CN106872692B - Competitive ELISA detection kit and its application in mercury ion food pollution - Google Patents
Competitive ELISA detection kit and its application in mercury ion food pollution Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention provides one kind to be based on Direct cELISA mercury ion detection kit, comprising: is coated with the detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package；The peridium concentration of the sheep anti-mouse igg secondary antibody is 50~200 μ g/mL；Mass concentration is the enzyme mark Hg-ITCBE chelating object haptens of 20~50 μ g/mL；Mass concentration is the anti-mercury ion monoclonal antibody solution of 10~20 μ g/mL；The anti-mercury ion monoclonal antibody solution is prepared by Hg-ITCBE-cBSA immunogen immune Balb/C mouse；The terminate liquid of 1.0~3.0mol/L；Sample diluting liquid；Substrate developing solution；Cleaning solution；Mercury ion standard solution；Mass concentration is the EDTA treatment fluid of 10~20mg/mL.The present invention, which provides kit, has the characteristics that high sensitivity, high specificity, while having the advantages that detection time is short.
Description
Technical field
The invention belongs to technical field of environmental detection, and in particular to competitive ELISA is examined in mercury ion food pollution
Test agent box and its application.
Background technique
Mercury is commonly called as mercury, is under room temperature, normal pressure uniquely with metal existing for liquid.The fast development of modern times society,
Mercury is increasingly used in agricultural, industry, pharmacy industry, metallurgy and dentistry, and the compound of mercuryvapour and mercury has severe toxicity more.
Along with applications in various fields, mercury forms the Tri-dimension Pollution circulation of atmosphere, water body and soil, and passes through plant, Fish
Interior enrichment enters human body using food chain, is enriched with again in the tissue such as liver, kidney, brain, inhibits human body enzymatic activity, upsets normal
Metabolism, or even deformity is caused, seriously threaten human health and food safety.Due to Hg2+With height electrophilicity, institute
To all have very strong attack to intracorporal most important sulfydryl, carbonyl, carboxyl, hydroxyl isoreactivity group, to make these
Active group loses activity, and has an immense impact on to organism physiology biochemical function.In addition, the methyl mercury in organic mercury is easiest to
Placenta is crossed, is transferred to fetus from parent, and fetus has very high accumulating capability to methyl mercury, since placental metastasis causes fetus
Generating serious fetal methylmercuric poisoning example has had a lot of reports.
Chronic mercury poisoning cardinal symptom shows as insanity, gingivitis, trembles with ephritis etc.；The acute main table of mercury poisoning
It is now acute corrosive stomatitis and gastroenteritis, acute renal failure, liver damage can occurs when serious.Skin contact mercury and
Its compound can cause contact dermatitis, have allergy property.Since the pollution and harm of mercury are larger, China has formulated soil
Earth, food, water body, cosmetics etc. relate to the national limit standard in mercury field, cow's milk mercury allowance standard (GB2762- in food
It 94) is≤0.01mg/Kg, regulation in groundwater quality standard (GB7468-2012) is primarily adapted for use in centralized domestic water water
The mercury ion limit standard of source and farmland irrigating water are≤0.01mg/L.
Hg in animal food and environmental water sample at present2+The method that predominantly detects of pollution includes traditional physical and chemical inspection and exempts from
Epidemiology examines two kinds of method.Physical and chemical inspection includes ultraviolet spectrophotometry (UV), electrochemical methods, atomic absorption light
Spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-
AES), Hydride Generation-atomic Fluorescence Spectrometry (HG-AFS) and Neutron activation analysiss (NAA) etc., they are current Hg2+It is dirty
The main method of dye detection, high sensitivity, as a result accurately, but due to needing expensive instrument and equipment, skilled practitioner, being tired of
It is trivial it is time-consuming, the period is long, it is costly, be unable to execute-in-place, the defects of sample screening amount is small, application is restricted.Immunology
The method of inspection is the analytical technology based on the specificity of antigen-antibody molecule, invertibity reaction, including Fluorescence Polarised Immunoassay
Measuring method (FPIA), enzyme-linked immunosorbent assay (ELISA), KinExA immunoassay (KIA), colloidal gold immunochromatographimethod examination
Measuring method (GICA) and micro-cantilever immunosensor (MIS) etc. are tested, since the combination of antigen-antibody has the selectivity of height
And sensibility, this technology have it is easy, quickly, the advantages such as sensitive, special, economy, screening amount be big, represent heavy metal ion
The developing direction of detection.Currently, the immunoassay technology research of metal mercury ions pollution both at home and abroad is very active, wherein notification number
A kind of mercury ion indirect competitive ELISA kit, detection sensitivity 10.31ng/ are disclosed for the patent of CN103472231B
ML, and detection time is longer.
Summary of the invention
Mercury ion reagent is detected based on Direct cELISA in view of this, the purpose of the present invention is to provide one kind
Box and its application make the kit have the advantages that detection time is short, while having the characteristics that high sensitivity, high specificity.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides one kind to be based on Direct cELISA mercury ion detection kit, comprising:
(1) detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package are coated with；The sheep anti-mouse igg two
Anti- peridium concentration is 50~200 μ g/mL；
(2) mass concentration is the enzyme mark Hg-ITCBE chelating object haptens solution of 20~50 μ g/mL；
(3) mass concentration is the anti-mercury ion monoclonal antibody solution of 10~20 μ g/mL；The anti-mercury ion monoclonal is anti-
Liquid solution is prepared by Hg-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate developing solution；
(7) cleaning solution；
(8) mercury ion standard solution；
(9) mass concentration is the EDTA treatment fluid of 10~20mg/mL.
