CN103472231B - Detect indirect competitive enzyme-linked immunosorbent kit of mercury ion and preparation method thereof - Google Patents
Detect indirect competitive enzyme-linked immunosorbent kit of mercury ion and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of for indirect competitive enzyme-linked immunosorbent kit detecting mercury ion and preparation method thereof.The ELISA Plate with mercury ion envelope antigen bag quilt, anti-mercury ion monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, mercury ion standard solution, washing lotion concentrate and sample treatment liquid is provided with in kit.Kit energy trace detection mercury ion of the present invention, for the mercury ion in testing environment, soil, water, food, medicine, cosmetics etc., there is quick, easy, responsive, special and economic dispatch characteristic, detecting step is few, save detection time, reduce operate miss, by force ageing, can Site Detection be carried out.This kit requires low to the pre-treatment of sample and processing procedure is simple, both can be used for the screening of gross sample, the quick detection of little batch sample can be carried out again, be not only environment, food security provides technical support, also for food import and export inspection, Food Inspection, environmental pollution monitoring evaluation etc. provide effective technological means.
Description
Technical field
The present invention relates to enzyme linked immunological and ion detection, particularly relate to a kind of for indirect competitive enzyme-linked immunosorbent kit detecting mercury ion and preparation method thereof.
Background technology
Mercury, is commonly called as mercury, is that stable chemical nature, can evaporate under mercury normal temperature, and the compound of mercuryvapour and mercury has severe toxicity more uniquely with the metal that liquid state exists under normal temperature, normal pressure.Mercury at occurring in nature ubiquity, China be mercury production, use and discharge big country, mercury pollution prevention and control situation is very severe.Heavy metal Hg is the lasting extremely toxic substance of environmental pollution, and toxicity is high, infringement human health, and mercury pollution has become global great environmental problem, also causes the great attention of the Chinese government, and mercury pollution control is listed in " 12 " planning key special subjects.Mercury poisoning with chronic be common, to suck caused by mercuryvapour and mercury compound dust mainly for a long time in production or use procedure.Mercuryvapour is easier in alveolus wall cell membrane, and the lipid binding with blood, is distributed to body tissue very soon.Mercury is oxidized to Hg in red blood cell and other tissue
2+, and accumulate with protein bound.Mercury metal absorbs hardly at intestines and stomach.Chronic mercury poisoning cardinal symptom shows as insanity, gingivitis, tremble and ephritis etc.; Acute mercury poisoning main manifestations is acute corrosive stomatitis and gastroenteritis, time serious, acute renal failure, liver damage can occur.Skin contact mercury and mercuric compounds can cause contact dermatitis, has allergic reaction character.
Due to the pollution of mercury and harmfulness larger, China has formulated soil, food, water body, cosmetics etc. and has related to national limit standard in mercury field, in food, cow's milk mercury allowance standard (GB2762-94) is≤0.01mg/kg, in groundwater quality standard (GB7468-2012), regulation, is mainly applicable to the mercury ion limitation≤0.001mg/L of centralized Drinking Water water source and farmland irrigating water.
At present, the method of testing environment mercury ion mainly contains (1) physics and chemistry analytical approach, comprises spectrophotometric method, atomic absorption spectrography (AAS) (AAS), inductively coupled plasma mass spectrometry (ICP-MS), ICP-AES (ICP-AES), atomic fluorescence spectrometry (AFS) etc.These methods respectively have relative merits, and spectrophotometric method is simple to operate, fast, disturb little, but its sensitivity are not high; Atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry, ICP-AES, atomic fluorescence spectrometry have the advantages such as selectivity is good, estimating precision is high, simple, quick, but instrument costly, can only detect in laboratory, and comparatively strict to operating personnel's technical requirement, limit it and widely use.(2) immune analysis method: comprise enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography.Enzyme linked immunosorbent assay (ELISA) is with competitiveness enzyme-linked immune response for Cleaning Principle, and measure light absorption value (OD) value by microplate reader after reaction solution and carry out result judgement, the method shortens detection time, heavy metal can carry out qualitative and quantitative detection.But ELISA method needs supporting microplate reader and matched reagent, and operating process is more complicated, and be domesticly now in development, the external valuable product of import, thus the application of ELISA method receives larger restriction.And commercial rapid detection apparatus, its sensitivity low (Monitoring lower-cut is 0.1mg/L), does not reach the limit value (sewage 0.05mg/L, potable water 0.001mg/L) of national regulations, and poor selectivity, cannot meet sensitive, testing requirement accurately.
