CN105021554B - arsenic-IgM chelate as well as preparation method and application thereof - Google Patents
arsenic-IgM chelate as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an arsenic-IgM chelate and a preparation method and application thereof, wherein the arsenic-IgM chelate is formed by chelating arsenic ions and IgM through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of arsenic-IgM chelate so as to quantitatively detect the application of arsenic-IgM chelate in evaluating the arsenic pollution degree of one area. arsenic-IgM chelate in serum of people in a region can be quantitatively detected, so that the arsenic pollution condition of people in the region can be indirectly reflected, and the arsenic pollution degree of the region can be indirectly reflected. The arsenic-IgM chelate quantitative detection method established by the invention greatly improves the accuracy and the repeatability of detection.
Description
Technical field
The present invention relates to detection fields, more specifically to a kind of arsenic-IgM chelate and its preparation method and application.
Background technique
IgM is to connect into pentamer by a J chain and disulfide bond by 5 monomers in serum, and molecular weight is maximum, is
970kD, sedimentation coefficient 19S, referred to as macroglobulin (maAsoglobulin).The hingeless sequence of IgM on molecular structure, C μ 2 can
It can be instead of the function of hinge area.IgM is the immunoglobulin occurred earliest during biological evolution.In ontogenetic process
In, either B cell Surface Ig (SmIg), or the Ig that synthesis is secreted into serum, IgM is the Ig occurred earliest,
The fetus in embryonic development advanced stage has the ability to generate IgM.During antigenic stimulus induction body fluid immune response, general IgM
It generates at first.IgM accounts for the 5%~10% of serum total Ig.Since IgM is generated in immune response early stage, and in complement presence
Strong 500 times of IgG of haemocylolysis ratio or more, and opsonic action, therefore IgM are played by segments such as C3b, C4b after complement activation
It is occupied an important position in the early immune protection of body.Natural blood group antibody (agglutinin) is IgM, and blood group is not inconsistent defeated
Serious hemolytic reaction easily occurs for blood.IgM cannot cross placenta, such as occur being directed to the IgM of certain pathogenic microorganism, table in bleeding of the umbilicus
Show that embryonic period, embryonic phase has corresponding pathogenic microorganism such as microspironema pallidum, rubeola or giant cell poison etc. to infect, referred to as fetal infection or vertical
Infection.Also contain yield monomer IgM in normal human serum.
Arsenic is common one of environmental poisonous substance, is widely present among water, soil, air and food.Due to its toxicity
And universal existence, U.S.'s poisonous substance and disease registration administration (UAsted States Agency for Toxic Substances
And Disease Registry, ATSDR) it is classified as first-class harmful substance (http://www.atsdr.cdc.gov/ always
SPL/index.html), while arsenic is also by many international organizations (including international cancer research institution (International
Agency for Research on Cancer, IARC) and Environmental Protection Agency (U.S.Environmental
Protection Agency, EPA)) it delimit as one of human carcinogen.Arsenic can generate acute or chronic toxicity to human body, anxious
Property arsenic poisoning will lead to the exacerbated events such as vomiting, dry, muscle cramp, abdominal pain, trick shouting pain, and arsenicalism is strong to the mankind
The influence of health also has become a significant problem concerned by people.It is two kinds by drinking water and air to contact most with arsenic in environment
Direct mode, it can induce cardiovascular disease, cause maladjusted nervous system and cause diabetes and lung cancer, skin lung cancer, wing
Guang cancer etc..And it is currently a popular disease learn display, the underground drinking water caused by arsenic existing for nature pollution, TaiWan, China,
The arsenic exposure of high dose is caused in multiple countries and regions such as Argentina, Chile, Bangladesh, India, China, becomes environmental pollution
With the global problem of harm health.At present the whole world had more than five million peoples be in the exposed threat with arsenicalism of arsenic it
In.
At present bleeding arsenic content, including ELISA, atomic absorption spectrum (Atomic can be detected with a variety of methods
Absorption Spectroscopy, AAS) quantitative assay, icp ms (inductively
Coupled plasma mass spectrometry, ICP-MS) quantitative assay etc., but there are still certain defects.
About the evaluation of arsenic poisoning, especially arsenicalism, it is only capable of by detecting blood arsenic content, in indirect reaction human body
Circulation arsenic content, can not further assess arsenic for the degree of injury of body function, and with the rapid development of science and technology,
The relationship of arsenic and human body is also increasingly close, thus finding one kind can be especially chronic from the evaluation arsenic poisoning of body function angle
Arsenic poisoning becomes more and more important for the evaluation method of the extent of damage of body.
Summary of the invention
For the serious problem of arsenic pollution, the purpose of the present invention is to provide a kind of arsenic-IgM chelate and its preparation sides
Method, and the qualitative and quantitative analysis method of arsenic-IgM chelate is established, so that quantitative detection arsenic-IgM chelate is evaluating a ground
The application of area's arsenic pollution degree.It can reflect this indirectly by arsenic-IgM chelate in quantitative detection one regional crowd's serum
The case where regional crowd is by arsenic pollution, to reflect this regional arsenic pollution degree indirectly.
The technical solution adopted by the present invention to solve the technical problems is: a kind of arsenic-IgM chelate is provided, arsenic ion with
IgM is chelated by sulfydryl or/and cysteine residues.
The present invention also provides a kind of preparation methods of above-mentioned arsenic-IgM chelate, comprising the following steps:
A) the chelatropic reaction of arsenic and IgM: arsenic ion is added in the IgM of source of people and carries out chelatropic reaction, obtains reaction solution；
B it) purifies the extraction of arsenic-IgM chelate: using immune-affinity chromatography, remove unreacted IgM in reaction solution
And extra arsenic ion is to get arsenic-IgM chelate.
Wherein, step B described in external synthetic method) specific as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), keeps arsenic-IgM chelate multiple
It is molten；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample to get arsenic-IgM chelate.
Wherein, the preparation method of above-mentioned arsenic-IgM chelate, further includes step C): the identification to arsenic-IgM chelate；
Wherein, step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of extraction purification, dilution buffer is added, and mix, so
After be loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, so
Afterwards again using ICP-MS method, AAS method or ELISA method detection whether containing arsenic and detect arsenic content.
