CN104987405B - A kind of arsenic IgG chelates and its preparation method and application - Google Patents

A kind of arsenic IgG chelates and its preparation method and application Download PDF

Info

Publication number
CN104987405B
CN104987405B CN201510411518.9A CN201510411518A CN104987405B CN 104987405 B CN104987405 B CN 104987405B CN 201510411518 A CN201510411518 A CN 201510411518A CN 104987405 B CN104987405 B CN 104987405B
Authority
CN
China
Prior art keywords
igg
arsenic
chelates
chromatographic column
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201510411518.9A
Other languages
Chinese (zh)
Other versions
CN104987405A (en
Inventor
张积仁
阳帆
董欣敏
吴婧
蔡睿
孙遥
赵乙木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Tenpai Medical Ltd By Share Ltd
Original Assignee
Guangdong Tenpai Medical Cci Capital Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Tenpai Medical Cci Capital Ltd filed Critical Guangdong Tenpai Medical Cci Capital Ltd
Priority to CN201510411518.9A priority Critical patent/CN104987405B/en
Publication of CN104987405A publication Critical patent/CN104987405A/en
Application granted granted Critical
Publication of CN104987405B publication Critical patent/CN104987405B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard

Landscapes

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

Abstract

The invention discloses a kind of arsenic IgG chelates and its preparation method and application, arsenic IgG chelates are that arsenic ion is formed with IgG by sulfydryl or/and cysteine residues chelating.The present invention establishes the method for qualitative and quantitative detection of arsenic IgG chelates, so that quantitative detection arsenic IgG chelates are evaluating the application of a regional arsenic pollution degree.Situation of this regional crowd by arsenic pollution can be reflected indirectly by arsenic IgG chelates in one regional crowd's serum of quantitative detection, so as to reflect this regional arsenic pollution degree indirectly.Arsenic IgG chelates quantitative detecting method its degree of accuracy that the present invention establishes greatly improves, and the repeatability of detection is greatly enhanced.