Preferably, the peridium concentration of the sheep anti-mouse igg secondary antibody is 80~150 μ g/mL.
Preferably, the mass concentration of the enzyme mark Hg-ITCBE chelating object haptens is 30~40 μ g/mL.
Preferably, the mass concentration of the anti-mercury ion monoclonal antibody solution is 13~18 μ g/mL.
Preferably, the sample diluting liquid is the HEPES buffer solution that mass concentration is 2~10g/L.
Preferably, the cleaning solution is the PBS solution comprising 0.05~0.1%Tween of mass concentration 20.
The present invention also provides the applications of mercury ion detection kit mercury ion in detection environment, including following step
It is rapid:
A, anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme mark Hg-ITCBE chelating object half is anti-
Original solution is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, and mixing is washed after incubation with cleaning solution；
B, substrate developing solution is added into detection plate, is incubated for, terminate liquid mixing is added, measures OD value；
C, according to the obtained OD value of the step b and scheduled standard curve, the concentration of mercury ion in sample to be tested is obtained,
The standard curve is the linear relationship curve between ion concentration of mercury and OD value.
Preferably, anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme mark in the detection plate hole
The volume ratio of Hg-ITCBE chelating object haptens solution is 1~2: 1~2: 1~2.
Preferably, the temperature being incubated in the step a and b independently is 30~40 DEG C.
Preferably, the time being incubated in the step a and b independently is 5~30min.
The present invention provides one kind to be based on Direct cELISA mercury ion detection kit, comprising: (1) is coated with
The detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package；The peridium concentration of the sheep anti-mouse igg secondary antibody is 50
~200 μ g/mL；(2) mass concentration is the enzyme mark Hg-ITCBE chelating object haptens of 20~50 μ g/mL；(3) mass concentration is 10
The anti-mercury ion monoclonal antibody solution of~20 μ g/mL；The anti-mercury ion monoclonal antibody solution is exempted from by Hg-ITCBE-cBSA
Epidemic focus is immunized Balb/C mouse and is prepared；The terminate liquid of (4) 1.0~3.0mol/L；(5) sample diluting liquid；(6) substrate develops the color
Liquid；(7) cleaning solution；(8) mercury ion standard solution；(9) mass concentration is the EDTA treatment fluid of 10~20mg/mL.The present invention
It is designed according to antigen-antibody direct competitive immunochromatography principle, by sheep anti-mouse igg secondary antibody pre-coated in detection plate, makes to resist
Mercury ion monoclonal antibody, measuring samples or mercury ion standard solution and three kinds of substances of enzyme mark haptens (Hg-ITCBE-HRP)
It can be added simultaneously, anti-mercury ion monoclonal antibody and pre-coated two anti-binding of sheep anti-mouse igg form compound, sample or standard
Mercury ion and enzyme mark haptens in product are competitively in conjunction with anti-mercury ion monoclonal antibody solution, when substrate developing solution is added
When, sample absorbance value and its remaining mercury ion content are negatively correlated, multiplied by its corresponding dilution compared with standard curve
Multiple, the content of mercury ion in you can get it sample.Kit provided by the invention reduces and is incubated for twice in conventional contention method
And washing times, the time 30min of incubation and washing is shortened, so that kit is quickly and easily obtained testing result, contracts significantly
Short detection cycle.Mercury ion detection kit minimum detection limit (LOD) provided by the invention is up to 0.12 μ g/L, detection range
For 0.23~46 μ g/L.
Detailed description of the invention
Fig. 1 is immunogene synthetic route chart in embodiment 1；
Fig. 2 is dELISA the and ciELISA standard curve established in embodiment 8.
Specific embodiment
The present invention provides one kind to be based on Direct cELISA mercury ion detection kit, comprising:
(1) detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package are coated with；The sheep anti-mouse igg two
Anti- peridium concentration is 50~200 μ g/mL；
(2) mass concentration is the enzyme mark Hg-ITCBE chelating object haptens solution of 20~50 μ g/mL；
(3) mass concentration is the anti-mercury ion monoclonal antibody solution of 10~20 μ g/mL；The anti-mercury ion monoclonal is anti-
Liquid solution is prepared by Hg-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate developing solution；
(7) cleaning solution；
(8) mercury ion standard solution；
(9) mass concentration is the EDTA treatment fluid of 10~20mg/mL.
Mercury ion detection kit provided by the invention includes the detection plate for being coated with sheep anti-mouse igg secondary antibody, the detection
Plate vacuum sealed package；The peridium concentration of the sheep anti-mouse igg secondary antibody be 50~200 μ g/mL, preferably 80~150 μ g/mL,
More preferably 100~120 μ g/mL.In the present invention, the source of the sheep anti-mouse igg secondary antibody is not particularly limited, using this field
Sheep anti-mouse igg secondary antibody known to technical staff.
In the present invention, the preparation method of the detection plate for being coated with sheep anti-mouse igg secondary antibody preferably includes following steps:
I. it is 50~200 μ g/mL sheep anti-mouse igg secondary antibody coating buffers 100 that peridium concentration is added into the detection hole of detection plate
~150 holes μ L/ are incubated for 1.5~3h under the conditions of 35~40 DEG C for the first time, remove coating buffer after incubation, are washed for the first time with cleaning solution
Plate 2~5 times；
II. it is 3~6% pig negative serum, 200~300 μ that mass concentration is added into detection plate obtained in the step I
The hole L/ is incubated for 1.5~3h second under the conditions of 35~40 DEG C, coating buffer is removed after incubation, with second of board-washing of cleaning solution 2~5
It is secondary, it will test plate naturally dry under the conditions of 23~27 DEG C, obtain the detection plate for being coated with sheep anti-mouse igg secondary antibody.