The present invention also provides a kind of examinations such as above-mentioned arsenic-IgM chelate arsenic-IgM chelate in preparation detection human body
Application in agent or kit.
The present invention also provides a kind of kits including at least such as above-mentioned arsenic-IgM chelate as reference substance.
It preferably, further include coating buffer in the kit, which contains the albumen that can capture IgM or capture the object of arsenic
Matter.
The present invention also provides a kind of methods of quantitative detection arsenic-IgM chelate, with the above-mentioned arsenic-IgM chela of known content
Object is closed as reference substance, is detected using a pair of sample of following methods: enzyme-linked immunization, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are with inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgM chelate in conjunction with enzyme linked immunological
Method, purification arsenic-IgM chelate and atomic absorption spectrum combined techniques, purification arsenic-IgM chelate and inductively coupled plasma constitution
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement arsenic-IgM chelate and its preparation method and application of the invention, has the advantages that
1. the present invention synthesizes arsenic-IgM chelate in vitro for the first time；
2. present invention firstly provides arsenic-IgM chelate can be used for preparing detection blood sample in arsenic-IgM chelate reagent or
Application in kit.
3. the present invention establishes the method for qualitative and quantitative detection of arsenic-IgM chelate, so as to quantitative detection arsenic-IgM chelate
In the application of evaluation one regional arsenic pollution degree.It can be with by arsenic-IgM chelate in the regional crowd's serum of quantitative detection one
Reflect the case where this regional crowd is by arsenic pollution indirectly, to reflect this regional arsenic pollution degree indirectly.The present invention establishes
Arsenic-IgM chelate quantitative detecting method its accuracy greatly improve, and the repeatability of detection is made to be greatly enhanced.
Detailed description of the invention
Fig. 1 is the non denatured electrophoretic band figure of arsenic-IgM chelate of the present invention；
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of arsenic-IgM chelate of the present invention；
Wherein, in Fig. 1, M Marker, 2 be arsenic-IgM chelate；In Fig. 2, abscissa is protein band position, indulges and sits
It is designated as arsenic energy in the protein band.
Specific embodiment
In the following, being described further in conjunction with specific embodiment to the present invention:
Unless otherwise indicated, the laboratory operating procedures being related to are the step of this field routine, agents useful for same, material to the present invention
Material as following cited, do not enumerate in the present invention come be reagent commonly used in the art or can be by commercially available mode
It obtains:
Extracting reagent is PEG solution, borate buffer solution etc. (using PEG method)；
Glue bed medium is one of Ago-Gel, polyacrylamide gel；
The filler that can be specifically bound with IgM in the present invention, has the silica gel for the albumen that can capture IgM for surface
Or resin；
The albumen (anti-IgM) that can capture IgM in the present invention, including but not limited to rabbit Anti- mankind IgM H&L,
Its brand is Abcam, model ab8505；
The filler that can be specifically bound with arsenic in the present invention, has silica gel or the tree of the substance that can capture arsenic for surface
Rouge；
The anti-As mAb of the substance that can capture arsenic in the present invention, including but not limited to mouse is bought from Guangzhou Ran Ke company, goods
Number be RK15728；
Enzyme labelled antibody is one of the antibody marked containing enzymes such as horseradish peroxidase, alkaline phosphatases；
Substrate is methyl biphenyl amine (TMB) solution；
Cleaning solution is to contain KH2PO40.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl 8.0mg/ml、KCl
The 0.15M PBS solution that the pH of 0.2mg/ml, 0.5%Tween-20 are 7.4；
Confining liquid is 1%-5% bovine serum albumin(BSA) or skimmed milk power；
Dilution buffer is Na containing 1.5mg/mL2CO3、2.93mg/ml NaHCO3PH be 9.6 0.05M phosphate it is slow
Fliud flushing；
Enzyme labelled antibody is HRP enzyme labelled antibody；
Terminate liquid are as follows: by the 2M H of 21.7ml2SO4It is settled to the ddH of 200ml2In O；
Eluent is the 0.1mol/L Tris-HCL buffer that the PH of the papain containing 1-2mg/ml is 8.0；
Acidulant is nitric acid；
Sample-loading buffer is to contain 1M Tris-HCl (pH 6.8) 15.5mL, 1% bromophenol blue 2.5mL, ddH2It is O7mL, sweet
The Sample buffer (5X) of propylhomoserin 25mL；
The ddH that electrophoretic buffer is 3mg/ml containing Tris, the PH of glycine 14.4mg/ml is 6.82O solution.
The present invention provides a kind of arsenic-IgM chelate, arsenic ion and IgM and is chelated by sulfydryl or/and cysteine residues
At.
The present invention also provides a kind of preparation methods of arsenic-IgM chelate, comprising the following steps:
A arsenic ion the) chelatropic reaction of arsenic and IgM: is added in the IgM of source of people or the IgM according to biological method recombination
Chelatropic reaction is carried out, reaction solution is obtained；
B it) purifies the extraction of arsenic-IgM chelate: using immune-affinity chromatography, remove unreacted IgM in reaction solution
And extra arsenic ion is to get arsenic-IgM chelate, the specific steps are as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), keeps arsenic-IgM chelate multiple
It is molten
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample to get arsenic-IgM chelate；
C) to the identification of arsenic-IgM chelate, the specific steps are as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of extraction purification, dilution buffer is added, and mix, so
After be loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, so
Afterwards again using ICP-MS method, AAS method or ELISA method detection whether containing arsenic and detect arsenic content.
The present invention also provides a kind of kits including at least such as above-mentioned arsenic-IgM chelate as reference substance.
It preferably, further include coating buffer in the kit, which contains the albumen that can capture IgM or can capture arsenic
Substance.
In the present invention, the kit for being able to achieve the object of the invention can be listed following several, and but it is not limited to this.
