CN105004685B - A kind of mercury IgE chelates and its preparation method and application - Google Patents
A kind of mercury IgE chelates and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of mercury IgE chelates and its preparation method and application, mercury IgE chelates are that mercury ion is formed with IgE by sulfydryl or/and cysteine residues chelating.The present invention establishes the method for qualitative and quantitative detection of mercury IgE chelates, so that quantitative detection mercury IgE chelates are evaluating the application of a regional mercury pollution degree.The mercury contaminated situation of this regional crowd can be reflected indirectly by mercury IgE chelates in one regional crowd's serum of quantitative detection, so as to reflect this regional mercury pollution degree indirectly.Mercury IgE chelates quantitative detecting method its degree of accuracy that the present invention establishes greatly improves, and the repeatability of detection is greatly enhanced.
Description
Technical field
The present invention relates to detection field, more specifically to a kind of mercury-IgE chelates and its preparation method and application.
Background technology
IgE is mainly produced by mucosa-associated lymphoid tissue, and wherein most is as synthesized by gastrointestinal lymphoid sample tissue, less
Part is synthesized by respiratory tract, salivary gland and genital tract mucosal tissue.Nursing period puerpera's glandular tissue contains a large amount of IgE and produces cell,
These cells are essentially from stomach and intestine.In the mankind, also a small amount of IgE comes from marrow.People birth after 4~June start synthesize IgE, 4
Content reaches adult levels in~12 years old serum, and 10% or so of the total Ig of serotype IgE, about 5~6 days half-life period.IgE have IgE1 and
Two subclass of IgE2.IgE1 is primarily present in serum, and the chain molecular weights of 85%, α 1 for accounting for IgE in serum are 56kD；IgE2 master
It is present in exocrine secretion, small part exists with serotype IgE, and the chains of 15%, α 2 for accounting for IgE in serum lack hinge area,
Molecular weight is 52kD.There are the forms such as the dimer being covalently attached to by J chains or tripolymer outside IgE demonomerizations form in serum.
Secretory IgE is made up of the binary and secretory component of J connections, is primarily present in colostrum, saliva, tear, gastro-intestinal Fluid, branch gas
Pipe secretion etc. is in exocrine secretion, is the most important factor of mucous membrane local immunity, secretory IgE by with corresponding pathogenic microorganism
(such as poliovirus) combines, and prevents it to be adsorbed onto on permissive cell, secretory IgE can also neutralize a toxin such as comma bacillus
Toxin and large intestine bar toxin etc..Neonate be susceptible to suffer from respiratory tract, alimentary infection may with IgE synthesize deficiency it is relevant.Chronic branch gas
The scorching breaking-out of pipe and the reduction of secretory IgE also have certain relation.Secretory IgE can be passed to baby by puerpera by colostrum, this
A kind of and important natural passive immunity.Eosinophil, neutrophil leucocyte and Expression of Macrophages Fc α R, serotype list
Body IgE can mediate opsonophagocytosis and ADCC to act on.In addition, secretory IgE has immune exclusion function, i.e. secretory IgE combines
The pyrogenic substances that substantial amounts of soluble antigen and normal intestinal flora or pathogenic microorganism are discharged in diet, prevent them from entering
Enter blood.
Mercury (Mercury, Hg) is a kind of ubiquitous heavy metal, is widely used in industry, agricultural, pharmaceutical sector, is one
The very important environmental contaminants of kind.2011, (UHgted States Agency for were affixed one's name in U.S.'s poisonous substance and disease registration
Toxic Substances and Disease Registry, ATSDR) it has been included in material preferred list
(Substance Priority List), and the World Health Organization (World Health OrgaHgzation, WHO) is also already
It is classified as one of chemicals of 10 big easily attractive health problems.In the U.S., Hg and Cr, Cd, Pb are considered as four overall situation
Pollutant, it drastically influence the health of people.Common mercury exposed population group includes being engaged in metal smelt, exploitation gold
Ore deposit, the crowd for burning coal, electric industry and wood producing, and for general population mainly by eating the seafood of mercury pollution, drinking into mercury
The mercury vapour of polluted source, suction fuel combustion or evaporation.In addition, the contact of mercury cosmetic, filling mercury alloy dental filler
The contact of thing, ethyl mercury, mercurous insecticide in contact vaccine preservative is all common mercury exposure chamber, the life of mercury and people
Work is closely related.2009, national health in nutrition test investigation with finding (National Health and Nutrition
Examination Study, NHANES), the mercury in 2% U.S.'s Women of Childbearing Age body is all exceeded, it is seen that mercury pollution is one
Urgent problem to be solved.
