complex
Step is as follows: (1) bag quilt: anti-Cd antibody dilution buffer be diluted to 12500 times (mass volume ratios), 37 DEG C of water-baths 3 hours, stores refrigerator;
(2) close: remove dilution buffer, and wash with washings, after washing completes, with 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid, and wash with washings for 37 DEG C;
(3) remove confining liquid, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, it is 2 μ g/mL that enzyme labelled antibody is diluted to concentration, and 37 DEG C act on 2 hours, itself and anti-Cd Ab are reacted;
(4) prepare elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 100ng/ml, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min;
(5) enzyme labelled antibody is removed, by dilution buffer, elutriant is diluted, make the concentration of the papoid in elutriant and the ratio of the concentration of enzyme labelled antibody be 1:80,1:40,1:20,1:10,1:5 (wherein each concentration does 3 multiple holes), be positioned over wash-out 1h, 2h, 3h at 37 DEG C of temperature respectively;
(6) remove elutriant, and wash with washings, after washing completes, add substrate, 37 DEG C of lucifuge effects 30 minutes;
(7) stop buffer is dropped to each micropore;
(8) get 405nm wavelength, after adding stop buffer, be placed in by elisa plate in microplate reader and detect, read respectively and often organize OD value, by comparing with PBS damping fluid group, compare optimal concentration and the elution time of elutriant, concrete outcome is see table 4.
Table 4:ELISA elutriant best effort concentration and elution time are determined
From table 4, we can find, as the ratio=1:20 of the concentration of the papoid in elutriant with the concentration of enzyme labelled antibody, each group of OD value is all lower than other several groups, this concentration elution effect optimum (reach maximum by the anti-Cd Ab-enzyme mark mixture wash-out degree that ELISA hole wall combines, thus OD value is minimum) is described; And no matter elution time is 1h, 2h, 3h, the change of each group OD value is all little, as seen along with the prolongation of time, enzyme activity weakens gradually, when enzyme concn is constant, extends digestion time and can not improve digestibility, so the elution time of elutriant is that 1-3h all can in this test.
application Example 1
Get and adopt ELISA method to detect cadmium-oxyphorase inner complex in 100 parts of sample whole bloods, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects cadmium-oxyphorase inner complex, detect in accordance with the following steps:
1) anti-HB Ab is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 times, adds in elisa plate micropore, 37 DEG C of water-baths 1 hour, store refrigerator;
2) get whole blood from the recycle system, make measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) material can catching cadmium is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and dilute 12500 times by dilution buffer, 37 DEG C act on 1 hour, the cadmium metal on anti-Cd Ab and oxyphorase are reacted;
6) enzyme conjugates incubation: remove Cadmium resistance antibody, and wash with washings, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, itself and enzyme labelled antibody are reacted;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: stop buffer is dropped to each micropore;
9) getting wavelength is 405nm, after adding stop buffer, is placed in by elisa plate in microplate reader and detects, and read measuring samples OD value (also can not use microplate reader, directly carry out qualitative detection by colour developing situation) respectively, concrete detected value is as shown in table 5.
The ELISA detected result of cadmium-oxyphorase inner complex in table 5:100 part blood sample
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the cadmium-oxyphorase inner complex in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the recycle system, make measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin or skim-milk as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) detect: sample from the micropore of elisa plate, detect the cadmium of chelating on oxyphorase in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 6.
The AAS detected result of cadmium-oxyphorase inner complex in table 6:100 part blood sample
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the cadmium-oxyphorase chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of oxyphorase can be caught, as anti-hemoglobin antibodies (anti-HB Ab) is coated on solid phase carrier: by dilution buffer, anti-HB Ab is diluted to 1000 times, adds in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get 100 parts of standard blood samples as measuring samples, add deionized water by whole blood, then add erythrocyte cracked liquid, the centrifugal 5-8 minute of 1000rmp, centrifugally discard precipitation;
3) close: remove dilution buffer, and wash with washings, after washing completes, add 2% bovine serum albumin as confining liquid, place 1 hour, remove confining liquid for 37 DEG C, and wash with washings, after having washed, elisa plate is placed 1 hour in 37 DEG C;
4) add measuring samples, and incubation: add step 2) in measuring samples, make standard substance with the cadmium of known content-oxyphorase inner complex; By dilution buffer, measuring samples is diluted to 20 times, adds in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, add the elutriant that Papain enzyme concn is 100ng/ml, wash-out 1-3 hour;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, and thorough acidifying, adds hydrogen peroxide, and acid is caught up with in heating;
7) detect: from step 6) sample the solution that obtains, under icp ms, detect chelating in the cadmium of oxyphorase, detected value is as shown in table 7.
The ICP-MS detected result of cadmium-oxyphorase inner complex in table 7:100 part blood sample
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.