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: the coating buffer containing the albumen that can capture IgM,
Confining liquid, cleaning solution, the substance for capturing arsenic as secondary antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, loading are slow
Fliud flushing, positive control, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: the coating buffer containing the albumen that can capture IgM,
Confining liquid, cleaning solution, eluent, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: the coating buffer containing the albumen that can capture IgM,
Confining liquid, cleaning solution, eluent, sample-loading buffer, acidulant, hydrogen peroxide, standard items, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: extract examination as needed for IgM in purification whole blood
Liquid, the coating buffer containing the substance that can capture arsenic, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution are redissolved in agent
Buffer, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: extract examination as needed for IgM in purification whole blood
Agent, redissolution liquid, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: extract examination as needed for IgM in purification whole blood
Agent, sample-loading buffer, redissolution liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: the coating buffer containing the albumen that can capture IgM,
Glue bed medium redissolves liquid, sample-loading buffer, dissolves liquid needed for the protein band containing arsenic on glue bed, contains the object that can capture arsenic
Coating buffer, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative control of matter etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: extract examination as needed for IgM in purification whole blood
Agent, glue bed medium redissolve liquid, sample-loading buffer, dissolve liquid, positive control, feminine gender needed for the protein band containing arsenic on glue bed
Control etc..
The kit of arsenic-IgM chelate in a kind of detection blood sample, comprising: extract examination as needed for IgM in purification whole blood
Agent, glue bed medium redissolve liquid, sample-loading buffer, dissolve liquid, acidulant, peroxidating needed for the protein band containing arsenic on glue bed
Hydrogen, positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with the IgM chelate of weight arsenic or is chelated with
The BSA chelate of weight arsenic；The negative control is dilution buffer.
Mentioned reagent box is used to detect the IgM of chelating arsenic, to improve the accuracy of detection, repeatability, and is allowed in clinic
In promoted.
The present invention also provides a kind of methods of quantitative detection arsenic-IgM chelate, with the above-mentioned arsenic-IgM chela of known content
Object is closed as reference substance, is detected using a pair of sample of following methods: enzyme-linked immunization, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are with inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgM chelate in conjunction with enzyme linked immunological
Method, purification arsenic-IgM chelate and atomic absorption spectrum combined techniques, purification arsenic-IgM chelate and inductively coupled plasma constitution
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention,
Having of can listing of method with detection arsenic chelating type immune complex is following several, but is not limited to following several.
Wherein, the reagent used in the method for above-mentioned quantitative detection arsenic-IgM chelate is as follows:
Method one:Enzyme-linked immunization (ELISA method) detects arsenic-IgM chelate, detects in accordance with the following steps:
1) substance that IgM will be captured, as human IgM antibody is coated on solid phase carrier: being diluted with dilution buffer anti-
IgM Ab to 500-4000 times, be added elisa plate micropore in, 4 DEG C overnight 16-18 hour or 37 DEG C water-bath 1-3 hours, store
Refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；It is diluted to corresponding multiple with dilution buffer dilution measuring samples, that is, dilutes 10-40 times, is added in micropore, 37 DEG C
Effect 1-2 hours；
5) substance that can capture arsenic is added, and incubates: removing measuring samples, and is washed with cleaning solution, it is to be washed
After the completion of washing, the dilution of addition dilution buffer with the substance that can capture arsenic or can react to form antigen antibody complex with arsenic
Anti- As Ab, 37 DEG C effect 1-2 hours, react anti-As Ab with the arsenic on IgM；
6) enzyme conjugates incubates: removing anti-arsenic antibody, and is washed with cleaning solution, after the completion of washing, is added with dilute
Release the diluted enzyme labelled antibody of buffer, make the concentration 2 μ g/ml of diluted enzyme labelled antibody, 37 DEG C effect 1-2 hours, make its with
Enzyme labelled antibody reaction；
7) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
8) it terminates reaction: terminate liquid is added dropwise to each micropore；
9) take wavelength 405nm, after adding terminate liquid, elisa plate is placed in microplate reader read respectively measuring samples and
The OD value of standard items, by drawing standard curve, the content for acquiring measuring samples (can also directly pass through dye without using microplate reader
Pornographic condition carries out qualitative detection).
This method utilizes ELISA principle, the specific IgM in whole blood can be extracted, the top IgM extracted
Divide and is chelated with weight arsenic, and the arsenic on the IgM of this part can be captured by the specific antibody of anti-arsenic, it later can be again by horseradish mistake
The antibody of the enzymes such as oxide enzyme, alkaline phosphatase label captures (the antibody nonrecognition coating protein), and the antibody in capture exists
Under the action of color developing agent and terminate liquid, OD value can be read under instrument, and without containing the IgM for chelating arsenic, then it will not be by anti-arsenic
Specific antibody captured, will not be captured with the antibody of the enzymes label such as horseradish peroxidase, alkaline phosphatase, and institute
With in reagent also without containing arsenic (negative control group result be feminine gender), thus when read OD value is as the result is shown the positive,
The i.e. provable arsenic for detecting to chelate on IgM.
Method two:Enzyme linked immunological is pressed with atomic absorption spectrum combined techniques (ELISA method+AAS method) detection arsenic-IgM chelate
It is detected according to following steps:
1) substance that IgM will be captured, as human IgM antibody is coated on solid phase carrier: being diluted with dilution buffer anti-
IgM Ab to 500-4000 times, be added elisa plate micropore in, 4 DEG C overnight 16-18 hour or 37 DEG C water-bath 1-3 hours, store
Refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rpm is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing coating buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% cow's serum
As confining liquid, 37 DEG C are placed 1 hour for albumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, have been washed
Cheng Hou, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；It is diluted to corresponding multiple with dilution buffer dilution measuring samples, that is, dilutes 10-40 times, is added in micropore, 37 DEG C
Effect 1-2 hours；
5) it elutes: removing measuring samples, and washed with cleaning solution, it is small with elution 1-3 after the completion of washing
When.
6) it detects: being sampled from ELISA micropore, in Atomic Absorption Spectrometer detection chelating in the arsenic on IgM, and draw mark
Directrix curve reads respective value；
The embodiment combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principle, utilizes atomic absorption light
Spectrometer detection chelating due to only containing IgM in solution, and is free of any arsenic (negative control group in the arsenic on IgM in agents useful for same
As a result be feminine gender), result will not be interfered, thus when it is read be as the result is shown positive when, i.e., it is provable to detect
The arsenic chelated on IgM.
