CN104987414A - Nickel-IgM chelate as well as preparation method and application thereof - Google Patents
Nickel-IgM chelate as well as preparation method and application thereof Download PDFInfo
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- CN104987414A CN104987414A CN201510413324.2A CN201510413324A CN104987414A CN 104987414 A CN104987414 A CN 104987414A CN 201510413324 A CN201510413324 A CN 201510413324A CN 104987414 A CN104987414 A CN 104987414A
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Abstract
The invention discloses a nickel-IgM chelate as well as a preparation method and application thereof. The nickel-IgM chelate is formed by chelating nickel ions with IgM through sulfydryl or/and cysteine residues. According to the invention, a qualitative and quantitative detection method of the nickel-IgM chelate is established for favorably quantitatively detecting the application of the nickel-IgM chelate to the evaluation of the nickel pollution degree of one region. By quantitatively detecting the nickel-IgM chelates in serums of people in one region, the condition that people in the region are polluted by nickel can be indirectly reflected, and further the nickel pollution degree of the region is indirectly reflected. According to the quantitative detection method for the nickel-IgM chelate, established by the invention, the accuracy is greatly improved, and the detection repeatability is greatly improved.
Description
Technical field
The present invention relates to detection field, more particularly, relate to a kind of nickel-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 (maNioglobulin).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.
Nickel common are noxious metals as one, is widely used among life, production, comprises and produces coin, jewelry, nickelalloy stainless steel, manufacture Ni-Cd battery etc., closely bound up with the life of people.Along with the progress of science and technology, people using nickel as catalyzer for the production of among the production of carbon nano-particle, deepened again the impact of nickel for the mankind further.For general population, nickel mainly takes in nickel by eating, drinking into modes such as, contacts, and certain smoking is also an important channel.Research finds, excessive nickel can cause body many places tissue and organ damage, cause the generation of various diseases, comprise respiratory system disease (pneumonia, bronchitis, pulmonary fibrosis, asthma, pulmonary edema etc.), cardiovascular disorder, kidney disease, dermatosis etc., even also can lead oncogenic generation.
Epidemiologic data shows, the workman of Long Term Contact nickel, and it suffers from probability of tumor in respiratory system increases greatly, and it also increases greatly because of the probability that tumor in respiratory system is lethal.The test of animal model, external model also finds, nickel can the generation of induced tumor.Nineteen ninety, international cancer research institution (International Agency forResearch on Cancer, IARC) using nickel compound as first kind carcinogenic substance (Group 1), using metallic nickel as 2B class carcinogenic substance (Group 2B).Visible, the health of nickel serious threat human body.
About nickel poisoning, the particularly evaluation of chronic nickel poisoning, only by detecting blood nickel content, circulation nickel content in indirect reaction human body, cannot assess the degree of injury of nickel for body function further, and along with the develop rapidly of science and technology, the relation of nickel 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 nickel poisoning, particularly chronic nickel poisoning for the evaluation method of the degree of damage of body.
Summary of the invention
For the problem that nickel contamination is serious, the object of the present invention is to provide a kind of nickel-IgM inner complex and preparation method thereof, and set up the qualitative and quantitative analysis method of nickel-IgM inner complex, so that detection by quantitative nickel-IgM inner complex is in the application of the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by nickel-IgM inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional nickel contamination degree.
The technical solution adopted for the present invention to solve the technical problems is: provide a kind of nickel-IgM inner complex, nickel ion and IgM by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of above-mentioned nickel-IgM inner complex, comprises the following steps:
A) chelatropic reaction of nickel and IgM: add nickel ion and carry out chelatropic reaction in the IgM in people source, obtain reaction soln;
B) extraction of purifying nickel-IgM inner complex: adopt immune-affinity chromatography, removes unreacted IgM and unnecessary nickel ion in reaction soln, obtains nickel-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, nickel-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 nickel specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(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 nickel-IgM inner complex.
Wherein, the preparation method of above-mentioned nickel-IgM inner complex, also comprises step C): to the qualification of nickel-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 nickel-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 nickeliferous protein band, 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 nickel and detection nickel.