Description

A kind of arsenic-IgG chelates and its preparation method and application
Technical field
The present invention relates to detection field, more specifically to a kind of arsenic-IgG chelates and its preparation method and application.
Background technology
IgG is mainly synthesized and secreted by the thick liquid cell in spleen, lymph node, is existed with monomeric form.In ontogenetic process Middle body synthesis IgG age will be later than IgM, start to synthesize within 3rd month after birth, 3~5 years old horizontal close to adult.IgG It is antibody component main in serum, accounts for the 75% of serum total Ig.IgG is uniquely can be by the Ig of placenta, naturally passive Played an important role in immune.In addition IgG also has opsonophagocytosis, ADCC and combines the effect such as SPA.Due to IgG These characteristics, IgG plays main effect in immunity of organism protection, and most of antibacterials, antiviral, t antibody belong to IgG classes and resisted Body.It is artificial passive using that can be carried out to third kind of the immune puerpera such as measles, hepatitis A or normal person or placental globulin It is immune, prevent corresponding communicable disease with having suburb.
Arsenic is one of common 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) first-class harmful substance (http is classified as always：//www.atsdr.cdc.gov/ 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)) delimit as one of human carcinogen.Arsenic can produce acute or chronic toxicity to human body, anxious Property arsenic poisoning can cause the exacerbated events such as vomiting, dry, muscle cramp, stomachache, trick shouting pain, and arsenicalism is good for the mankind The influence of health also turns into a significant problem of concern.It is two kinds by drinking water and air to contact most with arsenic in environment Direct mode, it can induce angiocardiopathy, cause maladjusted nervous system and cause diabetes and lung cancer, skin lung cancer, wing Guang cancer etc..And currently a popular disease learns display, polluted in underground drinking water caused by arsenic existing for nature, in Taiwan, A Gen The court of a feudal ruler, Chile, West Bengal, India, Bangladesh, the Inner Mongol, China etc. cause multiple countries and regions the arsenic of high dose to expose, As environmental pollution and the global problem of harm health.The whole world has had more than five million peoples and has been in arsenic exposure and chronic arsenic at present Among the threat of poisoning.
A variety of methods detection bleeding arsenic content, including ELISA, atomic absorption spectrum (Atomic can be used at present Absorption Spectroscopy, AAS) quantitative assay, icp mses (inductively Coupled plasma mass spectrometry, ICP-MS) quantitative assay etc., but still suffer from certain defect.
On the evaluation of arsenic poisoning, particularly arsenicalism, it is only capable of by detecting blood arsenic content, in indirect reaction human body Circulation arsenic content, can not further assess degree of injury of the arsenic for body function, and with the rapid development of science and technology, The relation of arsenic and human body is also increasingly close, thus searching one kind can evaluate arsenic poisoning from body function angle, particularly chronic Arsenic poisoning becomes more and more important for the evaluation method of the extent of damage of body.
The content of the invention
For arsenic pollution it is serious the problem of, it is an object of the invention to provide a kind of arsenic-IgG chelates and its preparation side Method, and the qualitative and quantitative analysis method of arsenic-IgG chelates is established, so that quantitative detection arsenic-IgG chelates are evaluating a ground The application of area's arsenic pollution degree.This can be reflected indirectly by arsenic-IgG chelates in one regional crowd's serum of quantitative detection Regional crowd by arsenic pollution situation, so as to reflecting this regional arsenic pollution degree indirectly.
The technical solution adopted for the present invention to solve the technical problems is：A kind of arsenic-IgG chelates are provided, arsenic ion with IgG is formed by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned arsenic-IgG chelates, comprises the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source and carries out chelatropic reaction, obtains reaction solution；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed And unnecessary arsenic ion, produce arsenic-IgG chelates.
Wherein, step B described in external synthetic method) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, answer arsenic-IgG chelates It is molten；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG The filler that the opposite sex combines, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG is with filling out Material specific binding；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid Salting liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces arsenic-IgG chelates.
Wherein, the preparation method of above-mentioned arsenic-IgG chelates, in addition to step C)：Identification to arsenic-IgG chelates；
Wherein, step C) in it is specific as follows：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, so After be loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, so Afterwards again using ICP-MS methods, AAS methods or ELISA method detection whether containing arsenic and detect arsenic content.
The present invention also provides a kind of arsenic-IgG chelates described above and is preparing the examination of arsenic-IgG chelates in detection human body Application in agent or kit.
The present invention, which also provides, a kind of comprises at least kit of the arsenic-IgG chelates described above as reference substance.
Preferably, coating buffer is also included in the kit, the coating buffer contains the albumen that can capture IgG or the thing for capturing arsenic Matter.
The present invention also provides a kind of method for quantitatively detecting arsenic-IgG chelates, with the above-mentioned arsenic-IgG chelas of known content Compound is detected as reference substance using a pair of samples of following methods：ELISA, enzyme linked immunological and atomic absorption light Spectrum combined techniques, enzyme linked immunological are combined with inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgG chelates with enzyme linked immunological Method, purification arsenic-IgG chelates and atomic absorption spectrum combined techniques, purification arsenic-IgG chelates and inductively coupled plasma matter Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement arsenic-IgG chelates of the present invention and its preparation method and application, have the advantages that：
1. arsenic-IgG chelates are extracted in present invention synthesis in vitro first from whole blood；
2. present invention firstly provides arsenic-IgG chelates can be used for prepare detection blood sample in arsenic-IgG chelates reagent or Application in kit.
3. the present invention establishes the method for qualitative and quantitative detection of arsenic-IgG chelates, so as to quantitative detection arsenic-IgG chelates Evaluating the application of a regional arsenic pollution degree.Can be with by arsenic-IgG chelates in one regional crowd's serum of quantitative detection Reflect situation of this regional crowd by arsenic pollution indirectly, so as to reflect this regional arsenic pollution degree indirectly.The present invention establishes Arsenic-IgG chelates quantitative detecting method its degree of accuracy greatly improve, and the repeatability of detection is greatly enhanced.
Brief description of the drawings
Fig. 1 is the non denatured electrophoretic band figure of arsenic-IgG chelates of the present invention；
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of arsenic-IgG chelates of the present invention；
Wherein, in Fig. 1, M Marker, 5 be arsenic-IgG chelates；In Fig. 2, abscissa is protein band position, indulges and sits It is designated as the energy of arsenic in the protein band.
Embodiment
Below, with reference to embodiment, the present invention is described further：
Unless otherwise indicated, the laboratory operating procedures being related to are the conventional step in this area to the present invention, agents useful for same, material Material as following cited, do not include in the present invention come be reagent commonly used in the art or can be by mode purchased in market Obtain：
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
Glue bed medium is one kind in Ago-Gel, polyacrylamide gel；
The filler that can be specifically bound with IgG in the present invention, has the silica gel for the albumen that can capture IgG for surface Or resin；
The albumen (anti-igg antibody) that can capture IgG in the present invention, including but not limited to rabbit Anti- IgGs H＆L, Its brand is Abcam, model ab6715；
The filler that can be specifically bound with arsenic in the present invention, have silica gel or the tree of the material that can capture arsenic for surface Fat；
The anti-As mAb of the material that can capture arsenic in the present invention, including but not limited to mouse, buy from Guangzhou Ran Ke companies, goods Number it is RK15728；
Enzyme labelled antibody is one kind in the antibody containing the enzymes such as horseradish peroxidase, alkaline phosphatase mark；
Substrate is methyl biphenyl amine (TMB) solution；