Method three:Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect arsenic-
IgM chelate detects in accordance with the following steps:
1) substance that IgM will be captured, as human IgM antibody is coated on solid phase carrier: being diluted with dilution buffer anti-
IgM Ab to 500-4000 times, be added elisa plate micropore in, 4 DEG C overnight 16-18 hour or 37 DEG C water-bath 1-3 hours, store
Refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；It is diluted to corresponding multiple with dilution buffer dilution measuring samples, that is, dilutes 10-40 times, is added in micropore, 37 DEG C
Effect 1-2 hours；
5) it elutes: removing measuring samples, and washed with cleaning solution, it is small with elution 1-3 after the completion of washing
When.
6) it is acidified: acidulant being added in the solution in step 5), solution is acidified, sealing overnight, is thoroughly acidified；
7) it detects: hydrogen peroxide is added, and heats and catches up with acid, and sampled from the solution eluted in ELISA agent plate, in
Detection chelating is in the arsenic of IgM under icp ms, and draws standard curve, reads respective value.
This method is on the basis of using ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption
Couple plasma mass spectrometer detection chelating is felt in the arsenic on IgM, is free of due to only containing IgM in solution, and in agents useful for same
Any arsenic (negative control group result is feminine gender), will not interfere result, thus working as read is as the result is shown the positive
When, i.e., the provable arsenic for detecting to chelate on IgM.
Method four:Purify arsenic-IgM chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detection arsenic-IgM chelating
Object detects in accordance with the following steps:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method is redissolved the IgM that the purification of blood comes out using whole blood extraction method, obtains the redissolution liquid of IgM；
2) anti-As Ab is coated on solid phase carrier: dilutes anti-As Ab to 25000-400000 times with dilution buffer,
Be added elisa plate micropore in, 4 DEG C overnight 16-18 hour or 37 DEG C water-bath 1-3 hours, storage refrigerator；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: being sampled from the redissolution liquid of the IgM of extraction, make measuring samples；With known content
Arsenic-IgM chelate make standard items；Corresponding multiple is diluted with dilution buffer, that is, dilutes 10-40 times, is added in micropore, 37 DEG C
Effect 1-2 hours；
5) enzyme conjugates incubates: removing the redissolution liquid of IgM, and is washed with cleaning solution, after the completion of washing, is added and uses
The diluted enzyme labelled antibody of dilution buffer makes the 2 μ g/ml of concentration of diluted enzyme labelled antibody, 37 DEG C effect 1-2 hours, make it
It is reacted with enzyme labelled antibody；
6) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
7) it terminates reaction: terminate liquid is added dropwise to each micropore；
8) take wavelength 405nm, after adding terminate liquid, elisa plate is placed in microplate reader read respectively measuring samples and
The OD value of standard items, by drawing standard curve, the content for acquiring measuring samples (can also directly pass through dye without using microplate reader
Pornographic condition carries out qualitative detection).
Method five:It purifies in arsenic-IgM chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detection blood sample
Arsenic-IgM chelate detects in accordance with the following steps:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgM that the purification of blood comes out from whole blood extraction method, obtains the redissolution liquid of IgM；
2) it detects: being sampled from the redissolution liquid in step 1), chelated in Atomic Absorption Spectrometer detection in the arsenic on IgM,
And standard curve is drawn, read respective value.
Method six:Purify arsenic-IgM chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method)
Arsenic-IgM chelate is detected, is detected in accordance with the following steps:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgM that the purification of blood comes out from whole blood extraction method, obtains the redissolution liquid of IgM；
2) it is acidified: being sampled from the redissolution liquid in step 1), acidulant (such as nitric acid) is added in the solution, solution is carried out
Acidification, sealing overnight, are thoroughly acidified；
3) it detects: hydrogen peroxide is added, and heats and catches up with acid, and sampled from corresponding solution, in inductively coupled plasma
Detection chelating is in the arsenic on IgM under constitution spectrometer, and draws standard curve, reads respective value.
Method seven:Electrophoresis and enzyme-linked immunization (electrophoresis-ELISA method) detect arsenic-IgM chelate, in accordance with the following steps
Detection:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgM that the purification of blood comes out from whole blood extraction method, obtains the redissolution liquid of IgM；
2) glue bed is prepared: selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gel as needed
Deng be used as medium, prepare glue bed；
3) it is loaded: taking the 8 μ L of redissolution liquid in step 1) that 2 μ L sample-loading buffers are added, mix, of short duration centrifugation；(pay attention to herein
Step cannot boil)
4) electrophoresis: connection electrophoresis plate is separated by electrophoresis；
5) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, which is redissolved, so
ELISA principle is recycled to detect the arsenic content being dissolved in liquid afterwards.Further, it is also possible to detect the IgM of chelating arsenic using the method
Isoelectric point, molecular weight and content etc..
Method eight:Electrophoresis and atomic absorption spectrography (AAS) (electrophoresis-AAS method) detect arsenic-IgM chelate, according to following step
Rapid detection:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgM that the purification of blood comes out from whole blood extraction method, obtains the redissolution liquid of IgM；
2) glue bed is prepared: selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gel as needed
Deng be used as medium, prepare glue bed；
3) it is loaded: being sampled from the redissolution liquid in step 1), sample-loading buffer is added, and mix, is then loaded onto sample
In slot；
4) electrophoresis: connection electrophoresis plate is separated by electrophoresis；
5) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, which is redissolved, so
AAS principle is recycled to detect the arsenic content being dissolved in liquid afterwards.Further, it is also possible to detect the IgM's of chelating arsenic using the method
Isoelectric point, molecular weight and content etc..
Method nine:Inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ICP-MS method) detect arsenic-IgM chelate, press
It is detected according to following steps:
1) IgM is extracted from whole blood: using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc.
Method redissolves the IgM that the purification of blood comes out from whole blood extraction method, obtains the redissolution liquid of IgM；
2) glue bed is prepared: selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gel as needed
Deng be used as medium, prepare glue bed；
3) it is loaded: being sampled from the redissolution liquid in step 1), sample-loading buffer is added, and mix, is then loaded onto sample
In slot；
4) electrophoresis: connection electrophoresis plate is separated by electrophoresis；
5) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, which is redissolved, so
ICP-MS principle is recycled to detect the arsenic content being dissolved in liquid afterwards.Further, it is also possible to detect the IgM of chelating arsenic using the method
Isoelectric point, molecular weight and content etc..
