CN105004683B - chromium-IgE chelate as well as preparation method and application thereof - Google Patents
chromium-IgE chelate as well as preparation method and application thereof Download PDFInfo
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- CN105004683B CN105004683B CN201510411517.4A CN201510411517A CN105004683B CN 105004683 B CN105004683 B CN 105004683B CN 201510411517 A CN201510411517 A CN 201510411517A CN 105004683 B CN105004683 B CN 105004683B
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- 238000001514 detection method Methods 0.000 claims abstract description 48
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a chromium-IgE chelate and a preparation method and application thereof, wherein the chromium-IgE chelate is formed by chelating chromium ions and IgE through sulfydryl or/and cysteine residues. The invention establishes a qualitative and quantitative detection method of chromium-IgE chelate so as to quantitatively detect the application of the chromium-IgE chelate in evaluating the chromium pollution degree of a region. The condition of chromium pollution of people in a region can be indirectly reflected by quantitatively detecting the chromium-IgE chelate in the serum of people in the region, so that the chromium pollution degree of the region can be indirectly reflected. The accuracy of the quantitative detection method of the chromium-IgE chelate is greatly improved, and the repeatability of detection is greatly improved.
Description
Technical field
The present invention relates to detection fields, more specifically to a kind of chromium-IgE chelate and its preparation method and application.
Background technique
IgE is a kind of Ig, the molecular weight 188kD of discovery in 1966, is distributed mainly on the mucous membrane tissue, outer at these positions
In juice and blood, exist with monomeric form.It is synthesized in ontogeny later.ε chain has 4 CH (C 1~C of ε ε 4), hingeless
Sequence contains more cysteine and methionine.To thermo-responsive, 56 DEG C, 30 minutes can make IgE lose biological activity.
IgE is mainly generated by the thick liquid cell of the proper mucous membranes such as pharynx nasalis, tonsillotome, bronchus, stomach and intestine, these positions are often allergens
The place that invasion and I allergic reaction type occur.IgE is cytophilic antibody, and C ε 2 and 3 functional areas C ε can be with basophilic granulocytes, fertilizer
High-affinity Fc ε R I is combined on maxicell film.Allergen is again introduced into body and has been fixed in basophilic granulocyte, mast cell
Upper IgE is combined, and can cause type Ⅰ allergy.When parasitic infection or allergic reaction break out, in local exocrine secretion and serum
IgE level is all significantly raised.
Chromium (chromium, Cr) is one of VI race of periodic table of elements transition metal, and quality is hard, and surface has light
There is higher fusing point in pool, has great economic value in the industrial production, has been widely used since a century, annual complete
Ball chromium output is up to 107 tons, thus chromium is a kind of qualified important meals pollutant.Currently, chromium is widely used in electricity
The various aspects such as plating, tanning, pigment, paint, alloy, printing and dyeing, it is closely bound up with people's lives, but equally also to the mankind's
Health has an immense impact on.In the U.S., Cr and Hg, Cd, Pb are referred to as four overall situation pollutants.According to statistics, have every year several
Ten hundred million common people are adding Li Fuliya to have up to 30% water resource dirty by chromium at the water source for drinking pollution of chromium
Dye, thus pollution of chromium drastically influences the health of people.
Chromium mainly exists in the form of trivalent and sexavalence in nature, and it is generally believed that trivalent chromium (Cr3+) is mankind institute
The microelement needed, and Cr VI (Cr6+) is only apparent toxicant.Enter cell since Cr VI easily passes through cell membrane,
And strong cytotoxicity and genotoxicity can be generated by series reaction, thus generally believe the toxicity of Cr VI
Higher than trivalent chromium, 10-100 times is reached.Scholar thinks that when the content of 6-valence Cr ions in water is more than 0.05mg/L be toxic.Cr VI
It can be drunk by skin contact, food intake, drinking water and enter human body into approach such as, respiratory tract suckings, for occupational exposure
Crowd mainly by respiratory tract suck Cr VI dust, and be then for general population by drink into polluted source, eat by
The modes such as food, the sucking of pollution take in Cr VI.Cr VI enter in vivo after, can trigger oxidative stress, chromosome aberration,
Apoptosis, a variety of DNA damages (including single-strand break, DNA- protein cross, DNA- amino acid crosslinks, Cr-DNA complex
Formed etc.), to cause body many places tissue, organ, system injury.Moreover, Cr VI is also considered to be a kind of and has by force
The carcinogenic substance of power studies tissue early in nineteen ninety international cancer.
It is in close relations between chromium and multiple proteins, the activity of multiple protein enzyme can be inhibited in conjunction with multiple proteins.
With going deep into chromium toxicity research, people gradually recognize that the interaction of chromium 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 chromium, therefore, the research to interact to chromium and protein is just
Seem ever more important.And the mechanism now concerning chromium and protein effect is only tip of the iceberg, there are also many protein and chromium it
Between relationship it is not clear, thus reinforce it is most important for the relationship between chromium and protein.
Summary of the invention
For the serious problem of pollution of chromium, the purpose of the present invention is to provide a kind of chromium-IgE chelate and its preparation sides
Method, and the qualitative and quantitative analysis method of chromium-IgE chelate is established, so that quantitative detection chromium-IgE chelate is evaluating a ground
The application of area's pollution of chromium degree.It can reflect this indirectly by chromium-IgE chelate in quantitative detection one regional crowd's serum
The case where regional crowd is by pollution of chromium, to reflect this regional pollution of chromium degree indirectly.
The technical solution adopted by the present invention to solve the technical problems is: a kind of chromium-IgE chelate is provided, chromium ion with
IgE is chelated by sulfydryl or/and cysteine residues.
The present invention also provides a kind of preparation methods of above-mentioned chromium-IgE chelate, comprising the following steps:
A) the chelatropic reaction of chromium and IgE: add in the IgE of human body or the IgE recombinated according to biological method in purification
Enter chromium ion and carry out chelatropic reaction, obtains reaction solution；
B it) purifies the extraction of chromium-IgE chelate: using immune-affinity chromatography, remove unreacted IgE in reaction solution
And extra chromium ion is to get chromium-IgE chelate.