The present invention also provides the application of a kind of nickel-IgM inner complex described above in preparation human body in the reagent of nickel-IgM inner complex or test kit.
The present invention also provides a kind of test kit at least comprising nickel-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 metallic nickel.
The present invention also provides a kind of method of detection by quantitative nickel-IgM inner complex, with the above-mentioned nickel-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, purifying nickel-IgM inner complex and enzyme linked immunological combined techniques, purifying nickel-IgM inner complex and atomic absorption spectrum combined techniques, purifying nickel-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 nickel-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 first or extract nickel-IgM inner complex from whole blood;
2. the present invention proposes nickel-IgM inner complex first and can be used for preparing application in the reagent or test kit detecting nickel-IgM inner complex in blood sample.
3. the present invention establishes the method for qualitative and quantitative detection of nickel-IgM inner complex, so that detection by quantitative nickel-IgM inner complex is in the application of the regional nickel contamination degree of evaluation one.Indirectly can reflect the situation of this regional crowd by nickel contamination by nickel-IgM inner complex in the regional crowd's serum of detection by quantitative one, thus indirectly reflect this regional nickel contamination degree.Its accuracy of nickel-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 nickel-IgM inner complex of the present invention;
Fig. 2 is the fluorometric analysis figure of the synchrotron radiation X line electrophoretic band of nickel-IgM inner complex of the present invention;
Wherein, in Fig. 1, M is Marker, and 2 is nickel-IgM inner complex; In Fig. 2, X-coordinate is protein band position, and ordinate zou is nickel 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 nickel specific binding, for surface is with the silica gel of material or the resin that can catch chromium;
The material catching nickel in the present invention, includes but not limited to that, into mouse-anti Ni mAb, buy from Guangzhou Ran Ke company, model is RK14419;
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 invention provides a kind of nickel-IgM inner complex, nickel ion and IgM by sulfydryl or/and cysteine residues chelating forms.
The present invention also provides a kind of preparation method of nickel-IgM inner complex, comprises the following steps:
A) chelatropic reaction of nickel and IgM: add nickel ion and carry out chelatropic reaction in the IgM in people source;
B) extraction of purifying nickel-IgM inner complex: adopt immune-affinity chromatography, remove unreacted IgM and unnecessary nickel ion in reaction soln, obtain nickel-IgM inner complex, concrete steps are as follows:
(1) sample dissolution: by above-mentioned steps A) in extract nickel-IgM inner complex be dissolved in physiological saline;
(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 albumen restore 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, dialysed overnight, 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 nickel specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(10) collect: collect the elutriant after step (9) wash-out, after collection, make albumen restore 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, dialysed overnight, collects sample, obtain nickel-IgM inner complex;
C) to the qualification of nickel-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 nickel-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 corresponding protein band, this protein band is taken out, protein band is redissolved, and then adopt ICP-MS, AAS whether to detect in this liquid containing nickel and the content detecting nickel.
The present invention also provides a kind of test kit at least comprising nickel-IgM inner complex described above product in contrast.
Preferably, also comprise coating buffer in this test kit, this coating buffer is catch the albumen of IgM or catch the material of metallic nickel.
The present invention also provides a kind of preparation method of nickel-IgM inner complex, comprises the following steps:
A) chelatropic reaction of nickel and IgM: add nickel 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 nickel-IgM inner complex: adopt immune-affinity chromatography, remove unreacted IgM and unnecessary nickel ion in reaction soln, obtain nickel-IgM inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains, nickel-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 nickel specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(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 nickel-IgM inner complex;
C) to the qualification of nickel-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 nickel-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 nickeliferous protein band, 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 nickel and detection nickel.
The present invention also provides a kind of test kit at least comprising nickel-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 metallic nickel.
In the present invention, the test kit that can realize the object of the invention can be listed following several, but is not limited to this.
Detect a test kit for nickel-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 nickel.
Detect a test kit for nickel-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 nickel-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 nickel-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 nickel.