In the present invention, the type of the detection plate is not particularly limited, ripe using those skilled in the art technical staff institute
The ELISA detection plate known.The hole count of the detection plate is not particularly limited, using inspection well-known to those skilled in the art
Drafting board, such as 24 holes, 48 holes or 96 holes.
In the present invention, the cleaning solution is PBST solution.The molar concentration of PBS solution is preferably 0.01~0.02mol/L,
More preferably 0.015mol/L.The pH of the PBS solution is preferably 7.4.Mass concentration of the Tween 20 in PBS solution
It is preferred that 0.05~0.1%, more preferably 0.08%.
In the present invention, the volume of the coating buffer is preferably 120 holes μ L/.The first time is incubated for and second of incubation
Temperature preferably stands alone as 37 DEG C.The first time is incubated for and second of the time being incubated for preferably stands alone as 2h.
In the present invention, the method for the removal coating buffer is not particularly limited, and use is well-known to those skilled in the art
Method.In the embodiment of the present invention, the method for the removal coating buffer is firmly to get rid of detection plate.
In the present invention, the number of the first time board-washing and second of board-washing is preferably 3~4 times.Washing and for the first time
The interval time of each board-washing is preferably 1.5~3min during secondary board-washing, more preferably 2min.
In the present invention, the source of the pig negative serum is not particularly limited, and use is well-known to those skilled in the art
Pig negative serum.The mass concentration of pig negative serum is preferably 5%.The addition volume of pig negative serum is preferably 220~
280 holes μ L/, the more preferable hole 260 μ L/.
In the present invention, the detection plate for being coated with sheep anti-mouse igg secondary antibody is vacuum-packed, is obtained vacuum-packed
It is coated with the detection plate of sheep anti-mouse igg secondary antibody.The vacuum-packed pressure is 600~1000Pa.
Mercury ion detection kit provided by the invention includes enzyme mark Hg-ITCBE chelating object haptens solution.The enzyme mark
The mass concentration of Hg-ITCBE chelating object haptens solution is 20~50 μ g/mL, preferably 30~40 μ g/mL, most preferably 35 μ
g/mL。
In the present invention, the type of the enzyme is not particularly limited using enzyme class well-known to those skilled in the art i.e.
It can.In the embodiment of the present invention, the type of the enzyme is horseradish peroxidase.
In the present invention, the preparation method of the enzyme mark Hg-ITCBE chelating object haptens preferably includes following steps:
A. isothiocycmatobenzyl ethylenediamine tetra-acetic acid (ITCBE) and dimethyl sulfoxide (DMSO) is mixed to form metal chelating
Agent solution；
B. by mercuric nitrate Hg (NO3)2It is mixed with HEPES buffer solution, forms Hg2+Solution；
C. by Hg obtained in metal cheating agents solution obtained in the step A and the step B2+Solution mixing, is adjusted
The pH value of mixed liquor is saved to 7.0~7.2, obtained mixed liquor 10~14h of oscillating reactions, it is anti-to form Hg-ITCBE chelating object half
It is former；
D. label enzyme is mixed with HEPES buffer solution, obtains enzyme solutions；
E. enzyme solutions obtained in the Hg-ITCBE chelating object haptens solution and the step D step C obtained
Mixing adjusts the pH value of mixture to 8.8~9.2, then by mixed liquor 22~26h of oscillating reactions, obtains enzyme mark Hg-ITCBE chela
Close object haptens；
There is no the limitation of time sequencing between the step A and B；It is suitable also without the time between D and A and B between C and D
The limitation of sequence.
In the present invention, the quality and dimethyl sulfoxide volume ratio of the ITCBE is preferably (5~15) mg: 1mL, more preferably
It is 10mg: 1mL.
In the present invention, the molar concentration for preparing enzyme mark Hg-ITCBE chelating object haptens HEPES buffer solution is preferably 8~
12mmol/L, more preferably 10mmol/L.The pH value of the HEPES buffer solution is preferably 7.8~8.5, and more preferably 8.0.
In the present invention, the mercuric nitrate Hg (NO3)2Quality and HEPES buffer solution volume ratio be preferably 8.5~
12.5mg: 1mL, more preferably 11.25mg: 1mL.
In the present invention, the metal cheating agents solution and the Hg2+The volume ratio of solution is preferably 0.8~1.2: 0.8~
1.2, more preferably 1: 1.
In the present invention, to contain metal-chelator and Hg2+The pH adjustment method of mixed liquor is not particularly limited, using this
Contain metal-chelator and Hg known to the technical staff of field2+The pH value Adjusted Option of mixed liquor.The embodiment of the present invention
In, the pH adjustment method of mixed liquor is carried out with the sodium hydroxide solution that mass concentration is 1%.
In the present invention, the time of mixed liquor oscillating reactions is preferably 12h in the step C.The mixed liquor oscillating reactions
Temperature be preferably 23~27 DEG C, more preferably 25 DEG C.
In the present invention, the quality of the label enzyme and the volume ratio of HEPES buffer solution are (80~120) mg: 1mL, more
Preferably 100mg: 1mL.
In the present invention, the Hg-ITCBE chelating object haptens liquor capacity and the enzyme solutions volume ratio are preferably 0.8
~1.2: 0.8~1.2, more preferably 1: 1.
In the present invention, the pH value of the mixture containing enzyme is preferably 9.0 in the step D.The mixed liquor containing enzyme
PH adjustment method with mass concentration be 1% sodium hydroxide solution carry out.The time of oscillating reactions is preferred in the step D
For for 24 hours.The temperature of the oscillating reactions is preferably 23~27 DEG C, and more preferably 25 DEG C.
