CN104987415A - Chromium-IgM chelate as well as preparation method and application thereof - Google Patents

Chromium-IgM chelate as well as preparation method and application thereof Download PDF

Info

Publication number
CN104987415A
CN104987415A CN201510413333.1A CN201510413333A CN104987415A CN 104987415 A CN104987415 A CN 104987415A CN 201510413333 A CN201510413333 A CN 201510413333A CN 104987415 A CN104987415 A CN 104987415A
Authority
CN
China
Prior art keywords
igm
chromium
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
CN201510413333.1A
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 CN201510413333.1A priority Critical patent/CN104987415A/en
Publication of CN104987415A publication Critical patent/CN104987415A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (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)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a chromium-IgM chelate as well as a preparation method and an application thereof. The chromium-IgM chelate is formed by chelating chromium ions and IgM by virtue of sulfydryl or/and cysteine residues. The invention establishes a quantitative and qualitative detection method of the chromium-IgM chelate, so as to perform a quantitative detection on the application of the chromium-IgM chelate in evaluating the chromium pollution level in an area. By performing the quantitative detection on the chromium-IgM chelate in sera of a group of people in the area, the condition of chromium-polluted people in the area can be indirectly reflected, so as to indirectly reflect the chromium pollution level of the area. The chromium-IgM 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 chromium-IgM inner complex and its preparation method and application
Technical field
The present invention relates to detection field, more particularly, relate to a kind of chromium-IgM inner complex and its preparation method and application.
Background technology
In serum, IgM connects into pentamer by 5 monomers by a J chain and disulfide linkage, and molecular weight is maximum, and be 970kD, settling ratio is 19S, is called macroglobulin (macroglobulin).The hingeless sequence of IgM on molecular structure, C μ 2 may instead of the function of hinge area.In organic evolution process, IgM is the immunoglobulin (Ig) occurred the earliest.In ontogenetic process, no matter be B cell Surface Ig (SmIg), or synthesis secretion is all the Ig occurred the earliest to the Ig in serum, IgM, at the fetus i.e. capable generation IgM in fetal development late period.In antigenic stimulation elicit humoral immune answering, general IgM also produces at first.IgM accounts for 5% ~ 10% of serum total Ig.Because IgM produces in early days in immunne response, and hemolytic action under complement participates in is stronger more than 500 times than IgG, and plays opsonization by the fragment such as C3b, C4b after complement activation, and therefore IgM occupies critical role in the early immune protection of body.Natural blood group antibody (lectin) is IgM, the blood transfusion that blood group is not inconsistent, and easily serious hemolytic reaction occurs.IgM can not cross placenta, as there is the IgM for certain pathogenic micro-organism in bleeding of the umbilicus, representing there is corresponding pathogenic micro-organism embryonic stage as infection such as treponema pallidum, rubella or giant cells poison, being called fetal infection or vertical infection.Also containing output monomer I gM in normal human serum.
Chromium (chromium, Cr) be a kind of transition metal in the periodic table of elements VI race, its quality is hard, surface has gloss, there is higher fusing point, there is great economic worth in the industrial production, be widely used since the century, annual global chromium turnout is up to 107 tons, and thus chromium is a kind of genuine important meals pollutent.At present, chromium is widely used in all respects such as plating, tanning, pigment, paint, alloy, printing and dyeing, closely bound up with the life of people, but equally also has an immense impact on to the healthy of the mankind.In the U.S., Cr and Hg, Cd, Pb are called as four overall situation pollutents.According to statistics, have the common people of tens hundred million every year all at the water source drinking pollution of chromium, and markon's welfare Asia have up to 30% water resources all by pollution of chromium, thus pollution of chromium drastically influence the healthy of people.
Occurring in nature chromium mainly exists with the form of trivalent and sexavalence, and it is generally acknowledged, trivalent chromium is the trace element of necessary for human, and sexavalent chrome is only obvious toxicant.Because sexavalent chrome easily enters cell by cytolemma, and can series reaction be passed through, produce strong cytotoxicity and genotoxicity, thus generally believe that chromic toxicity is higher than trivalent chromium, reaches 10-100 times.Scholar thinks when namely the content of 6-valence Cr ions in water is poisonous more than 0.05mg/L.Sexavalent chrome can be drunk by skin contact, food intake, tap water and enter human body into approach such as, respiratory tract suctions, mainly sucking sexavalent chrome dust by respiratory tract for occupational exposed population group, is then take in sexavalent chrome by drinking into polluted source, the modes such as contaminated food, suction of eating for general population.Sexavalent chrome enters after in body, can initiated oxidation stress, chromosome aberration, apoptosis, multiple DNA damage (comprising single-strand break, DNA-protein cross, DNA-amino acid crosslinks, the formation of Cr-DNA title complex etc.), thus cause body many places tissue, organ, system injury.Moreover, sexavalent chrome is also considered to a kind of strong carcinogens, as far back as nineteen ninety international cancer research organization (InternationalAgency for Research on Cancer, IARC) just using sexavalent chrome as a kind of human carcinogen.The problem of current extensive research has been become about chromic cytotoxicity, genotoxicity, carinogenicity.