Cleaning solution is to contain KH2PO4 0.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl 8.0mg/ml、KCl 0.2mg/ml, 0.5%Tween-20 pH are 7.4 0.15M PBS solutions；
Confining liquid is 1%-5% bovine serum albumin(BSA)s or skimmed milk power；
Sample diluting liquid is Na containing 1.5mg/mL2CO3、2.93mg/ml NaHCO3PH be 9.6 0.05M phosphate delay Fliud flushing；
Enzyme labelled antibody is HRP enzyme labelled antibodies；
Terminate liquid is：By 21.7ml 2M H2SO4It is settled to 200ml ddH2In O；
Eluent is the 0.1mol/L Tris-HCL buffer solutions that the PH 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 Propylhomoserin 25mL Sample buffer (5X)；
Electrophoretic buffer is the ddH that 3mg/ml containing Tris, glycine 14.4mg/ml PH are 6.82O solution.
The present invention provides a kind of arsenic-IgG chelates, arsenic ion and IgG by sulfydryl or/and cysteine residues chelate and Into.
The present invention also provides a kind of preparation method of arsenic-IgG chelates, comprises the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source and carries out chelatropic reaction；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed And unnecessary arsenic ion, arsenic-IgG chelates are produced, are comprised the following steps that：
(1) sample dissolution：By above-mentioned steps A) in extraction arsenic-IgG chelates be dissolved in physiological saline；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG The filler that the opposite sex combines, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG is with filling out Material specific binding；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, dialysed overnight, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid Salting liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, dialysed overnight, sample is collected, produces arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, so After be loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：Corresponding protein band is found out on glue bed, the protein band is taken out, protein band is redissolved, then Detect in the liquid whether containing arsenic and detect the content of arsenic using ICP-MS, AAS again.
The present invention, which also provides, a kind of comprises at least kit of the arsenic-IgG chelates described above as reference substance.
Preferably, coating buffer is also included in the kit, the coating buffer is capture IgG albumen or the material of capture arsenic.
The present invention also provides a kind of preparation method of arsenic-IgG chelates, comprises the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source or the IgG according to biological method restructuring Chelatropic reaction is carried out, obtains reaction solution；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed And unnecessary arsenic ion, arsenic-IgG chelates are produced, are comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, answer arsenic-IgG chelates It is molten
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG The filler that the opposite sex combines, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG is with filling out Material specific binding；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid Salting liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, so After be loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, so Afterwards again using ICP-MS methods, AAS methods or ELISA method detection whether containing arsenic and detect arsenic content.
The present invention, which also provides, a kind of comprises at least kit of the arsenic-IgG chelates described above as reference substance.
Preferably, coating buffer is also included in the kit, the coating buffer contains the albumen that can capture IgG or can capture arsenic Material.
In the present invention, can realize the kit of the object of the invention can list following several, but be not limited to this.
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG, Confining liquid, cleaning solution, the material for capturing arsenic as secondary antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, loading are delayed Fliud flushing, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG, Confining liquid, cleaning solution, eluent, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG, Confining liquid, cleaning solution, eluent, sample-loading buffer, acidulant, hydrogen peroxide, standard items, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood Agent, redissolve liquid, the coating buffer containing the material that can capture arsenic, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution Buffer solution, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood Agent, redissolve liquid, sample-loading buffer, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG in purification whole blood Agent, sample-loading buffer, redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Coating buffer containing the albumen that can capture IgG, Glue bed medium, redissolve liquid, sample-loading buffer, liquid needed for the protein band containing arsenic on dissolving glue bed, contain the thing that can capture arsenic The coating buffer of matter, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative control etc..
The kit of arsenic-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG 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-IgG chelates in a kind of detection blood sample, including：Examination is extracted as needed for IgG 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 IgG chelates of weight arsenic or is chelated with The BSA chelates of weight arsenic；The negative control is dilution buffer.
Mentioned reagent box is used for the IgG for detecting chelating arsenic, to improve the accuracy of detection, repeatability, and is allowed in clinic In be promoted.
The present invention also provides a kind of method for quantitatively detecting arsenic-IgG chelates, with the above-mentioned arsenic-IgG chelas of known content Compound is detected as reference substance using a pair of samples of following methods：ELISA, enzyme linked immunological and atomic absorption light Spectrum combined techniques, enzyme linked immunological are combined with inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgG chelates with enzyme linked immunological Method, purification arsenic-IgG chelates and atomic absorption spectrum combined techniques, purification arsenic-IgG chelates and inductively coupled plasma matter 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 above-mentioned method for quantitatively detecting arsenic-IgG chelates is as follows：
Method one：ELISA (ELISA method) detects arsenic-IgG chelates, detects in accordance with the following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with sample diluting liquid dilution IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 are small When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；Corresponding multiple is diluted to sample diluting liquid dilution measuring samples, that is, dilutes 10-40 times, adds in micropore, 37 DEG C Act on 1-2 hours；
5) material of arsenic can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed After the completion of washing, addition sample diluting liquid dilutes and can capture the material of arsenic or can react to form antigen antibody complex with arsenic Anti- As Ab, 37 DEG C of effect 1-2 hours, react the arsenic on anti-As Ab and IgG；
6) enzyme conjugates incubates：Anti- arsenic antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition sample The enzyme labelled antibody of product diluted, the concentration for making the enzyme labelled antibody of dilution are 2 μ g/ml, 37 DEG C of effect 1-2 hours, make its with Enzyme labelled antibody reacts；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore in measuring samples；
9) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA and reads measuring samples and mark respectively The OD values of quasi- product, by drawing standard curve, try to achieve the content of measuring samples (also can directly pass through dyeing without using ELIASA Situation carries out qualitative detection).
This method utilizes ELISA principles, can extract the specific IgG in whole blood, the IgG tops extracted Divide and be chelated with weight arsenic, and the arsenic on the IgG of this part can be captured by the specific antibody of anti-arsenic, afterwards can be again by horseradish mistake The antibody of the enzymes such as oxide enzyme, alkaline phosphatase mark captures (the antibody nonrecognition coating protein), and the antibody in capture exists In the presence of developer and terminate liquid, OD values can be read under instrument, and do not contain the IgG of chelating arsenic, then will not be by anti-arsenic Specific antibody captured, will not also be captured with the antibody of the enzyme such as horseradish peroxidase, alkaline phosphatase mark, and institute With not containing arsenic (negative control group result for feminine gender) in reagent, thus when the OD value results read are shown as the positive yet, The i.e. provable arsenic for detecting to chelate on IgG.