In the present invention, preferably Hg-ITCBE- enzyme haptens solution is placed in after mixed liquor oscillating reactions in the step E
Analysis bag successively dialyses in distilled water and dialyses in PBS solution, the enzyme mark Hg-ITCBE chelate haptens purified.?
In the present invention, the time dialysed in the distilled water is preferably 2d；The time dialysed in the PBS solution is preferably 3d.
Mercury ion detection kit provided by the invention includes anti-mercury ion monoclonal antibody solution.The anti-mercury ion list
The mass concentration of clonal antibody solution is 10~20 μ g/mL, preferably 13~18 μ g/mL, more preferably 15 μ g/mL.
In the present invention, the anti-mercury ion monoclonal antibody solution is small by Hg-ITCBE-cBSA immunogen immune Balb/C
Mouse is prepared, and the preparation method that the present invention fights mercury ion monoclonal antibody is not particularly limited, using those skilled in the art
The preparation method of anti-mercury ion monoclonal antibody known to member.
In the present invention, the preparation method of the Hg-ITCBE-cBSA immunogene the following steps are included:
(1) be according to the quality of ITCBE and the volume ratio of dimethyl sulfoxide by ITCBE and dimethyl sulfoxide (DMSO) (8~
12) mg: 1mL mixing forms metal cheating agents solution；
(2) by mercuric nitrate Hg (NO3)2It is mixed with HEPES buffer solution according to 11.25mg: 1mL ratio, forms Hg2+It is molten
Liquid；
(3) by Hg obtained in metal cheating agents solution obtained in the step (1) and the step (2)2+Solution is pressed
It is mixed according to volume ratio for 1: 1, adjusting mixed liquor pH value to 7.0, then 10~14h of oscillating reactions, shape under the conditions of 23~27 DEG C
At Hg-ITCBE chelating object haptens；
(4) BSA, EDC and PBS buffer solution are mixed to obtain mixed liquor, BSA mass, EDC mass and PBS buffer solution
Volume ratio be 66mg: 11.6mg: 5mL, be according to mixeding liquid volume and ethylenediamine mass ratio by the mixed liquor and ethylenediamine
5mL: 7mg ratio mixing, 37 DEG C of oscillating reactions 2h obtain reaction solution；Reaction solution is with PBS dialysis 4d, the carrier protein activated
BSA；
(5) HEPES buffer solution that the carrier protein BSA and pH value of the activation are 8.0 is mixed, forming concentration is
The carrier protein solution of 30mg/mL；
(6) load for obtaining Hg-ITCBE chelating object haptens solution obtained in the step (3) and the step (5)
Body protein solution is mixed according to the ratio that volume ratio is 1: 1, and adjusting pH value to oscillating reactions 22 under the conditions of 9.0,23~27 DEG C~
26h, by obtained reaction solution be packed into bag filter, first with distilled water dialyse 2d, then with PBS dialyse 3d, that is, form Hg-ITCBE-
CBSA immunogene.
The synthetic route of the Hg-ITCBE-cBSA immunogene is shown in Fig. 1.
Mercury ion detection kit provided by the invention includes terminate liquid.The molar concentration of the terminate liquid be 1.0~
3.0mol/L, more preferably 1.5~2.5mol/L, most preferably 2.0mol/L.The terminate liquid is preferably H2SO4Solution.
Mercury ion detection kit provided by the invention includes sample diluting liquid.The sample diluting liquid is HEPES buffering
Liquid.The mass concentration of HEPES buffer solution is preferably 2~10g/L, more preferably 4~8g/L, most preferably 5g/L.The HEPES
The pH value of buffer is preferably 7.8~8.5, and more preferably 8.0.
Mercury ion detection kit provided by the invention includes substrate developing solution.The concentration 1 of the substrate developing solution~
2mg/mL, more preferably 1.27mg/mL.The type of the substrate developing solution is determined according to the type of label enzyme.The present invention is real
It applies in example, when the type of enzyme is HRP, the substrate developing solution is tetramethyl benzidine (TMB).
Mercury ion detection kit provided by the invention includes cleaning solution.The cleaning solution is mole comprising Tween20
Concentration is the PBS solution of 0.01~0.02mol/L.The pH value of the PBS solution is 7.0~8.0, more preferably 7.4.
Mass concentration of the Tween 20 in PBS solution is preferably 0.05~0.1%, and more preferably 0.08%.
Mercury ion detection kit provided by the invention includes EDTA treatment fluid.The mass concentration of EDTA treatment fluid be 10~
20mg/mL, more preferably 15mg/mL.When EDTA treatment fluid uses preferably by sample solution, sample diluting liquid and EDTA processing
The volume ratio of liquid is 5: 5: 1.The effect of EDTA treatment fluid is to combine EDTA and mercury ion for sample pre-treatments, form mercury
Ion-EDTA huge legendary turtle polymer solution, the identification convenient for antibody to mercury ion.
Mercury ion detection kit provided by the invention includes mercury ion standard solution.The mercury ion standard solution
For Hg2+- EDTA solution.The Hg2+The source of-EDTA solution is not particularly limited, and use is well-known to those skilled in the art
Hg2+- EDTA solution.In the embodiment of the present invention, Hg2+- EDTA solution is laboratory self-control.The Hg2+- EDTA solution system
Preparation Method is not particularly limited, using Hg well-known to those skilled in the art2+- EDTA solution manufacturing method.