Wherein, step B described in external synthetic method) specific as follows:
(1) sample dissolution: physiological saline is added into the reaction solution obtained by step A) keeps chromium-IgE chelate multiple
It is molten；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgE spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgE with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted, and eluent I is obtained；
(5) it collects: collecting eluent I；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted, and eluent II is obtained；
(10) it collects: collecting eluent II；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample to get chromium-IgE chelate.
Wherein, the preparation method of above-mentioned chromium-IgE chelate, further includes step C): the identification to chromium-IgE chelate；
Wherein, step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained chromium-IgE chelate of extraction purification, dilution buffer is added, and mix, so
After be loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing chromium on glue bed, which is taken out and dissolved, is then used again
ELISA method or ICP-MS method or AAS method detect whether containing chromium and detect the content of chromium.
The present invention also provides a kind of examinations such as above-mentioned chromium-IgE chelate chromium-IgE chelate in preparation detection human body
Application in agent or kit.
The present invention also provides a kind of kits including at least such as above-mentioned chromium-IgE chelate as standard items.
It preferably, further include coating buffer in the kit, the substance which contains capture IgE or the object for capturing chromium
Matter.
The present invention also provides a kind of methods of quantitative detection chromium-IgE chelate, with the above-mentioned chromium-IgE chela of known content
Object is closed as standard items, is detected using a pair of sample of following methods: enzyme-linked immunization, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are with inductivity coupled plasma mass spectrometry combined techniques, purification chromium-IgE chelate in conjunction with enzyme linked immunological
Method, purification chromium-IgE chelate and atomic absorption spectrum combined techniques, purification chromium-IgE chelate and inductively coupled plasma constitution
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.
Implement chromium-IgE chelate and its preparation method and application of the invention, has the advantages that
1. the present invention synthesizes chromium-IgE chelate in vitro for the first time；
2. present invention firstly provides chromium-IgE chelate can be used for preparing detection blood sample in chromium-IgE chelate reagent or
Application in kit.
3. the present invention establishes the method for qualitative and quantitative detection of chromium-IgE chelate, so as to quantitative detection chromium-IgE chelate
In the application of evaluation one regional pollution of chromium degree.It can be with by chromium-IgE chelate in the regional crowd's serum of quantitative detection one
Reflect the case where this regional crowd is by pollution of chromium indirectly, to reflect this regional pollution of chromium degree indirectly.The present invention establishes
Chromium-IgE chelate quantitative detecting method its accuracy greatly improve, and the repeatability of detection is made to be greatly enhanced.
Detailed description of the invention
Fig. 1 is the non denatured electrophoretic band figure of chromium-IgE chelate of the present invention；
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium-IgE chelate of the present invention.
Specific embodiment
In the following, being described further in conjunction with specific embodiment to the present invention:
Unless otherwise indicated, the laboratory operating procedures being related to are the step of this field routine, agents useful for same, material to the present invention
Material as following cited, do not enumerate in the present invention come be reagent commonly used in the art or can be by commercially available mode
It obtains:
Phosphate solution is 0.05-0.1mol/L Na2HPO4Solution or 0.5-1mol/L Na2HPO4Solution；
Extracting reagent is PEG solution, borate buffer solution etc. (using PEG method)；
Glue bed medium is one of Ago-Gel, polyacrylamide gel；
Capture the substance purchase of IgE certainlyAmy victory Science and Technology Ltd.Model Abcam ab171396 rabbit Anti-
IgE antibody；Wherein, substance, anti-IgE antibodies, the anti-IgE antibodies of the present invention with IgE specific binding are capture IgE
Substance；
The substance of capture chromium buys the anti-chromium antibody from the model RK10641 of Guangzhou Ran Ke Biotechnology Co., Ltd；
Wherein, substance, anti-Cr antibody, the anti-chromium antibody of the present invention with chromium specific binding is the substance for capturing chromium；
Enzyme labelled antibody is one of the antibody marked containing enzymes such as horseradish peroxidase, alkaline phosphatases；
Substrate is methyl biphenyl amine (TMB) solution；
Cleaning solution is to contain KH2PO40.2mg/ml、Na2HPO4·12H2O 2.90mg/ml、NaCl 8.0mg/ml、KCl
The 0.15M PBS solution that the pH of 0.2mg/ml, 0.5%Tween-20 are 7.4；
Confining liquid is 1%-5% bovine serum albumin(BSA) or skimmed milk power；
Dilution buffer is Na containing 1.5mg/mL2CO3、2.93mg/ml NaHCO3PH be 9.6 0.05M carbonate it is slow
Fliud flushing；
Enzyme labelled antibody is HRP enzyme labelled antibody；
Terminate liquid are as follows: by the 2M H of 21.7ml2SO4It is settled to the ddH of 200ml2In O；
Eluent is the 0.1mol/L Tris-HCL buffer that the PH 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
The Sample buffer (5X) of propylhomoserin 25mL；
The ddH that electrophoretic buffer is 3mg/ml containing Tris, the PH of glycine 14.4mg/ml is 6.82O solution；
The present invention provides a kind of chromium-IgE chelate, chromium ion and IgE and is chelated by sulfydryl or/and cysteine residues
At.
The present invention also provides a kind of preparation methods of chromium-IgE chelate, comprising the following steps:
A) the chelatropic reaction of chromium and IgE: chromium ion is added in the IgE of source of people and carries out chelatropic reaction, obtains reaction solution；
B it) purifies the extraction of chromium-IgE chelate: using immune-affinity chromatography, remove unreacted IgE in reaction solution
And extra chromium ion is to get chromium-IgE chelate, the specific steps are as follows:
(1) sample dissolution: physiological saline is added into the reaction solution obtained by step A) keeps chromium-IgE chelate multiple
It is molten；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgE spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；It is described to be filled out with what IgE was specifically bound
Material is silica gel or resin containing anti-IgE antibodies；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgE with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted, and eluent I is obtained；
(5) it collects: collecting eluent I；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling column；It is described to be filled out with what chromium was specifically bound
Material is silica gel or resin containing anti-chromium antibody；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted, and eluent II is obtained；
(10) it collects: collecting eluent II；
(11) it dialyses: by the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalination, changes water
After three times, 4 DEG C of dialysed overnights collect sample to get chromium-IgE chelate；
C) to the identification of chromium-IgE chelate, the specific steps are as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained chromium-IgE chelate of extraction purification, dilution buffer is added, and mix, so
After be loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing chromium on glue bed, which is taken out and dissolved, is then used again
ELISA method or ICP-MS method or AAS method detect whether containing chromium and detect the content of chromium.
