CN106841627A - One kind detects molybdenum ion kit and its application based on Direct cELISA - Google Patents
One kind detects molybdenum ion kit and its application based on Direct cELISA Download PDFInfo
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- CN106841627A CN106841627A CN201710099809.8A CN201710099809A CN106841627A CN 106841627 A CN106841627 A CN 106841627A CN 201710099809 A CN201710099809 A CN 201710099809A CN 106841627 A CN106841627 A CN 106841627A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
Direct cELISA molybdenum ion detection kit is based on the invention provides one kind, including：It is coated with the detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package；The coating concentration of the sheep anti-mouse igg secondary antibody is 50~200 μ g/mL；Mass concentration is the enzyme mark Mo ITCBE chelant thing haptens of 20~50 μ g/mL；Mass concentration is the anti-molybdenum ion monoclonal antibody solution of 10~20 μ g/mL；The anti-molybdenum ion monoclonal antibody solution is prepared by Mo ITCBE cBSA immunogen immune Balb/C mouse；The terminate liquid of 1.0~3.0mol/L；Sample diluting liquid；Substrate nitrite ion；Cleaning solution；Molybdenum ion standard solution；Mass concentration is the EDTA treatment fluids of 10~20mg/mL.The characteristics of present invention offer kit has sensitivity high, 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 one kind based on Direct cELISA detection molybdenum from
Sub- kit and its preparation method and application.
Background technology
Molybdenum has important effect to organism as a kind of transiting state metal element, has during organism fixed nitrogen
Catalytic action, plays a part of transmission electronics, catalytic substrate in vivo during redox reaction, participate in xanthine oxidation
The composition of enzyme/dehydrogenase, aldehyde oxidase and sulfite oxidase, while have keeping Global Carbon, sulphur, the work(of nitrogen metabolism balance
Energy.Molybdenum is safe from harm to organism in its natural state, but because molybdenum is widely used in special steel, machinery, petrochemical industry, national defence
The fields such as aviation, electronic apparatus and nuclear industry, cause molybdenum ore largely to be exploited, and sexavalence molybdenum ion enters nature in different forms,
Environmental pollution sharp deterioration, cumulative effect and biotic recovery effect in addition, environmental pollution and human health constitute serious prestige
The side of body.
Ferguson in 1938 reports that molybdenum content in herbage is too high to cause the nosotoxicosis such as sheep persistent diarrhea and depilation first
Shape." calf diarrhea " of Britain, Zelanian " ox peat rushes down ", " the ox endemic hematuria disease " of Australia and China 1981
Farm cattle " red skin white hair disease " that south jiangxi area occurs etc., are all the typical cases of sexavalence molybdenosis.Therefore, strengthen to environment soil
Earth, water source, food and in feed sexavalence molybdenum ion content Clinical significance of detecting it is great.
Mo in current animal food and environmental water sample6+The method that predominantly detects of pollution includes traditional physical and chemical inspection and exempts from
Epidemiology checks 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), ICP-AES (ICP-
AES), Hydride Generation-atomic Fluorescence Spectrometry (Mo-AFS) and Neutron activation analysiss (NAA) etc., they are current Mo6+It is dirty
The main method of detection is contaminated, sensitivity is high, as a result accurately, but due to needing expensive instrument and equipment, skilled practitioner, being tired of
The defect such as the trivial time-consuming, cycle is long, costly, be unable to execute-in-place, sample screening amount is small, its 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
Determination method (FPIA), enzyme-linked immunosorbent assay (ELISA), KinExA immunoassays (KIA), colloidal gold immunochromatographimethod examination
Determination method (GICA) and micro-cantilever immunosensor (MIS) etc. are tested, there is the selectivity of height due to the combination of antigen-antibody
And sensitiveness, this technology advantage such as have easy, quick, sensitive, special, economy, screening amount big, represent heavy metal ion
The developing direction of detection.At present, domestic and international heavy metal molybdenum ionic soil immunoassay technology research is very active, wherein notification number
For the patent of CN103412127B discloses a kind of enzyme linked immunological kit for detecting sexavalence molybdenum ion and its establishment and inspection
Survey method, its detection sensitivity is 1ng/mL, and detection time is more long.
The content of the invention
In view of this, molybdenum ion reagent is detected based on Direct cELISA it is an object of the invention to provide one kind
Box and its application, make the kit have the advantages that detection time is short, while the characteristics of having sensitivity high, high specificity.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
Direct cELISA molybdenum ion detection kit is based on the invention provides one kind, including：
(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- coating concentration is 50~200 μ g/mL；
(2) mass concentration is the enzyme mark Mo-ITCBE chelant thing haptens solution of 20~50 μ g/mL；
(3) mass concentration is the anti-molybdenum ion monoclonal antibody solution of 10~20 μ g/mL；The anti-molybdenum ion monoclonal resists
Liquid solution is prepared by Mo-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate nitrite ion；
(7) cleaning solution；
(8) molybdenum ion standard solution；
(9) mass concentration is the EDTA treatment fluids of 10~20mg/mL.