Mercury typically contains three kinds of forms：Metal mercury element, inorganic mercury, organic mercury, people's dominant touch organic mercury and metal
Mercury element, but the mercury of both forms can change inorganic mercury into biological living is built-in.Mercury metal is generally in a manner of suction
Into internal, it can be oxidized by hydrogen-hydrogen peroxide-hydrogen peroxide enzymatic pathway, then form inorganic mercury.It is inorganic in brain
Mercury is attached to selenides to form mercury selenide, and mercury selenide is insoluble, will all the year round stagnate in intracerebral, and will cause for a long time serious
Brain damage.Inorganic mercury, it is generally difficult to be ingested in vivo, only in rare cases, the mercury for having up to 40% passes through stomach and intestine
Road absorbs.Once inorganic mercury enters blood flow, various tissues are just distributed in, it is not easy to pass through blood-brain barrier and placental barrier, mainly
Deposited in renal tissue, therefore easily cause kidney damage for a long time.Compared with inorganic mercury, organic mercury is most readily by intestines and stomach suction
Receive.With the difference of organic mercury species, the effect that it is absorbed by skin is different, and dimethylmercury can absorb rapidly through skin, and
Methyl mercury is then more difficult.Most organic mercuries all have stronger toxicity, such as methyl mercury (Methylmercury, MeHg) easily passes through
Blood-brain barrier and placental barrier, it is now recognized that it is likely to result in fetus learning disability, cognitive ability is low, suppresses fetus
Immune system, fetus is caused to suffer from the nervous system disease etc..
It is in close relations between mercury and multiple proteins, it can be combined with multiple proteins, suppress the activity of multiple protein enzyme.
As what Hg toxicity was studied gos deep into, people gradually recognize that the interaction of mercury and albumen is played the part of emphatically during its intoxicating
The role wanted, it is considered to be understand the key point of the toxicity mechanism of mercury, therefore, the research to mercury and protein interaction is just
Seem ever more important.And the mechanism now concerning mercury and protein effect is only tip of the iceberg, also many protein and mercury it
Between relation it is not clear, thus strengthen it is most important for the relation between mercury and protein.
The content of the invention
For mercury pollution it is serious the problem of, it is an object of the invention to provide a kind of mercury-IgE chelates and its preparation side
Method, and the qualitative and quantitative analysis method of mercury-IgE chelates is established, so that quantitative detection mercury-IgE chelates are evaluating a ground
The application of area's mercury pollution degree.This can be reflected indirectly by mercury-IgE chelates in one regional crowd's serum of quantitative detection
The mercury contaminated situation of regional crowd, so as to reflect this regional mercury pollution degree indirectly.
The technical solution adopted for the present invention to solve the technical problems is：A kind of mercury-IgE chelates are provided, mercury ion with
IgE is formed by sulfydryl or/and cysteine residues chelating.
The present invention also provides a kind of preparation method of above-mentioned mercury-IgE chelates, comprises the following steps：
A) mercury and IgE chelatropic reaction：Add in the IgE that purification comes from the IgE of human body or is recombinated according to biological method
Enter mercury ion and carry out chelatropic reaction, obtain reaction solution；
B the extraction of mercury-IgE chelates) is purified：Using immune-affinity chromatography, unreacted IgE in reaction solution is removed
And unnecessary mercury ion, produce mercury-IgE 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 answers mercury-IgE 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 IgE
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, IgE 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, and obtains eluent I；
(5) collect：Collect eluent I；
(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 mercury 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, and obtains eluent II；
(10) collect：Collect eluent II；
(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 mercury-IgE chelates.
Wherein, the preparation method of above-mentioned mercury-IgE chelates, in addition to step C)：Identification to mercury-IgE 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 mercury-IgE 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：Mercurous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
The present invention also provides a kind of mercury-IgE chelates described above and is preparing the examination of mercury-IgE 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 mercury-IgE chelates described above as standard items.
Preferably, coating buffer is also included in the kit, the coating buffer contains capture IgE material or the thing of capture mercury
Matter.
The present invention also provides a kind of method for quantitatively detecting mercury-IgE chelates, with the above-mentioned mercury-IgE chelas of known content
Compound is detected as standard items 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 mercury-IgE chelates with enzyme linked immunological
Method, purification mercury-IgE chelates and atomic absorption spectrum combined techniques, purification mercury-IgE 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 mercury-IgE chelates of the present invention and its preparation method and application, have the advantages that：
1. the present invention synthesizes mercury-IgE chelates in vitro first；
2. present invention firstly provides mercury-IgE chelates can be used for prepare detection blood sample in mercury-IgE chelates reagent or
Application in kit.
3. the present invention establishes the method for qualitative and quantitative detection of mercury-IgE chelates, so as to quantitative detection mercury-IgE chelates
Evaluating the application of a regional mercury pollution degree.Can be with by mercury-IgE chelates in one regional crowd's serum of quantitative detection
Reflect the mercury contaminated situation of this regional crowd indirectly, so as to reflect this regional mercury pollution degree indirectly.The present invention establishes
Mercury-IgE 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 mercury-IgE chelates of the present invention；
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of mercury-IgE chelates of the present invention.
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：
Phosphate solution is 0.05-0.1mol/L Na2HPO4Solution or 0.5-1mol/L Na2HPO4Solution；
Extracts reagent is (using PEG methods) such as PEG solution, borate buffer solutions；
Glue bed medium is one kind in Ago-Gel, polyacrylamide gel；
IgE material purchase is captured from the model Abcam ab171396 of Amy victory Science and Technology Ltd. rabbit Anti-
IgE antibody；Wherein, material, anti-IgE antibodies, the anti-IgE antibodies of the present invention with IgE specific bindings are capture IgE
Material；
The material for capturing mercury is the model AP7014 of Ba Ao get bio tech ltd production anti-mercury antibody；Its
In, material, the Hg of the specific binding of the present invention with mercury be anti-, anti-Hg antibody, anti-mercury antibody are the material for capturing mercury；
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；
Dilution buffer is Na containing 1.5mg/mL2CO3、2.93mg/mL NaHCO3PH be 9.6 0.05M carbonate 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 containing papain 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 mercury-IgE chelates, mercury ion and IgE by sulfydryl or/and cysteine residues chelate and
Into.