IgM in method seven to nine can come out (such as supercentrifugation, high pressure liquid chromatography (HPLC) with a variety of Methods For Purifications
Method, gel-filtration chromatography, gel electrophoresis, ELISA method etc.), the IgM that purification is come out redissolves, and obtains the redissolution of IgM
Liquid takes a certain amount of IgM, using charge shifting principle, carries out electrophoresis (electrophoresis, EP), gel slab (can root
According to needing using different medium) on can run out of different bands according to molecular weight, isoelectric point etc. are different, search out the phase rich in arsenic
Band is answered, the corresponding double solvents of the protein in gel is redissolved in solution, it can detect correlation IgM at a particular wavelength
Content, also can use the principles such as ELISA, AAS, ICP-MS and detect to chelate in the arsenic content on IgM, due in solution only
Result will not be interfered containing IgM, and in agents useful for same without any arsenic (negative control group result is feminine gender), thus
When it is read be as the result is shown positive when, i.e., the provable arsenic for detecting to chelate on IgM.
Embodiment 1: the preparation method of arsenic-IgM chelate, comprising the following steps:
A) the chelatropic reaction of arsenic and IgM: arsenic ion is added in the IgM of source of people and carries out chelatropic reaction, obtains reaction solution；
Preparation of reagents:
1) borate buffer solution (0.01M): weighing 0.31g boric acid and be dissolved in 400ml ultrapure water, is adjusted with the NaOH of 0.1M
PH to 9.0, is settled to 500mL.
2) it IgM solution: weighs 4.0mgIgM and is dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, sufficiently vibrate molten
Solution, is configured to the protein solution of 1.0mg/mL；
3)5mmol/L EDTA+200mmol/LNaHCO3Solution: EDTA2H is weighed2O 1.86g、NaHCO316.8g is molten
In 900mL ultrapure water, 1000ml, high pressure sterilization, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
4) ITCBE (the certainly Japanese colleague's chemistry institute of purchase, article No. M030)
5) bag filter (molecular cut off 14000) (Bioshop Inc)
Preparation step specifically:
1) bag filter the processing of bag filter: is put into the 5mmol/L of 500ml (according to the convertible dosage of beaker volume) volume
EDTA+200mmol/L NaHCO3In solution, 10min is boiled；Tipping EDTA/NaHCO3Liquid is gently rinsed with ultrapure water, then used
500ml 5mmol/L EDTA boils 10min；Boiling liquid is discarded, is thoroughly cleaned with ultrapure water, a large amount of ultrapure water is added and impregnates
4 DEG C of bag filter overnight.In use, wearing gloves, bag filter is taken out, with a large amount of its surfaces externally and internally of ultrapure water cleaning down；
2) 2.0mg ITCBE is taken to be dissolved in 2ml DMSO；
3) 4.0mg IgM is taken to be dissolved in 4.0ml borate buffer solution in (0.01M pH9.0)；
4) liquid for slowly preparing step 2 is added in IgM solution, shakes when being added dropwise, in 25 DEG C, 100r/min's is shaken
It is acted on for 24 hours in bed, then for 24 hours with bag filter dialysis, removes the ITCBE not in conjunction with IgM；
5) the resulting liquid 1mol/L HCl that will dialyse adjusts pH value to 7.0, and 80 μ l are then slowly gradually added dropwise
1mmol/L arsenic ion soln is vibrated when being added dropwise, in case arsenic ion precipitates albuminous degeneration；
6) solution added is reacted into 2h in 25 DEG C, the shaking table of 100r/min, is dialysed with the bag filter handled well
24h；
7) liquid after dialysis is saved in -20 DEG C of packing.
B it) purifies the extraction of arsenic-IgM chelate: using immune-affinity chromatography, remove reaction solution (i.e. 7 in step A)
Dialysis after liquid) in unreacted IgM and extra arsenic ion to get arsenic-IgM chelate, the specific steps are as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), keeps arsenic-IgM chelate multiple
It is molten；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample to get arsenic-IgM chelate；
C) to the identification of arsenic-IgM chelate, the specific steps are as follows:
(1) it prepares glue bed: preparing glue bed using Ago-Gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of 8 μ L extraction purifications, 2 μ L sample-loading buffers are added, and
It mixes, is then loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate is added electrophoretic buffer and carries out electrophoresis；In electrophoresis process, electric current is 22mA constant current,
Environment temperature is 4 degree；Stop electrophoresis when moving to glue bottom to bromophenol blue；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, so
The content of arsenic is detected whether containing arsenic and detected again afterwards using AAS method.
D) testing result
(1) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of arsenic-IgG chelate of the present invention.
(2) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
4W1 " of the SRXRF analysis of micronutrient levels in Beijing Positive and Negative Electronic Collider (BEPC) is synchronous in protein band
It is completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200
Type, Beijing are stood upright Han Guang company) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into
Penetrate facula position, moving step length 0.0025mm.The X-ray gone out from electromagnetic radiation is by Si (Li) detector (PGT Inc.LS
It 30143-DS) detects, pop one's head in coplanar with incidence SR line and is mutually perpendicular to, away from sample irradiation point 20mm, signal PGT multiple tracks point
Analyzer (MCA4000) obtains output.Sample is excited with the monochromatic synchrotron radiation light of 11.5keV, is adjusted launching spot (1mmx3mm)
Position is allowed in band one end, and in the minute of 300s, hot spot is uniformly slowly moved along band always, at the end of counting
Hot spot moves on to the band other end.Along electrophoresis direction, every 1mm takes a spectrum.Using AX IL software data processing, and with deriving from
Air and the Ar signal peak of content constant carry out normalization to other element peaks, to offset beam intensity variation to signal strength or weakness
The influence of generation.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way at identical conditions.
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of arsenic-IgM chelate of the present invention, cross in figure
Coordinate is protein band position, and ordinate is energy (content) value of arsenic in the protein band.
(3) in the arsenic-IgG chelate for using graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination the present embodiment to obtain
Arsenic content, content be 92.401 μ g/L.