Preferably, the coating concentration of the sheep anti-mouse igg secondary antibody is 80~150 μ g/mL.
Preferably, the mass concentration of the enzyme mark Mo-ITCBE chelant thing haptens is 30~40 μ g/mL.
Preferably, the mass concentration of the anti-molybdenum ion monoclonal antibody solution is 13~18 μ g/mL.
Preferably, the sample diluting liquid is 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.
Present invention also offers the application of molybdenum ion detection kit molybdenum ion in environment is detected, including following step
Suddenly：
It is a, anti-molybdenum ion monoclonal antibody solution, the testing sample solution of dilution and enzyme mark Mo-ITCBE chelants thing half is anti-
Original solution, is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, mixing, is washed with cleaning solution after incubation；
B, in detection plate add substrate nitrite ion, be incubated, add terminate liquid mixing, determine OD values；
C, the OD values obtained according to the step b and predetermined standard curve, obtain the concentration of molybdenum ion in testing sample,
The standard curve is the linear relationship curve between molybdenum ion concentration and OD values.
Preferably, anti-molybdenum ion monoclonal antibody solution, the testing sample solution and enzyme mark of dilution in the detection plate hole
The volume ratio of Mo-ITCBE chelant thing 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.
Direct cELISA molybdenum ion detection kit is based on the invention provides one kind, including：(1) it is coated with
The detection plate of sheep anti-mouse igg secondary antibody, the detection plate vacuum sealed package；The coating concentration of the sheep anti-mouse igg secondary antibody is 50
~200 μ g/mL；(2) mass concentration is the enzyme mark Mo-ITCBE chelant thing haptens of 20~50 μ g/mL；(3) mass concentration is 10
The anti-molybdenum ion monoclonal antibody solution of~20 μ g/mL；The anti-molybdenum ion monoclonal antibody solution is exempted from by Mo-ITCBE-cBSA
Immune Balb/C mouse are prepared epidemic focus；The terminate liquid of (4) 1.0~3.0mol/L；(5) sample diluting liquid；(6) substrate colour developing
Liquid；(7) cleaning solution；(8) molybdenum ion standard solution；(9) mass concentration is the EDTA treatment fluids of 10~20mg/mL.The present invention
According to antigen-antibody direct competitive immunochromatography principle design, by the pre-coated sheep anti-mouse igg secondary antibody in detection plate, make to resist
Molybdenum ion monoclonal antibody, measuring samples or molybdenum ion standard solution and three kinds of materials of enzyme mark haptens (Mo-ITCBE-HRP)
Can add simultaneously, anti-molybdenum ion monoclonal antibody combines to form compound, sample or standard with pre-coated sheep anti-mouse igg secondary antibody
Molybdenum ion and enzyme mark haptens in product are competitively combined with anti-molybdenum ion monoclonal antibody solution, when addition substrate nitrite ion
When, sample absorbance value is negatively correlated with the molybdenum ion content that it is remained, and compares multiplied by with its corresponding dilution with standard curve
Multiple, you can draw the content of molybdenum ion in sample.The kit that the present invention is provided, reducing be incubated twice in conventional contention method
And washing times, the time 30min for being incubated and washing is shortened, kit is quickly and easily obtained testing result, contract significantly
Short detection cycle.Molybdenum ion detection kit LDL (LOD) that the present invention is provided is up to 0.13 μ g/L, detection range
It is 0.16~32.9 μ g/L.
Brief description of the drawings
Fig. 1 is immunogene synthetic route chart in embodiment 1；
Fig. 2 is dELISA the and ciELISA standard curves of foundation in embodiment 8.
Specific embodiment
Direct cELISA molybdenum ion detection kit is based on the invention provides one kind, including：
(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- coating concentration is 50~200 μ g/mL；
(2) mass concentration is the enzyme mark Mo-ITCBE chelant thing haptens solution of 20~50 μ g/mL；
(3) mass concentration is the anti-molybdenum ion monoclonal antibody solution of 10~20 μ g/mL；The anti-molybdenum ion monoclonal resists
Liquid solution is prepared by Mo-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate nitrite ion；
(7) cleaning solution；
(8) molybdenum ion standard solution；
(9) mass concentration is the EDTA treatment fluids of 10~20mg/mL.
The molybdenum ion detection kit that the present invention is provided is coated with the detection plate of sheep anti-mouse igg secondary antibody, the detection
Plate vacuum sealed package；The coating 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 area
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, is 50~200 μ g/mL sheep anti-mouse igg secondary antibodies coating buffers 100 to coating concentration is added in the detection hole of detection plate
~150 μ L/ holes, are incubated 1.5~3h for the first time under the conditions of 35~40 DEG C, coating buffer is removed after incubation, are washed for the first time with cleaning solution
Plate 2~5 times；
II, is the μ of 3~6% pig negative serum 200~300 to mass concentration is added in the detection plate obtained in the step I
L/ holes, second 1.5~3h of incubation, removes coating buffer, with second board-washing of cleaning solution 2~5 under the conditions of 35~40 DEG C after incubation
It is secondary, detection plate is dried naturally under the conditions of 23~27 DEG C, obtain being coated with the detection plate of sheep anti-mouse igg secondary antibody.