The present invention also provides a kind of preparation method of mercury-IgE chelates, comprises the following steps：
A) mercury and IgE chelatropic reaction：Mercury ion is added in the IgE in people source and carries out chelatropic reaction, obtains reaction solution；
B the extraction of mercury-IgE chelates) is purified：Using immune-affinity chromatography, unreacted IgE in reaction solution is removed
And unnecessary mercury ion, mercury-IgE 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 answers mercury-IgE 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 IgE
The filler that the opposite sex combines, after filling post, it is continuing with dilution buffer balance chromatographic column；It is described to be filled out with what IgE was specifically bound
Expect for silica gel or resin containing anti-IgE antibodies；
(3) loading：After column equilibration is chromatographed, with sample in dilution buffer dilution step (1), then upper prop, IgE 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, and obtains eluent I；
(5) collect：Collect eluent I；
(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 mercury specific binding, chromatographic column is balanced with dilution buffer again after filling post；It is described to be filled out with what mercury was specifically bound
Expect for silica gel or resin containing anti-mercury antibody；
(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, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water
After three times, 4 DEG C of dialysed overnights, sample is collected, produces mercury-IgE chelates；
C) to the identification of mercury-IgE 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 mercury-IgE 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：Mercurous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
The present invention, which also provides, a kind of comprises at least kit of the mercury-IgE chelates described above as standard items.
Preferably, coating buffer is also included in the kit, the coating buffer contains capture IgE material or the thing of capture mercury
Matter.
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 mercury-IgE chelates in a kind of detection blood sample, including the coating buffer of the material containing capture IgE, closing
Liquid, cleaning solution, the material for capturing mercury as secondary antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control,
Negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including the coating buffer of the material containing capture IgE, closing
Liquid, cleaning solution, eluent, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including the coating buffer of the material containing capture IgE, closing
Liquid, cleaning solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including as extracts reagent needed for IgE in purifying blood plasma,
Redissolve liquid, the coating buffer containing the material for capturing mercury, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer
Liquid, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including as extracts reagent needed for IgE in purifying blood plasma,
Redissolve liquid, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including as extracts reagent needed for IgE in purifying blood plasma,
Redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including as IgE extracts reagent, glue in purifying blood plasma
Bed medium, redissolve liquid, sample-loading buffer, dissolve liquid needed for protein band mercurous on glue bed, containing the material for capturing mercury
Coating buffer, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative control etc..
The kit of mercury-IgE chelates in a kind of detection blood sample, including as extracts reagent needed for IgE in purifying blood plasma,
Glue bed medium, redissolve liquid, sample-loading buffer, dissolve liquid, positive control, negative control needed for protein band mercurous on glue bed
Deng.
The kit of mercury-IgE chelates in a kind of detection blood sample, including as extracts reagent needed for IgE in purifying blood plasma,
Glue bed medium, redissolve liquid, sample-loading buffer, liquid needed for mercurous protein band on dissolving glue bed, acidulant, hydrogen peroxide,
Positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with IgE chelates or the chela of heavy metal Hg
Closing has the BSA chelates of heavy metal Hg；The negative control is the dilution buffer for not containing standard items.
Mentioned reagent box is used for the IgE for detecting chelating mercury, 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 mercury-IgE chelates, with the above-mentioned mercury-IgE chelas of known content
Compound is detected as standard items 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 mercury-IgE chelates with enzyme linked immunological
Method, purification mercury-IgE chelates and atomic absorption spectrum combined techniques, purification mercury-IgE 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 mercury-IgE chelates is following several, but is not limited to following several.
Wherein, the reagent used in the above-mentioned method for quantitatively detecting mercury-IgE chelates is as follows：
Method one：ELISA (ELISA method) detects mercury-IgE chelates, detects in accordance with the following steps：
1) IgE material will can be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier：It is slow with dilution
Fliud flushing to diluting 500-4000 times, adds anti-IgE Ab in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-baths
1-3 hours, store refrigerator；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) material of mercury 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 is diluted to 2500-20000 times of anti-Hg Ab with dilution buffer, 37 DEG C of effect 1-2 hours, makes anti-Hg
Mercury metal on Ab and IgE reacts to form antigen antibody complex；
6) enzyme conjugates incubates：Anti- mercury antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute
Release the enzyme labelled antibody of buffer solution dilution, the concentration for making the enzyme labelled antibody of dilution is 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；
9) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively
With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA
Colour developing situation carries out qualitative detection).