A kind of determination of the testing conditions of the method for quantitative detection arsenic-IgM chelate of the present invention:
1. the determination of the optimum diluting multiple of complement protein best effort concentration and blood plasma
Steps are as follows:
(1) by anti-IgM Ab dilution buffer according to following mass volume ratio (dilution) 1:500,1:1000,1:
2000,1:4000 is diluted, and is added in elisa plate micropore, anti-IgM Ab is coated on solid phase carrier, each concentration coating
Three rows, 4 DEG C are stayed overnight 18 hours；
(2) dilution buffer is removed, and is washed with cleaning solution, after the completion of washing, is made with 2% bovine serum albumin(BSA)
For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) measuring samples are carried out with dilution buffer according to following mass volume ratio (dilution) 1:10,1:20,1:40 dilute
It releases, is added in micropore, according to above-mentioned coated anti-As Ab concentration, the anti-As Ab's of the same concentration is separately added into different dilutions
Blood plasma is spent, 37 DEG C act on 1 hour；
(4) measuring samples are removed, and are washed with cleaning solution, after the completion of washing, anti-As Ab are added, anti-As Ab is used
Dilution buffer carries out dilute according to following mass volume ratio (dilution) 1:25000,1:50000,1:100000,1:200000
It releases, according to each identical anti-IgM Ab, serum diluted concentration, the anti-As Ab of various concentration respectively adds 2 holes, and 37 DEG C act on 1 hour,
React anti-As Ab with the arsenic on IgM；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2ng/ml removes anti-As antibody, and washed with cleaning solution,
After the completion of washing, addition dilution buffer dilutes HRP enzyme labelled antibody, and 37 DEG C act on 1 hour, makes HRP enzyme labelled antibody and arsenic-
The reaction of IgM chelate；
(6) enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, substrate is added, 37 DEG C are protected from light effect
30 minutes；
(7) terminate liquid is added dropwise to each micropore with speed identical with substrate solution is added and sequence；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed in microplate reader and reads each hole OD value respectively.
According to each hole OD value numerical value, anti-IgM Ab, the best effort concentration of anti-As-Ab and the optimum diluting multiple of blood plasma are selected.
In test simultaneously using made reference substance as positive control, select IgM antibody+closing+As resist+enzyme mark+substrate (i.e.
Detection sample is not added) it is right as feminine gender as negative control 1, IgM antibody+closing+blood plasma+enzyme mark+substrate (it is anti-that As is not added)
According to 2, IgM antibody+closing+enzyme mark+substrate (detection sample is not added and As is anti-) as negative control 3, IgM antibody+closing+blood
Slurry+As is anti-+ and substrate (i.e. not enzyme mark) is used as negative control 4, and closing+blood plasma+As is anti-+ enzyme mark+substrate (IgM antibody is not added)
As blank control 1, only add substrate and PBS as blank control 2；Testing result is shown in Table 1-2.
Table 1: the determination of anti-IgM Ab and As antibody best effort concentration and diluted plasma multiple
Table 2:ELISA positive control and negative control ELISA testing result
It is shown by table 1-2 data, we can see that when the dilution of human IgM antibody is 1:1000, whole blood dilution is
When the anti-dilution of 1:10, As is 1:25000, OD value is maximum, although negative control group of the OD value less than 0.8, corresponding to it
OD value is all less than 0.1, and the positive controls corresponding to it, and OD value is greater than 0.8, so concentration corresponding to this value is selected to make
For best effort concentration (i.e. human IgM antibody concentration is 1:1000, and whole blood dilution is 1:10, and As concentration is 1:25000).
2.ELISA eluent best effort concentration and time determine
Steps are as follows: (1) being diluted to 1000 times (mass volume ratios) human IgM antibody with dilution buffer, ELISA is added
In plate micropore, 37 DEG C water-bath 3 hours, store refrigerator；
(2) dilution buffer is removed, and is washed with cleaning solution, after the completion of washing, is made with 2% bovine serum albumin(BSA)
For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) confining liquid is removed, and is washed with cleaning solution, after the completion of washing, is added diluted with dilution buffer
HRP enzyme labelled antibody, 37 DEG C act on 2 hours, react it with human IgM antibody；
(4) prepare eluent: by papain with pH 8.0,0.1mol/L Tris-HCI buffer at 1-
2mg/ml adds 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT) (DTT)；
(5) enzyme labelled antibody is removed, eluent is diluted with dilution buffer, keeps the papain in eluent dense
Degree: enzyme labelled antibody concentration ratio=1:80,1:40,1:20,1:10,1:5, wherein each concentration makees 3 multiple holes, is respectively placed in
1h, 2h, 3h are eluted at a temperature of 37 DEG C；
(6) eluent is removed, and is washed with cleaning solution, after the completion of washing, substrate is added, 37 DEG C are protected from light effect 30
Minute；
(7) terminate liquid is added dropwise to each micropore with speed identical with substrate solution is added and sequence；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed in microplate reader and reads every group of OD value respectively,
By compared with PBS buffer solution group, comparing the optimum concentration and elution time of eluent, concrete outcome is referring to table 3.
Table 3:ELISA eluent best effort concentration and elution time determine
| 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 it can be found that the papain in eluent concentration and enzyme labelled antibody the ratio between concentration=
When 1:20, i.e. when the concentration 100ng/ml of papain, each group OD value is below other several groups, illustrates that the concentration eluent is washed
De- effect is optimal, and (human IgM antibody that will combine on ELISA hole wall-enzyme mark compound elution degree reaches maximum, thus OD value
It is minimum)；And action time either 1h, 2h, 3h, each group OD value variation it is little, it is seen that with the extension of time, enzyme activity by
Decrescence weak, in the case where enzyme concentration is constant, digestibility can not be improved by extending digestion time, so eluent in this experiment
Action time is that 1-3h all may be used.