The present invention also provides the applications of mercury ion detection kit mercury ion in detection environment, including following step
It is rapid:
A, anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme mark Hg-ITCBE chelating object half is anti-
Original is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, and mixing is washed after incubation with cleaning solution；
B, substrate developing solution is added into detection plate, is incubated for, terminate liquid mixing is added, measures OD value；
C, according to the obtained OD value of the step b and scheduled standard curve, the concentration of mercury ion in sample to be tested is obtained,
The standard curve is detected with mercury ion standard solution, and obtained OD value has with the building of mercury ion standard concentration
The curve of linear relationship.
The present invention is by anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme mark Hg-ITCBE chelating object
Haptens is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, and mixing is washed after incubation with cleaning solution.
In the present invention, preferably it is diluted before the sample solution inspection.The diluted multiple is preferably 100~1000
Times, more preferably 200~800 times, most preferably 500 times.The dilution is handled using sample diluting liquid.The sample is dilute
EDTA treatment fluid is preferably added to after releasing liquid dilute sample.It is 5 by the volume ratio of sample solution, sample diluting liquid and EDTA treatment fluid
∶5∶1。
In the present invention, anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme in the detection plate hole
The volume ratio for marking haptens solution is preferably 1~2: 1~2: 1~2, and more preferably 1: 1: 1.
In the present invention, the method that the mixed method preferably uses pipettor gun head pressure-vaccum is mixed.
In the present invention, the temperature of the incubation is preferably 30~40 DEG C, and more preferably 35 DEG C.The time of incubation is preferably 5
~30min, more preferably 20min.
In the present invention, the method for the washing is not particularly limited, using washing side well-known to those skilled in the art
Method.
After washing in the step a, substrate developing solution is added into obtained detection plate by the present invention, is incubated for, adds
Terminate liquid mixing, measures OD value.
In the present invention, the volume of the substrate developing solution is preferably 60~120 holes μ L/, more preferably 80~100 μ L.?
In the present invention, it is preferably 4~8min that the time being incubated for after developing solution, which is added, more preferably 5min.The temperature of the incubation is preferred
It is 23~28 DEG C, more preferably 25 DEG C.
In the present invention, solution in detection plate is preferably discarded before terminate liquid is added.The method for discarding solution in detection plate
It is preferred that the mode dried carries out.
In the present invention, the addition volume of the terminate liquid is preferably 80~120 holes μ L/, more preferably 100 μ L.Mixing
Time is preferably 2~4min, more preferably 3min.
In the present invention, the wavelength of the OD value measurement is preferably 450nm.
After obtaining OD value, the present invention obtains mercury ion in sample to be tested according to scheduled standard curve and the OD value
Concentration.In the present invention, the inspection method of the titer is identical as sample solution detection method.With inhibiting rate B/B0(%) is vertical
Coordinate (B0For Hg2+Absorbance value when-EDTA is 0 concentration, B Hg2+Absorbance value when-EDTA various concentration), with Hg2+-
The concentration of EDTA standard items is abscissa, draws standard curve, derives regression equation according to curvilinear trend.According to after dilution to
OD value substitutes into regression equation in sample solution, obtains the concentration of mercury ion in sample.
Below with reference to embodiment to competitive ELISA detection reagent in mercury ion food pollution provided by the invention
Box and its application are described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The synthesis (Fig. 1) of Hg-ITCBE-cBSA immunogene
(1) it weighs 10mg ITCBE and is dissolved in formation metal cheating agents solution in 1mL dimethyl sulfoxide (DMSO)；It weighs
11.25mg mercuric nitrate Hg (NO3)2The HEPES buffer solution for being dissolved in 1mL pH 8.0 (acts on cytotoxic.It is a kind of hydrogen from
Sub- buffer can control constant pH range the long period) (10mmol/L) middle formation Hg2+Solution；By metal cheating agents solution
And Hg2+Then solution mixing, and adjust pH value to 7.0 with NaOH is placed on shake bed reaction 12 hours, i.e. shape at room temperature
At Hg-ITCBE chelating object haptens.
(2) weigh 66mg BSA, 11.6mg EDC is dissolved in 5mL PBS buffer solution, under stirring condition, be slowly added to 7mg
Ethylenediamine (is dissolved in 3mL PBS+DMF solution) in advance, 37 DEG C of oscillating reactions 2h.Reaction solution PBS dialysis 4d, the carrier of activation
Protein B SA freeze-drying saves, as cBSA.
(3) it weighs 30mg cBSA and is dissolved in the load for forming 30mg/mL in the HEPES buffer solution (10mmol/L) of 1mL pH8.0
Body protein solution；1mL Hg-ITCBE chelating object haptens solution is taken to be added in 1mL carrier protein solution and adjusted with NaOH
To 9.0, room temperature shaker reacts 24 hours pH value, and reaction solution is packed into bag filter after reaction, is first dialysed 2d with distilled water, then
With PBS dialysis 3d, that is, Hg-ITCBE-cBSA immunogene is formed, synthetic route is shown in Fig. 1.
Embodiment 2
The preparation of anti-mercury ion monoclonal antibody
The anti-mercury ion monoclonal antibody specific is prepared by Hg-ITCBE-cBSA immunogen immune Balb/C mouse
, it is realized by following steps:
(1) mouse immune: 6-8 week old female Balb/C mouse 5 is immunized with Hg-ITCBE-cBSA, dosage is 60 μ g/
Only, volume 0.2mL.Head exempts to be emulsified completely with the diluted immunogene of PBS with isometric FCA, later every 4w booster immunization one
It is secondary, use FIA emulsification instead.7d docking blood sampling separation serum after 5 times immune, with indirect ELISA and indirect competitive ELISA
(ciELISA) screening potency is high, and the good mouse of barrier effect is as the spare mouse of fusion.3d is super before merging exempts from mouse, tail vein and
50 μ g immunogenes are respectively injected in abdominal cavity, and volume is 100 μ L.