This method utilizes ELISA principles, can extract the specific IgE in blood plasma, the IgE tops extracted
Divide and be chelated with heavy metal Hg, and the mercury on the IgE of this part is captured by anti-mercury antibody, afterwards can be again by horseradish peroxidase
The antibody of the enzymes such as enzyme, alkaline phosphatase mark captures (the antibody nonrecognition coating protein), and the antibody in capture is in developer
And in the presence of terminate liquid, OD values can be read under instrument, and do not contain the IgE of chelated mineral mercury, then will not be by anti-mercury
Specific antibody is captured, and the antibody that will not be also marked with enzymes such as horseradish peroxidase, alkaline phosphatases is captured, and used
Mercury metal (negative control group result is feminine gender) is not contained in reagent, thus works as read OD value results and is shown as positive yet
When, you can prove to detect the mercury metal chelated on IgE.
Method two：Enzyme linked immunological is pressed with atomic absorption spectrum combined techniques (ELISA method+AAS methods) detection mercury-IgE chelates
Detected according to following steps：
1) IgE material will can be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier：It is slow with dilution
Anti- IgE Ab are diluted to 500-4000 times by fliud flushing, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-baths
1-3 hours, store refrigerator；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rpm centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C
Lower elution 1-3 hours.
6) detect：Sample, chelated in Atomic Absorption Spectrometer detection in the mercury on IgE, and paint from the micropore of elisa plate
Standard curve processed, readout value；
This method combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principles, utilizes atomic absorption spectrum
Instrument detection is chelated in the mercury on IgE, due to only containing IgE in solution, and any heavy metal (negative control is free of in agents useful for same
Group result is feminine gender), result will not be interfered, thus when the result read is shown as the positive, you can prove detection
Go out the mercury metal chelated on IgE.
Method three：Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS methods) detection mercury-
IgE chelates detect in accordance with the following steps：
1) IgE material will can be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier：It is slow with dilution
Anti- IgE Ab are diluted to 500-4000 times by fliud flushing, are added in elisa plate micropore, 4 DEG C of overnight 16-18 hours, or 37 DEG C of water-baths
1-3 hours, store refrigerator；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2 hours；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, eluent are added, at 37 DEG C
Lower elution 1-3 hours.
6) it is acidified：Acidulant is added in solution in step 5) to be acidified solution, sealing overnight, is thoroughly acidified,
Hydrogen peroxide is added, and heats and catches up with acid；
7) detect：0.5mL liquid is taken from the solution of the elisa plate of step 6), in icp mses
Lower detection chelates the mercury in IgE, and draws standard curve, readout 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 mercury on IgE, due to only containing IgE in solution, and is free of in agents useful for same
Any heavy metal (negative control group result is feminine gender), will not interfere to result, thus works as read result and be shown as
When positive, you can prove to detect the mercury metal chelated on IgE.
Method four：Mercury-IgE chelates are purified to chelate with enzyme linked immunological combined techniques (method of purification+ELISA method) detection mercury-IgE
Thing, detect in accordance with the following steps：
1) IgE is extracted from blood plasma：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel filtration
The methods of from the IgE in blood plasma extraction purification blood, and IgE is redissolved in solution, obtains IgE solution；
2) anti-Hg Ab are coated on solid phase carrier：Anti- Hg Ab are diluted to 2500-20000 times with dilution buffer,
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：Dilution buffer 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 solution of step 1), make measuring samples；With the mercury of known content-
IgE chelates make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, added in micropore, 37 DEG C of effect 1-2
Hour；
5) enzyme conjugates incubates：The measuring samples in step 4) are removed, and are washed with cleaning solution, treat that washing is completed
Afterwards, the enzyme labelled antibody that addition is diluted with dilution buffer, the concentration for making the enzyme labelled antibody of dilution are 2 μ g/mL, 37 DEG C of effect 1-2
Hour, it is 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；
8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads measuring samples respectively
With the OD values of standard items, by drawing standard curve, the content for trying to achieve measuring samples (also can directly pass through without using ELIASA
Colour developing situation carries out qualitative detection).
Method five：Mercury-IgE chelates are purified to detect in blood sample with atomic absorption spectrum combined techniques (method of purification+AAS methods)
Mercury-IgE chelates, are detected in accordance with the following steps：
1) IgE is extracted from blood plasma：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel filtration
The methods of from the IgE in blood plasma extraction purification blood, and IgE is redissolved in solution, obtains IgE solution；
2) detect：Sample in the solution obtained from step 1), chelated in Atomic Absorption Spectrometer detection in the mercury on IgE,
And standard curve is drawn, readout value.
Method six：Purify mercury-IgE chelates and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS methods)
Mercury-IgE chelates are detected, are detected in accordance with the following steps：
1) IgE is extracted from blood plasma：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel filtration
The methods of from the IgE in blood plasma extraction purification blood, and IgE is redissolved in solution, obtains IgE solution；
2) it is acidified：Sampled in the solution obtained from step 1), add acidulant (such as nitric acid) in the solution and solution is carried out
Acidifying, sealing overnight, thoroughly acidifying, add hydrogen peroxide, and heat and catch up with acid；
3) detect：0.5mL liquid is taken in the solution obtained from step 2), is detected under icp mses
Chelate in the mercury on IgE, and draw standard curve, readout value.