About chromium poisoning, the particularly evaluation of chronic chromium poisoning, only by detecting blood chromium content, circulation chromium content in indirect reaction human body, cannot assess the degree of injury of chromium for body function further, and along with the develop rapidly of science and technology, the relation of chromium and human body is also day by day tight, thus find one to become more and more important from the evaluation of body function angle chromium poisoning, particularly chronic chromium poisoning for the evaluation method of the degree of damage of body.
Summary of the invention
For the problem that pollution of chromium is serious, the object of the present invention is to provide a kind of chromium-IgM inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of chromium-IgM inner complex, so that detection by quantitative chromium-IgM inner complex is in the application of the regional pollution of chromium degree of evaluation one.Indirectly can reflect the situation of this regional crowd by pollution of chromium by chromium-IgM inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional pollution of chromium degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of chromium-IgM inner complex, chromium ion and IgM by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned chromium-IgM inner complex, comprises the following steps:
A) chelatropic reaction of chromium and IgM: add chromium ion and carry out chelatropic reaction in the IgM in people source, obtain reaction soln;
B) extraction of purifying chromium-IgM inner complex: adopt immune-affinity chromatography, removes unreacted IgM and unnecessary chromium ion in reaction soln, obtains chromium-IgM 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, chromium-IgM 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 IgM 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), IgM 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;
(5) collect: collect the elutriant after step (4) wash-out, after collection, make protein renaturation immediately;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium 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;
(10) collect: collect the elutriant after step (9) wash-out, after collection, make protein renaturation immediately;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-IgM inner complex.
Wherein, the preparation method of above-mentioned chromium-IgM inner complex, also comprises step C): to the qualification of chromium-IgM 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 the chromium-IgM inner complex that obtains 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 chromium, this protein band is taken out, protein band is redissolved, and then adopt ICP-MS method, AAS method or ELISA method to detect the content whether containing chromium and detection chromium.
The present invention also provides the application of a kind of chromium-IgM inner complex described above in preparation human body in the reagent of chromium-IgM inner complex or test kit.
The present invention also provides a kind of test kit at least comprising chromium-IgM inner complex described above product in contrast.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the albumen can catching IgM or the material of catching chromium metal.
The present invention also provides a kind of method of detection by quantitative chromium-IgM inner complex, with the above-mentioned chromium-IgM inner complex product in contrast of known content, 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 chromium-IgM inner complex and enzyme linked immunological combined techniques, purification chromium-IgM inner complex and atomic absorption spectrum combined techniques, purification chromium-IgM 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 chromium-IgM inner complex of the present invention and its preparation method and application, there is following beneficial effect:
1. the present invention's external synthesis chromium-IgM inner complex first;
2. the present invention proposes chromium-IgM inner complex first and can be used for preparing application in the reagent or test kit detecting chromium-IgM inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of chromium-IgM inner complex, so that detection by quantitative chromium-IgM inner complex is in the application of the regional pollution of chromium degree of evaluation one.Indirectly can reflect the situation of this regional crowd by pollution of chromium by chromium-IgM inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional pollution of chromium degree.Its accuracy of chromium-IgM inner complex quantitative detecting method that the present invention sets up improves greatly, and the repeatability of detection is greatly enhanced.
Accompanying drawing explanation
Fig. 1 is the non denatured electrophoretic band figure of chromium-IgM inner complex of the present invention;
Fig. 2 is the fluorometric analysis figure of the synchrotron radiation X line electrophoretic band of chromium-IgM inner complex of the present invention;
Wherein, in Fig. 1, M is Marker, and 2 is chromium-IgM inner complex; In Fig. 2, X-coordinate is protein band position, and ordinate zou is chromium metal energy in this protein band.
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:
Extracting reagent is PEG solution, borate buffer solution etc. (adopting PEG method);
Glue bed medium is the one in sepharose, polyacrylamide gel;
In the present invention described can with the filler of IgM specific binding, for surface is with the silica gel of albumen or the resin that can catch IgM;
The albumen (anti-IgM) catching IgM in the present invention, include but not limited to rabbit Anti-mankind IgM H & L, its brand is Abcam, model is ab8505;
In the present invention can with the filler of chromium specific binding, for surface is with the silica gel of material or the resin that can catch chromium;
The material catching chromium in the present invention, includes but not limited to mouse-anti Cr mAb, buy from Guangzhou Ran Ke company, article No. be RK10641;
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;
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 phosphate buffered saline buffer;
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 1-2mg/ml 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 present invention also provides a kind of preparation method of chromium-IgM inner complex, comprises the following steps:
A) chelatropic reaction of chromium and IgM: add chromium ion in the IgM in people source or the IgM that recombinates according to biological method and carry out chelatropic reaction, obtain reaction soln;
B) extraction of purifying chromium-IgM inner complex: adopt immune-affinity chromatography, remove unreacted IgM and unnecessary chromium ion in reaction soln, obtain chromium-IgM inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains, chromium-IgM 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 IgM 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), IgM 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;
(5) collect: collect the elutriant after step (4) wash-out, after collection, make protein renaturation immediately;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium 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;
(10) collect: collect the elutriant after step (9) wash-out, after collection, make protein renaturation immediately;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-IgM inner complex;
C) to the qualification of chromium-IgM 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 the chromium-IgM inner complex that obtains 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 chromium, this protein band is taken out, protein band is redissolved, and then adopt ICP-MS method, AAS method or ELISA method to detect the content whether containing chromium and detection chromium.