The present invention also provides a kind of kits including at least such as above-mentioned chromium-IgE chelate as standard items.
It preferably, further include coating buffer in the kit, the substance which contains capture IgE or the object for capturing chromium
Matter.
In the present invention, the kit for being able to achieve the object of the invention can be listed following several, and but it is not limited to this.
The kit of chromium-IgE chelate in a kind of detection blood sample, coating buffer, closing including the substance containing capture IgE
Liquid, cleaning solution, the substance for capturing chromium as secondary antibody, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control,
Negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, coating buffer, closing including the substance containing capture IgE
Liquid, cleaning solution, eluent, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, coating buffer, closing including the substance containing capture IgE
Liquid, cleaning solution, eluent, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, including as extraction reagent needed for IgE in purifying blood plasma,
Redissolve liquid, coating buffer, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer containing the substance for capturing chromium
Liquid, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, including as extraction reagent needed for IgE in purifying blood plasma,
Redissolve liquid, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, including as extraction reagent needed for IgE in purifying blood plasma,
Redissolve liquid, acidulant, hydrogen peroxide, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, including extraction reagent, the glue as IgE in purifying blood plasma
Bed medium redissolves liquid, sample-loading buffer, dissolves liquid needed for the protein band containing chromium on glue bed, containing the substance for capturing chromium
Coating buffer, confining liquid, cleaning solution, enzyme labelled antibody, substrate, terminate liquid, dilution buffer, positive control, negative control etc..
The kit of chromium-IgE chelate in a kind of detection blood sample, including as extraction reagent needed for IgE in purifying blood plasma,
Glue bed medium redissolves liquid, sample-loading buffer, dissolves liquid, positive control, negative control needed for the protein band containing chromium on glue bed
Deng.
The kit of chromium-IgE chelate in a kind of detection blood sample, including as extraction reagent needed for IgE in purifying blood plasma,
Glue bed medium, redissolve liquid, sample-loading buffer, liquid, acidulant needed for the protein band containing chromium on dissolution glue bed, hydrogen peroxide,
Positive control, negative control etc..
In above-mentioned several kits, the positive control is standard items, that is, is chelated with IgE chelate or the chela of heavy metal chromium
Close the BSA chelate for having heavy metal chromium；The negative control is the dilution buffer without containing standard items.
Mentioned reagent box is used to detect the IgE of chelated chromium, to improve the accuracy of detection, repeatability, and is allowed in clinic
In promoted.
The present invention also provides a kind of methods of quantitative detection chromium-IgE chelate, with the above-mentioned chromium-IgE chela of known content
Object is closed as standard items, is detected using a pair of sample of following methods: enzyme-linked immunization, enzyme linked immunological and atomic absorption light
Spectrum combined techniques, enzyme linked immunological are with inductivity coupled plasma mass spectrometry combined techniques, purification chromium-IgE chelate in conjunction with enzyme linked immunological
Method, purification chromium-IgE chelate and atomic absorption spectrum combined techniques, purification chromium-IgE chelate and inductively coupled plasma constitution
Compose combined techniques, electrophoresis and enzyme linked immunological or atomic absorption spectrum or inductivity coupled plasma mass spectrometry combined techniques.In the present invention,
Having of can listing of method with detection chromium-IgE chelate is following several, but is not limited to following several.
Wherein, the reagent used in the method for above-mentioned quantitative detection chromium-IgE chelate is as follows:
Method one:Enzyme-linked immunization (ELISA method) detects chromium-IgE chelate, detects in accordance with the following steps:
1) substance that IgE will be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier: slow with dilution
Anti- IgE Ab to 1000-8000 times of dilution is added in elisa plate micropore fliud flushing, 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 is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, are added in micropore, 37 DEG C effect 1-2 hours；
5) substance that can capture chromium is added, and incubates: removing measuring samples, and is washed with cleaning solution, it is to be washed
After the completion of washing, addition is diluted to 5000-40000 times of anti-Cr Ab with dilution buffer, 37 DEG C effect 1-2 hours, make anti-Cr
Ab reacts to form antigen antibody complex with the crome metal on IgE；
6) enzyme conjugates incubates: removing anti-chromium antibody, and is washed with cleaning solution, after the completion of washing, is added with dilute
Release the diluted enzyme labelled antibody of buffer, make the concentration 2 μ g/mL of diluted enzyme labelled antibody, 37 DEG C effect 1-2 hours, make its with
Enzyme labelled antibody reaction；
7) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
8) it terminates reaction: terminate liquid is added dropwise to each micropore；
9) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed in microplate reader and is detected, reads measuring samples respectively
With the OD value of standard items, by drawing standard curve, the content for acquiring measuring samples (can also directly pass through without using microplate reader
The situation that develops the color carries out qualitative detection).
This method utilizes ELISA principle, the specific IgE in blood plasma can be extracted, the top IgE extracted
Divide and is chelated with heavy metal chromium, and the chromium on the IgE of this part is captured by anti-chromium antibody, it later can be again by horseradish peroxidase
The antibody of the enzymes such as enzyme, alkaline phosphatase label captures (the antibody nonrecognition coating protein), and the antibody in capture is in color developing agent
And under the action of terminate liquid, OD value can be read under instrument, and does not contain the IgE of chelated mineral chromium, then it will not be by anti-chromium
Specific antibody is captured, and the antibody that will not be marked with enzymes such as horseradish peroxidase, alkaline phosphatases is captured, and used
Also without containing crome metal (negative control group result is feminine gender) in reagent, thus when read OD value is positive as the result is shown
When, i.e., the provable crome metal for detecting to chelate on IgE.