Method seven：Electrophoresis and ELISA or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques
(electrophoresis-ELISA method/AAS methods/ICP-MS methods) detects mercury-IgE chelates, detects in accordance with the following steps：
1) IgE is extracted from blood plasma：Using the polyethylene glycol PEG precipitation method or ultracentrifugation or molecule ultrafiltration or gel filtration
The methods of from the IgE in blood plasma extraction purification blood, and IgE is redissolved in solution, obtains IgE solution；
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 conventionally prepared；
3) it is loaded：The μ L of solution 8 for taking step 1) to obtain 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 mercury is found out on glue bed, the band is taken out, after treatment by the albumen one
Band is dissolved among liquid, is then utilized respectively ELISA or ICP-MS again or AAS detects the mercury content being dissolved in liquid.In addition, also
IgE isoelectric point, molecular weight and content of the method detection chelating mercury etc. can be utilized.
IgE in method seven can be come out with a variety of Methods For Purifications (such as supercentrifugation or HPLC or
Gel-filtration chromatography or gel electrophoresis or ELISA method etc.), IgE out will be purified and redissolved in solution, taken a certain amount of
IgE, using electric charge shifting principle, carry out electrophoresis (electrophoresis, EP), (can be as needed using not in gel slab
Same medium) on can run out of different bands according to molecular weight, isoelectric point etc. are different, search out the respective strap rich in mercury, will be solidifying
Protein in glue is redissolved in solution, you can to detect related IgE content at a particular wavelength, can also utilize ELISA,
The principles such as AAS, ICP-MS detect to chelate in the mercury content on IgE, due to only containing IgE in solution, and in agents useful for same not
Containing any heavy metal (negative control group result is feminine gender), result will not be interfered, thus work as read result and show
For the positive when, you can prove to detect the mercury metal chelated on IgE.
Embodiment 1：The preparation method of mercury-IgE chelates, comprises the following steps：
A) mercury and IgE chelatropic reaction：Mercury ion is added in the IgE 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) IgE solution：Weigh 4.0mg IgE 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) EDTA-NaHCO3 mixed solutions：Weigh EDTA2H2O 1.86g、NaHCO316.8g is dissolved in 900mL ultra-pure waters
In, it is settled to 1000mL, autoclaving, room temperature preservation with 1.0M NaOH adjustment pH to 8.0；
4) ITCBE is bought from Japanese colleague's chemistry institute, article No. M030；
5) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of mercury-IgE chelates：
1) processing of bag filter：Bag filter is put into 500mL EDTA-NaHCO3In mixed solution, 10min is boiled；Incline
Abandon EDTA/NaHCO3Liquid, gently rinsed with ultra-pure water, then 10min is boiled with 500mL 5mmol/L EDTA solution；Discard and boil
Liquid is boiled, is thoroughly cleaned with ultra-pure water, adds 4 DEG C of ultra-pure water immersion bag filter overnight.In use, putting on one's gloves, bag filter is taken out,
With 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 IgE are taken to be dissolved in 4.0mL borate buffer solutions；
4) liquid for slowly preparing step 2) is added in the IgE solution that step 3) is prepared, and is shaken when being added dropwise, is placed in temperature
Spend for 25 DEG C, rotating speed is to react 24h in 100r/min shaking table, then with bag filter dialysis 24h, removes what is do not combined with IgE
ITCBE；
5) liquid obtained by step 4) is adjusted into pH value to 7.0 with 1mol/L HCl, is then slowly gradually added dropwise 80 μ l's
1mmol/L mercury ion solution, vibrated when being added dropwise, in case mercury ion precipitates albuminous degeneration；
6) solution obtained by step 5) is placed in temperature as 25 DEG C, rotating speed is to react 2h in 100r/min shaking table, with saturating
Analysis bag carries out dialysis 24h；
7) liquid after step 6) is dialysed preserves in -20 DEG C of packing, obtains reaction solution.
B the extraction of mercury-IgE chelates) is purified：Using immune-affinity chromatography, removing reaction solution, (i.e. step 7) obtains
Reaction solution) in unreacted IgE and unnecessary mercury ion, produce mercury-IgE chelates, comprise the following steps that：
(1) sample dissolution：Physiological saline is added into the reaction solution obtained by step 7) answers mercury-IgE 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 IgE
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, IgE 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, and obtains eluent I；
(5) collect：Collect eluent I；
(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 mercury 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, and obtains eluent II；
(10) collect：Collect eluent II；
(11) dialyse：By the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalinations, change water
After three times, 4 DEG C of dialysed overnights, sample is collected, produces mercury-IgE chelates；
C) to the identification of mercury-IgE 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 mercury-IgE 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 DEG C；Electrophoresis to bromophenol blue stops electrophoresis when moving to glue bottom；
(4) detect：Mercurous protein band is found out on glue bed, the protein band is taken out and dissolved, is then used again
ELISA method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
D) testing result
(1) content of beary metal in graphite furnace atomic absorption spectrometry (AAS) Preliminary Determination IgE：It the results are shown in Table 1
Table 1 is content of beary metal (μ g/L) in IgE
| Sample name | Hg(μg/L) |
| IgE | 60.716 |
| (NH4)2SO4 | 0.369 |
| N.S | 0 |
| N.S | 0 |
(2) electrophoresis result：
Wherein, Fig. 1 is the non denatured electrophoretic band figure of mercury-IgE chelates of the present invention.