Claims (8)

1. cadmium-oxyphorase inner complex, is characterized in that, cadmium ion and oxyphorase by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for cadmium as claimed in claim 1-oxyphorase inner complex, is characterized in that, comprise the following steps:
A) chelatropic reaction of cadmium and oxyphorase: add cadmium ion in the oxyphorase coming from human body or the oxyphorase of recombinating according to biological method and carry out chelatropic reaction purifying, obtain reaction soln;
B) extraction of purifying cadmium-oxyphorase inner complex: adopt immune-affinity chromatography, removes unreacted oxyphorase and unnecessary cadmium ion in reaction soln, obtains cadmium-oxyphorase inner complex.
3. preparation method according to claim 2, is characterized in that,
Described step B) specific as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains cadmium-oxyphorase inner complex is redissolved;
(2) balance chromatography column: use dilution buffer to rinse the pipeline of chromatography column, load in chromatography column can with the filler of oxyphorase specific binding, after dress post, continue to use dilution buffer balance chromatography column;
(3) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (1), oxyphorase and filler specific binding;
(4) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.05-0.1mol/L to carry out wash-out, obtain elutriant I;
(5) collect: collect elutriant I
(6) dialyse: by the elutriant I of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of cadmium specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out, obtain elutriant II;
(10) collect: collect elutriant II;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, after changing water three times, at 4 DEG C of dialysed overnight nights, collects sample, obtains cadmium-oxyphorase inner complex.
4. the preparation method of cadmium according to claim 2-oxyphorase inner complex, is characterized in that, also comprise step C): to the qualification of cadmium-oxyphorase inner complex;
Wherein, step C) in specific as follows:
(1) glue bed is prepared: prepare glue bed using the one in sepharose, polyacrylamide gel as medium;
(2) application of sample: get step B) in cadmium-oxyphorase inner complex of obtaining of extraction purification, add dilution buffer, and mix, then application of sample is in sample cell;
(3) electrophoresis: connect electrophoresis plate, carry out electrophoresis;
(4) detect: on glue bed, find out the protein band containing cadmium, 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 cadmium and detection cadmium.
5. the application of cadmium as claimed in claim 1-oxyphorase inner complex in the reagent or test kit preparing to detect cadmium-oxyphorase inner complex in blood sample.
6. one kind at least comprises the test kit of cadmium as claimed in claim 1-oxyphorase inner complex as standard substance.
7. test kit according to claim 6, is characterized in that, also comprises coating buffer, and this coating buffer contains the material of catching oxyphorase or the material of catching cadmium.
8. the method for detection by quantitative cadmium-oxyphorase inner complex, it is characterized in that, using the cadmium according to claim 1-oxyphorase inner complex of known content as standard substance, a pair sample of following methods is adopted to detect: euzymelinked immunosorbent assay (ELISA), enzyme linked immunological and atomic absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification cadmium-oxyphorase inner complex and enzyme linked immunological combined techniques, purification cadmium-oxyphorase inner complex and atomic absorption spectrum combined techniques, purification cadmium-oxyphorase inner complex and inductivity coupled plasma mass spectrometry combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
CN201510413021.0A 2015-07-14 2015-07-14 Cadmium-hemoglobin chelate as well as preparation method and application thereof Pending CN104987401A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510413021.0A CN104987401A (en) 2015-07-14 2015-07-14 Cadmium-hemoglobin chelate as well as preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510413021.0A CN104987401A (en) 2015-07-14 2015-07-14 Cadmium-hemoglobin chelate as well as preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN104987401A true CN104987401A (en) 2015-10-21

Family

ID=54299316

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510413021.0A Pending CN104987401A (en) 2015-07-14 2015-07-14 Cadmium-hemoglobin chelate as well as preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN104987401A (en)

Citations (1)

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

Patent Citations (1)

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

Non-Patent Citations (2)

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

Similar Documents

Publication Publication Date Title
CN105137059B (en) A kind of hydrargyrum chelating type immune complex and its preparation method and application
CN104987400A (en) Mercury-hemoglobin chelate as well as preparation method and application thereof
CN105021548B (en) A kind of arsenic fibrinogen chelate and its preparation method and application
CN104987390A (en) Cadmium-albumin chelate as well as preparation method and application thereof
CN104987401A (en) Cadmium-hemoglobin chelate as well as preparation method and application thereof
CN105001321A (en) Mercury-fibrinogen chelate and preparation method therefor and application thereof
CN104987411A (en) Cadmium-IgA chelate as well as preparation method and application thereof
CN104987402A (en) Lead-hemoglobin chelate as well as preparation method and application thereof
CN104987412A (en) Arsenic-IgE chelate as well as preparation method and application thereof
CN104987409A (en) Lead-IgG chelate as well as preparation method and application thereof
CN105004684A (en) Nickel-IgE chelate and its preparation method and use
CN105001324A (en) Chromium-haemoglobin chelate and preparation method therefor and application thereof
CN104987403A (en) Nickel-hemoglobin chelate as well as preparation method and application thereof
CN104987407A (en) Cadmium-IgG chelate as well as preparation method and application thereof
CN104987387A (en) Chromium-fibrinogen chelate as well as preparation method and application thereof
CN105037533A (en) Arsenic-ferrohemoglobin chelate, and preparation method and application of chelate
CN104987414A (en) Nickel-IgM chelate as well as preparation method and application thereof
CN104987415A (en) Chromium-IgM chelate as well as preparation method and application thereof
CN104987404A (en) Nickel-IgG chelate as well as preparation method and application thereof
CN104987388A (en) Lead-albumin chelate as well as preparation method and application thereof
CN105044004B (en) A kind of mercury IgM chelates and its preparation method and application
CN105044006B (en) A kind of mercury IgG chelates and its preparation method and application
CN104987391A (en) Mercury-albumin chelate as well as preparation method and application thereof
CN105037536A (en) Cadmium-IgE chelate and preparation method therefor and application thereof
CN104961824A (en) Lead and high-density lipoprotein chelate as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20151021