Method two:Enzyme linked immunological is pressed with atomic absorption spectrum combined techniques (ELISA method+AAS method) detection chromium-IgE chelate
It is detected according to following steps:
1) substance that IgE will be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier: slow with dilution
Anti- IgE Ab is diluted to 1000-8000 times by fliud flushing, is 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 is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, are added in micropore, 37 DEG C effect 1-2 hours；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, eluent is added, at 37 DEG C
Lower elution 1-3 hours.
6) it detects: being sampled from the micropore of elisa plate, in Atomic Absorption Spectrometer detection chelating in the chromium on IgE, and drawn
Standard curve processed, readout value；
This method combines atomic absorption spectrum (AAS) principle on the basis of utilizing ELISA principle, utilizes atomic absorption spectrum
Instrument detection chelating due to only containing IgE in solution, and is free of any heavy metal (negative control in the chromium on IgE in agents useful for same
Group result is feminine gender), result will not be interfered, thus when it is read be as the result is shown positive when, i.e., provable detection
The crome metal chelated on IgE out.
Method three:Enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method) detect chromium-
IgE chelate detects in accordance with the following steps:
1) substance that IgE will be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier: slow with dilution
Anti- IgE Ab is diluted to 1000-8000 times by fliud flushing, is 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 is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10-40 times with dilution buffer, are added in micropore, 37 DEG C effect 1-2 hours；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, eluent is added, at 37 DEG C
Lower elution 1-3 hours.
6) it being acidified: acidulant being added in the solution in step 5), solution is acidified, sealing overnight, is thoroughly acidified,
Hydrogen peroxide is added, and heats and catches up with acid；
7) it detects: 0.5ml liquid is taken from the solution of the elisa plate of step 6), in icp ms
Lower detection chelating draws standard curve in the chromium of IgE, readout value.
This method is on the basis of using ELISA principle, in conjunction with sense coupled plasma mass spectrometry (ICP-MS) principle, electricity consumption
Couple plasma mass spectrometer detection chelating is felt in the chromium on IgE, is free of due to only containing IgE in solution, and in agents useful for same
Any heavy metal (negative control group result is feminine gender), will not interfere result, thus be as the result is shown when read
When positive, i.e., the provable crome metal for detecting to chelate on IgE.
Method four:Purify chromium-IgE chelate and enzyme linked immunological combined techniques (method of purification+ELISA method) detection chromium-IgE chelating
Object 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 by IgE redissolve in solution, obtain IgE solution；
2) anti-Cr Ab is coated on solid phase carrier: anti-Cr Ab is diluted to 5000-40000 times with dilution buffer,
Be added elisa plate micropore in, 4 DEG C overnight 16-18 hour or 37 DEG C water-bath 1-3 hours, storage refrigerator；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, be added with 1%-5% ox
As confining liquid, 37 DEG C are placed 1 hour for seralbumin or skimmed milk power, are removed confining liquid, and washed with cleaning solution, are washed
After the completion of washing, elisa plate in 36.5-37.5 DEG C placement 1-2 hours；
4) add measuring samples, and incubate: being sampled from the solution of step 1), make measuring samples；With the chromium-of known content
IgE chelate makees standard items；Measuring samples are diluted to 10-40 times with dilution buffer, are added in micropore, 37 DEG C of effect 1-2
Hour；
5) enzyme conjugates incubates: removing the measuring samples in step 4), and is washed with cleaning solution, is completed wait wash
Afterwards, it is added and uses the diluted enzyme labelled antibody of dilution buffer, make concentration 2 the μ g/mL, 37 DEG C of effect 1-2 of diluted enzyme labelled antibody
Hour, react it with enzyme labelled antibody；
6) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
7) it terminates reaction: terminate liquid is added dropwise to each micropore；
8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed in microplate reader and is detected, reads measuring samples respectively
With the OD value of standard items, by drawing standard curve, the content for acquiring measuring samples (can also directly pass through without using microplate reader
The situation that develops the color carries out qualitative detection).
Method five:It purifies in chromium-IgE chelate and atomic absorption spectrum combined techniques (method of purification+AAS method) detection blood sample
Chromium-IgE chelate 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 by IgE redissolve in solution, obtain IgE solution；
2) it detects: being sampled from the solution that step 1) obtains, chelated in Atomic Absorption Spectrometer detection in the chromium on IgE,
And standard curve is drawn, readout value.
Method six:Purify chromium-IgE chelate and inductivity coupled plasma mass spectrometry combined techniques (method of purification+ICP-MS method)
Chromium-IgE chelate is detected, is 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 by IgE redissolve in solution, obtain IgE solution；
2) it is acidified: being sampled from the solution that step 1) obtains, acidulant (such as nitric acid) is added in the solution, solution is carried out
Acidification, sealing overnight, are thoroughly acidified, and hydrogen peroxide are added, and heat and catch up with acid；
3) it detects: taking 0.5ml liquid from the solution that step 2) obtains, detected under icp ms
It chelates in the chromium on IgE, and draws standard curve, readout value.
Method seven:Electrophoresis and enzyme-linked immunization or atomic absorption spectrography (AAS) or inductivity coupled plasma mass spectrometry combined techniques
(electrophoresis-ELISA method/AAS method/ICP-MS method) detects chromium-IgE chelate, 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 by IgE redissolve in solution, obtain IgE solution；
2) glue bed is prepared: selection Ago-Gel or polyacrylamide gel or SDS- polyacrylamide gel as needed
Deng be used as medium, conventionally prepare glue bed；
3) be loaded: 2 μ L sample-loading buffers are added in the 8 μ L of solution for taking step 1) to obtain, and mix, of short duration centrifugation；(pay attention to herein
Step cannot boil)
4) electrophoresis: connection electrophoresis plate is separated by electrophoresis；
5) it detects: finding out the protein band containing chromium on glue bed, which 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 chromium content being dissolved in liquid.In addition, also
It can use isoelectric point, molecular weight and the content of IgE etc. of the method detection chelated chromium.