Native polyacrylamide gel electrophoresis (Native-PAGE) or for active electrophoresis be be added without SDS and dredge
Under conditions of the denaturants such as base ethanol, polyacrylamide gel electrophoresis is carried out to the protein for keeping activity, is usually used in the mirror of enzyme
Fixed, Isozyme Analysis and purification.Not plus SDS native polyacrylamide gel electrophoresis can make large biological molecule in electrophoresis process
Middle its natural shape of holding and electric charge, their separation are made according to the difference of its electrophoretic mobility and the molecular sieve of gel
With, thus higher resolution ratio can be obtained, especially remain to keep the large biological molecule such as protein and enzyme after electrophoretic separation
Bioactivity, therefore left lane M is denaturation Marker, is served only for detecting, does not mark effect, swimming lane 4 for IgE bar
Band.
(3) 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 synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of mercury-IgE chelates of the present invention, horizontal in figure
Coordinate is protein band position, and ordinate is each metal energy of the band (content) value.
A kind of determination of the testing conditions for the method for quantitatively detecting mercury-IgE chelates of the present invention：
1. the determination of the optimum diluting multiple of anti-IgE antibodies, anti-mercury antibody best effort concentration and blood plasma
Step is as follows：
(1) it is 1 according to anti-IgE Ab and the mass volume ratio of dilution buffer by anti-IgE Ab dilution buffers:500、
1:1000、1:2000、1:4000 are diluted, and add in elisa plate micropore, anti-IgE Ab are coated on solid phase carrier, often
Individual concentration is coated with three rows, and 4 DEG C are stayed overnight 18 hours；
(2) dilution buffer 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 are 1 by measuring samples and the mass volume ratio of dilution buffer:10、1:20、1:40 progress are dilute
Release, add in micropore, according to above-mentioned coated anti-IgE Ab concentration, being separately added into for the anti-IgE 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-Hg Ab, anti-Hg Ab are pressed
Anti- Hg Ab and the mass volume ratio of dilution buffer are 1:2500、1:5000、1:10000、1:20000 are diluted, according to every
One identical anti-IgE Ab, diluted plasma concentration, the anti-Hg Ab of various concentrations respectively add 2 holes, and 37 DEG C act on 1 hour, make anti-Hg Ab
Reacted with the mercury metal on IgE；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2 μ g/mL, removes anti-Hg 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 mercury-
IgE 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) terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads each hole OD respectively
Value.According to each hole OD value numerical value, anti-IgE Ab, anti-Hg Ab best effort concentration and blood plasma optimum diluting multiple are selected.
Simultaneously using made standard items as positive control in experiment, anti-IgE Ab+ closings+anti-Hg Ab+ enzymes mark+bottom is selected
Thing (being not added with measuring samples) is used as negative control 1；Anti- IgE Ab+ closings+blood plasma+enzyme mark+substrate (being not added with anti-Hg Ab) is made
For negative control 2；Anti- IgE Ab+ closings+enzyme mark+substrate (being not added with measuring samples and anti-Hg Ab) are used as negative control 3；It is anti-
IgE Ab+ closings+blood plasma+anti-Hg Ab+ substrates (i.e. not enzyme-added mark) are used as negative control 4；Closing+blood plasma+anti-Hg Ab+ enzyme marks
+ substrate (being not added with anti-IgE Ab captures IgE) is used as blank control 1；Only add substrate as blank control 2 and PBS as blank
Control 3.Testing result is shown in Table 2-3.
Table 2：Checkerboard titration method determines anti-IgE Ab, anti-Hg Ab best effort concentration and blood plasma optimal dilution times
Several data (each data are all average value)
Table 3：ELISA positive controls and negative control ELISA testing results
Shown by table 2-3 data, we can see that when the dilution factor of anti-IgE antibodies is 1:1000th, dilution rate of blood plasma is
1:20th, the anti-dilution factors of Hg are 1:When 2500, OD values are maximum, so selecting the concentration corresponding to this value as best effort concentration.
2. quantitatively detect in the method for mercury-IgE chelates eluent best effort concentration and elution time in method two, three
Determination
Step is as follows：(1) it is coated with：Anti- Hg Ab are diluted to 2500 times (mass volume ratios) with dilution buffer, added
In elisa plate micropore, 37 DEG C of water-baths 3 hours, refrigerator is stored；
(2) close：Dilution buffer is removed, and is washed with cleaning solution, is waited after the completion of washing, it is pure with 2% ox blood
As confining liquid, 37 DEG C are placed 1 hour albumen, 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
Enzyme labelled antibody, enzyme labelled antibody are diluted to concentration as 2 μ g/mL, and 37 DEG C act on 2 hours, it is reacted with anti-Hg Ab；
(4) eluent is prepared：By papain with pH 8.0,0.1mol/L Tris-HCI buffers into
100ng/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 papain in eluent
The ratio between concentration and the concentration of enzyme labelled antibody are 1:80、1:40、1:20、1:10、1:5 (wherein each concentration makees 3 multiple holes), respectively
1h, 2h, 3h are eluted at a temperature of being positioned over 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) terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed on ELIASA and detected, reads every group of OD respectively
Value, by compared with PBS group, comparing the optimum concentration and elution time of eluent, concrete outcome is referring to table 4.