Application Example 1
It takes using the arsenic-IgM chelate in 100 parts of sample blood plasma of ELISA method detection, follows the steps below detection:
Enzyme-linked immunization (ELISA method) detects arsenic-IgM chelate, detects in accordance with the following steps:
1) anti-IgM Ab is coated on solid phase carrier: with anti-IgM Ab to 1000 times of dilution buffer dilution, is added
In elisa plate micropore, 37 DEG C water-bath 1 hour, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, add toluene, dissolve cell membrane；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, add 2% bovine serum albumin
It is white be used as confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate in
37 DEG C are placed 1 hour；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；It is diluted to corresponding multiple with dilution buffer dilution measuring samples, that is, dilutes 10 times, is added in micropore, 37 DEG C of works
With 1 hour；
5) substance that can capture arsenic is added, and incubates: removing measuring samples, and is washed with cleaning solution, it is to be washed
After the completion of washing, addition dilution buffer dilutes 25000 times, and 37 DEG C act on 1 hour, reacts anti-As Ab with the arsenic on IgM；
6) enzyme conjugates incubates: removing anti-arsenic antibody, and is washed with cleaning solution, after the completion of washing, is added with dilute
The diluted HRP enzyme labelled antibody of buffer is released, 37 DEG C act on 1 hour, react it with enzyme labelled antibody；
7) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
8) it terminates reaction: terminate liquid is added dropwise to each micropore；
9) take wavelength 405nm, after adding terminate liquid, elisa plate is placed in microplate reader read respectively measuring samples and
The OD value (microplate reader can also not used, directly carry out qualitative detection by staining conditions) of standard items, testing result such as 4 institute of table
Show.
The ELISA testing result of arsenic-IgM chelate in 4:100 parts of blood samples of table
Application Example 2
Using in 100 minute mark this blood sample of enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detection
Arsenic-IgM chelate detects in accordance with the following steps:
1) substance that IgM will be captured, as anti-IgM (anti-IgM Ab) is coated on solid phase carrier: slow with dilution
It anti-IgM Ab to 1000 times of fliud flushing dilution, is added in elisa plate micropore, 4 DEG C are stayed overnight 18 hours；
2) whole blood is taken from the circulatory system, makees measuring samples, add toluene, dissolve cell membrane；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, it is pure that 2% ox blood is added
Albumen or skimmed milk power are as confining liquid, and 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution, and washing is completed
Afterwards, elisa plate is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；10 times are diluted to dilution buffer dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, be added with papain
Concentration be 100ng/ml eluent, elute 3 hours；
6) it detects: being sampled from ELISA micropore, in Atomic Absorption Spectrometer detection chelating in the arsenic on IgM, detected value is such as
Shown in table 5.
The AAS testing result of arsenic-IgM chelate in 5:100 parts of blood samples of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μg/L | 2.963 | 3.28 | 2.38 | 0.841 | 0.13 | 3.85 | 3.144 | 0.516 | 3.543 | 1.58 |
| Number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 4.282 | 1.879 | 0.671 | 3.969 | 1.845 | 3.381 | 2.699 | 3.309 | 0.972 | 2.533 |
| Number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 2.198 | 0.371 | 1.281 | 3.73 | 1.452 | 3.261 | 4.157 | 0.4 | 1.798 | 2.3 |
| Number | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 2.325 | 1.504 | 3.829 | 0.281 | 1.787 | 2.73 | 0.249 | 1.861 | 0.58 | 0.289 |
| Number | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 0.167 | 4.209 | 1.412 | 1.663 | 0.738 | 0.147 | 3.521 | 3.07 | 2.305 | 3.814 |
| Number | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 4.146 | 4.029 | 0.224 | 2.23 | 2.132 | 2.669 | 4.089 | 4.283 | 3.603 | 2.853 |
| Number | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 0.773 | 1.267 | 0.078 | 1.988 | 3.749 | 0.685 | 0.994 | 2.07 | 1.106 | 0.365 |
| Number | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 3.023 | 2.235 | 2.149 | 1.927 | 3.638 | 3.763 | 0.944 | 2.83 | 2.838 | 2.326 |
| Number | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 2.855 | 3.312 | 3.334 | 0.74 | 4.297 | 3.73 | 3.88 | 1.223 | 4.106 | 3.52 |
| Number | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 4.136 | 1.718 | 4.227 | 0.723 | 2.917 | 1.288 | 2.172 | 3.08 | 0.963 | 3.959 |
Application Example 3
100 minute marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method)
Arsenic-IgM chelate content in this blood sample detects in accordance with the following steps:
1) substance that IgM will be captured, as anti-IgM (anti-IgM Ab) is coated on solid phase carrier: slow with dilution
It anti-IgM Ab to 1000 times of fliud flushing dilution, is added in elisa plate micropore, 4 DEG C are stayed overnight 18 hours；
2) it takes 100 parts of standard blood samples as measuring samples, adds toluene, dissolve cell membrane；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, it is pure that 2% ox blood is added
As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate
It is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate: the measuring samples in step 2), the arsenic-IgM chelate with known content are added
Make standard items；10 times are diluted to dilution buffer dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, be added with papain
Concentration be 100ng/ml eluent, elute 3 hours；
6) it is acidified: nitric acid being added in the solution in step 5), solution is acidified, sealing overnight, is thoroughly acidified；
7) it detects: hydrogen peroxide is added, and heats and catches up with acid, and sampled from corresponding solution, in inductively coupled plasma
For detection chelating in the arsenic of IgM, detected value is as shown in table 6 under constitution spectrometer.