Method two：Enzyme linked immunological is pressed with atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection arsenic-IgG chelates Detected according to following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with sample diluting liquid dilution IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 are small When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rpm centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Coating buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% cow's serums As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, have been washed for albumin or skimmed milk power Cheng Hou, elisa plate place 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；Corresponding multiple is diluted to sample diluting liquid dilution measuring samples, that is, dilutes 10-40 times, adds in micropore, 37 DEG C Act on 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, add eluent, elute 1- 3 hours.
6) detect：Sample, chelated in Atomic Absorption Spectrometer detection in the arsenic on IgG, and draw mark from ELISA micropores Directrix curve, read respective value；
The embodiment combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principles, utilizes atomic absorption light Spectrometer detection is chelated in the arsenic on IgG, due to only containing IgG in solution, and any arsenic (negative control group is free of in agents useful for same As a result it is feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can prove to detect The arsenic chelated on IgG.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection arsenic- IgG chelates detect in accordance with the following steps：
1) IgG material will can be captured, as human IgG antibody is coated on solid phase carrier：It is anti-with sample diluting liquid dilution IgG Ab to 500000-4000000 times, add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 are small When, store refrigerator；
2) whole blood is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；Corresponding multiple is diluted to sample diluting liquid dilution measuring samples, that is, dilutes 10-40 times, adds in micropore, 37 DEG C Act on 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, add eluent, elute 1- 3 hours.
6) it is acidified：Add acidulant in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified；
7) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled in the solution eluted from ELISA agent plates, in Detection chelates the arsenic in IgG under icp mses, and draws standard curve, reads respective value.
This method is on the basis of using ELISA principles, with reference to sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption Sense couple plasma mass spectrometer detection is chelated in the arsenic on IgG, due to only containing IgG in solution, and is free of in agents useful for same Any arsenic (negative control group result is feminine gender), will not interfere to result, thus works as read result and be shown as positive When, you can prove to detect the arsenic chelated on IgG.
Method four：Arsenic-IgG chelates are purified to chelate with enzyme linked immunological combined techniques (method of purification+ELISA method) detection arsenic-IgG Thing, detect in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method is redissolved the IgG of the purification of blood out using whole blood extraction method, obtains IgG redissolution liquid；
2) anti-As Ab are coated on solid phase carrier：Anti- As Ab to 25000-200000 times is diluted with sample diluting liquid, Add in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-bath 1-3 hours, store refrigerator；
3) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with 1%-5% oxen As confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, are washed for seralbumin or skimmed milk power After the completion of washing, elisa plate places 1-2 hours in 36.5-37.5 DEG C；
4) add measuring samples, and incubate：Sampled from the IgG of extraction redissolution liquid, make measuring samples；With known content Arsenic-IgG chelates make standard items；Corresponding multiple is diluted with sample diluting liquid, that is, dilutes 10-40 times, is added in micropore, 37 DEG C Act on 1-2 hours；
5) enzyme conjugates incubates：IgG redissolution liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, adds and uses The enzyme labelled antibody of sample diluting liquid dilution, the concentration for making the enzyme labelled antibody of dilution is 2 μ g/ml, 37 DEG C of effect 1-2 hours, makes it Reacted with enzyme labelled antibody；
6) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
7) terminating reaction：Terminate liquid is added dropwise to each micropore in measuring samples；
8) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA and reads measuring samples and mark respectively The OD values of quasi- product, by drawing standard curve, try to achieve the content of measuring samples (also can directly pass through dyeing without using ELIASA Situation carries out qualitative detection).
Method five：Arsenic-IgG chelates are purified to detect in blood sample with atomic absorption spectrum combined techniques (method of purification+AAS methods) Arsenic-IgG chelates, are detected in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) detect：Sample in redissolution liquid from step 1), chelated in Atomic Absorption Spectrometer detection in the arsenic on IgG, And standard curve is drawn, read respective value.
Method six：Purify arsenic-IgG chelates and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS methods) Arsenic-IgG chelates are detected, are detected in accordance with the following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) it is acidified：Sampled in redissolution liquid from step 1), add acidulant (such as nitric acid) in the solution and solution is carried out Acidifying, sealing overnight, are thoroughly acidified；
3) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled from solution, in inductively coupled plasma matter Detection is chelated in the arsenic on IgG under spectrometer, and draws standard curve, reads respective value.
Method seven：Electrophoresis and ELISA (electrophoresis-ELISA method) detection arsenic-IgG chelates, in accordance with the following steps Detection：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) glue bed is prepared：Selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gels as needed Deng medium is used as, corresponding glue bed is conventionally prepared；
3) it is loaded：Take the μ L of redissolution liquid 8 in step 1) to add 2 μ L sample-loading buffers, mix, of short duration centrifugation；(pay attention to herein Step can not boil)
4) electrophoresis：Electrophoresis plate is connected, is separated by electrophoresis；
5) detect：The protein band containing arsenic is found out on glue bed, the band is taken out, after treatment by the albumen one Band redissolves, and then recycles the detection of ELISA principles to be dissolved in the arsenic content in liquid.Further, it is also possible to detect chela using the method Close IgG isoelectric point, molecular weight and content of arsenic etc..
Method eight：Electrophoresis and atomic absorption spectrography (AAS) (electrophoresis-AAS methods) detection arsenic-IgG chelates, according to following step Rapid detection：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) glue bed is prepared：Selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gels as needed Deng medium is used as, glue bed is prepared；
3) it is loaded：Sampled in redissolution liquid from step 1), add sample-loading buffer, and mixed, be then loaded onto sample In groove；
4) electrophoresis：Electrophoresis plate is connected, is separated by electrophoresis；
5) detect：The protein band containing arsenic is found out on glue bed, the band is taken out, after treatment by the albumen one Band redissolves, and then recycles the detection of AAS principles to be dissolved in the arsenic content in liquid.Chelated further, it is also possible to be detected using the method The IgG of arsenic isoelectric point, molecular weight and content etc..
Method nine：Inductivity coupled plasma mass spectrometry combined techniques (electrophoresis-ICP-MS methods) detects arsenic-IgG chelates, presses Detected according to following steps：
1) IgG is extracted from whole blood：Using the polyethylene glycol PEG precipitation method, ultracentrifugation, molecule ultrafiltration, gel filtration etc. Method redissolves the IgG of the purification of blood out from whole blood extraction method, obtains IgG redissolution liquid；
2) glue bed is prepared：Selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gels as needed Deng medium is used as, glue bed is prepared；
3) it is loaded：Sampled in redissolution liquid from step 1), add sample-loading buffer, and mixed, be then loaded onto sample In groove；
4) electrophoresis：Electrophoresis plate is connected, is separated by electrophoresis；
5) detect：The protein band containing arsenic is found out on glue bed, the band is taken out, after treatment by the albumen one Band redissolves, and then recycles the detection of ICP-MS principles to be dissolved in the arsenic content in liquid.Further, it is also possible to detect chela using the method Close IgG isoelectric point, molecular weight and content of arsenic etc..
IgG 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.), IgG out will be purified and redissolved, obtain IgG redissolution Liquid, a certain amount of IgG is taken, using electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), (can root in gel slab According to needing to use 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 protein in gel is redissolved in solution with corresponding double solvents, you can to detect related IgG at a particular wavelength Content, the principle such as ELISA, AAS, ICP-MS can also be utilized to detect to chelate in the arsenic content on IgG, due in solution only Containing IgG, and result will not be interfered without any arsenic (negative control group result is feminine gender) in agents useful for same, thus When the result read is shown as the positive, you can prove to detect the arsenic chelated on IgG.