Table 4：ELISA eluent best effort concentration and elution time determine
We are it can be found that when the concentration of the papain in eluent and the ratio between the concentration of enzyme labelled antibody from table 4
=1:When 20, each group OD values are below other several groups, illustrate that the concentration elution effect is optimal (i.e. by ELISA hole walls
With reference to anti-Hg Ab- enzyme marks compound elution degree reach maximum, thus OD values are minimum)；And elution time either 1h, 2h,
3h, each group OD value changes are little, it is seen that with the extension of time, enzyme activity gradually weakens, in the case where enzyme concentration is constant,
Digestibility can not be improved by extending digestion time, so the elution time of eluent all may be used for 1-3h in this experiment.
Application Example 1
Take using the mercury-IgE chelates in 100 parts of sample blood plasma of ELISA method detection, follow the steps below detection：
ELISA (ELISA method) detects mercury-IgE chelates, detects in accordance with the following steps：
1) anti-IgE Ab are coated on solid phase carrier：Anti- IgE Ab are diluted to 1000 times with dilution buffer, added
In elisa plate micropore, 37 DEG C of water-baths 1 hour, refrigerator is stored；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) material of mercury 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 dilution buffer dilutes 2500 times, and 37 DEG C act on 1 hour, make anti-Hg Ab and the mercury metal on IgE anti-
Should；
6) enzyme conjugates incubates：Anti- mercury antibody is removed, and is washed with cleaning solution, is waited after the completion of washing, is added with dilute
The HRP enzyme labelled antibodies of buffer solution dilution are released, 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；
9) it is 405nm to take wavelength, after adding terminate liquid, elisa plate is placed on ELIASA and detected, and reads treat sample respectively
Product OD values (also directly can carry out qualitative detection) without using ELIASA by the situation that develops the color, and specific detected value is as shown in table 5.
Table 5：The ELISA testing results of mercury-IgE chelates in 100 parts of blood samples
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD405 | 0.552 | 0.671 | 0.604 | 0.664 | 0.329 | 0.582 | 0.444 | 0.746 | 0.68 | 0.595 |
| Numbering | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| OD405 | 0.778 | 0.777 | 0.306 | 0.383 | 0.727 | 0.513 | 0.581 | 0.708 | 0.336 | 0.552 |
| Numbering | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| OD405 | 0.411 | 0.519 | 0.688 | 0.41 | 0.581 | 0.345 | 0.742 | 0.459 | 0.56 | 0.468 |
| Numbering | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| OD405 | 0.378 | 0.473 | 0.606 | 0.615 | 0.357 | 0.474 | 0.738 | 0.305 | 0.717 | 0.471 |
| Numbering | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| OD405 | 0.605 | 0.555 | 0.396 | 0.615 | 0.434 | 0.703 | 0.638 | 0.509 | 0.625 | 0.52 |
| Numbering | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| OD405 | 0.628 | 0.501 | 0.617 | 0.412 | 0.689 | 0.365 | 0.655 | 0.367 | 0.409 | 0.752 |
| Numbering | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| OD405 | 0.366 | 0.681 | 0.441 | 0.307 | 0.685 | 0.726 | 0.378 | 0.577 | 0.545 | 0.654 |
| Numbering | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| OD405 | 0.367 | 0.375 | 0.415 | 0.333 | 0.63 | 0.341 | 0.59 | 0.619 | 0.551 | 0.442 |
| Numbering | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| OD405 | 0.687 | 0.325 | 0.701 | 0.63 | 0.41 | 0.382 | 0.301 | 0.6 | 0.615 | 0.442 |
| Numbering | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| OD405 | 0.422 | 0.775 | 0.739 | 0.354 | 0.535 | 0.486 | 0.763 | 0.507 | 0.659 | 0.649 |
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
Mercury-IgE chelates, are detected in accordance with the following steps：
1) IgE material will can be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier：It is slow with dilution
Anti- IgE Ab are diluted to 1000 times by fliud flushing, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 20 times with dilution buffer dilution, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain
The eluent for 100ng/mL is spent, elutes 1-3 hours；
6) detect：Sample from the micropore of elisa plate, chelated in Atomic Absorption Spectrometer detection in the mercury on IgE, detection
Value is as shown in table 6.
Table 6：The AAS testing results of mercury-IgE chelates in 100 parts of blood samples
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)
Mercury-IgE chelate contents in this blood sample, are detected in accordance with the following steps：
1) IgE material will can be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier：It is slow with dilution
Anti- IgE Ab are diluted to 1000 times by fliud flushing, are added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) taking 100 parts of standard blood samples, 1000rmp centrifugation 5-8 minutes, centrifugation discards precipitation as measuring samples；
3) close：Dilution buffer 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 mercury-IgE chelates with known content added in step 2)
Make standard items；Measuring samples are diluted to 20 times with dilution buffer, added in micropore, 37 DEG C act on 1 hour；
5) elute：Measuring samples are removed, and are washed with cleaning solution, are waited after the completion of washing, it is dense to add papain
The eluent for 100ng/mL is spent, elutes 1-3 hours；
6) it is acidified：Add nitric acid in solution in step 5) to be acidified solution, overnight, thoroughly acidifying, adds for sealing
Enter hydrogen peroxide, and heat and catch up with acid；
7) detect：Sampled in the solution obtained from step 6), under icp mses detection chelate in
IgE mercury, detected value are as shown in table 7.