The present invention also provides a kind of test kit at least comprising chromium-IgM inner complex described above product in contrast.
Preferably, also comprise coating buffer in this test kit, this coating buffer contains the material that the albumen can catching IgM maybe can catch chromium metal.
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 chromium-IgM inner complex in blood sample, comprising: containing catching the coating buffer of albumen of IgM, confining liquid, washings, material, enzyme labelled antibody, substrate, stop buffer, dilution buffer, sample-loading buffer, positive control, negative control etc. as two anti-caught chromium.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: the coating buffer, confining liquid, washings, elutriant, sample-loading buffer, positive control, negative control etc. that contain the albumen can catching IgM.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: the coating buffer, confining liquid, washings, elutriant, sample-loading buffer, souring agent, hydrogen peroxide, standard substance, negative control etc. that contain the albumen can catching IgM.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: as extracting reagent in purification whole blood needed for IgM, redissolve liquid, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, sample-loading buffer, positive control, negative control etc. of material can catching chromium.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: as extracting reagent, redissolution liquid, sample-loading buffer, positive control, negative control etc. in purification whole blood needed for IgM.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: extract reagent, sample-loading buffer, redissolution liquid, souring agent, hydrogen peroxide, positive control, negative control etc. needed for IgM as in purification whole blood.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: containing the coating buffer of albumen of IgM, glue bed medium can be caught, to redissolve on liquid, sample-loading buffer, dissolving glue bed containing liquid needed for the protein band of chromium, containing the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of material can catching chromium.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: as extracting reagent, glue bed medium in purification whole blood needed for IgM, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, positive control, negative control etc. needed for the protein band of chromium.
Detect a test kit for chromium-IgM inner complex in blood sample, comprising: as extracting reagent, glue bed medium in purification whole blood needed for IgM, redissolve liquid, sample-loading buffer, on dissolving glue bed containing liquid, souring agent, hydrogen peroxide, positive control, negative control etc. needed for the protein band of chromium.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the IgM inner complex of heavy metal chromium or is chelated with the BSA inner complex of heavy metal chromium; Described negative control is dilution buffer.
Mentioned reagent box is for detecting the IgM of chelated chromium, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides a kind of method of detection by quantitative chromium-IgM inner complex, with the above-mentioned chromium-IgM inner complex product in contrast of known content, 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 chromium-IgM inner complex and enzyme linked immunological combined techniques, purification chromium-IgM inner complex and atomic absorption spectrum combined techniques, purification chromium-IgM 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 can listing of the method detecting chromium chelating type immunocomplex, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative chromium-IgM inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-IgM inner complex, detects in accordance with the following steps:
1) material of IgM can be caught, as human IgM antibody is coated on solid phase carrier: dilute anti-IgM Ab to 500-4000 doubly by dilution buffer, 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, 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 chromium-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching chromium is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add with dilution buffer dilution and the material of chromium can be caught or can react with chromium the anti-cr Ab forming immune complex, 37 DEG C of effect 1-2 hour, make the chromium metal on anti-cr Ab and IgM react;
6) enzyme conjugates incubation: remove anti-chromium 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: drip stop buffer to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in OD value microplate reader reading respectively measuring samples and standard substance, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by staining conditions) of measuring samples.
The method utilizes ELISA principle, specific IgM in whole blood can be extracted, the IgM upper part extracted is chelated with heavy metal chromium, and chromium on this part IgM can catch by the specific antibody of anti-chromium, 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 IgM not containing chelated mineral chromium, then can not catch by the specific antibody of anti-chromium, also can not with horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase caught, and also not containing chromium 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 chromium metal detecting chelating on IgM.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect chromium-IgM inner complex and detect in accordance with the following steps:
1) material of IgM can be caught, as human IgM antibody is coated on solid phase carrier: dilute anti-IgM Ab to 500-4000 doubly by dilution buffer, 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, the centrifugal 5-8 minute of 1000rpm, centrifugally discard precipitation;
3) close: remove coating 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 chromium-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, with elution 1-3 hour.
6) detect: sample from ELISA micropore, detect the chromium of chelating on IgM in Atomic Absorption Spectroscopy AAS, and drawing standard curve, read respective value;
In conjunction with atomic absorption spectrum (AAS) principle on the basis that this embodiment utilizes ELISA principle, Atomic Absorption Spectroscopy AAS is utilized to detect the chromium of chelating on IgM, owing to only containing IgM 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 chromium metal detecting chelating on IgM.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect chromium-IgM inner complex and detect in accordance with the following steps:
1) material of IgM can be caught, as human IgM antibody is coated on solid phase carrier: dilute anti-IgM Ab to 500-4000 doubly by dilution buffer, 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, 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 chromium-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
5) wash-out: remove measuring samples, and wash with washings, after washing completes, with elution 1-3 hour.