In the present invention, the species of the detection plate is not particularly limited, ripe using those skilled in the art technical staff institute
The ELISA detection plates 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 concentrations 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 μ L/ holes.The first time is incubated and second incubation
Temperature preferably stands alone as 37 DEG C.The time that the first time is incubated and second is incubated preferably stands alone as 2h.
In the present invention, the method for the removal coating buffer is not particularly limited, using 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 times of the first time board-washing and second 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, more preferably 2min during secondary board-washing.
In the present invention, the source of the pig negative serum is not particularly limited, using 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 μ L/ holes, more preferably 260 μ L/ holes.
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.
The molybdenum ion detection kit that the present invention is provided includes enzyme mark Mo-ITCBE chelant thing haptens solution.The enzyme mark
The mass concentration of Mo-ITCBE chelant thing 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 species of the enzyme is not particularly limited using enzyme class well-known to those skilled in the art i.e.
Can.In the embodiment of the present invention, the species of the enzyme is horseradish peroxidase.
In the present invention, the preparation method of the enzyme mark Mo-ITCBE chelant thing haptens preferably includes following steps：
A. isothiocycmatobenzyl ethylenediamine tetra-acetic acid (ITCBE) and dimethyl sulfoxide (DMSO) (DMSO) are mixed to form metal chelant
Agent solution；
B. by molybdic acid H2MoO4Mix with HEPES buffer solution, form Mo6+Solution；
C. the Mo that will be obtained in the metal cheating agents solution obtained in the step A and the step B6+Solution mixes, and adjusts
The pH value of mixed liquor is saved to 7.0~7.2, the mixed liquor 10~14h of oscillating reactions for obtaining, Mo-ITCBE chelants thing half is formed anti-
It is former；
D. mark enzyme is mixed with HEPES buffer solution, obtains enzyme solutions；
E. the enzyme solutions obtained in the Mo-ITCBE chelants thing haptens solution for the step C being obtained and the step D
Mixing, adjusts the pH value of mixture to 8.8~9.2, then by mixed liquor 22~26h of oscillating reactions, obtains enzyme mark Mo-ITCBE chelas
Compound 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, quality and the dimethyl sulfoxide (DMSO) volume ratio of the ITCBE are preferably (5~15) mg：1mL, more preferably
It is 10mg：1mL.
In the present invention, the molar concentration for preparing enzyme mark Mo-ITCBE chelant thing haptens HEPES buffer solutions is preferably 8~
12mmol/L, more preferably 10mmol/L.The pH value of the HEPES buffer solution is preferably 7.8~8.5, more preferably 8.0.
In the present invention, the molybdic acid H2MoO4Quality and the volume ratio of HEPES buffer solution be preferably 7.5~11.5mg：
1mL, more preferably 8.38mg：1mL.
In the present invention, the metal cheating agents solution and the Mo6+The volume ratio of solution is preferably 0.8~1.2:0.8~
1.2, more preferably 1:1.
In the present invention, to containing metal-chelator and Mo6+The pH adjustment method of mixed liquor is not particularly limited, using this
Contain metal-chelator and Mo known to art personnel6+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 mark enzyme is (80~120) mg with the volume ratio of HEPES buffer solution：1mL, more
Preferably 100mg：1mL.
In the present invention, the Mo-ITCBE chelants thing haptens liquor capacity is preferably 0.8 with the enzyme solutions volume ratio
~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 carried out with the sodium hydroxide solution that mass concentration is 1%.The time of oscillating reactions is preferred in the step D
It is 24h.The temperature of the oscillating reactions is preferably 23~27 DEG C, more preferably 25 DEG C.
In the present invention, preferably Mo-ITCBE- enzyme haptens solution is placed in after mixed liquor oscillating reactions in the step E
Dialysis and the dialysis, the enzyme mark Mo-ITCBE chelate haptens for being purified in PBS solution in distilled water successively of analysis bag.
In the present invention, the time dialysed in the distilled water is preferably 2d；The time dialysed in the PBS solution is preferably 3d.
The molybdenum ion detection kit that the present invention is provided includes anti-molybdenum ion monoclonal antibody solution.The anti-molybdenum ion list
The mass concentration of clonal antibody solution is 10~20 μ g/mL, more preferably preferably 13~18 μ g/mL, 15 μ g/mL.
In the present invention, the anti-molybdenum ion monoclonal antibody solution is small by Mo-ITCBE-cBSA immunogen immunes Balb/C
Mouse is prepared, and the preparation method of present invention confrontation molybdenum ion monoclonal antibody is not particularly limited, using people in the art
The preparation scheme of the anti-molybdenum ion monoclonal antibody known to member.