Therefore, study a kind of can easy, responsive, special, economy, the quick testing product of mercury ion that screening amount is large, be current technical matters in the urgent need to address, for minimizing environmental pollution, ensure food safety significant.
Summary of the invention
The technical problem to be solved in the present invention: for the deficiency of existing detection mercury ion technology, prepare the monoclonal antibody of the hypersensitivity of anti-mercury ion, high-affinity, high specific, based on this for the preparation of the indirect competitive enzyme-linked immunosorbent kit and the construction method thereof that detect mercury ion, the mercury ion in the detection sample that this kit can be quick, easy, responsive, special.
Technical scheme of the present invention:
A kind of indirect competitive enzyme-linked immunosorbent kit detecting mercury ion, comprise box body, be provided with in box body with mercury ion envelope antigen bag by the monoclonal antibody of the ELISA Plate crossed, anti-mercury ion, ELIAS secondary antibody, substrate nitrite ion, stop buffer, mercury ion standard solution, washing lotion concentrate and sample treatment liquid.
described ELIAS secondary antibody is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or rabbit against murine ELIAS secondary antibody RaMIgG-HRP, and concentration is 100ng/mL; Described substrate nitrite ion is made up of developer A and developer B, and developer A is urea peroxide, and developer B is tetramethyl benzidine (TMB); Described stop buffer is the sulfuric acid solution of 2mol/L; Described mercury ion standard solution is the series concentration solution that phosphate buffer (PBS) that mercury ion is dissolved in 0.01mol/L, pH7.4 obtains; Described washing lotion concentrate is the phosphate buffer of 0.1mol/L, pH7.4, wherein contains the Tween-20(PBST of 0.05%); Described sample treatment liquid is ethylenediamine tetraacetic acid (EDTA) solution of 0.1mol/L; Solid phase material for the preparation of described ELISA Plate is polystyrene, tygon or polypropylene.
Described mercury ion envelope antigen is prepared by following methods:
(1) take 20mg ovalbumin (OVA) and be dissolved in that 1mL concentration is 0.01mol/L, pH value is in the HEPES damping fluid of 9.0, form carrier protein solution;
(2) take 10mg isothiocycmatobenzyl ethylenediamine tetraacetic acid intercalating agent (ITCBE) and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO);
(3) be added in carrier protein solution by 0.5mL metal-chelator dropwise, add the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, makes OVA-ITCBE solution;
(4) get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in OVA-ITCBE solution, adjust ph to 7.4, and at room temperature shaking table hatches 4h, then moves in bag filter and to dialyse 10d with PBS, namely form Hg
2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen.
Described ELISA Plate wraps quilt as follows: envelope antigen is Hg
2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, the pH that bag is 0.05moL/L by solution is the carbonate buffer solution of 9.6, coating agent amount is 100 μ L/ holes, and 37 DEG C of incubation 2h or room temperature 8h, wash 3 times with washing lotion concentrate PBST, then close with the Swine serum of 5%, every hole 250 μ L, 37 DEG C of incubation 1h, then wash 3 times with PBST.