The ICP-MS testing result of 6 100 parts of sample blood sample arsenic-IgM chelates of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| μg/L | 0.524 | 1.81 | 0.517 | 3.665 | 2.601 | 4.01 | 0.746 | 0.795 | 1.071 | 2.139 |
| Number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| μg/L | 2.056 | 4.052 | 2.831 | 2.599 | 1.707 | 3.431 | 1.921 | 2.507 | 3.661 | 1.623 |
| Number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| μg/L | 1.588 | 1.171 | 0.647 | 2.67 | 0.976 | 1.503 | 0.085 | 3.469 | 2.677 | 2.337 |
| Number | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| μg/L | 3.967 | 2.416 | 3.542 | 2.03 | 3.043 | 1.452 | 0.604 | 3.998 | 0.767 | 1.216 |
| Number | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| μg/L | 1.626 | 3.342 | 3.688 | 0.749 | 3.826 | 4.008 | 2.844 | 2.982 | 3.391 | 1.176 |
| Number | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| μg/L | 0.064 | 3.25 | 3.55 | 3.034 | 4.224 | 2.383 | 3.765 | 1.838 | 4.018 | 1.821 |
| Number | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| μg/L | 1.795 | 4.12 | 2.278 | 0.083 | 2.317 | 4.103 | 0.507 | 2.3 | 1.019 | 2.556 |
| Number | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| μg/L | 0.547 | 0.255 | 0.053 | 3.255 | 1.195 | 2.06 | 1.818 | 0.523 | 0.014 | 0.163 |
| Number | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| μg/L | 0.333 | 3.166 | 4.249 | 4.188 | 0.597 | 2.456 | 1.65 | 2.325 | 0.591 | 3.886 |
| Number | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| μg/L | 1.666 | 0.564 | 2.937 | 2.759 | 1.416 | 2.815 | 1.989 | 2.251 | 2.625 | 0.948 |
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (4)
1. a kind of application of arsenic-IgM chelate in preparation detection blood sample in the reagent or kit of arsenic-IgM chelate, special
Sign is that the arsenic-IgM chelate is chelated with IgM by sulfydryl or/and cysteine residues by arsenic ion；
The arsenic-IgM chelate is through the following steps that be made:
A) the chelatropic reaction of arsenic and IgM: arsenic ion is added in the IgM recombinated according to biological method and carries out chelatropic reaction, obtains
To reaction solution；
B) purify arsenic-IgM chelate extraction: use immune-affinity chromatography, remove reaction solution in unreacted IgM and
Extra arsenic ion is to get arsenic-IgM chelate；
C): the identification to arsenic-IgM chelate；The step B) specific as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), redissolves arsenic-IgM chelate；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM specificity
In conjunction with filler, fill column after, continue to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM and filler are special
The opposite sex combines；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphate of 0.05-0.1mol/L
Solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4 DEG C
Dialysed overnight collects sample；
(7) it balances chromatographic column: using new chromatographic column, with dilution buffer flushing pipeline, energy and arsenic are packed into the chromatographic column
The filler of specific binding balances chromatographic column with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, it is then molten using the phosphate of 0.5-1.0mol/L
Liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4
DEG C dialysed overnight collects sample to get arsenic-IgM chelate；
Step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of extraction purification, dilution buffer is added, and mix, then plus
Sample is in sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, then again
Using ICP-MS method, AAS method or ELISA method detection whether containing arsenic and detect arsenic content.
2. a kind of kit including at least arsenic-IgM chelate as standard items, which is characterized in that the arsenic-IgM chelate is
It is chelated with IgM by sulfydryl or/and cysteine residues by arsenic ion:
The arsenic-IgM chelate is through the following steps that be made:
A) the chelatropic reaction of arsenic and IgM: arsenic ion is added in the IgM recombinated according to biological method and carries out chelatropic reaction, obtains
To reaction solution；
B) purify arsenic-IgM chelate extraction: use immune-affinity chromatography, remove reaction solution in unreacted IgM and
Extra arsenic ion is to get arsenic-IgM chelate；
C): the identification to arsenic-IgM chelate；The step B) specific as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), redissolves arsenic-IgM chelate；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM specificity
In conjunction with filler, fill column after, continue to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM and filler are special
The opposite sex combines；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphate of 0.05-0.1mol/L
Solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4 DEG C
Dialysed overnight collects sample；
(7) it balances chromatographic column: using new chromatographic column, with dilution buffer flushing pipeline, energy and arsenic are packed into the chromatographic column
The filler of specific binding balances chromatographic column with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, it is then molten using the phosphate of 0.5-1.0mol/L
Liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4
DEG C dialysed overnight collects sample to get arsenic-IgM chelate；
Step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of extraction purification, dilution buffer is added, and mix, then plus
Sample is in sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, then again
Using ICP-MS method, AAS method or ELISA method detection whether containing arsenic and detect arsenic content.
3. kit according to claim 2, which is characterized in that further include coating buffer, which, which contains, can capture IgM
Albumen or the substance of arsenic can be captured.
4. a kind of method of quantitative detection arsenic-IgM chelate, which is characterized in that with the described in claim 1 of known content
Arsenic-IgM chelate is detected as standard items using a pair of sample of following methods: enzyme-linked immunization, enzyme linked immunological and original
Sub- absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgM chelate with it is enzyme-linked
Immune combined techniques, purification arsenic-IgM chelate and atomic absorption spectrum combined techniques, purification arsenic-IgM chelate and inductive coupling etc.
Gas ions mass spectrum combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques:
The arsenic-IgM chelate is through the following steps that be made:
A) the chelatropic reaction of arsenic and IgM: arsenic ion is added in the IgM recombinated according to biological method and carries out chelatropic reaction, obtains
To reaction solution；
B) purify arsenic-IgM chelate extraction: use immune-affinity chromatography, remove reaction solution in unreacted IgM and
Extra arsenic ion is to get arsenic-IgM chelate；
C): the identification to arsenic-IgM chelate；The step B) specific as follows:
(1) sample dissolution: being added physiological saline into the reaction solution obtained by step A), redissolves arsenic-IgM chelate；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgM specificity
In conjunction with filler, fill column after, continue to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgM and filler are special
The opposite sex combines；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphate of 0.05-0.1mol/L
Solution is eluted；
(5) it collects: collecting the eluent after step (4) elution, make protein renaturation after collection immediately；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4 DEG C
Dialysed overnight collects sample；
(7) it balances chromatographic column: using new chromatographic column, with dilution buffer flushing pipeline, energy and arsenic are packed into the chromatographic column
The filler of specific binding balances chromatographic column with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, it is then molten using the phosphate of 0.5-1.0mol/L
Liquid is eluted；
(10) it collects: collecting the eluent after step (9) elution, make protein renaturation after collection immediately；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4
DEG C dialysed overnight collects sample to get arsenic-IgM chelate；
Step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained arsenic-IgM chelate of extraction purification, dilution buffer is added, and mix, then plus
Sample is in sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing arsenic on glue bed, which is taken out, protein band is redissolved, then again
Using ICP-MS method, AAS method or ELISA method detection whether containing arsenic and detect arsenic content.
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
| JP2000321263A (en) * | 1999-05-14 | 2000-11-24 | Murata Mfg Co Ltd | Quantitative determination method for trivalent titanium ions and divalent iron ions |
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