Detect a test kit for nickel-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 nickel-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 nickel-IgM inner complex in blood sample, comprising: contain liquid needed for protein band nickeliferous on the coating buffer of the albumen can catching IgM, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed, contain the coating buffer, confining liquid, washings, enzyme labelled antibody, substrate, stop buffer, dilution buffer, positive control, negative control etc. of the material can catching nickel.
Detect a test kit for nickel-IgM inner complex in blood sample, comprising: as extracting liquid, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for IgM.
Detect a test kit for nickel-IgM inner complex in blood sample, comprising: as extracting liquid, souring agent, hydrogen peroxide, positive control, negative control etc. needed for protein band nickeliferous on reagent, glue bed medium, redissolution liquid, sample-loading buffer, dissolving glue bed in purification whole blood needed for IgM.
In above-mentioned several test kit, described positive control is standard substance, is namely chelated with the IgM inner complex of heavy metal nickel or is chelated with the BSA inner complex of heavy metal nickel; Described negative control is dilution buffer.
Mentioned reagent box is for detecting the IgM of chelating nickel, to improve the accuracy of detection, repeated, and makes it to be promoted in clinical.
The present invention also provides a kind of method of detection by quantitative nickel-IgM inner complex, with the above-mentioned nickel-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, purifying nickel-IgM inner complex and enzyme linked immunological combined techniques, purifying nickel-IgM inner complex and atomic absorption spectrum combined techniques, purifying nickel-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 nickel chelating type immunocomplex, but be not limited to following several.
Wherein, the reagent adopted in the method for above-mentioned detection by quantitative nickel-IgM inner complex is as follows:
method one:euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-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 nickel-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 20-80 doubly, add in micropore, 37 DEG C of effect 1-2 hour;
5) material can catching nickel is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add with dilution buffer dilution with can catch the material of nickel or the anti-Ni Ab of immune complex can be formed with nickel reactant, 37 DEG C of effect 1-2 hour, make the metallic nickel on anti-Ni Ab and IgM react;
6) enzyme conjugates incubation: remove anti-nickel antibody, and wash with washings, after washing completes, add the enzyme labelled antibody with dilution buffer dilution, make the concentration of the enzyme labelled antibody of dilution be 2 μ g/ml, 37 DEG C of effect 1-2 hour, make itself and enzyme labelled antibody react;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: drip stop buffer to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed at 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 nickel, and nickel on this part IgM can catch by the specific antibody of anti-nickel, 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 nickel, then can not catch by the specific antibody of anti-nickel, also can not with horseradish peroxidase, the antibody of the enzyme labellings such as alkaline phosphatase caught, and also not containing metallic nickel (negative control group result is negative) in agents useful for same, thus when read OD value result is shown as the positive, the i.e. provable metallic nickel detecting chelating on IgM.
method two:enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect nickel-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 nickel-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 20-80 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 nickel 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 nickel 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 metallic nickel detecting chelating on IgM.
method three:enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect nickel-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 nickel-IgM inner complex of known content; Be diluted to corresponding multiple with dilution buffer dilution measuring samples, namely dilute 20-80 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 nickel 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 nickel 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 metallic nickel detecting chelating on IgM.
method four:purifying nickel-IgM inner complex and enzyme linked immunological combined techniques (method of purification+ELISA method) detect nickel-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-Ni Ab is coated on solid phase carrier: dilute anti-Ni Ab to 50000-400000 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 nickel-IgM inner complex of known content; Dilute corresponding multiple by dilution buffer, namely dilute 20-80 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 at 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:purifying nickel-IgM inner complex and atomic absorption spectrum combined techniques (method of purification+AAS method) detect nickel-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 nickel of chelating on IgM in Atomic Absorption Spectroscopy AAS, and drawing standard curve, read respective value.
method six:purifying nickel-IgM inner complex and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method) detect nickel-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 nickel of chelating on IgM, and drawing standard curve, read respective value.
method seven:electrophoresis and euzymelinked immunosorbent assay (ELISA) (electrophoretic method-ELISA method) detect nickel-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 nickel, this band is taken out, this protein band is redissolved, and then utilize ELISA principle to detect the nickel content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelating nickel, molecular weight and content etc. can also be utilized.