Described anti-mercury ion monoclonal antibody is prepared by the following method:
(1) artificial immunity antigen synthesis: take that 20mg bovine serum albumin(BSA) (BSA) is dissolved in 1mL, concentration be 10mmol/L, pH is in the HEPES damping fluid of 9.0, form BSA carrier protein solution;
Take 10mgITCBE and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO); Be added in BSA carrier protein solution by 0.5mL metal-chelator dropwise, after mixing, add the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, adjust ph to 7.4, and at room temperature shaking table hatches 4h, then moves in bag filter and to dialyse 10d with PBS, namely form immunizing antigen Hg
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 5 only as immune animal, dorsal sc branch is injected, immunizing dose is every only each protein content 50 μ g, volume 0.2mL, head exempts from Freund's complete adjuvant, booster immunization incomplete Freund's adjuvant, within 3 weeks, carries out second time immunity after head exempts from, the immunity in 2 weeks of later interval once, is total to immunity 5 times;
(3) Fusion of Cells mouse for subsequent use is selected: indirect ELISA detects Hg in antiserum
2+polyclonal antibody (the Hg of-EDTA chelate
2+-EDTApAb) to tire, stop band restrain method detects Hg
2+-EDTA is to Hg
2+the half-inhibition concentration of-ITCBE-BSA
iC 50, select tire the highest,
iC 50minimum mouse, surpasses and exempts from for Fusion of Cells;
(4) positive hybridoma cell is prepared: get NS0 myeloma cell and immune mouse spleen cell PEG carries out Fusion of Cells, carry out the screening of positive hybridoma cell strain by indirect elisa method and stop band restrain method, 3 limited dilution clonings are carried out to the cell of screening; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA measures antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Hg
2+-EDTA monoclonal antibody (Hg
2+-EDTAmAb), get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freund's adjuvant (FIA) 0.5mL/ only, uses after 10 ~ 15 days; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned supernatant, and collecting cell precipitates; Cell precipitation is suspended with the PBS of sterilizing, mixes, cell number is adjusted to 10
6/ mL, lumbar injection 0.5mL/ only, produce ascites after inoculating cell 7-10 days, carry out collection ascites, in 37 DEG C of water-bath 30min, 4 DEG C of placements are spent the night, the centrifugal 5min of 12000r/min, discards upper-layer fat, FIA and lower sediment thing, carries out purifying with saturated ammonium sulfate salting out method, measure IgG content and tire ,-20 DEG C save backup.
Detect a construction method for the indirect competitive enzyme-linked immunosorbent kit of mercury ion, kit comprises Hg
2+the ELISA Plate that-ITCBE-OVA bag is closed by the Swine serum also with 5%; C1 liquid: working concentration is the monoclonal antibody of the anti-mercury ion of 1: 10000; C2 liquid: working concentration is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or the rabbit against murine ELIAS secondary antibody RaMIgG-HRP of 1: 1000, and wherein enzyme is horseradish peroxidase (HRP); C3 liquid: developer A is urea peroxide; C4 liquid: developer B, is tetramethyl benzidine (TMB); C5 liquid: stop buffer is the sulfuric acid solution of 2moL/L; Mercury ion standard solution be mercuric nitrate with 2% nitric acid dilute the solution of the series concentration obtained; Washing lotion concentrate PBST is the phosphate buffer of 0.1mol/L, pH7.4, wherein contains the Tween-20 of 0.05%; Sample treatment liquid is the EDTA solution of 0.1mol/L.
Described ELISA Plate wraps quilt as follows: envelope antigen is Hg
2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, the pH that bag is 0.05moL/L by solution is the carbonate buffer solution of 9.6, coating agent amount is 100 μ L/ holes, and 37 DEG C of incubation 2h or room temperature 8h, wash 3 times with washing lotion concentrate PBST, then close with the Swine serum of 5%, every hole 250 μ L, 37 DEG C of incubation 1h, then wash 3 times with PBST.
Described anti-mercury ion monoclonal antibody is prepared by the following method:
(1) artificial immunity antigen synthesis: take that 20mg bovine serum albumin(BSA) (BSA) is dissolved in 1mL, concentration be 10mmol/L, pH is in the HEPES damping fluid of 9.0, form BSA carrier protein solution;
Take 10mgITCBE and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO); Be added in BSA carrier protein solution by 0.5mL metal-chelator dropwise, after mixing, add the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, adjust ph to 7.4, and at room temperature shaking table hatches 4h, then moves in bag filter and to dialyse 10d with PBS, namely form immunizing antigen Hg
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 5 only as immune animal, dorsal sc branch is injected, immunizing dose is every only each 50 μ g, head exempts from Freund's complete adjuvant, booster immunization incomplete Freund's adjuvant, within 3 weeks, carries out second time immunity after head exempts from, the immunity in 2 weeks of later interval once, is total to immunity 5 times;
(3) Fusion of Cells mouse for subsequent use is selected: indirect ELISA detects Hg in antiserum
2+polyclonal antibody (the Hg of-EDTA chelate
2+-EDTApAb) to tire, stop band restrain method detects Hg
2+-EDTA is to Hg
2+the half-inhibition concentration of-ITCBE-BSA
iC 50, select tire the highest,
iC 50minimum mouse, surpasses and exempts from for Fusion of Cells;
(4) positive hybridoma cell is prepared: get NS0 myeloma cell and immune mouse spleen cell PEG carries out Fusion of Cells, carry out the screening of positive hybridoma cell strain by indirect elisa method and stop band restrain method, selection strong positive, the hole that inhibiting rate is higher, Growth of Cells is vigorous carry out 3 limited dilution clonings; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA measures antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Hg
2+-EDTA monoclonal antibody (Hg
2+-EDTAmAb), get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freund's adjuvant (FIA) 0.5mL/ only, uses after 10 ~ 15 days; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned supernatant, and collecting cell precipitates; Cell precipitation is suspended with the PBS of sterilizing, mixes, cell number is adjusted to 10
6/ mL, lumbar injection Balb/C mouse 0.5mL/ only, produce ascites after inoculating cell 7-10 days, collect ascites, in 37 DEG C of water-bath 30min, 4 DEG C of placements are spent the night, the centrifugal 5min of 12000r/min, discards upper-layer fat, FIA and lower sediment thing, carries out purifying with saturated ammonium sulfate salting out method, measure IgG content and tire ,-20 DEG C save backup.