In the present invention, the preparation method of the Mo-ITCBE-cBSA immunogenes is comprised the following steps：
(1) by ITCBE and dimethyl sulfoxide (DMSO) (DMSO) according to the quality of ITCBE and the volume ratio of dimethyl sulfoxide (DMSO) for (8~
12)mg：1mL mixes, and forms metal cheating agents solution；
(2) by molybdic acid (H2MoO4) with HEPES buffer solution according to 8.38mg：The ratio mixing of 1mL, forms Mo6+Solution；
(3) Mo that will be obtained in the metal cheating agents solution obtained in the step (1) and the step (2)6+Solution is pressed
It is 1 according to volume ratio:1 mixing, adjusts mixed liquor pH value to 7.0, then 10~14h of oscillating reactions, shape under the conditions of 23~27 DEG C
Into Mo-ITCBE chelant thing haptens；
(4) BSA, EDC and PBS are mixed and obtains mixed liquor, BSA mass, EDC mass and PBS
Volume ratio be 66mg：11.6mg：5mL, be according to mixeding liquid volume and ethylenediamine mass ratio with ethylenediamine by the mixed liquor
5mL：The ratio mixing of 7mg, 37 DEG C of oscillating reactions 2h obtain reaction solution；Reaction solution PBS 4d, the carrier protein for being 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 that the Mo-ITCBE chelants thing haptens solution and the step (5) that will be obtained in the step (3) are obtained
Body protein solution is 1 according to volume ratio：1 ratio mixing, regulation pH value to 9.0, oscillating reactions 22 under the conditions of 23~27 DEG C~
26h, the reaction solution that will be obtained loads bag filter, first with distilled water dialysis 2d, then uses PBS 3d, that is, form Mo-ITCBE-
CBSA immunogenes.
The synthetic route of the Mo-ITCBE-cBSA immunogenes is shown in Fig. 1.
The molybdenum ion detection kit that the present invention is provided 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.
The molybdenum ion detection kit that the present invention is provided includes sample diluting liquid.The sample diluting liquid is buffered for HEPES
Liquid.The mass concentration of HEPES buffer solution is preferably 2~10g/L, most preferably more preferably 4~8g/L, 5g/L.The HEPES
The pH value of buffer solution is preferably 7.8~8.5, more preferably 8.0.
The molybdenum ion detection kit that the present invention is provided includes substrate nitrite ion.The concentration of the substrate nitrite ion be 1~
2mg/mL, more preferably 1.27mg/mL.The species of the substrate nitrite ion determines according to the species of mark enzyme.It is of the invention real
Apply in example, when the species of enzyme is HRP, the substrate nitrite ion is tetramethyl benzidine (TMB).
The molybdenum ion detection kit that the present invention is provided includes cleaning solution.The cleaning solution be comprising Tween 20 mole
Concentration is the PBS solution of 0.01~0.02mol/L.It is 7.0~8.0 that the pH value of the PBS solution is preferably, more preferably 7.4.
Mass concentrations of the Tween 20 in PBS solution is preferably 0.05~0.1%, more preferably 0.08%.
The molybdenum ion detection kit that the present invention is provided includes EDTA treatment fluids.The mass concentration of EDTA treatment fluids be 10~
20mg/mL, more preferably 15mg/mL.When EDTA treatment fluids are used preferably by sample solution, sample diluting liquid and EDTA treatment
The volume ratio of liquid is 5：5：1.The effect of EDTA treatment fluids is, for sample pre-treatments, to combine EDTA and molybdenum ion, forms molybdenum
Ion-EDTA huge legendary turtle polymer solutions, are easy to identification of the antibody to molybdenum ion.
The molybdenum ion detection kit that the present invention is provided includes molybdenum ion standard solution.The molybdenum ion standard solution
It is Mo6+- EDTA solution.The Mo6+The source of-EDTA solution is not particularly limited, using well-known to those skilled in the art
Mo6+- EDTA solution.In the embodiment of the present invention, Mo6+- EDTA solution is made by oneself for laboratory.The Mo6+- EDTA solution systems
Preparation Method is not particularly limited, using Mo well-known to those skilled in the art6+- EDTA solution manufacturing methods.
Present invention also offers the application of molybdenum ion detection kit molybdenum ion in environment is detected, including following step
Suddenly：
It is a, anti-molybdenum ion monoclonal antibody solution, the testing sample solution of dilution and enzyme mark Mo-ITCBE chelants thing half is anti-
Original is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, mixing, is washed with cleaning solution after incubation；
B, in detection plate add substrate nitrite ion, be incubated, add terminate liquid mixing, determine OD values；
C, the OD values obtained according to the step b and predetermined standard curve, obtain the concentration of molybdenum ion in testing sample,
The standard curve is detected with molybdenum ion standard solution, and the OD values for obtaining build with molybdenum ion standard concentration to be had
The curve of linear relationship.