6) acidifying: in step 5) in solution in add respective acids agent acidifying carried out to solution, sealing is spent the night, thorough acidifying;
7) detect: add hydrogen peroxide, and acid is caught up with in heating, and from ELISA agent plate wash-out solution in sample, under icp ms, detect chelating in the chromium of IgM, and drawing standard curve, read respective value.
The method is on the basis utilizing ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, the chromium of chelating on IgM is detected with icp ms, owing to only containing IgM 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 chromium metal detecting chelating on IgM.
method four:purification chromium-IgM inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect chromium-IgM inner complex, detect in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to adopt whole blood extraction method to be redissolved by the purification of blood IgM out, obtain the redissolution liquid of IgM;
2) anti-cr Ab is coated on solid phase carrier: dilute anti-cr Ab to 5000-40000 doubly by dilution buffer, 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;
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) measuring samples is added, and incubation: sample from the redissolution liquid of the IgM extracted, make measuring samples; Standard substance are made with the chromium-IgM inner complex of known content; Dilute corresponding multiple by dilution buffer, namely dilute 10-40 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
5) enzyme conjugates incubation: the redissolution liquid removing IgM, 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;
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: drip stop buffer to each micropore;
8) wavelength 405nm is got, after adding stop buffer, elisa plate is placed in OD value microplate reader reading respectively measuring samples and standard substance, by drawing standard curve, try to achieve the content (also microplate reader can not be used, directly carry out qualitative detection by staining conditions) of measuring samples.
method five:purification chromium-IgM inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect chromium-IgM inner complex in blood sample, detect in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to be redissolved by the purification of blood IgM out from whole blood extraction method, obtain the redissolution liquid of IgM;
2) detect: from step 1) redissolution liquid in sample, detect the chromium of chelating on IgM in Atomic Absorption Spectroscopy AAS, and drawing standard curve, read respective value.
method six:purification chromium-IgM inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect chromium-IgM inner complex, detect in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to be redissolved by the purification of blood IgM out from whole blood extraction method, obtain the redissolution liquid of IgM;
2) acidifying: from step 1) redissolution liquid in sample, add respective acids agent (as nitric acid) in the solution and carry out acidifying to solution, sealing is spent the night, thorough acidifying;
3) detect: add hydrogen peroxide, and acid is caught up with in heating, and sample from corresponding solution, under icp ms, detect the chromium of chelating on IgM, and drawing standard curve, read respective value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) (electrophoretic method-ELISA method) detect chromium-IgM inner complex, detect in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to be redissolved by the purification of blood IgM out from whole blood extraction method, obtain the redissolution liquid of IgM;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, conventionally prepare corresponding glue bed;
3) application of sample: get step 1) in redissolution liquid 8 μ L add 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 chromium, this band is taken out, by this protein band, and then utilize ELISA principle to detect the chromium content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelated chromium, molecular weight and content etc. can also be utilized.
method eight:electrophoresis and atomic absorption spectrometry (electrophoretic method-AAS method) detect chromium-IgM inner complex, detect in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to be redissolved by the purification of blood IgM out from whole blood extraction method, obtain the redissolution liquid of IgM;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, prepare glue bed;
3) application of sample: from step 1) redissolution liquid in sample, add sample-loading buffer, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: on glue bed, find out the protein band containing chromium, this band is taken out, by this protein band, and then utilize AAS principle to detect the chromium content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelated chromium, molecular weight and content etc. can also be utilized.
method nine:inductivity coupled plasma mass spectrometry combined techniques (electrophoretic method-ICP-MS method) detects chromium-IgM inner complex, detects in accordance with the following steps:
1) from whole blood, IgM is extracted: adopt the method such as the polyoxyethylene glycol PEG precipitator method, ultracentrifugation, molecule ultrafiltration, gel-filtration to be redissolved by the purification of blood IgM out from whole blood extraction method, obtain the redissolution liquid of IgM;
2) glue bed is prepared: select sepharose or polyacrylamide gel or SDS-polyacrylamide gel etc. as medium as required, prepare glue bed;
3) application of sample: from step 1) redissolution liquid in sample, add sample-loading buffer, and mix, then application of sample is in sample cell;
4) electrophoresis: connect electrophoresis plate, carry out electrophoretic separation;
5) detect: on glue bed, find out the protein band containing chromium, this band is taken out, by this protein band, and then utilize ICP-MS principle to detect the chromium content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelated chromium, molecular weight and content etc. can also be utilized.