(2) cell fusion: complete medium (RPMI- containing 15%FBS of the 4-5d containing 8-anaguanine before merging
1640) secondary culture NS0 cell；Preceding 1d cultivates trophocyte with HAT；Sinus is taken a blood sample under socket of the eye when fusion, takes off the lethal mouse of neck.It is sterile
Spleen is taken to prepare splenocyte, it, will be fused with NS0 cell fusion (cell quantity is than about 10: 1) under PEG-1500 effect
Cell suspension is added in 96 porocyte plates for being covered with trophocyte, HAT culture.
(3) screening of monoclonal cell strain: 10-14d indirect ELISA and ciELISA screening strong positive, inhibition after fusion
The hybridoma cell strain that rate is high, growth conditions are good carries out 3 subclones with limiting dilution assay.Then the quasi- product solution of mercury ion is used
The monoclonal source cell strain of screening sensitivity height, high specificity, obtains 3 plants altogether.Wherein, H3C6 cell strain sensitivity highest, it is special
It is anisotropic most strong,
(4) preparation of monoclonal antibody: H3C6 cell strain is transferred to expand in 24 porocyte plates and 50mL cell bottle and is trained
It supports.The hybridoma concentration of screening reaches about 107When/mL, to 10d before through atoleine it is processed through produce female rat it is intraperitoneal
Inject monoclonal cell 108/ only.Ascites is extracted after 10-12d, it is anti-that saturation amine sulfate method purifies anti-mercury ion specific monoclonal
Body.
Embodiment 3
Indirect ELISA measures mercury ion antibody potency
The first step, with the diluted Hg-ITCBE-cOVA coating antigen wrapper sheet of the 0.05mol/L carbonate buffer solution of pH 9.6,
2 μ g/mL of peridium concentration, 100 μ L of the every hole of package amount, 37 DEG C of incubation 2h, PBST board-washing 4 times, every minor tick 3min；Second step is used
The closing of 5% pig negative serum, 280 μ L of every hole, 37 DEG C of incubation 1h, PBST board-washing 4 times, every minor tick 3min；Third step, add mercury from
Sub polyclonal or monoclonal antibody, every 50 μ L of hole, with confining liquid doubling dilution, if negative control (negative control,
NC) and blank control (blank control, BC), 37 DEG C of incubation 15min, PBST board-washing 4 times, every minor tick 3min；4th
Step, adds GaMIgG-HRP, 1: 1000 times of dilution, 50 μ L of every hole, 37 DEG C of incubation 25min, and PBST board-washing 4 times, every minor tick 3min；
5th step, plus enzyme substrate TMB developing solution, every 60 μ L of hole react at room temperature 5-10min；6th step, color development stopping reaction, every hole adds
Enter terminate liquid 2mol/L H2SO4100 μ L read A with microplate reader450nmValue；7th step, as a result judges, to gaging hole A450nm≥
NCA450nm2.1 times (P/N >=2.1) when, the positive is judged to, with maximum dilution multiple calculating antibody potency.
Embodiment 4
The identification of antibody specificity result
DELISA standard curve is established with anti-mercury ion monoclonal antibody prepared by H3C6 cell strain, is tried using cross reaction
Authenticate its fixed specificity.Cross reaction test selects the huge legendary turtle polymer solution of cadmium, lead, copper, zinc, cobalt, molybdenum, chromium and EDTA as inhibition
Agent, standard huge legendary turtle polymer solution preparation method are as follows: EDTA solution are configured to 10mM HEPES buffer solution, by above-mentioned heavy metal
Ion standard reserving solution is diluted to 0,0.01,0.02,0.04,0.08,0.16,0.32,0.64,1.28,2.56,5.12,
10.24,20.48,40.96,81.92,163.84 μ g/L, the two use NaHCO after mixing3Adjusting pH value is 6.0, and room temperature shaker is anti-
It answers 24 hours, that is, forms heavy metal ion-EDTA huge legendary turtle polymer solution.Antibody specificity is indicated with cross reacting rate (CR), is calculated
Formula is: CR%=Hg2+The IC of-EDTA50/ other competitor IC50× 100, Hg ion concentration are lower, and antibody specificity is stronger,
Its experiment the results are shown in Table one.Table one is it is found that Hg2+The association reaction of-EDTA and its mAb have very strong specificity, remove and Cu2+-
EDTA has outside 8.9% cross reaction, with EDTA and other basic no cross reactions of heavy metal ion chelating object.
1 Hg of table2+The cross reaction of-EDTA mAb and other heavy metal ion chelating agent
Embodiment 5
The synthesis of Hg-ITCBE-HRP enzyme mark haptens
(1) it weighs 10mg ITCBE and is dissolved in formation metal cheating agents solution in 1mL dimethyl sulfoxide (DMSO)；It weighs
11.25mg mercuric nitrate Hg (NO3)2It is dissolved in the HEPES buffer solution (10mmol/L) of 1mL pH 8.0 and forms Hg2+Solution；It will be golden
Belong to chelating agent solution and Hg2+Solution mixing, and pH value is adjusted to 7.0 with NaOH, it is then placed at room temperature on shaking table anti-
It answers 12 hours, that is, forms Hg-ITCBE chelating object haptens.