method eight:electrophoresis and atomic absorption spectrometry (electrophoretic method-AAS method) detect nickel-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 nickel, this band is taken out, this protein band is redissolved, and then utilize AAS principle to detect the nickel content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelating nickel, molecular weight and content etc. can also be utilized.
method nine:inductivity coupled plasma mass spectrometry combined techniques (electrophoretic method-ICP-MS method) detects nickel-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 nickel, this band is taken out, this protein band is redissolved, and then utilize ICP-MS principle to detect the nickel content be dissolved in liquid.In addition, the iso-electric point of the IgM of this method detection chelating nickel, 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 nickel, 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 nickel 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 metallic nickel detecting chelating on IgM.
embodiment 1: the preparation method of nickel-IgM inner complex, comprises the following steps:
A) chelatropic reaction of nickel and IgM: add nickel 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 nickel ion solution gradually, dropping limit, limit vibrates, in order to avoid nickel 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 nickel-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 nickel ion, obtain nickel-IgM inner complex, concrete steps are as follows:
(1) sample dissolution: to through steps A) add physiological saline in the reaction soln that obtains, nickel-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 nickel specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(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 nickel-IgM inner complex;
C) to the qualification of nickel-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 nickel-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 nickeliferous protein band, this protein band is taken out, protein band is redissolved, and then adopt AAS method to detect the content whether containing nickel and detection nickel.
D) detected result
(1) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of nickel-IgG 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 nickel-IgM inner complex of the present invention, and in figure, X-coordinate is protein band position, and ordinate zou is nickel metal energy (content) value in this protein band.
(3) the nickel content in the nickel-IgG inner complex adopting graphite furnace atomic absorption spectrometry (AAS) rough determination the present embodiment to obtain, its content is 187.111 μ g/L.
the determination of the testing conditions of the method for a kind of detection by quantitative nickel 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 dilution buffer is 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-Ni Ab, anti-Ni Ab dilution buffer is 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-Ni Ab of different concns respectively adds 2 holes, 37 DEG C act on 1 hour, the metallic nickel on anti-Ni Ab and IgM are reacted;
(5) enzyme labelled antibody selects the suitableeest working concentration, i.e. 2ng/ml, removes anti-Ni antibody, and wash with washings, after washing completes, add with dilution buffer dilution HRP enzyme labelled antibody, 37 DEG C act on 1 hour, HRP enzyme labelled antibody and nickel-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-Ni-Ab and the optimum diluting multiple of blood plasma.
Simultaneously using made reference substance as positive control in test, IgM antibody+closed+Ni 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 Ni to resist) as negative control 2, IgM antibody+close+enzyme mark+substrate (namely do not add detect sample and Ni resist) is as negative control 3, IgM antibody+close+blood plasma+Ni is anti-+ and substrate (i.e. not enzyme-added mark) is as negative control 4, close+blood plasma+Ni 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 Ni 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, Ni is 1:5000, 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 1:20, Ni is 1:5000).