Embodiment 1：The preparation method of arsenic-IgG chelates, comprises the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source and carries out chelatropic reaction, obtains reaction solution；
Preparation of reagents：
1) borate buffer solution (0.01M)：Weigh 0.31g boric acid to be dissolved in 400ml ultra-pure waters, adjusted with 0.1M NaOH PH to 9.0, is settled to 500mL.
2) IgG solution：Weigh 4.0mgIgG to be dissolved in 4.0mL 0.01M pH9.0 borate buffer solutions, fully vibrate molten Solution, it is configured to 1.0mg/mL protein solution；
3)5mmol/L EDTA+200mmol/LNaHCO3Solution：Weigh EDTA2H2O 1.86g、NaHCO316.8g is molten In 900mL ultra-pure waters, 1000ml, autoclaving, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
4) ITCBE (buying from Japanese colleague's chemistry institute, article No. M030)
5) bag filter (molecular cut off 14000) (Bioshop Inc)
Preparation process is specially：
1) processing of bag filter：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, gently rinsed, then used with ultra-pure water 500ml 5mmol/L EDTA boil 10min；Boiling liquid is discarded, is thoroughly cleaned with ultra-pure water, adds substantial amounts of ultra-pure water immersion 4 DEG C of bag filter is overnight.In use, putting on one's gloves, bag filter is taken out, with substantial amounts of its surfaces externally and internally of ultra-pure water cleaning down；
2) 2.0mg ITCBE are taken to be dissolved in 2ml DMSO；
3) 4.0mg IgG are taken to be dissolved in 4.0ml borate buffer solutions in (0.01M pH9.0)；
4) liquid for slowly preparing step 2 is added in IgG solution, is shaken when being added dropwise, in 25 DEG C, 100r/min's shakes 24h is acted in bed, then with bag filter dialysis 24h, removes the ITCBE not combined with IgG；
5) liquid for gained of dialysing is adjusted into pH value to 7.0 with 1mol/L HCl, 80 μ l is then slowly gradually added dropwise 1mmol/L arsenic ion solns, vibrated when being added dropwise, in case arsenic ion precipitates albuminous degeneration；
6) solution added is reacted into 2h in 25 DEG C, 100r/min shaking table, dialysed with the bag filter handled well 24h；
7) liquid after dialysis is preserved in -20 DEG C of packing.
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, unreacted IgG in reaction solution is removed And unnecessary arsenic ion, arsenic-IgG chelates are produced, are comprised the following steps that：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, answer arsenic-IgG chelates It is molten；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be special with IgG The filler that the opposite sex combines, after filling post, it is continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG is with filling out Material specific binding；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphorus Acid salt solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, load energy in the chromatographic column With the filler of arsenic specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.5-1.0mol/L phosphoric acid Salting liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, change water three times Afterwards, 4 DEG C of dialysed overnights, sample is collected, produces arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates, comprise the following steps that：
(1) glue bed is prepared：Glue bed is prepared using Ago-Gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of 8 μ L extraction purifications, add 2 μ L sample-loading buffers, and Mix, be then loaded onto in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, electrophoretic buffer is added and carries out electrophoresis；In electrophoresis process, electric current is 22mA constant currents, Environment temperature is 4 degree；Stop electrophoresis when moving to glue bottom to bromophenol blue；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, so The content of arsenic is detected whether containing arsenic and detected again afterwards using AAS methods.
D) testing result
(1) electrophoresis result：
Wherein, Fig. 1 is the non denatured electrophoretic band figure of arsenic-IgG chelates of the present invention.
(2) Synchrotron Radiation X-Ray Fluorescence Anal ysis：
4W1 " of the SRXRF analyses of micronutrient levels in BEPC (BEPC) is synchronous in protein band Completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200 Type, Beijing are stood upright Han Guang companies) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into Penetrate facula position, moving step length 0.0025mm.From the X ray that electromagnetic radiation goes out by Si (Li) detector (PGT Inc.LS 30143-DS) detect, probe is with incident SR lines copline and being mutually perpendicular to, and away from sample irradiation point 20mm, signal is divided with PGT multiple tracks Analyzer (MCA4000) obtains output.Sample, regulation launching spot (1mmx3mm) are excited with 11.5keV monochromatic synchrotron radiation light Position is allowed to be in band one end, and in 300s minute, hot spot uniformly slowly moves along band always, at the end of counting Hot spot moves on to the band other end.Along electrophoresis direction a spectrum is taken per 1mm.Using AX IL software data processings, and with deriving from Air and the Ar signal peaks of content constant carry out normalization to other element peaks, to offset beam intensity change to signal strength Caused influence.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way under the same conditions.
Fig. 2 is the fluorescence analysis figure of the synchrotron radiation X line electrophoretic band of arsenic-IgG chelates of the present invention, horizontal in figure Coordinate is protein band position, and ordinate is energy (content) value of arsenic in the protein band.
(3) use in the arsenic-IgG chelates that graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination the present embodiment obtains Arsenic content, its content is 102.006 μ g/L.
A kind of determination of the testing conditions for the method for quantitatively detecting arsenic-IgG chelates of the present invention：
1. the determination of the optimum diluting multiple of complement protein best effort concentration and blood plasma
Step is as follows：
(1) by IgG Ab sample diluting liquids according to mass volume ratio (dilution factor) 1:500000、1:1000000、1: 2000000、1:4000000 are diluted, and add in elisa plate micropore, anti-igg Ab are coated on solid phase carrier, Mei Genong Degree three rows of coating, 4 DEG C are stayed overnight 18 hours；
(2) sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, is made with 2% bovine serum albumin(BSA) For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) measuring samples and dilution buffer are according to following mass volume ratio (dilution factor) 1:10、1:20、1:40 progress are dilute Release, add in micropore, according to above-mentioned coated anti-igg Ab concentration, being separately added into for the anti-igg Ab of same concentration is different dilute Degree of releasing blood plasma, 37 DEG C act on 1 hour；
(4) measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, add anti-As Ab, anti-As Ab with Dilution buffer is according to following mass volume ratio (dilution factor) 1:25000、1:50000、1:100000、1:200000 progress are dilute Release, according to each identical anti-igg Ab, serum-dilution concentration, the anti-As Ab of various concentrations respectively add 2 holes, and 37 DEG C act on 1 hour, React the arsenic on anti-As Ab and IgG；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2ng/ml, removes anti-As antibody, and is washed with cleaning solution, Wait after the completion of washing, addition dilution buffer dilutes HRP enzyme labelled antibodies, and 37 DEG C act on 1 hour, make HRP enzyme labelled antibodies and arsenic- IgG chelates react；
(6) enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects 30 minutes；
(7) with adding substrate solution same speed and order that terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and reads each hole OD values respectively.Root According to each hole OD value numerical value, anti-igg Ab, anti-As-Ab best effort concentration and the optimum diluting multiple of blood plasma are selected.
In experiment simultaneously using made reference substance as positive control, select IgG antibody+closing+As resist+enzyme mark+substrate (i.e. It is not added with detecting sample) it is right as feminine gender as negative control 1, IgG antibody+closing+blood plasma+enzyme mark+substrate (being not added with As to resist) According to 2, IgG antibody+closing+enzyme mark+substrate (be not added with detecting sample and As resists) as negative control 3, IgG antibody+closing+blood Slurry+As is anti-+ and substrate (i.e. not enzyme-added mark) is used as negative control 4, and closing+blood plasma+As resists+enzyme mark+substrate (being not added with IgG antibody) 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-igg Ab and As antibody best effort concentration and diluted plasma multiple
Table 2：ELISA positive controls and negative control ELISA testing results