IgE in method seven can be come out with a variety of Methods For Purifications (such as supercentrifugation or high pressure liquid chromatography or
Gel-filtration chromatography or gel electrophoresis or ELISA method etc.), the IgE that purification is come out redissolves in solution, takes a certain amount of
IgE carried out electrophoresis (electrophoresis, EP) using charge shifting principle, gel slab (can be as needed using not
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 chromium, will coagulate
Protein in glue redissolves in solution, it can the content for detecting correlation IgE at a particular wavelength, also can use ELISA,
The principles such as AAS, ICP-MS detect to chelate in the chromium 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 be feminine gender), result will not be interfered, thus when it is read as the result is shown
When for the positive, i.e., the provable crome metal for detecting to chelate on IgE.
Embodiment 1: the preparation method of chromium-IgE chelate, comprising the following steps:
A) the chelatropic reaction of chromium and IgE: chromium ion is added in the IgE of source of people and carries out chelatropic reaction, obtains reaction solution；
Preparation of reagents:
1) borate buffer solution (0.01M): weighing 0.31g boric acid and be dissolved in 400ml ultrapure water, is adjusted with the NaOH of 0.1M
PH to 9.0, is settled to 500mL.
2) it IgE solution: weighs 4.0mg IgE and is dissolved in 4.0mL 0.01M pH9.0 borate buffer solution, sufficiently vibrate molten
Solution, is configured to the protein solution of 1.0mg/mL；
3) EDTA-NaHCO3 mixed solution: EDTA2H is weighed2O 1.86g、NaHCO316.8g is dissolved in 900mL ultrapure water
In, 1000ml, high pressure sterilization, room temperature preservation are settled to 1.0M NaOH adjustment pH to 8.0；
4) the certainly Japanese colleague's chemistry institute of ITCBE purchase, article No. M030；
5) bag filter is purchased from Bioshop Inc, molecular cut off 14000.
The chelation step of chromium-IgE chelate:
1) bag filter the processing of bag filter: is put into the EDTA-NaHCO of 500ml3In mixed solution, 10min is boiled；Incline
Abandon EDTA/NaHCO3Liquid is gently rinsed with ultrapure water, then boils 10min with the 5mmol/L EDTA solution of 500ml；It discards and boils
Liquid is boiled, is thoroughly cleaned with ultrapure water, ultrapure water is added and impregnates 4 DEG C of bag filter overnight.In use, wearing gloves, bag filter is taken out,
With its surfaces externally and internally of ultrapure water cleaning down；
2) 2.0mg ITCBE is taken to be dissolved in 2ml DMSO；
3) 4.0mg IgE is taken to be dissolved in 4.0ml borate buffer solution；
4) liquid for slowly preparing step 2) is added in the IgE solution that step 3) is prepared, and shakes when being added dropwise, is placed in temperature
Degree is 25 DEG C, is reacted for 24 hours in the shaking table that revolving speed is 100r/min, then for 24 hours with bag filter dialysis, is removed not in conjunction with IgE
ITCBE；
5) step 4) resulting liquid 1mol/L HCl is adjusted into pH value to 7.0, is then slowly gradually added dropwise 80 μ l's
1mmol/L chromium ion solution is vibrated when being added dropwise, in case chromium ion precipitates albuminous degeneration；
6) the resulting solution of step 5) is placed in temperature is 25 DEG C, 2h is reacted in the shaking table that revolving speed is 100r/min, with saturating
Analysis bag is dialysed for 24 hours；
7) liquid after step 6) dialysis is saved in -20 DEG C of packing, obtains reaction solution.
B it) purifies the extraction of chromium-IgE chelate: using immune-affinity chromatography, (i.e. step 7) obtains removal reaction solution
Reaction solution) in unreacted IgE and extra chromium ion to get chromium-IgE chelate, the specific steps are as follows:
(1) sample dissolution: physiological saline is added into the reaction solution obtained by step 7) keeps chromium-IgE chelate multiple
It is molten；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgE spy
The filler that the opposite sex combines after filling column, continues to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgE with fill out
Material specific binding；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphorus of 0.05-0.1mol/L
Acid salt solution is eluted, and eluent I is obtained；
(5) it collects: collecting eluent I；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, changes water three times
Afterwards, 4 DEG C of dialysed overnights collect sample；
(7) it balances chromatographic column: energy is packed into the chromatographic column with dilution buffer flushing pipeline using new chromatographic column
With the filler of chromium specific binding, chromatographic column is balanced with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphoric acid of 0.5-1.0mol/L
Salting liquid is eluted, and eluent II is obtained；
(10) it collects: collecting eluent II；
(11) it dialyses: by the eluent of the collection in step (10), bag filter is filled, with 2 liters of ddH2O dialysis desalination, changes water
After three times, 4 DEG C of dialysed overnights collect sample to get chromium-IgE chelate；
C) to the identification of chromium-IgE chelate, the specific steps are as follows:
(1) it prepares glue bed: preparing glue bed using Ago-Gel as medium；
(2) be loaded: taking step B) in the obtained chromium-IgE chelate of 8 μ L extraction purifications, 2 μ L sample-loading buffers are added, and
It mixes, is then loaded onto sample cell；
(3) electrophoresis: connection electrophoresis plate is added electrophoretic buffer and carries out electrophoresis；In electrophoresis process, electric current is 22mA constant current,
Environment temperature is 4 DEG C；Electrophoresis to bromophenol blue stops electrophoresis when moving to glue bottom；
(4) it detects: finding out the protein band containing chromium on glue bed, which is taken out and dissolved, is then used again
ELISA method or ICP-MS method or AAS method detect whether containing chromium and detect the content of chromium.
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 | Cr(μg/L) |
| IgE | 201.217 |
| (NH4)2SO4 | 0.054 |
| N.S | 0.412 |
| N.S | 0 |
(2) electrophoresis result:
Wherein, Fig. 1 is the non denatured electrophoretic band figure of chromium-IgE chelate 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 ethyl alcohol, to keeping active protein to carry out polyacrylamide gel electrophoresis, it is usually used in the mirror of enzyme
Fixed, Isozyme Analysis and purification.Not plus the native polyacrylamide gel electrophoresis of SDS can make large biological molecule in electrophoresis process
Middle holding its natural shape and charge, their separation are made according to the difference of its electrophoretic mobility and the molecular sieve of gel
With, thus available higher resolution ratio, the large biological molecules such as protein and enzyme are still able to maintain especially 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 item
Band.