IgM in method seven to nine can by multiple Methods For Purification out (such as ultracentrifugation, HPLC, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the IgM purified out is redissolved, obtain the redissolution liquid of IgM, get a certain amount of IgM, 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 chromium, the corresponding double solvents of protein in gel is redissolved in solution, namely the content of relevant IgM can be detected at a particular wavelength, also ELISA can be utilized, AAS, the principles such as ICP-MS detect the chromium content of chelating on IgM, owing to only containing IgM 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 chromium metal detecting chelating on IgM.
embodiment 1: the preparation method of chromium-IgM inner complex, comprises the following steps:
A) chelatropic reaction of chromium and IgM: add chromium ion and carry out chelatropic reaction in the IgM 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) IgM solution: take 4.0mgIgM 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) 5mmol/L EDTA+200mmol/LNaHCO 3solution: 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 (buying from Japanese colleague's chemistry institute, article No. M030)
5) dialysis tubing (molecular weight cut-off 14000) (Bioshop Inc)
Preparation process is specially:
1) process of dialysis tubing: 5mmol/L EDTA+200mmol/L NaHCO dialysis tubing being put into 500ml (according to the convertible consumption of beaker volume) volume 3in solution, boil 10min; Tipping EDTA/NaHCO 3liquid, with ultrapure water rinsing gently, then boils 10min with 500ml 5mmol/L EDTA; Discard boiling liquid, thoroughly with ultrapure water cleaning, add a large amount of ultrapure water immersion dialysis tubings 4 DEG C and spend the night.During use, put on one's gloves, take out dialysis tubing, with a large amount of its surfaces externally and internallies of ultrapure water cleaning down;
2) getting 2.0mg ITCBE is dissolved in 2ml DMSO;
3) getting 4.0mg IgM to be dissolved in 4.0ml borate buffer solution in (0.01M pH9.0);
4) liquid slowly step 2 prepared adds in IgM solution, and dropping limit, limit is shaken, and in 25 DEG C, acts on 24h in the shaking table of 100r/min, then to dialyse 24h with dialysis tubing, removes the ITCBE be not combined with IgM;
5) by the liquid 1mol/L HCl adjust ph to 7.0 of dialysis gained, then slowly drip 80 μ l 1mmol/L chromium ion solution gradually, dropping limit, limit vibrates, in order to avoid chromium ion makes protein denaturation precipitate;
6) by the solution that added at 25 DEG C, react 2h in the shaking table of 100r/min, carry out dialysis 24h with the dialysis tubing handled well;
7) liquid after dialysis is preserved in-20 DEG C of packing.
B) extraction of purifying chromium-IgM inner complex: adopt immune-affinity chromatography, remove the liquid after the dialysis of reaction soln (namely in steps A 7)) in unreacted IgM and unnecessary chromium ion, obtain chromium-IgM inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains, chromium-IgM 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 IgM 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), IgM 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;
(5) collect: collect the elutriant after step (4) wash-out, after collection, make protein renaturation immediately;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium 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;
(10) collect: collect the elutriant after step (9) wash-out, after collection, make protein renaturation immediately;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-IgM inner complex;
C) to the qualification of chromium-IgM 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 the chromium-IgM inner complex that obtains 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 degree; Electrophoresis is stopped when moving to bottom glue to tetrabromophenol sulfonphthalein;
(4) detect: on glue bed, find out the protein band containing chromium, this protein band is taken out, protein band is redissolved, and then adopt AAS method to detect the content whether containing chromium and detection chromium.
D) detected result
(1) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of chromium-IgM inner complex of the present invention.
(2) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
In protein band, the SRXRF of micronutrient levels analyzes and " synchrotron radiation bunch completes at the 4W1 of Beijing electron positron collider (BEPC).In storage ring, beam current energy is 2.2GeV, beam intensity 100mA.Sample transfer table (TSA200 type, Beijing stand upright Han Guang company) can move up to change launching spot position along X, Y two-dimensional square under computer-controlled step motor drives, and moving step length is 0.0025mm.The X-ray gone out from electromagnetic radiation is detected by Si (Li) detector (PGT Inc.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 fluorometric analysis figure of the synchrotron radiation X line electrophoretic band of chromium-IgM inner complex of the present invention, and in figure, X-coordinate is protein band position, and ordinate zou is chromium metal energy (content) value in this protein band.
(3) the chromium content in the chromium-IgG inner complex adopting graphite furnace atomic absorption spectrometry (AAS) rough determination the present embodiment to obtain, its content is 173.242 μ g/L.
the determination of the testing conditions of the method for a kind of detection by quantitative chromium of the present invention-IgM inner complex:
1. the determination of the optimum diluting multiple of complement proteins best effort concentration and blood plasma
Step is as follows:
(1) anti-IgM Ab dilution buffer is diluted according to following mass volume ratio (extent of dilution) 1:500,1:1000,1:2000,1:4000, add in elisa plate micropore, anti-IgM 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 and dilution buffer are diluted according to following mass volume ratio (extent of dilution) 1:10,1:20,1:40, add in micropore, according to the anti-IgM Ab concentration of above-mentioned bag quilt, the anti-IgMAb of same concentration adds different extent of dilution blood plasma respectively, 37 DEG C of effects 1 hour;
(4) measuring samples is removed, and wash with washings, after washing completes, add anti-cr Ab, anti-cr Ab and dilution buffer are diluted according to following mass volume ratio (extent of dilution) 1:5000,1:10000,1:20000,1:40000, and according to each identical anti-IgM Ab, serum-dilution concentration, the anti-cr Ab of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the chromium metal on anti-cr Ab and IgM are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2ng/ml, removes anti-cr 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 chromium-IgM 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 dripped to each micropore with the speed identical with adding substrate solution and order;
(8) get wavelength 405nm, after adding stop buffer, elisa plate is placed in microplate reader and reads each hole OD value respectively.According to each hole OD value numerical value, select the best effort concentration of anti-IgM Ab, anti-cr-Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made reference substance as positive control in test, IgM antibody+closed+cr is selected to resist+enzyme mark+substrate (namely do not add and detect sample) as negative control 1, IgM antibody+close+blood plasma+enzyme mark+substrate (namely not adding cr to resist) as negative control 2, IgM antibody+close+enzyme mark+substrate (namely do not add detect sample and cr resist) is as negative control 3, IgM antibody+close+blood plasma+cr is anti-+ and substrate (i.e. not enzyme-added mark) is as negative control 4, close+blood plasma+cr is anti-+ and enzyme mark+substrate (namely not adding IgM antibody) is as blank 1, only add substrate and PBS as blank 2, detected result is in Table 1-2.