Shown by table 1-2 data, we can see that when the dilution factor of human IgG antibody is 1:1000000th, whole blood dilution factor For 1:20th, the anti-dilution factors of As are 1:When 5000, OD values are maximum, although OD values are less than 0.8, the negative control group corresponding to it OD values are all less than 0.1, and the positive controls corresponding to it, and OD values are more than 0.8, so selecting the concentration corresponding to this value to make For best effort concentration, (i.e. human IgG antibody's concentration is 1:1000000, whole blood dilution factor is 1:20, As anti-diluted concentrations are 1: 25000)。
2. ELISA eluent best effort concentration and time determine
Step is as follows：(1) human IgG antibody is diluted to 1000000 times (mass volume ratios) with sample diluting liquid, added In elisa plate micropore, 37 DEG C of water-baths 3 hours, refrigerator is stored；
(2) sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, is made with 2% bovine serum albumin(BSA) For confining liquid, 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution；
(3) confining liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, addition is diluted with dilution buffer HRP enzyme labelled antibodies, 37 DEG C act on 2 hours, it is reacted with human IgG antibody；
(4) eluent is prepared：By papain with pH 8.0,0.1mol/L Tris-HCI buffers into 1- 2mg/ml, add 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT)s (DTT)；
(5) enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes 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, is waited after the completion of washing, adds substrate, 37 DEG C of lucifuge effects 30 Minute；
(7) with adding substrate solution same speed and order that terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed on ELIASA and reads every group of OD value respectively, is led to The optimum concentration and elution time for compared with PBS group, comparing eluent are crossed, 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 papain in eluent concentration and enzyme labelled antibody the ratio between concentration= 1:When 20, i.e. during the concentration 100ng/ml of papain, each group OD values are below other several groups, illustrate that the concentration eluent is washed De- effect is optimal (to reach maximum, thus OD values by the human IgG antibody combined on ELISA hole walls-enzyme mark compound elution degree It is minimum)；And action time either 1h, 2h, 3h, each group OD value changes are 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
Take using the arsenic-IgG chelates in 100 parts of sample blood plasma of ELISA method detection, follow the steps below detection： ELISA (ELISA method) detects arsenic-IgG chelates, detects in accordance with the following steps：
1) anti-igg Ab is coated on solid phase carrier：Anti-igg Ab to 1000000 times is diluted with sample diluting liquid, is added In elisa plate micropore, 37 DEG C of water-baths 1 hour, refrigerator is stored；
2) whole blood is taken from the circulatory system, makees measuring samples, add toluene, dissolve cell membrane；
3) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, adds 2% bovine serum albumin It is white be used as confining liquid, 37 DEG C of placements 1 hour, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate in 37 DEG C are placed 1 hour；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；Corresponding multiple is diluted to sample diluting liquid dilution measuring samples, that is, dilutes 20 times, adds in micropore, 37 DEG C of works With 1 hour；
5) material of arsenic can be captured by adding, and be incubated：Measuring samples are removed, and are washed with cleaning solution, it is to be washed After the completion of washing, addition sample diluting liquid dilutes 25000 times, and 37 DEG C act on 1 hour, reacts the arsenic on anti-As Ab and IgG；
6) enzyme conjugates incubates：Anti- arsenic antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition sample The HRP enzyme labelled antibodies of product diluted, 37 DEG C act on 1 hour, it is reacted with enzyme labelled antibody；
7) substrate incubates：Enzyme labelled antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, addition substrate, 37 DEG C Lucifuge acts on 30 minutes；
8) terminating reaction：Terminate liquid is added dropwise to each micropore in measuring samples；
9) take wavelength 405nm, after adding terminate liquid, elisa plate is placed on ELIASA and reads measuring samples and mark respectively The OD values (also directly can carry out qualitative detection by staining conditions without using ELIASA) of quasi- product, testing result is as shown in table 4.
Table 4：The ELISA testing results of arsenic-IgG chelates in 100 parts of blood samples
Numbering 1 2 3 4 5 6 7 8 9 10
OD405 0.55 0.461 0.381 0.318 0.596 0.492 0.29 0.426 0.763 0.165
Numbering 11 12 13 14 15 16 17 18 19 20
OD405 0.183 0.349 0.268 0.576 0.6 0.293 0.206 0.407 0.493 0.709
Numbering 21 22 23 24 25 26 27 28 29 30
OD405 0.735 0.723 0.481 0.418 0.588 0.264 0.732 0.339 0.165 0.454
Numbering 31 32 33 34 35 36 37 38 39 40
OD405 0.621 0.496 0.736 0.54 0.757 0.66 0.241 0.451 0.194 0.486
Numbering 41 42 43 44 45 46 47 48 49 50
OD405 0.663 0.211 0.607 0.234 0.537 0.225 0.142 0.661 0.615 0.475
Numbering 51 52 53 54 55 56 57 58 59 60
OD405 0.185 0.198 0.443 0.283 0.572 0.203 0.258 0.524 0.157 0.51
Numbering 61 62 63 64 65 66 67 68 69 70
OD405 0.132 0.346 0.392 0.37 0.385 0.252 0.534 0.452 0.419 0.607
Numbering 71 72 73 74 75 76 77 78 79 80
OD405 0.542 0.122 0.222 0.33 0.223 0.104 0.317 0.477 0.575 0.163
Numbering 81 82 83 84 85 86 87 88 89 90
OD405 0.653 0.35 0.773 0.218 0.317 0.48 0.773 0.758 0.475 0.219
Numbering 91 92 93 94 95 96 97 98 99 100
OD405 0.402 0.52 0.213 0.276 0.266 0.76 0.658 0.629 0.331 0.241
Application Example 2
Using in enzyme linked immunological and minute mark this blood sample of atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection 100 Arsenic-IgG chelates, are detected in accordance with the following steps：
1) IgG material will can be captured, as anti-igg antibody (anti-igg Ab) is coated on solid phase carrier：It is dilute with sample Release liquid and dilute anti-igg Ab to 1000000 times, add 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) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood Albumen or skimmed milk power are as confining liquid, and 37 DEG C are placed 1 hour, remove confining liquid, and are washed with cleaning solution, and washing is completed Afterwards, elisa plate is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；20 times are diluted to sample diluting liquid dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, waits after the completion of washing, adds with papain Concentration be 100ng/ml eluent, elute 3 hours；
6) detect：Sample, chelated in Atomic Absorption Spectrometer detection in the arsenic on IgG, detected value is such as from ELISA micropores Shown in table 5.
Table 5：The AAS testing results of arsenic-IgG chelates in 100 parts of blood samples
Numbering 1 2 3 4 5 6 7 8 9 10
μg/L 0.325 0.324 0.978 0.361 1.926 0.16 1.056 1.054 1.063 1.148
Numbering 11 12 13 14 15 16 17 18 19 20
μg/L 1.001 1.099 1.306 0.714 0.813 0.456 0.671 1.692 0.028 1.739
Numbering 21 22 23 24 25 26 27 28 29 30
μg/L 1.686 0.234 0.564 2.182 0.342 0.408 2.186 1.585 0.064 1.749
Numbering 31 32 33 34 35 36 37 38 39 40
μg/L 1.998 0.67 0.317 0.18 1.284 0.716 1.325 1.199 2.151 0.974
Numbering 41 42 43 44 45 46 47 48 49 50
μg/L 1.433 1.22 1.472 1.677 2.095 1.553 2.103 1.579 1.385 1.431
Numbering 51 52 53 54 55 56 57 58 59 60
μg/L 0.241 1.929 1.547 0.725 1.372 0.87 1.441 1.629 0.206 1.764
Numbering 61 62 63 64 65 66 67 68 69 70
μg/L 1.056 1.8 2.049 1.978 0.503 0.528 2.116 2.142 0.366 1.235
Numbering 71 72 73 74 75 76 77 78 79 80
μg/L 0.451 1.404 0.24 0.934 0.08 0.433 1.866 0.503 1.556 0.198
Numbering 81 82 83 84 85 86 87 88 89 90
μg/L 1.194 1.807 0.688 0.865 1.467 1.5 1.205 0.34 0.786 0.726
Numbering 91 92 93 94 95 96 97 98 99 100
μg/L 0.982 0.916 0.549 0.233 0.354 0.747 1.418 1.542 0.86 0.6
Application Example 3
100 minute marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) Arsenic-IgG chelate contents in this blood sample, are detected in accordance with the following steps：
1) IgG material will can be captured, as anti-igg antibody (anti-igg Ab) is coated on solid phase carrier：It is dilute with sample Release liquid and dilute anti-igg Ab to 1000000 times, add in elisa plate micropore, 4 DEG C are stayed overnight 18 hours；
2) 100 parts of standard blood samples are taken to add toluene as measuring samples, dissolve cell membrane；
3) close：Sample diluting liquid is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure to add 2% ox blood As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and are washed with cleaning solution, after the completion of washing, elisa plate Placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate：Measuring samples, the arsenic-IgG chelates with known content added in step 2) Make standard items；20 times are diluted to sample diluting liquid dilution measuring samples, is added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, waits after the completion of washing, adds with papain Concentration be 100ng/ml eluent, elute 3 hours；
6) it is acidified：Add nitric acid in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified；
7) detect：Hydrogen peroxide is added, and heats and catches up with acid, and is sampled from solution, in inductively coupled plasma matter Detection chelates the arsenic in IgG under spectrometer, and detected value is as shown in table 6.
The ICP-MS testing results of 6 100 parts of sample blood sample arsenic-IgG chelates of table
It will be apparent to those skilled in the art that technical scheme that can be as described above and design, make other various Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention Within.