(2) it weighs 100mg HRP and is dissolved in formation 100mg/mL in the HEPES buffer solution (10mmol/L) of 1mL pH 8.0
HRP horseradish peroxidase solution；1mL Hg-ITCBE chelating object haptens solution is taken to be added to 1mL horseradish peroxidase molten
PH value is adjusted to 9.0 in liquid and with NaOH, and room temperature shaker reacts 24 hours, reaction solution is packed into bag filter after reaction, first
With distilled water dialyse 2d, then with PBS dialyse 3d, that is, form Hg-ITCBE-HRP enzyme mark haptens.
Embodiment 6
Direct competive ELISA (dELISA) detecting step
The first step uses chessboard method optimization sheep anti mouse secondary antibody (GaMIgG) peridium concentration for 10 holes μ g/, the every hole of package amount
100 μ L, 37 DEG C of incubation 2h, PBST board-washing 3 times, every minor tick 2min；Second step is closed, every 280 μ of hole with 5% pig negative serum
L, 37 DEG C of incubation 1h, PBST board-washing 3 times, every minor tick 2min；Third step, according to the anti-mercury ion Dan Ke after potency addition dilution
Grand antibody, the standard items of sample or doubling dilution, enzyme mark haptens, after three mixes, 37 DEG C of incubation 25min, PBST board-washing 3
Secondary, every minor tick 2min, reaction sets NC and BC；4th step, plus enzyme substrate TMB developing solution, every 60 μ L of hole react at room temperature 5min；
5th step, color development stopping reaction, terminate liquid 2mol/L H is added in every hole2SO4100 μ L read A with microplate reader450nmValue.
Embodiment 7
Indirect competitive ELISA (ciELISA) detecting step
The first step, with the diluted Hg-ITCBE-cOVA coating antigen wrapper sheet of the 0.05mol/L carbonate buffer solution of pH 9.6,
2 μ g/mL of peridium concentration, 100 μ L of the every hole of package amount, 37 DEG C of incubation 2h, PBST board-washing 4 times, every minor tick 3min；Second step is used
The closing of 5% pig negative serum, 280 μ L of every hole, 37 DEG C of incubation 1h, PBST board-washing 4 times, every minor tick 3min；Third step, addition mark
Quasi- product: ELISA Plate is first with 50uL confining liquid shop fixtures, and then the Hg of 500 μ g/L is added in the 1st hole2+- EDTA chelating object standard solution,
And doubling dilution is carried out backward, reaction sets NC and BC；4th step, according to indirect ELISA antibody titer, after addition confining liquid dilution
Anti- mercury ion pAb or mAb, 50 μ L of every hole, 37 DEG C of incubation 15min, PBST board-washing 4 times, every minor tick 3min；5th step, adds
GaMIgG-HRP is diluted with 1: 1000 times of confining liquid, 50 μ L of every hole, 37 DEG C of incubation 25min, and PBST board-washing 4 times, every minor tick
3min；6th step, plus enzyme substrate TMB developing solution, every 60 μ L of hole react at room temperature 5-10min；7th step, color development stopping reaction, often
Terminate liquid 2mol/L H is added in hole2SO4100 μ L read A with microplate reader450nmValue.
Embodiment 8
In embodiment 6 in dELISA and embodiment 7 ciELISA detection performance comparison
With H3C6 cell strain prepare anti-mercury ion monoclonal antibody establish ELISA detection method, and carry out dELISA and
The comparison of ciELISA detection performance.With inhibiting rate B/B0(%) is ordinate (B0For Hg2+Absorbance when-EDTA is 0 concentration
Value, B Hg2+Absorbance value when-EDTA various concentration), with the Hg of various concentration2+- EDTA standard items are abscissa, are drawn
Standard curve derives regression equation according to curvilinear trend, carries out correlation analysis.Sensitivity is with half-inhibitory concentration (IC50Value) table
Show；Quantitative detection limits (IC20-IC80) standard items are represented to maximum signal (B0) inhibition range；Detection limit is with IC15Table
Show, the result is shown in Fig. 2.
The regression equation of dELISA standard curve is the (R of y=-6.129Ln (x)+57.9862=0.9623), IC50Value is
1.45μg/L；The regression equation of ciELISA standard curve is the (R of y=-6.612Ln (x)+56.3372=0.9655), IC50Value
For 1.65 μ g/L.DELISA standard curve is calculated from the formula in PBS to Hg2+The linear detection range of-EDTA is 0.23
~46 μ g/L, detection limit (LOD) are 0.12 μ g/L；CiELISA standard curve is in PBS to Hg2+The linear detection range of-EDTA
It is 0.16 μ g/L for 0.31~58 μ g/L, LOD.