Table 1: the determination of anti-IgM Ab and cr antibody best effort concentration and diluted plasma multiple
Table 2:ELISA positive control and negative control ELISA detected result
By table 1-2 data presentation, when we can find out that the extent of dilution when human IgM antibody is 1:500, whole blood extent of dilution be the anti-extent of dilution of 1:20, cr is 1:10000, OD value is maximum, although OD value is less than 0.8, but the negative control group OD value corresponding to it is all less than 0.1, and the positive controls corresponding to it, OD value is greater than 0.8, so select the concentration corresponding to this value, as best effort concentration, (namely human IgM antibody concentration is 1:500, whole blood extent of dilution is the anti-weaker concn of 1:10, cr is 1:10000).
2.ELISA elutriant best effort concentration and time are determined
Step is as follows: human IgM antibody dilution buffer is diluted to 500 times (mass volume ratios) by (1), adds in elisa plate micropore, 37 DEG C of water-baths 3 hours, stores refrigerator;
(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) remove confining liquid, and wash with washings, after washing completes, add the HRP enzyme labelled antibody with dilution buffer dilution, 37 DEG C act on 2 hours, itself and human IgM antibody are reacted;
(4) prepare elutriant: papoid pH 8.0,0.1mol/L Tris-HCI buffer is become 1-2mg/ml, then add 1mmol/L dithiothreitol (DTT) (DTT) 37 DEG C and hatch 30min;
(5) enzyme labelled antibody is removed, by dilution buffer, elutriant is diluted, make the Papain enzyme concn in elutriant: enzyme labelled antibody concentration ratio=1:80,1:40,1:20,1:10,1:5, wherein, each concentration does 3 multiple holes, is 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 dripped to each micropore with the speed identical with adding substrate solution and order;
(8) get 405nm wavelength, after adding stop buffer, be placed in by elisa plate in microplate reader and read often group OD value respectively, by comparing with PBS damping fluid group, compare optimal concentration and the elution time of elutriant, concrete outcome is see table 3.
Table 3:ELISA elutriant best effort concentration and elution time are determined
1:5 1:10 1:20 1:40 1:80
1h 0.281 0.168 0.081 0.114 0.469
2h 0.250 0.115 0.050 0.183 0.438
3h 0.225 0.106 0.100 0.196 0.441
From table 4, we can find, during ratio=the 1:20 of the concentration of the papoid in elutriant with the concentration of enzyme labelled antibody, during the concentration 100ng/ml of i.e. papoid, each group of OD value is all lower than other several groups, this concentration elution effect optimum (reach maximum by the human IgM antibody that ELISA hole wall combines-enzyme mark mixture wash-out degree, thus OD value is minimum) is described; And no matter action 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, extend digestion time can not improve digestibility, so in this experiment the action time of elutriant be that 1-3h all can.
application Example 1
Get and adopt ELISA method to detect chromium-IgM inner complex in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects chromium-IgM inner complex, detect in accordance with the following steps:
1) anti-IgM Ab is coated on solid phase carrier: dilute anti-IgM Ab to 500 times by dilution buffer, 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, then add toluene, dissolved cell film;
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 chromium-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 20 times, add in micropore, 37 DEG C act on 1 hour;
5) material can catching chromium is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and dilute 10000 times by dilution buffer, 37 DEG C act on 1 hour, the chromium metal on anti-cr Ab and IgM are reacted;
6) enzyme conjugates incubation: remove anti-chromium 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: drip stop buffer to each micropore;
9) get wavelength 405nm, after adding stop buffer, elisa plate is placed in the OD value (also can not use microplate reader, directly carry out qualitative detection by staining conditions) microplate reader reading respectively measuring samples and standard substance, detected result is as shown in table 4.