Claims (4)

1. a kind of application of arsenic-IgG chelates in the reagent or kit of arsenic-IgG chelates in preparing detection blood sample, it is special Sign is that the arsenic-IgG chelates are that arsenic ion is formed with IgG by sulfydryl or/and cysteine residues chelating；
The preparation method of the arsenic-IgG chelates, it is characterised in that comprise the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source or according to the IgG of biological method restructuring to carry out Chelatropic reaction, obtain reaction solution；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, remove in reaction solution unreacted IgG and Unnecessary arsenic ion, produce arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates；
Wherein, step B) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, redissolve arsenic-IgG chelates；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with IgG specificity With reference to filler, fill post after, be continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG are special with filler The opposite sex combines；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate Solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C Dialysed overnight, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, energy and arsenic are loaded in the chromatographic column The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, it is then molten using 0.5-1.0mol/L phosphate Liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C dialysed overnight, sample is collected, produces arsenic-IgG chelates；
Wherein, step C) in it is specific as follows：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, Ran Houjia Sample is in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, Ran Houzai Using ICP-MS methods, AAS methods or ELISA method detection whether containing arsenic and detect arsenic content.
2. a kind of comprise at least kit of the arsenic-IgG chelates as standard items, it is characterised in that the arsenic-IgG chelates Formed for arsenic ion and IgG by sulfydryl or/and cysteine residues chelating；
The preparation method of the arsenic-IgG chelates, it is characterised in that comprise the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG in people source or according to the IgG of biological method restructuring to carry out Chelatropic reaction, obtain reaction solution；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, remove in reaction solution unreacted IgG and Unnecessary arsenic ion, produce arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates；
Wherein, step B) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, redissolve arsenic-IgG chelates；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with IgG specificity With reference to filler, fill post after, be continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG are special with filler The opposite sex combines；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate Solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C Dialysed overnight, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, energy and arsenic are loaded in the chromatographic column The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, it is then molten using 0.5-1.0mol/L phosphate Liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C dialysed overnight, sample is collected, produces arsenic-IgG chelates；
Wherein, step C) in it is specific as follows：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, Ran Houjia Sample is in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, Ran Houzai Using ICP-MS methods, AAS methods or ELISA method detection whether containing arsenic and detect arsenic content.
3. kit according to claim 2, it is characterised in that also including coating buffer, the coating buffer, which contains, can capture IgG Albumen or the material of arsenic can be captured.
4. it is a kind of quantitatively detect arsenic-IgG chelates method, it is characterised in that using the arsenic-IgG chelates of known content as Standard items, detected using a pair of samples of following methods：ELISA, enzyme linked immunological are combined with atomic absorption spectrum Method, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification arsenic-IgG chelates and enzyme linked immunological combined techniques, purification Arsenic-IgG chelates are combined with atomic absorption spectrum combined techniques, purification arsenic-IgG chelates with inductivity coupled plasma mass spectrometry Method, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques；
Arsenic-IgG the chelates are that arsenic ion is formed with IgG by sulfydryl or/and cysteine residues chelating；
The preparation method of the arsenic-IgG chelates, it is characterised in that comprise the following steps：
A) arsenic and IgG chelatropic reaction：Arsenic ion is added in the IgG recombinated according to biological method and carries out chelatropic reaction, is obtained To reaction solution；
B the extraction of arsenic-IgG chelates) is purified：Using immune-affinity chromatography, remove in reaction solution unreacted IgG and Unnecessary arsenic ion, produce arsenic-IgG chelates；
C) to the identification of arsenic-IgG chelates；
Wherein, step B) it is specific as follows：
(1) sample dissolution：To passing through step A) physiological saline is added in obtained reaction solution, redissolve arsenic-IgG chelates；
(2) chromatographic column is balanced：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with IgG specificity With reference to filler, fill post after, be continuing with dilution buffer balance chromatographic column；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgG are special with filler The opposite sex combines；
(4) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate Solution is eluted；
(5) collect：The eluent after step (4) elution is collected, makes protein renaturation after collection immediately；
(6) dialyse：By the eluent of the collection in step (5), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C Dialysed overnight, collect sample；
(7) chromatographic column is balanced：Using new chromatographic column, with dilution buffer flushing line, energy and arsenic are loaded in the chromatographic column The filler of specific binding, chromatographic column is balanced with dilution buffer again after filling post；
(8) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (6), then upper prop；
(9) elute：Chromatographic column to baseline is rinsed using dilution buffer to balance, it is then molten using 0.5-1.0mol/L phosphate Liquid is eluted；
(10) collect：The eluent after step (9) elution is collected, makes protein renaturation after collection immediately；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, uses ddH2O dialysis desalinations, after changing water three times, 4 DEG C dialysed overnight, sample is collected, produces arsenic-IgG chelates；
Wherein, step C) in it is specific as follows：
(1) glue bed is prepared：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；
(2) it is loaded：Take step B) in the obtained arsenic-IgG chelates of extraction purification, add dilution buffer, and mix, Ran Houjia Sample is in sample cell；
(3) electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；
(4) detect：The protein band containing arsenic is found out on glue bed, the protein band is taken out, protein band is redissolved, Ran Houzai Using ICP-MS methods, AAS methods or ELISA method detection whether containing arsenic and detect arsenic content.
CN201510411518.9A 2015-07-14 2015-07-14 A kind of arsenic IgG chelates and its preparation method and application Expired - Fee Related CN104987405B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510411518.9A CN104987405B (en) 2015-07-14 2015-07-14 A kind of arsenic IgG chelates and its preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510411518.9A CN104987405B (en) 2015-07-14 2015-07-14 A kind of arsenic IgG chelates and its preparation method and application