(3) Synchrotron Radiation X-Ray Fluorescence Anal ysis:
4W1 " of the SRXRF analysis of micronutrient levels in Beijing Positive and Negative Electronic Collider (BEPC) is synchronous in protein band
It is completed on radiation bunch.Beam current energy is 2.2GeV, beam intensity 100mA in storage rings.Sample mobile station (TSA200
Type, Beijing are stood upright Han Guang company) can be moved up under the stepper motor driving that computer controls along X, Y two-dimensional square with change into
Penetrate facula position, moving step length 0.0025mm.The X-ray gone out from electromagnetic radiation is by Si (Li) detector (PGT Inc.LS
It 30143-DS) detects, pop one's head in coplanar with incidence SR line and is mutually perpendicular to, away from sample irradiation point 20mm, signal PGT multiple tracks point
Analyzer (MCA 4000) obtains output.Sample is excited with the monochromatic synchrotron radiation light of 11.5keV, adjusts launching spot (1mmx
3mm) position is allowed in band one end, and in the minute of 300s, hot spot is uniformly slowly moved along band always, counts knot
Hot spot moves on to the band other end when beam.Along electrophoresis direction, every 1mm takes a spectrum.Using AX IL software data processing, and it is used to
Derived from air and the Ar signal peak of content constant carries out normalization to other element peaks, to offset beam intensity variation to signal
The influence that power generates.Measure the fluorescence Spectra of quantitative criterion dry glue film in the same way at identical conditions.
Fig. 2 is the synchrotron radiation X line fluorescence analysis figure of the electrophoretic band of chromium-IgE chelate of the present invention, cross 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 of the method for quantitative detection chromium-IgE chelate of the present invention:
1. the determination of the optimum diluting multiple of anti-IgE antibodies, anti-chromium antibody best effort concentration and blood plasma
Steps are as follows:
(1) it is 1 according to anti-IgE Ab and the mass volume ratio of dilution buffer by anti-IgE Ab dilution buffer:
1000,1:2000,1:4000,1:8000 are diluted, and are added in elisa plate micropore, anti-IgE Ab is coated in solid phase carrier
On, each 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, 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 that 1:10,1:20,1:40 progress are dilute by measuring samples and the mass volume ratio of dilution buffer
It releases, is added in micropore, according to above-mentioned coated anti-IgE Ab concentration, being separately added into for the anti-IgE Ab of the 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, after the completion of washing, anti-Cr Ab are added, anti-Cr Ab is pressed
Anti- Cr Ab and the mass volume ratio of dilution buffer are that 1:5000,1:10000,1:20000,1:40000 are diluted, according to
Each identical anti-IgE Ab, diluted plasma concentration, the anti-Cr Ab of various concentration respectively add 2 holes, and 37 DEG C act on 1 hour, make anti-Cr
Ab is reacted with the crome metal on IgE；
(5) enzyme labelled antibody selects most suitable working concentration, i.e. 2 μ g/mL remove anti-Cr antibody, and washed with cleaning solution,
After the completion of washing, addition dilution buffer dilutes HRP enzyme labelled antibody, and 37 DEG C act on 1 hour, makes HRP enzyme labelled antibody and chromium-
The reaction of IgE chelate；
(6) enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, substrate is added, 37 DEG C are protected from light effect
30 minutes；
(7) terminate liquid is added dropwise to each micropore；
(8) wavelength 405nm is taken, after adding terminate liquid, elisa plate is placed in microplate reader and is detected, reads each hole OD respectively
Value.According to each hole OD value numerical value, the optimum diluting multiple of anti-IgE Ab, the best effort concentration of anti-Cr Ab and blood plasma are selected.
Simultaneously using made standard items as positive control in test, anti-IgE Ab+ closing+anti-Cr Ab+ enzyme mark+bottom is selected
Object (measuring samples are not added) is used as negative control 1；Anti- IgE Ab+ closing+blood plasma+enzyme mark+substrate (anti-Cr Ab is not added) is made
For negative control 2；Anti- IgE Ab+ closing+enzyme mark+substrate (measuring samples and anti-Cr Ab are not added) is used as negative control 3；It is anti-
IgE Ab+ closing+blood plasma+anti-Cr Ab+ substrate (i.e. not enzyme mark) is used as negative control 4；Closing+blood plasma+anti-Cr Ab+ enzyme mark
+ substrate (anti-IgE Ab capture IgE is not added) is 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 the best dilution times of anti-IgE Ab, the best effort concentration of anti-Cr Ab and blood plasma
Several data (each data are all average value)
Table 3:ELISA positive control and negative control ELISA testing result
It is shown by table 2-3 data, we can see that the dilution of anti-IgE antibodies is 1:2000, dilution rate of blood plasma 1:
10, when the anti-dilution of Cr is 1:10000, OD value is maximum, so selecting concentration corresponding to this value as best effort concentration.
2. eluent best effort concentration and elution time in method two, three in the method for quantitative detection chromium-IgE chelate
Determination
Steps are as follows: (1) being coated with: anti-Cr Ab is diluted to 10000 times (mass volume ratios) with dilution buffer, be added
In elisa plate micropore, 37 DEG C water-bath 3 hours, store refrigerator；
(2) it closes: removing dilution buffer, and washed with cleaning solution, it is pure with 2% ox blood after the completion of washing
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, after the completion of washing, is added diluted with dilution buffer
Enzyme labelled antibody, it is 2 μ g/mL that enzyme labelled antibody, which is diluted to concentration, and 37 DEG C act on 2 hours, react it with anti-Cr Ab；
(4) prepare eluent: by papain with pH 8.0,0.1mol/L Tris-HCI buffer at
100ng/ml adds 37 DEG C of incubation 30min of 1mmol/L dithiothreitol (DTT) (DTT)；
(5) enzyme labelled antibody is removed, eluent is diluted with dilution buffer, makes the 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 placed in 37 DEG C；
(6) eluent is removed, and is washed with cleaning solution, after the completion of washing, substrate is added, 37 DEG C are protected from light effect 30
Minute；
(7) terminate liquid is added dropwise to each micropore；
(8) 405nm wavelength is taken, after adding terminate liquid, elisa plate is placed in microplate reader and is detected, reads every group of OD respectively
Value, by compared with PBS buffer solution 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
From in table 4 we it can be found that the papain in the eluent the ratio between concentration and the concentration of enzyme labelled antibody
When=1:20, each group OD value is below other several groups, and illustrating that the concentration elution effect is optimal (i.e. will be on ELISA hole wall
In conjunction with anti-Cr Ab- enzyme mark compound elution degree reach maximum, thus OD value is minimum)；And elution time either 1h, 2h,
3h, the variation of each group OD value 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 is that 1-3h all may be used in this test.