The ELISA detected result of chromium-IgM inner complex in table 4:100 part blood sample
Numbering 1 2 3 4 5 6 7 8 9 10
OD 405 0.634 0.519 0.417 0.571 0.536 0.459 0.707 0.366 0.319 0.475
Numbering 11 12 13 14 15 16 17 18 19 20
OD 405 0.606 0.65 0.389 0.47 0.712 0.391 0.584 0.44 0.459 0.374
Numbering 21 22 23 24 25 26 27 28 29 30
OD 405 0.461 0.397 0.684 0.697 0.535 0.355 0.75 0.733 0.553 0.669
Numbering 31 32 33 34 35 36 37 38 39 40
OD 405 0.512 0.412 0.458 0.663 0.414 0.493 0.603 0.569 0.742 0.621
Numbering 41 42 43 44 45 46 47 48 49 50
OD 405 0.62 0.746 0.704 0.721 0.347 0.459 0.621 0.547 0.506 0.684
Numbering 51 52 53 54 55 56 57 58 59 60
OD 405 0.413 0.63 0.329 0.557 0.656 0.699 0.537 0.49 0.334 0.699
Numbering 61 62 63 64 65 66 67 68 69 70
OD 405 0.495 0.515 0.361 0.555 0.702 0.608 0.651 0.744 0.456 0.515
Numbering 71 72 73 74 75 76 77 78 79 80
OD 405 0.325 0.577 0.541 0.321 0.491 0.46 0.376 0.367 0.616 0.623
Numbering 81 82 83 84 85 86 87 88 89 90
OD 405 0.483 0.725 0.357 0.664 0.375 0.613 0.466 0.413 0.666 0.549
Numbering 91 92 93 94 95 96 97 98 99 100
OD 405 0.621 0.468 0.49 0.508 0.533 0.732 0.48 0.378 0.708 0.341
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the chromium-IgM inner complex in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of IgM can be caught, as anti-IgM (anti-IgM Ab) is coated on solid phase carrier: dilute anti-IgM Ab to 500 times by dilution buffer, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get whole blood from the recycle system, make measuring samples, then add toluene, dissolved cell film;
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 chromium-IgM inner complex of known content; Be diluted to 20 times with dilution buffer dilution measuring samples, add in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and washing with washings, after washing completes, the elutriant that to add with the concentration of papoid be 100ng/ml, wash-out 3 hours;
6) detect: sample from ELISA micropore, detect the chromium of chelating on IgM in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 5.
The AAS detected result of chromium-IgM inner complex in table 5:100 part blood sample
Numbering 1 2 3 4 5 6 7 8 9 10
μg/L 6.541 5.024 4.8 3.625 6.651 16.556 6.798 5.988 4.379 6.984
Numbering 11 12 13 14 15 16 17 18 19 20
μg/L 3.107 13.621 5.456 3.293 13.378 4.384 6.449 15.757 3.505 5.182
Numbering 21 22 23 24 25 26 27 28 29 30
μg/L 3.457 6.345 13.161 6.03 4.27 16.046 6.079 6.934 4.575 6.362
Numbering 31 32 33 34 35 36 37 38 39 40
μg/L 3.135 14.043 4.991 6.165 3.364 15.417 5.167 4.907 6.665 6.317
Numbering 41 42 43 44 45 46 47 48 49 50
μg/L 5.643 13.45 6.034 6.082 16.343 4.741 3.51 4.689 4.72 6.244
Numbering 51 52 53 54 55 56 57 58 59 60
μg/L 16.048 4.341 6.338 5.886 5.051 4.181 15.914 5.057 5.356 3.39
Numbering 61 62 63 64 65 66 67 68 69 70
μg/L 14.238 3.755 6.421 3.547 13.86 6.867 4.978 14.586 6.486 6.946
Numbering 71 72 73 74 75 76 77 78 79 80
μg/L 6.136 14.598 5.732 6.914 3.855 15.942 3.723 4.519 3.751 4.395
Numbering 81 82 83 84 85 86 87 88 89 90
μg/L 13.802 4.516 4.855 13.958 3.323 6.803 13.585 4.562 3.347 14.493
Numbering 91 92 93 94 95 96 97 98 99 100
μg/L 4.633 6.635 6.945 14.814 4.454 6.89 5.657 14.011 4.949 6.964
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the chromium-IgM chelate content in 100 points of sample blood samples, detect in accordance with the following steps:
1) material of IgM can be caught, as anti-IgM (anti-IgM Ab) is coated on solid phase carrier: dilute anti-IgM Ab to 500 times by dilution buffer, add in elisa plate micropore, 4 DEG C are spent the night 18 hours;
2) get 100 parts of standard blood samples as measuring samples, then add toluene, dissolved cell film;
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 chromium-IgM inner complex of known content; Be diluted to 20 times with dilution buffer dilution measuring samples, add in micropore, 37 DEG C act on 1 hour;
5) wash-out: remove measuring samples, and washing with washings, after washing completes, the elutriant that to add with the concentration of papoid be 100ng/ml, wash-out 3 hours;
6) acidifying: in step 5) in solution in add nitric acid acidifying carried out to solution, sealing is spent the night, thorough acidifying;
7) detect: add hydrogen peroxide, and acid is caught up with in heating, and sample from corresponding solution, under icp ms, detect chelating in the chromium of IgM, detected value is as shown in table 6.