Positive beneficial effect of the present invention:
1. the construction method of kit of the present invention, has prepared the artificial antigen of mercury ion, obtains appropriate molecule in conjunction with the artificial immunity antigen of ratio and envelope antigen, and obtains high-titer, sensitivity and special antiserum, for the establishment of kit provides necessary condition.
2. the construction method of kit of the present invention, has prepared the monoclonal antibody of anti-mercury ion, and the titer of ascites of antibody is 1: 6.9 × 10
5, affinity costant
kabe 1.52 × 10
10l/mol, is less than 1% with the cross reacting rate of other heavy metal ion, and this antibody has the characteristics such as high-titer, high-affinity, high specificity, and the trace for mercury ion detects fast and provides guarantee.
3. mercury ion indirect competitive enzyme-linked immunosorbent kit of the present invention, energy trace detection mercury ion, have quick, easy, responsive, special and economic dispatch characteristic, detecting step is few, saves detection time, reduces operate miss.Fast, 45 ~ 50min goes out result, greatly saves time than Physico-chemical tests method (3d); Easy, without any need for auxiliary instrumentation and reagent, people can operate per capita; Sensitivity, detection sensitivity is 0.5ng/mL, meets national limit standard requirement, suitable with the sensitivity of Physico-chemical tests method; Special, with other heavy metal ion no cross reaction; Economy, compared with Physico-chemical tests method, testing cost is less than 1/20 of physico-chemical analysis method; That detects is by force ageing, can carry out Site Detection.
4. mercury ion indirect competitive enzyme-linked immunosorbent kit of the present invention, can be used for testing environment, soil, water, food, medicine, mercury ion in cosmetics etc., require low to the pre-treatment of sample and processing procedure is simple, both can be used for the screening of gross sample, the quick detection of little batch sample can be carried out again, be not only environment, food security provides technical support, also be food import and export inspection, Food Inspection, environmental pollution monitoring evaluation etc. provides effective technological means and detection method, for raising food security, ensure that the people are physically and mentally healthy, environmental friendliness and sustainable development is kept to have important practical significance, the popularization of this technology will have significant economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 is artificial immunizing antigen Hg
2+the Technology Roadmap of-ITCBE-Protein synthesis;
Fig. 2 is the canonical plotting detecting mercury ion enzyme linked immunological kit.
Embodiment
Illustrate the present invention by embodiment below, but do not represent any limitation of the invention, as being not particularly illustrated, percentage composition is wherein weight percentage.
the preparation of embodiment one, mercury ion artificial immunity antigen
Adopt different sulphur hydrocyanic ester legal system for artificial complete antigen, comprise immunizing antigen Hg
2+-ITCBE-BSA and envelope antigen Hg
2+-ITCBE-OVA.With envelope antigen Hg
2+-ITCBE-OVA is example, and its preparation method is as follows.
(1) take 20mg ovalbumin OVA and be dissolved in that 1mL concentration is 0.01mol/L, pH value is in the HEPES damping fluid of 9.0, form carrier protein solution;
(2) take 10mg isothiocycmatobenzyl ethylenediamine tetraacetic acid intercalating agent (ITCBE) and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO);
(3) be added in carrier protein solution by 0.5mL metal-chelator dropwise, add the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, makes OVA-ITCBE solution;
(4) get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in OVA-ITCBE solution, adjust ph to 7.4, and at room temperature shaking table hatches 4h, then moves in bag filter and to dialyse 10d with PBS, namely form Hg
2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen.