Application Example 1
It takes using the chromium-IgE chelate in 100 parts of sample blood plasma of ELISA method detection, follows the steps below detection:
Enzyme-linked immunization (ELISA method) detects chromium-IgE chelate, detects in accordance with the following steps:
1) anti-IgE Ab is coated on solid phase carrier: anti-IgE Ab is diluted to 2000 times with dilution buffer, is added
In elisa plate micropore, 37 DEG C water-bath 1 hour, store refrigerator；
2) blood plasma is taken from the circulatory system, makees measuring samples, 1000rmp is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, add 2% bovine serum albumin
It is white be used as confining liquid, 37 DEG C place 1 hour, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate in
37 DEG C are placed 1 hour；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10 times with dilution buffer, are added in micropore, 37 DEG C act on 1 hour；
5) substance that can capture chromium is added, and incubates: removing measuring samples, and is washed with cleaning solution, it is to be washed
After the completion of washing, addition dilution buffer dilutes 10000 times, and 37 DEG C act on 1 hour, keeps the crome metal on anti-Cr Ab and IgE anti-
It answers；
6) enzyme conjugates incubates: removing anti-chromium antibody, and is washed with cleaning solution, after the completion of washing, is added with dilute
The diluted HRP enzyme labelled antibody of buffer is released, 37 DEG C act on 1 hour, react it with enzyme labelled antibody；
7) substrate incubates: enzyme labelled antibody is removed, and is washed with cleaning solution, after the completion of washing, and addition substrate, 37 DEG C
It is protected from light effect 30 minutes；
8) it terminates reaction: terminate liquid is added dropwise to each micropore；
9) taking wavelength is 405nm, after adding terminate liquid, elisa plate is placed in microplate reader and is detected, read respectively to sample
Product OD value (can also not use microplate reader, directly carry out qualitative detection by colour developing situation), and specific detected value is as shown in table 5.
The ELISA testing result of chromium-IgE chelate in 5:100 parts of blood samples of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD405 | 0.433 | 0.758 | 0.308 | 0.347 | 0.797 | 0.562 | 0.308 | 0.347 | 0.71 | 0.599 |
| Number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| OD405 | 0.65 | 0.775 | 0.447 | 0.719 | 0.572 | 0.716 | 0.575 | 0.666 | 0.587 | 0.576 |
| Number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| OD405 | 0.79 | 0.622 | 0.648 | 0.476 | 0.595 | 0.807 | 0.361 | 0.738 | 0.614 | 0.455 |
| Number | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| OD405 | 0.742 | 0.528 | 0.425 | 0.765 | 0.505 | 0.444 | 0.607 | 0.586 | 0.369 | 0.786 |
| Number | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 |
| OD405 | 0.729 | 0.757 | 0.564 | 0.678 | 0.681 | 0.572 | 0.43 | 0.398 | 0.634 | 0.402 |
| Number | 51 | 52 | 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 |
| OD405 | 0.746 | 0.446 | 0.338 | 0.476 | 0.4 | 0.403 | 0.756 | 0.66 | 0.672 | 0.399 |
| Number | 61 | 62 | 63 | 64 | 65 | 66 | 67 | 68 | 69 | 70 |
| OD405 | 0.619 | 0.647 | 0.54 | 0.404 | 0.446 | 0.464 | 0.665 | 0.625 | 0.584 | 0.539 |
| Number | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | 79 | 80 |
| OD405 | 0.769 | 0.483 | 0.321 | 0.686 | 0.626 | 0.451 | 0.366 | 0.46 | 0.485 | 0.6 |
| Number | 81 | 82 | 83 | 84 | 85 | 86 | 87 | 88 | 89 | 90 |
| OD405 | 0.449 | 0.308 | 0.464 | 0.751 | 0.651 | 0.473 | 0.321 | 0.301 | 0.788 | 0.802 |
| Number | 91 | 92 | 93 | 94 | 95 | 96 | 97 | 98 | 99 | 100 |
| OD405 | 0.622 | 0.494 | 0.505 | 0.569 | 0.749 | 0.644 | 0.757 | 0.681 | 0.776 | 0.72 |
Application Example 2
Using in 100 minute mark this blood sample of enzyme linked immunological and atomic absorption spectrum combined techniques (ELISA method+AAS method) detection
Chromium-IgE chelate detects in accordance with the following steps:
1) substance that IgE will be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier: slow with dilution
Anti- IgE Ab is diluted to 2000 times by fliud flushing, is 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 is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, it is pure that 2% ox blood is added
Albumen or skimmed milk power are as confining liquid, and 37 DEG C are placed 1 hour, remove confining liquid, and washed with cleaning solution, and washing is completed
Afterwards, elisa plate is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10 times with dilution buffer dilution, are added in micropore, 37 DEG C act on 1 hour；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, it is dense that papain is added
Degree is the eluent of 100ng/ml, is eluted 1-3 hours；
6) it detects: being sampled from the micropore of elisa plate, in Atomic Absorption Spectrometer detection chelating in the chromium on IgE, detection
Value is as shown in table 6.