The present invention by anti-molybdenum ion monoclonal antibody solution, dilution testing sample solution and enzyme mark Mo-ITCBE chelant things
Haptens is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, mixing, is washed with cleaning solution after incubation.
In the present invention, preferably it is diluted before the sample solution inspection.The multiple of the dilution is preferably 100~1000
Times, more preferably 200~800 times, most preferably 500 times.The dilution is processed using sample diluting liquid.The sample is dilute
EDTA treatment fluids are preferably added to after releasing liquid dilute sample.Volume ratio by sample solution, sample diluting liquid and EDTA treatment fluids is
5：5：1.
In the present invention, anti-molybdenum ion monoclonal antibody solution, the testing sample solution and enzyme of dilution in the detection plate hole
The volume ratio for marking haptens solution is preferably 1~2：1~2：1~2, more preferably 1：1：1.
In the present invention, the method for the mixing is preferably mixed using the method for pipettor gun head pressure-vaccum.
In the present invention, the temperature of the incubation is preferably 30~40 DEG C, 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, the present invention is incubated to substrate nitrite ion is added in the detection plate for obtaining, and adds
Terminate liquid mixes, and determines OD values.
In the present invention, the volume of the substrate nitrite ion is preferably 60~120 μ L/ holes, more preferably 80~100 μ L.
In the present invention, the time being incubated after nitrite ion is added to be preferably 4~8min, 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, preferably solution in detection plate is discarded before adding terminate liquid.The method for discarding solution in detection plate
It is preferred that the mode for drying is carried out.
In the present invention, the addition volume of the terminate liquid is preferably 80~120 μ L/ holes, more preferably 100 μ L.Mixing
Time is preferably 2~4min, more preferably 3min.
In the present invention, the wavelength that the OD values are determined is preferably 450nm.
After obtaining OD values, the present invention obtains molybdenum ion in testing sample according to predetermined standard curve and the OD values
Concentration.In the present invention, the inspection method of the titer is identical with sample solution detection method.With inhibiting rate B/B0(%) is vertical
Coordinate (B0It is Mo6+Absorbance when-EDTA is 0 concentration, B is Mo6+Absorbance during-EDTA various concentrations), with Mo6+-
The concentration of EDTA standard items is abscissa, draws standard curve, and regression equation is derived according to curvilinear trend.Treating after according to dilution
During OD values substitute into regression equation in survey sample solution, the concentration of molybdenum ion in sample is obtained.
Direct cELISA is based on to one kind that the present invention is provided with reference to embodiment and detects molybdenum ion reagent
Box and its application are described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The synthesis (Fig. 1) of Mo-ITCBE-cBSA immunogenes
(1) weigh during 10mg ITCBE are dissolved in 1mL dimethyl sulfoxide (DMSO)s (DMSO) and form metal cheating agents solution；Weigh
8.38mg molybdic acids (H2MoO4) HEPES buffer solution of 1mLpH 8.0 is dissolved in (to cytotoxic effect.It is that a kind of hydrogen ion delays
Electuary, can control constant pH scopes the long period) (10mmol/L) middle formation Mo6+Solution；By metal cheating agents solution and Mo6 +Solution mixes, and adjusts pH value to 7.0 with NaOH, is then placed at ambient temperature and shakes bed reaction 12 hours, that is, formed
Mo-ITCBE chelant thing haptens.
(2) 66mg BSA, 11.6mg EDC are weighed to be dissolved in 5mLPBS buffer solutions, under stirring condition, 7mg second is slowly added to
Diamines (is dissolved in 3mLPBS+DMF solution) in advance, 37 DEG C of oscillating reactions 2h.Reaction solution PBS 4d, the carrier egg of activation
White BSA is lyophilized to be preserved, as cBSA.
(3) load that 30mg cBSA are dissolved in formation 30mg/mL in the HEPES buffer solution (10mmol/L) of 1mLpH8.0 is weighed
Body protein solution；1mL Mo-ITCBE chelant thing haptens solution is taken to be added in 1mL carrier protein solutions and adjusted with NaOH
To 9.0, room temperature shaker is reacted 24 hours pH value, and reaction solution is loaded bag filter by reaction after terminating, first with distilled water dialysis 2d, then
PBS 3d is used, that is, forms Mo-ITCBE-cBSA immunogenes, its synthetic route is shown in Fig. 1.