Table 7：The ICP-MS testing results of mercury-IgE chelates in 100 parts of blood samples
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 (6)
- A kind of 1. mercury-IgE chelates, it is characterised in that mercury ion and IgE by sulfydryl or/and cysteine residues chelate and Into；The preparation method of the mercury-IgE chelates, comprises the following steps：A）The chelatropic reaction of mercury and IgE：Mercury is added in purification comes from the IgE of human body or the IgE according to biological method restructuring Ion carries out chelatropic reaction, obtains reaction solution；The step A）It is specific as follows：1）The processing of bag filter：Bag filter is put into 500mL EDTA-NaHCO3In mixed solution, 10min is boiled；Tipping EDTA/NaHCO3Liquid, gently rinsed with ultra-pure water, then 10min is boiled with 500mL 5mmol/L EDTA solution；Discard and boil Liquid, thoroughly cleaned with ultra-pure water, add 4 DEG C of ultra-pure water immersion bag filter overnight；In use, putting on one's gloves, bag filter is taken out, is used 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 IgE are taken to be dissolved in 4.0mL borate buffer solutions；4）Slowly by step 2）The liquid of preparation adds step 3）In the IgE solution of preparation, shaken when being added dropwise, being placed in temperature is 25 DEG C, rotating speed is to react 24h in 100r/min shaking table, then with bag filter dialysis 24h, removes what is do not combined with IgE ITCBE；5）By step 4）The liquid of gained adjusts pH value to 7.0 with 1mol/L HCl, is then slowly gradually added dropwise 80 μ l's 1mmol/L mercury ion solution, vibrated when being added dropwise, in case mercury ion precipitates albuminous degeneration；6）By step 5）The solution of gained is placed in temperature as 25 DEG C, and rotating speed is to react 2h in 100r/min shaking table, uses bag filter Carry out dialysis 24h；7）By step 6）Liquid after dialysis preserves in -20 DEG C of packing, obtains reaction solution；B）Purify the extraction of mercury-IgE chelates：Using immune-affinity chromatography, remove in reaction solution unreacted IgE and Unnecessary mercury ion, produce mercury-IgE chelates；The step B）It is specific as follows：（1）Sample dissolution：To by step A）Physiological saline is added in obtained reaction solution redissolves mercury-IgE chelates；（2）Balance chromatographic column：The pipeline of chromatographic column is rinsed using dilution buffer, loading in chromatographic column can be with IgE specificity With reference to filler, fill post after, be continuing with dilution buffer balance chromatographic column；（3）Loading：After column equilibration is chromatographed, with dilution buffer dilution step（1）Middle sample, then upper prop, IgE are special with filler The opposite sex combines；（4）Elution：Chromatographic column to baseline is rinsed using dilution buffer to balance, then using 0.05-0.1mol/L phosphate Solution is eluted, and obtains eluent I；（5）Collect：Collect eluent I；（6）Dialysis：By step（5）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C Dialysed overnight, collect sample；（7）Balance chromatographic column：Using new chromatographic column, with dilution buffer flushing line, energy and mercury 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 dilution buffer dilution step（6）Middle sample, then upper prop；（9）Elution：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, and obtains eluent II；（10）Collect：Collect eluent II；（11）Dialysis：By step（10）In collection eluent, fill bag filter, use ddH2O dialysis desalinations, after changing water three times, 4 DEG C dialysed overnight, sample is collected, produces mercury-IgE chelates.
- 2. a kind of preparation method of mercury-IgE chelates as claimed in claim 1, it is characterised in that also including step C）：It is right The identification of mercury-IgE chelates；Wherein, step C）In it is specific as follows：（1）Prepare glue bed：Glue bed is prepared using one kind in Ago-Gel, polyacrylamide gel as medium；（2）Sample-adding：Take step B）Mercury-IgE the chelates that middle extraction purification obtains, dilution buffer is added, and mixed, Ran Houjia Sample is in sample cell；（3）Electrophoresis：Electrophoresis plate is connected, carries out electrophoresis；（4）Detection：Mercurous protein band is found out on glue bed, the protein band is taken out and dissolved, then uses ELISA again Method or ICP-MS methods or AAS methods detect whether containing mercury and detected the content of mercury.
- 3. a kind of mercury-IgE chelates as claimed in claim 1 are preparing the reagent for detecting mercury-IgE chelates in blood sample or examination Application in agent box.
- 4. a kind of comprise at least kit of the mercury-IgE chelates as claimed in claim 1 as standard items.
- 5. kit as claimed in claim 4, it is characterised in that also including coating buffer, the coating buffer contains capture IgE thing Matter or the material for capturing mercury.
- A kind of 6. method for quantitatively detecting mercury-IgE chelates, it is characterised in that with described in the claim 1 of known content Mercury-IgE chelates are detected as standard items using a pair of samples of following methods：ELISA, enzyme linked immunological and original Sub- absorption spectrum combined techniques, enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques, purification mercury-IgE chelates with it is enzyme-linked Immune combined techniques, purification mercury-IgE chelates and atomic absorption spectrum combined techniques, purification mercury-IgE chelates and inductive etc. Gas ions mass spectrum combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
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| WO1994022490A1 (en) * | 1993-03-29 | 1994-10-13 | Immunomedics, Inc. | Conjugates of proteins and bifunctional ligands |
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