The AAS testing result of chromium-IgE chelate in 6:100 parts of blood samples of table
Application Example 3
100 minute marks are detected using enzyme linked immunological and inductivity coupled plasma mass spectrometry combined techniques (ELISA method+ICP-MS method)
Chromium-IgE chelate content in this blood sample detects in accordance with the following steps:
1) substance that IgE will be captured, as anti-IgE antibodies (anti-IgE Ab) are coated on solid phase carrier: slow with dilution
Anti- IgE Ab is diluted to 2000 times by fliud flushing, is added in elisa plate micropore, and 4 DEG C are stayed overnight 18 hours；
2) take 100 parts of standard blood samples as measuring samples, 1000rmp is centrifuged 5-8 minutes, and centrifugation discards precipitating；
3) it closes: removing dilution buffer, and washed with cleaning solution, after the completion of washing, it is pure that 2% ox blood is added
As confining liquid, 37 DEG C are placed 1 hour albumen, remove confining liquid, and washed with cleaning solution, after the completion of washing, elisa plate
It is placed 1 hour in 37 DEG C；
4) add measuring samples, and incubate: the measuring samples in step 2), the chromium-IgE chelate with known content are added
Make standard items；Measuring samples are diluted to 10 times with dilution buffer, are added in micropore, 37 DEG C act on 1 hour；
5) it elutes: removing measuring samples, and washed with cleaning solution, after the completion of washing, it is dense that papain is added
Degree is the eluent of 100ng/ml, is eluted 1-3 hours；
6) it is acidified: nitric acid being added in the solution in step 5), solution is acidified, sealing overnight, is thoroughly acidified, adds
Enter hydrogen peroxide, and heats and catch up with acid；
7) detect: sampled from the solution that step 6) obtains, under icp ms detection chelating in
The chromium of IgE, detected value are as shown in table 7.
The ICP-MS testing result of chromium-IgE chelate in 7:100 parts of blood samples of table
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (5)
1. a kind of preparation method of chromium-IgE chelate, which comprises the following steps:
A chromium the) chelatropic reaction of chromium and IgE: is added in the IgE of human body or the IgE recombinated according to biological method in purification
Ion carries out chelatropic reaction, obtains reaction solution；
Step A) specific as follows:
1) bag filter the processing of bag filter: is put into the EDTA-NaHCO of 500mL3In mixed solution, 10min is boiled；Tipping
EDTA/NaHCO3Liquid is gently rinsed with ultrapure water, then boils 10min with the 5mmol/L EDTA solution of 500mL；It discards and boils
Liquid is thoroughly cleaned with ultrapure water, and ultrapure water is added and impregnates 4 DEG C of bag filter overnight；
In use, wearing gloves, bag filter is taken out, with its surfaces externally and internally of ultrapure water cleaning down；
2) 2.0mg ITCBE is taken to be dissolved in 2mL DMSO；
3) 4.0mg IgE is taken to be dissolved in 4.0mL borate buffer solution；
4) liquid for slowly preparing step 2 is added in the IgE solution that step 3) is prepared, and shakes when being added dropwise, being placed in temperature is
25 DEG C, then for 24 hours with bag filter dialysis revolving speed, removes not in conjunction with IgE to react for 24 hours in the shaking table of 100r/min
ITCBE；
5) step 4) resulting liquid 1mol/L HCl is adjusted into pH value to 7.0, is then slowly gradually added dropwise 80 μ l's
1mmol/L chromium ion solution is vibrated when being added dropwise；
6) the resulting solution of step 5) is placed in temperature is 25 DEG C, reacts 2h in the shaking table that revolving speed is 100r/min, uses bag filter
It is dialysed for 24 hours；
7) liquid after step 6) being dialysed is saved in -20 DEG C of packing, obtains reaction solution；
B) purify chromium-IgE chelate extraction: use immune-affinity chromatography, remove reaction solution in unreacted IgE and
Extra chromium ion is to get chromium-IgE chelate；
The step B) specific as follows:
(1) sample dissolution: physiological saline, which is added, into the reaction solution obtained by step A) redissolves chromium-IgE chelate；
(2) it balances chromatographic column: rinsing the pipeline of chromatographic column using dilution buffer, being packed into chromatographic column can be with IgE specificity
In conjunction with filler, fill column after, continue to use dilution buffer balance chromatographic column；
(3) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (1), then upper prop, IgE and filler are special
The opposite sex combines；
(4) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, then use the phosphate of 0.05-0.1mol/L
Solution is eluted, and eluent I is obtained；
(5) it collects: collecting eluent I；
(6) it dialyses: by the eluent of the collection in step (5), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4 DEG C
Dialysed overnight collects sample；
(7) it balances chromatographic column: using new chromatographic column, with dilution buffer flushing pipeline, energy and chromium are packed into the chromatographic column
The filler of specific binding balances chromatographic column with dilution buffer again after filling column；
(8) loading: after chromatographing column equilibration, with sample in dilution buffer dilution step (6), then upper prop；
(9) it elutes: rinsing chromatographic column to baseline using dilution buffer and balance, it is then molten using the phosphate of 0.5-1.0mol/L
Liquid is eluted, and eluent II is obtained；
(10) it collects: collecting eluent II；
(11) it dialyses: by the eluent of the collection in step (10), filling bag filter, use ddH2O dialysis desalination, after changing water three times, 4
DEG C dialysed overnight collects sample to get chromium-IgE chelate.
2. the preparation method of chromium-IgE chelate according to claim 1, which is characterized in that further include step C): to chromium-
The identification of IgE chelate；
Wherein, step C) in it is specific as follows:
(1) it prepares glue bed: preparing glue bed using one of Ago-Gel, polyacrylamide gel as medium；
(2) be loaded: taking step B) in the obtained chromium-IgE chelate of extraction purification, dilution buffer is added, and mix, then plus
Sample is in sample cell；
(3) electrophoresis: connection electrophoresis plate carries out electrophoresis；
(4) it detects: finding out the protein band containing chromium on glue bed, which is taken out and dissolved, then use ELISA again
Method or ICP-MS method or AAS method detect whether containing chromium and detect the content of chromium.
3. a kind of chromium-IgE chelate in preparation detection blood sample of chromium-IgE chelate obtained by method as claimed in claim 1
Application in reagent or kit.
4. a kind of including at least kit of the chromium-IgE chelate as standard items obtained by method as claimed in claim 1.
5. kit according to claim 4, which is characterized in that further include coating buffer, which contains capture IgE's
Substance or the substance for capturing chromium.
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| 《六价铬间接竞争ELISA检测试剂盒的研制及新型一步法竞争ELISA的建立》;邹军辉;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;CNKI;20121015(第10期);正文第13-16页 * |
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