Embodiment 2
The preparation of anti-molybdenum ion monoclonal antibody
Described anti-molybdenum ion monoclonal antibody specific is prepared by Mo-ITCBE-cBSA immunogen immune Balb/C mouse
, realized by following steps：
(1) mouse immune：With the immune 6-8 week old female Balb/C mouse 5 of Mo-ITCBE-cBSA, dosage is 60 μ g/
Only, volume is 0.2mL.The immunogene that head exempts to be diluted with PBS is emulsified completely with isometric FCA, later every 4w booster immunizations one
It is secondary, use FIA emulsifications instead.7d dockings blood sampling separates serum after immune 5 times, with indirect ELISA and indirect competitive ELISA
(ciELISA) screening potency is high, and the good mouse of barrier effect is used as the standby mouse of fusion.3d is super before fusion 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-s containing 15%FBS of the 4-5d containing 8-anaguanine before fusion
1640) Secondary Culture NS0 cells；Preceding 1d cultivates trophocyte with HAT；Sinus blood sampling under socket of the eye, takes off the lethal mouse of neck during fusion.It is aseptic
Take spleen and prepare splenocyte, with NS0 cell fusions (cell quantity ratio about 10 under PEG-1500 effects：1), by after fusion
Cell suspension is added in 96 porocyte plates for being covered with trophocyte, HAT cultures.
(3) screening of monoclonal cell strain：10-14d indirect ELISAs and ciELISA screenings strong positive, suppression after fusion
The hybridoma cell strain that rate is high, growth conditions are good, 3 subclones are carried out with limiting dilution assay.Then the quasi- product solution of molybdenum ion is used
Screening sensitivity is high, high specificity monoclonal source cell strain, and 8 plants are obtained to obtain altogether.Wherein, G2D7 cell lines sensitivity highest,
Specificity is most strong.
(4) preparation of monoclonal antibody：Expand training during G2D7 cell lines are transferred into 24 porocyte plates and 50mL cell bottles
Support.The hybridoma concentration of screening reaches about 107During/mL, to before 10d through atoleine treat through produce dams it is intraperitoneal
Injection monoclonal cell 108/ only.Ascites is extracted after 10-12d, saturation amine sulfate method purifies anti-molybdenum ion specific monoclonal and resists
Body.
Embodiment 3
Indirect ELISA determines molybdenum ion antibody titer
The first step, the Mo-ITCBE-cOVA coating antigen wrapper sheets diluted with the 0.05mol/L carbonate buffer solutions of pH 9.6,
The coating μ g/mL of concentration 2, package amount is per hole 100 μ L, 37 DEG C of incubation 2h, PBST board-washings 4 times, per minor tick 3min；Second step, uses
5% pig negative serum is closed, per hole 280 μ L, 37 DEG C of incubation 1h, PBST board-washings 4 times, per minor tick 3min；3rd step, plus molybdenum from
Sub polyclonal or monoclonal antibody, per the μ L of hole 50, uses confining liquid doubling dilution, if negative control (negative control,
NC) and blank (blank control, BC), 37 DEG C of incubation 15min, PBST board-washings 4 times, per minor tick 3min；4th
Step, plus GaMIgG-HRP, 1:1000 times of dilutions, per hole 50 μ L, 37 DEG C of incubation 25min, PBST board-washings 4 times, per minor tick 3min；
5th step, plus enzyme substrate TMB nitrite ions, per hole 60 μ L, room temperature reaction 5-10min；6th step, color development stopping reaction, adds per hole
Enter terminate liquid 2mol/L H2SO4100 μ L, A is read with ELIASA450nmValue；7th step, as a result judges, to treat 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
Antibody specificity result is identified
The anti-molybdenum ion monoclonal antibody prepared with G2D7 cell lines sets up dELISA standard curves, is tried using cross reaction
Authenticate fixed its specificity.The huge legendary turtle polymer solution of cross reaction experiment selection cadmium, copper, mercury, zinc, cobalt, lead, chromium and EDTA is used as suppression
Agent, its standard huge legendary turtle polymer solution compound method is as follows：EDTA solution is configured to 10mM HEPES buffer solutions, 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,
10.24th, 20.48,40.96,81.92,163.84 μ g/L, both use NaHCO after mixing3Regulation pH value is 6.0, and room temperature shaker is anti-
Answer 24 hours, that is, form heavy metal ion-EDTA huge legendary turtle polymer solutions.Antibody specificity is represented with cross reacting rate (CR), calculated
Formula is：CR%=Mo6+The IC of-EDTA50/ other competitor IC50× 100, Mo ion concentration are lower, and antibody specificity is stronger,
Its experiment the results are shown in Table one.Knowable to table one, Mo6+The association reaction of-EDTA and its mAb has very strong specificity, except with Cr3+-
EDTA has outside 6.7% cross reaction, with EDTA and other basic no cross reactions of heavy metal ion chelating thing.
The Mo of table 16+- EDTAmAb and the cross reaction of other heavy metal ion chelating agent
Embodiment 5
The synthesis of Mo-ITCBE-HRP enzyme mark haptens
(1) weigh during 10mg ITCBE are dissolved in 1mL dimethyl sulfoxide (DMSO)s (DMSO) and form metal cheating agents solution；Weigh
8.38mg molybdic acids (H2MoO4) it is dissolved in formation Mo in the HEPES buffer solution (10mM/L) of 1mLpH 8.06+Solution；By metal chelant
Agent solution and Mo6+Solution mixes, and adjusts pH value to 7.0 with NaOH, is then placed on that to shake bed reaction 12 small at ambient temperature
When, that is, form Mo-ITCBE chelant thing haptens.