Embodiment 9
The operating procedure of mercury ion pollution detection dELISA kit in environmental water sample
The first step takes out required reagent from cold storage environment, is placed in (20~25 DEG C) balance 30min of room temperature, pays attention to every
Kind liquid reagent must shake up before；Second step adds 0.4mL sample with suction pipe acquisition water sample 0.5mL in centrifuge tube
EDTA treatment fluid 0.1mL is added in dilution after mixing；Third step 50 μ L of sample treatment liquid is added in micropore of enzyme marker plate item, together
When be added dilution after anti-50 μ L of mercury ion monoclonal antibody, the 50 μ L of enzyme mark haptens after dilution, mix stand 25min after wash
Plate notices that detection sets three repetitions, and NC and BC control is arranged；4th step adds substrate developing solution A liquid and each 30 μ L of B liquid, stands
Board-washing after 5min；H is added in 5th step2SO4100 μ L of terminate liquid, microplate reader reads absorbance value after standing 5min；6th step, will
Absorbance value substitutes into the regression equation formula of dELISA standard curve, and mercury ion content in water sample is calculated.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (5)
1. one kind is based on Direct cELISA mercury ion detection kit, comprising:
(1) detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package are coated with；The sheep anti-mouse igg secondary antibody
Peridium concentration is 80~150 μ g/mL；
(2) mass concentration is the enzyme mark Hg-ITCBE chelating object haptens solution of 30~40 μ g/mL；
(3) mass concentration is the anti-mercury ion monoclonal antibody solution of 13~18 μ g/mL；The anti-mercury ion monoclonal antibody is molten
Liquid is prepared by Hg-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate developing solution；
(7) cleaning solution；
(8) mercury ion standard solution；
(9) mass concentration is the EDTA treatment fluid of 10~20mg/mL；
The sample diluting liquid is the HEPES buffer solution that mass concentration is 2~10g/L；
The cleaning solution is the PBS solution comprising 0.05~0.1%Tween of mass concentration 20；
The preparation method of the Hg-ITCBE-cBSA immunogene the following steps are included:
It (1) is (8~12) mg: 1mL according to the quality of ITCBE and the volume ratio of dimethyl sulfoxide by ITCBE and dimethyl sulfoxide
Mixing forms metal cheating agents solution；
(2) by mercuric nitrate Hg (NO3)2It is mixed with HEPES buffer solution according to 11.25mg: 1mL ratio, forms Hg2+Solution；
(3) by Hg obtained in metal cheating agents solution obtained in the step (1) and the step (2)2+Solution is according to body
Product adjusts mixed liquor pH value to 7.0, then 10~14h of oscillating reactions under the conditions of 23~27 DEG C, forms Hg- than mixing for 1: 1
ITCBE chelating object haptens；
(4) BSA, EDC and PBS buffer solution are mixed to obtain mixed liquor, the body of BSA mass, EDC mass and PBS buffer solution
The mixed liquor and ethylenediamine according to mixeding liquid volume and ethylenediamine mass ratio are 5mL than being 66mg: 11.6mg: 5mL by product:
The ratio of 7mg mixes, and 37 DEG C of oscillating reactions 2h obtain reaction solution；Reaction solution PBS dialysis 4d, the carrier protein BSA activated；
(5) HEPES buffer solution that the carrier protein BSA and pH value of the activation are 8.0 is mixed, forming concentration is 30mg/mL
Carrier protein solution；
(6) the carrier egg for obtaining Hg-ITCBE chelating object haptens solution obtained in the step (3) and the step (5)
White solution is mixed according to the ratio that volume ratio is 1: 1, adjusting pH value to 22~26h of oscillating reactions under the conditions of 9.0,23~27 DEG C,
By obtained reaction solution be packed into bag filter, first with distilled water dialyse 2d, then with PBS dialyse 3d, that is, form Hg-ITCBE-cBSA and exempt from
Epidemic focus；
The preparation method of the enzyme mark Hg-ITCBE chelating object haptens, comprising the following steps:
A. ITCBE and dimethyl sulfoxide are mixed to form metal cheating agents solution；
B. by mercuric nitrate Hg (NO3)2It is mixed with HEPES buffer solution, forms Hg2+Solution；
C. by Hg obtained in metal cheating agents solution obtained in the step A and the step B2+Solution mixing is adjusted mixed
The pH value of liquid is closed to 7.0~7.2, obtained mixed liquor 10~14h of oscillating reactions, forms Hg-ITCBE chelating object haptens；
D. label enzyme is mixed with HEPES buffer solution, obtains enzyme solutions；
E. the Hg-ITCBE chelating object haptens solution that the step C is obtained is mixed with enzyme solutions obtained in the step D,
The pH value of mixture is adjusted to 8.8~9.2, then by mixed liquor 22~26h of oscillating reactions, obtains enzyme mark Hg-ITCBE chelate half
Antigen；
There is no the limitation of time sequencing between the step A and B；Between C and D, also without time sequencing between D and A and B
Limitation.
2. mercury ion detection kit described in claim 1 detection environment in mercury ion application, which is characterized in that including with
Lower step:
A, anti-mercury ion monoclonal antibody solution, diluted testing sample solution and enzyme mark Hg-ITCBE chelating object haptens is molten
Liquid is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, and mixing is washed after incubation with cleaning solution；
B, substrate developing solution is added into detection plate, is incubated for, terminate liquid mixing is added, measures OD value；
C, according to the obtained OD value of the step b and scheduled standard curve, the concentration of mercury ion in sample to be tested is obtained, it is described
Standard curve is the linear relationship curve between ion concentration of mercury and OD value.
3. application according to claim 2, which is characterized in that anti-mercury ion monoclonal antibody is molten in the detection plate hole
The volume ratio of liquid, diluted testing sample solution and enzyme mark Hg-ITCBE chelating object haptens solution is 1~2: 1~2: 1~2.
4. application according to claim 2, which is characterized in that the temperature being incubated in the step a and b independently is 30~
40℃。
5. application according to claim 2 or 4, which is characterized in that the time being incubated in the step a and b independently is 5
~30min.
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| CN1707267A (en) * | 2004-06-11 | 2005-12-14 | 中国兽医药品监察所 | Enzyme-linked immunologic reagent kit for detecting streptomycin drug |
| CN103472231A (en) * | 2013-09-28 | 2013-12-25 | 郑州大学 | Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof |
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| CN1707267A (en) * | 2004-06-11 | 2005-12-14 | 中国兽医药品监察所 | Enzyme-linked immunologic reagent kit for detecting streptomycin drug |
| CN103472231A (en) * | 2013-09-28 | 2013-12-25 | 郑州大学 | Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof |
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