The ICP-MS detected result of table 6 100 parts of sample blood sample chromium-IgM inner complexs
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. a chromium-IgM inner complex, is characterized in that, chromium ion and IgM by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for chromium-IgM inner complex as claimed in claim 1, is characterized in that, comprise the following steps:
A) chelatropic reaction of chromium and IgM: people source IgM or the IgM that recombinates according to biological method in add chromium ion and carry out chelatropic reaction, obtain reaction soln;
B) extraction of purifying chromium-IgM inner complex: adopt immune-affinity chromatography, removes unreacted IgM and unnecessary chromium ion in reaction soln, obtains chromium-IgM 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, chromium-IgM 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 IgM 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), IgM 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;
(5) collect: collect the elutriant after step (4) wash-out, after collection, make protein renaturation immediately;
(6) dialyse: by the elutriant of the collection in step (5), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample;
(7) balance chromatography column: adopt new chromatography column, use dilution buffer flushing pipeline, in this chromatography column load can with the filler of chromium 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;
(10) collect: collect the elutriant after step (9) wash-out, after collection, make protein renaturation immediately;
(11) dialyse: by the elutriant of the collection in step (10), dress dialysis tubing, uses ddH 2o dialyses desalination, and after changing water three times, 4 DEG C of dialysed overnight, collect sample, obtain chromium-IgM inner complex.
4. the preparation method of chromium-IgM inner complex according to claim 2, is characterized in that, also comprise step C): to the qualification of chromium-IgM 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 the chromium-IgM inner complex that obtains 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 chromium, this protein band is taken out, protein band is redissolved, and then adopt ICP-MS method, AAS method or ELISA method to detect the content whether containing chromium and detection chromium.
5. the application of chromium-IgM inner complex as claimed in claim 1 in the reagent or test kit preparing to detect chromium-IgM inner complex in blood sample.
6. one kind at least comprises the test kit of chromium-IgM inner complex as claimed in claim 1 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 that the albumen can catching IgM maybe can catch chromium metal.
8. the method for a detection by quantitative chromium-IgM inner complex, it is characterized in that, using the chromium-IgM inner complex according to claim 1 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 chromium-IgM inner complex and enzyme linked immunological combined techniques, purification chromium-IgM inner complex and atomic absorption spectrum combined techniques, purification chromium-IgM 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.
CN201510413333.1A 2015-07-14 2015-07-14 Chromium-IgM chelate as well as preparation method and application thereof Pending CN104987415A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510413333.1A CN104987415A (en) 2015-07-14 2015-07-14 Chromium-IgM chelate as well as preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510413333.1A CN104987415A (en) 2015-07-14 2015-07-14 Chromium-IgM chelate as well as preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN104987415A true CN104987415A (en) 2015-10-21

Family

ID=54299330

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510413333.1A Pending CN104987415A (en) 2015-07-14 2015-07-14 Chromium-IgM chelate as well as preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN104987415A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands
CN101422621A (en) * 2007-10-31 2009-05-06 韩国科学技术研究院 Method for the production of bio-imaging nanoparticles with high yield by early introduction of irregular structure

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022490A1 (en) * 1993-03-29 1994-10-13 Immunomedics, Inc. Conjugates of proteins and bifunctional ligands
CN101422621A (en) * 2007-10-31 2009-05-06 韩国科学技术研究院 Method for the production of bio-imaging nanoparticles with high yield by early introduction of irregular structure

Similar Documents

Publication Publication Date Title
CN104987415A (en) Chromium-IgM chelate as well as preparation method and application thereof
CN104987409A (en) Lead-IgG chelate as well as preparation method and application thereof
CN104987400A (en) Mercury-hemoglobin chelate as well as preparation method and application thereof
CN104987414A (en) Nickel-IgM chelate as well as preparation method and application thereof
CN105004686A (en) Chromium-albumin chelate as well as preparation method and application thereof
CN104961824A (en) Lead and high-density lipoprotein chelate as well as preparation method and application thereof
CN105044004B (en) Mercury-IgM chelate as well as preparation method and application thereof
CN105001324A (en) Chromium-hemoglobin chelate as well as preparation method and application thereof
CN105044007B (en) cadmium-IgM chelate as well as preparation method and application thereof
CN104987412A (en) Arsenic-IgE chelate as well as preparation method and application thereof
CN105021548A (en) Arsenic-fibrinogen chelate as well as preparation method and application thereof
CN105021551A (en) chromium-IgG chelate as well as preparation method and application thereof
CN104987390A (en) Cadmium-albumin chelate as well as preparation method and application thereof
CN105153299A (en) lead-IgM chelate as well as preparation method and application thereof
CN105115914B (en) Cadmium-low density lipoprotein chelate as well as preparation method and application thereof
CN105004684A (en) Nickel-IgE chelate as well as preparation method and application thereof
CN104987404A (en) Nickel-IgG chelate as well as preparation method and application thereof
CN105004683B (en) chromium-IgE chelate as well as preparation method and application thereof
CN105044009B (en) Chromium-very low density lipoprotein chelate and preparation method and application thereof
CN105021554A (en) arsenic-IgM chelate as well as preparation method and application thereof
CN104987407A (en) Cadmium-IgG chelate as well as preparation method and application thereof
CN104987405A (en) Arsenic-IgG chelate as well as preparation method and application thereof
CN104987387A (en) Chromium-fibrinogen chelate as well as preparation method and application thereof
CN104987401A (en) Cadmium-hemoglobin chelate as well as preparation method and application thereof
CN104987402A (en) Lead-hemoglobin 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