(2) weigh during 100mg HRP are dissolved in the HEPES buffer solution (10mmol/L) of 1mLpH 8.0 and form 100mg/mL's
HRP horseradish peroxidase solution；Taking 1mLMo-ITCBE chelant thing haptens solution, to be added to 1mL horseradish peroxidases molten
PH value to 9.0 is adjusted in liquid and with NaOH, room temperature shaker is reacted 24 hours, and reaction solution is loaded bag filter by reaction after terminating, first
Dialysed 2d with distilled water, then use PBS 3d, that is, form Mo-ITCBE-HRP enzyme mark haptens.
Embodiment 6
Direct competive ELISA (dELISA) detecting step
The first step, it is 10 μ g/ holes to use chessboard method to optimize sheep anti mouse secondary antibody (GaMIgG) coating concentration, and package amount is per hole
100 μ L, 37 DEG C of incubation 2h, PBST board-washings 3 times, per minor tick 2min；Second step, is closed with 5% pig negative serum, per the μ of hole 280
L, 37 DEG C of incubation 1h, PBST board-washings 3 times, per minor tick 2min；3rd step, according to the anti-molybdenum ion Dan Ke after potency addition dilution
The standard items of grand antibody, sample or doubling dilution, enzyme mark haptens, after three mixes, 37 DEG C of incubation 25min, PBST board-washings 3
Secondary, per minor tick 2min, reaction sets NC and BC；4th step, plus enzyme substrate TMB nitrite ions, per hole 60 μ L, room temperature reaction 5min；
5th step, color development stopping reaction, terminate liquid 2mol/L H are added per hole2SO4100 μ L, A is read with ELIASA450nmValue.
Embodiment 7
Indirect competitive ELISA (ciELISA) detecting step
The first step, the Mo-ITCBE-cOVA coating antigen wrapper sheets diluted with the 0.05mol/L carbonate buffer solutions of pH 9.6,
The coating μ g/mL of concentration 2, package amount is per hole 100 μ L, 37 DEG C of incubation 2h, PBST board-washings 4 times, per minor tick 3min；Second step, uses
5% pig negative serum is closed, per hole 280 μ L, 37 DEG C of incubation 1h, PBST board-washings 4 times, per minor tick 3min；3rd step, addition mark
Quasi- product：ELISA Plate elder generation adds the Mo of 500 μ g/L with 50uL confining liquid shop fixtures, then the 1st hole6+- EDTA chelant thing standard solutions,
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- molybdenum ion pAb, or mAb, per hole 50 μ L, 37 DEG C of incubation 15min, PBST board-washings 4 times, per minor tick 3min；5th step, plus
GaMIgG-HRP, with confining liquid 1:1000 times of dilutions, per hole 50 μ L, 37 DEG C of incubation 25min, PBST board-washings 4 times, per minor tick
3min；6th step, plus enzyme substrate TMB nitrite ions, per hole 60 μ L, room temperature reaction 5-10min；7th step, color development stopping reaction, often
Hole adds terminate liquid 2mol/LH2SO4100 μ L, A is read with ELIASA450nmValue.
Embodiment 8
CiELISA detects the comparing of performance in dELISA and embodiment 7 in embodiment 6
The anti-molybdenum ion monoclonal antibody prepared with G2D7 cell lines sets up ELISA detection method, and carry out dELISA and
CiELISA detects the comparing of performance.With inhibiting rate B/B0(%) is ordinate (B0It is Mo6+Absorbance when-EDTA is 0 concentration
Value, B is Mo6+Absorbance during-EDTA various concentrations), with the Mo of various concentrations6+- EDTA standard items are abscissa, are drawn
Standard curve, regression equation is derived according to curvilinear trend, carries out correlation analysis.Sensitivity is with half-inhibition concentration (IC50Value) table
Show；Quantitative determination limits (IC20–IC80) standard items are represented to maximum signal (B0) suppression scope；Test limit is with IC15Table
Show, its result is shown in Fig. 2.
The regression equation of dELISA standard curves is the (R of y=-7.089Ln (x)+58.2212=0.9514), IC50Value
It is 0.68 μ g/L；The regression equation of ciELISA standard curves is the (R of y=-5.699Ln (x)+57.1052=0.9713),
IC50It is 0.77 μ g/L to be worth.DELISA standard curves are calculated in PBS to Mo according to formula6+The linear detection range of-EDTA
It is 0.16~32.9 μ g/L, test limit (LOD) is 0.13 μ g/L；CiELISA standard curves are in PBS to Mo6+- EDTA's is linear
Detection range is that 0.21~37.6 μ g/L, LOD is 0.16 μ g/L.