Publications (2)

Publication Number Publication Date
CN104987405A CN104987405A (en) 2015-10-21
CN104987405B true CN104987405B (en) 2018-01-23

Family

ID=54299320

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510411518.9A Expired - Fee Related CN104987405B (en) 2015-07-14 2015-07-14 A kind of arsenic IgG chelates and its preparation method and application

Country Status (1)

Country Link
CN (1) CN104987405B (en)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5612016A (en) * 1988-04-01 1997-03-18 Immunomedics, Inc. Conjugates of antibodies and bifunctional ligands

Also Published As

Publication number Publication date
CN104987405A (en) 2015-10-21

Similar Documents

Publication Publication Date Title
CN104987405B (en) A kind of arsenic IgG chelates and its preparation method and application
CN104987409B (en) lead-IgG chelate as well as preparation method and application thereof
CN105044006B (en) Mercury-IgG chelate and preparation method and application thereof
CN105044324B (en) Arsenic-chelated immune complex and preparation method and application thereof
CN105044004B (en) Mercury-IgM chelate as well as preparation method and application thereof
CN104987407B (en) cadmium-IgG chelate as well as preparation method and application thereof
CN105021554B (en) arsenic-IgM chelate as well as preparation method and application thereof
CN105021548B (en) Arsenic-fibrinogen chelate as well as preparation method and application thereof
CN105004684B (en) Nickel-IgE chelate as well as preparation method and application thereof
CN105044007B (en) cadmium-IgM chelate as well as preparation method and application thereof
CN105004685B (en) Mercury-IgE chelate and preparation method and application thereof
CN105044005B (en) chromium-IgA chelate as well as preparation method and application thereof
CN105021550B (en) Mercury-high density lipoprotein chelate and preparation method and application thereof
CN105004683B (en) chromium-IgE chelate as well as preparation method and application thereof
CN105044008B (en) Cadmium-high density lipoprotein chelate as well as preparation method and application thereof
CN104987412A (en) Arsenic-IgE chelate as well as preparation method and application thereof
CN105044009B (en) Chromium-very low density lipoprotein chelate and preparation method and application thereof
CN104987391B (en) Mercury-albumin chelate and preparation method and application thereof
CN105004686B (en) Chromium-albumin chelate as well as preparation method and application thereof
CN104987404A (en) Nickel-IgG chelate as well as preparation method and application thereof
CN104987414A (en) Nickel-IgM chelate as well as preparation method and application thereof
CN104987400A (en) Mercury-hemoglobin chelate as well as preparation method and application thereof
CN104987411A (en) Cadmium-IgA chelate as well as preparation method and application thereof
CN105021549B (en) Nickel-albumin chelate as well as preparation method and application thereof
CN105021552B (en) Nickel-high density lipoprotein chelate as well as preparation method and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20161017

Address after: 511500 Guangdong Qingcheng District Qingyuan City, Heng he Street Lion Lake Road No. 1 Administrative Building Room 301

Applicant after: Guangdong tenpai medical Cci Capital Ltd

Address before: 200000 Shanghai, Pudong New Area, China (Shanghai) free trade zone, new Jinqiao Road, No. 13, building 2, floor 27

Applicant before: SHANGHAI BAIHAO BIOTECHNOLOGY CO., LTD.

GR01 Patent grant
GR01 Patent grant
CP03 Change of name, title or address
CP03 Change of name, title or address

Address after: 511500, B, 101, commercial street, fisherman's Wharf, 1 Lion Lake Road, Guangdong, Qingyuan

Patentee after: Guangdong tenpai medical Limited by Share Ltd

Address before: 511500 Guangdong Qingcheng District Qingyuan City, Heng he Street Lion Lake Road No. 1 Administrative Building Room 301

Patentee before: Guangdong tenpai medical Cci Capital Ltd

CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20180123

Termination date: 20210714