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 at 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 nickel-IgM inner complex in 100 parts of sample blood plasma, detect according to following steps: euzymelinked immunosorbent assay (ELISA) (ELISA method) detects nickel-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 nickel-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 nickel is added, and incubation: remove measuring samples, and wash with washings, after washing completes, add and dilute 5000 times by dilution buffer, 37 DEG C act on 1 hour, the metallic nickel on anti-NiAb and IgM are reacted;
6) enzyme conjugates incubation: remove anti-nickel antibody, and wash with washings, after washing completes, adds the HRP enzyme labelled antibody with dilution buffer dilution, and 37 DEG C act on 1 hour, itself and enzyme labelled antibody are reacted;
7) substrate incubation: remove enzyme labelled antibody, and wash with washings, after washing completes, adds substrate, 37 DEG C of lucifuge effects 30 minutes;
8) termination reaction: drip stop buffer to each micropore;
9) wavelength 405nm is got, after adding stop buffer, elisa plate is placed at 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 nickel-IgM inner complex in table 4:100 part blood sample
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD 405 | 0.473 | 0.369 | 0.6 | 0.682 | 0.527 | 0.312 | 0.533 | 0.557 | 0.635 | 0.715 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| OD 405 | 0.727 | 0.381 | 0.697 | 0.718 | 0.665 | 0.764 | 0.746 | 0.477 | 0.417 | 0.394 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| OD 405 | 0.684 | 0.641 | 0.706 | 0.47 | 0.461 | 0.638 | 0.354 | 0.705 | 0.39 | 0.686 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| OD 405 | 0.382 | 0.423 | 0.723 | 0.736 | 0.611 | 0.433 | 0.506 | 0.493 | 0.584 | 0.766 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| OD 405 | 0.583 | 0.56 | 0.412 | 0.726 | 0.654 | 0.325 | 0.659 | 0.492 | 0.711 | 0.542 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| OD 405 | 0.567 | 0.351 | 0.367 | 0.694 | 0.743 | 0.37 | 0.326 | 0.644 | 0.752 | 0.477 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| OD 405 | 0.622 | 0.681 | 0.775 | 0.474 | 0.627 | 0.517 | 0.694 | 0.69 | 0.62 | 0.395 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| OD 405 | 0.755 | 0.363 | 0.491 | 0.531 | 0.683 | 0.398 | 0.575 | 0.312 | 0.422 | 0.414 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| OD 405 | 0.402 | 0.346 | 0.693 | 0.555 | 0.697 | 0.536 | 0.587 | 0.421 | 0.333 | 0.487 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| OD 405 | 0.592 | 0.43 | 0.596 | 0.389 | 0.375 | 0.725 | 0.623 | 0.607 | 0.698 | 0.772 |
application Example 2
Employing enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detect the nickel-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 nickel-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 nickel of chelating on IgM in Atomic Absorption Spectroscopy AAS, detected value is as shown in table 5.
The AAS detected result of nickel-IgM inner complex in table 5:100 part blood sample
application Example 3
Employing enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect the nickel-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 nickel-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 nickel of IgM, detected value is as shown in table 6.
The ICP-MS detected result of table 6 100 parts of sample blood sample nickel-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 nickel-IgM inner complex, is characterized in that, nickel ion and IgM by sulfydryl or/and cysteine residues chelating forms.
2. a preparation method for nickel-IgM inner complex as claimed in claim 1, is characterized in that, comprise the following steps:
A) chelatropic reaction of nickel and IgM: people source IgM or the IgM that recombinates according to biological method in add nickel ion and carry out chelatropic reaction, obtain reaction soln;
B) extraction of purifying nickel-IgM inner complex: adopt immune-affinity chromatography, removes unreacted IgM and unnecessary nickel ion in reaction soln, obtains nickel-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, nickel-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 nickel specific binding, dress post after balance chromatography column by dilution buffer again;
(8) loading: after chromatography column balance, with sample, then upper prop in dilution buffer dilution step (6);
(9) wash-out: use dilution buffer to rinse chromatography column and balance to baseline, then use the phosphate solution of 0.5-1.0mol/L to carry out wash-out;
(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 nickel-IgM inner complex.
4. the preparation method of nickel-IgM inner complex according to claim 2, is characterized in that, also comprise step C): to the qualification of nickel-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 nickel-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 nickeliferous protein band, 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 nickel and detection nickel.
5. the application of nickel-IgM inner complex as claimed in claim 1 in the reagent or test kit preparing to detect nickel-IgM inner complex in blood sample.
6. one kind at least comprises the test kit of nickel-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 metallic nickel.
8. the method for a detection by quantitative nickel-IgM inner complex, it is characterized in that, using the nickel-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, purifying nickel-IgM inner complex and enzyme linked immunological combined techniques, purifying nickel-IgM inner complex and atomic absorption spectrum combined techniques, purifying nickel-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.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
Non-Patent Citations (2)
| Title |
|---|
| 何春燕、喻红主编: "《医学生物化学实验指导》", 30 September 2010 * |
| 邹军辉: "六价铬间接竞争 ELISA 检测试剂盒的研制及新型一步法竞争 ELISA的建立", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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Application publication date: 20151021 |