Embodiment 9
The operating procedure of molybdenum ion pollution detection dELISA kits in environmental water sample
The first step, required reagent is taken out from cold storage environment, is placed in (20~25 DEG C) balance 30min of room temperature, notes every
Planting liquid reagent must shake up before；Second step, water sample 0.5mL is gathered in centrifuge tube with suction pipe, adds 0.4mL samples
Dilution, adds EDTA treatment fluids 0.1mL after mixing；3rd step, by the μ L of sample treatment liquid 50 add ELISA Plate capillary strip in, together
When add dilution after the μ L of anti-molybdenum ion monoclonal antibody 50, dilution after the μ L of enzyme mark haptens 50, mix stand 25min after wash
Plate, notices that detection sets three repetitions, and set NC and BC controls；4th step, plus substrate nitrite ion A liquid and each 30 μ L of B liquid, stand
Board-washing after 5min；5th step, adds H2SO4The μ L of terminate liquid 100, ELIASA reads absorbance after standing 5min；6th step, will
Absorbance substitutes into the regression equation formula of dELISA standard curves, is calculated molybdenum ion content in water sample.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of based on Direct cELISA molybdenum ion detection kit, including：
(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
Coating concentration is 50~200 μ g/mL；
(2) mass concentration is the enzyme mark Mo-ITCBE chelant thing haptens solution of 20~50 μ g/mL；
(3) mass concentration is the anti-molybdenum ion monoclonal antibody solution of 10~20 μ g/mL；The anti-molybdenum ion monoclonal antibody is molten
Liquid is prepared by Mo-ITCBE-cBSA immunogen immune Balb/C mouse；
The terminate liquid of (4) 1.0~3.0mol/L；
(5) sample diluting liquid；
(6) substrate nitrite ion；
(7) cleaning solution；
(8) molybdenum ion standard solution；
(9) mass concentration is the EDTA treatment fluids of 10~20mg/mL.
2. it is according to claim 1 to detect molybdenum ion kit, it is characterised in that the coating of the sheep anti-mouse igg secondary antibody
Concentration is 80~150 μ g/mL.
3. it is according to claim 1 to detect molybdenum ion kit, it is characterised in that the enzyme mark Mo-ITCBE chelants thing half
The mass concentration of antigenic solution is 30~40 μ g/mL.
4. it is according to claim 1 to detect molybdenum ion kit, it is characterised in that the anti-molybdenum ion monoclonal antibody is molten
The mass concentration of liquid is 13~18 μ g/mL.
5. it is according to claim 1 to detect molybdenum ion kit, it is characterised in that the sample diluting liquid is mass concentration
It is the HEPES buffer solution of 2~10g/L.
6. it is according to claim 1 to detect molybdenum ion kit, it is characterised in that the cleaning solution is comprising mass concentration
The PBS solution of 0.05~0.1%Tween 20.
7. the application of the molybdenum ion in environment is detected of molybdenum ion detection kit described in claim 1~6 any one, its feature
It is to comprise the following steps：
A, by anti-molybdenum ion monoclonal antibody solution, dilution testing sample solution and enzyme mark Mo-ITCBE chelant thing haptens it is molten
Liquid is added in the detection plate hole for being coated with sheep anti-mouse igg secondary antibody, mixing, is washed with cleaning solution after incubation；
B, in detection plate add substrate nitrite ion, be incubated, add terminate liquid mixing, determine OD values；
C, the OD values obtained according to the step b and predetermined standard curve, obtain the concentration of molybdenum ion in testing sample, described
Standard curve is the linear relationship curve between molybdenum ion concentration and OD values.
8. application according to claim 7, it is characterised in that anti-molybdenum ion monoclonal antibody is molten in the detection plate hole
The volume ratio of liquid, the testing sample solution of dilution and enzyme mark Mo-ITCBE chelant thing haptens solution is 1~2：1~2：1~2.
9. application according to claim 7, it is characterised in that the temperature being incubated in the step a and b independently is 30~
40℃。
10. the application according to claim 7 or 9, it is characterised in that the time being incubated in the step a and b independently is
5~30min.
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| CN110887795A (en) * | 2019-11-15 | 2020-03-17 | 吉林大学 | Molybdenum monoatomic nanoenzyme material and application thereof in xanthine colorimetric sensing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103412127A (en) * | 2013-08-25 | 2013-11-27 | 河南科技学院 | Enzyme linked immunosorbent assay kit for detecting hexavalent molybdenum ions as well as establishment and detection methods thereof |
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2017
- 2017-02-23 CN CN201710099809.8A patent/CN106841627B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103412127A (en) * | 2013-08-25 | 2013-11-27 | 河南科技学院 | Enzyme linked immunosorbent assay kit for detecting hexavalent molybdenum ions as well as establishment and detection methods thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110887795A (en) * | 2019-11-15 | 2020-03-17 | 吉林大学 | Molybdenum monoatomic nanoenzyme material and application thereof in xanthine colorimetric sensing |
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