With legal system for immunizing antigen Hg
2+-ITCBE-BSA.
embodiment two, mercury ion artificial immunity Antigen Identification
Bicinchoninic acid method is adopted to measure Hg
2+carrier protein BSA concentration in-ITCBE-BSA.Using BSA as standard protein, be made into the concentration gradient of 0 μ g/mL, 10 μ g/mL, 20 μ g/mL, 40 μ g/mL, 60 μ g/mL, 80 μ g/mL, build Concentration Testing typical curve by bicinchoninic acid method.The linear equation of BSA concentration standard curve is: y=0.0035x+0.0065, R
2=0.9981, wherein y is sample is the absorbance at 562nm place at wavelength, and x is sample protein concentration.
ICP-AES method is adopted to measure Hg
2+hg in-ITCBE-BSA
2+concentration.By the Hg of 100 μ g/mL
2+hg in-ITCBE-BSA
2+the standard reserving solution nitric acid of 2% is diluted to the concentration gradient of 0 μ g/mL, 0.1 μ g/mL, 0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 0.8 μ g/mL, the automatic drawing standard curve of instrument software, and draw equation of linear regression Y=0.0174X+0.0019, related coefficient 0.9989; Measure under the optimum optimization experiment condition of 253.7nm wavelength, instrument software automatic analysis result.
the preparation of embodiment three, anti-mercury ion monoclonal antibody
(1) immune animal.Use Hg
2+-ITCBE-BSA immunity Balb/c mouse 5, immunizing dose is 50 μ g0.2mL/, dorsal sc multi-point injection.Head exempts from, and dilutes Hg with sterile phosphate damping fluid (PBS)
2+-ITCBE-BSA, with equivalent Freund's complete adjuvant (CFA) mixing and emulsifying; Booster immunization, dilutes Hg with sterilizing PBS
2+-ITCBE-BSA, with equivalent incomplete Freund's adjuvant (IFA) mixing and emulsifying, second time immunity within 3 weeks, is carried out after head exempts from, the immunity in 3 weeks of later interval once, exempts from 5 times altogether, and blood is got in third time immunity docking in latter 10 days, 37 DEG C of water-bath 30min, 4 DEG C of placements are spent the night, and 800r/min4 DEG C of centrifugal 5min, after getting supernatant ,-20 DEG C save backup.
(2) Fusion of Cells mouse for subsequent use is selected.Indirect ELISA detects Hg in antiserum
2+with ethylenediamine tetraacetic acid (EDTA) chelate Hg
2+the polyclonal antibody (pAb) of-EDTA is tired, and stop band restrain detects Hg
2+-EDTApAb is to Hg
2+the half-inhibition concentration of-EDTA (
iC 50), select tire the highest,
iC 50minimum mouse, surpasses and exempts from for Fusion of Cells.
(3) screening of Fusion of Cells and positive hybridoma cell strain.Fusion of Cells, PEG solution, GNK solution are preheated to 40 DEG C, by the splenocyte prepared and NS0 myeloma cell in 10: 1 ratio be mixed in 50mL centrifuge tube, add GNK solution to 40mL, the centrifugal 10min of 1000r/min, abandon supernatant, break up cell mass, this fusion pipe is moved in 40 DEG C of water-baths.With the 50%PEG(pH8.0 of 1mL suction pipe by preheating) be added drop-wise in fusion pipe, limit edged shakes fusion pipe gently, adds in 1min, and continues slowly to shake fusion pipe 1.5min in a water bath; Then slowly add GNK solution to 40mL, 37 DEG C of water-baths leave standstill 5min, and the centrifugal 10min of 1000r/min, abandons supernatant; Break up cell mass, add 40mLHAT piping and druming mixing, be added on the 96 porocyte culture plates containing feeder cells, every hole 100 μ L, put 37 DEG C, the CO of 5%
2cultivate in incubator.
The screening of positive hybridoma cell, carries out the screening of positive hybridoma cell strain by indirect elisa method and stop band restrain method.Selection strong positive, the hole that inhibition is good, Growth of Cells is vigorous carry out 3 limited dilution clonings, respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect elisa method measures antibody titer, investigates the anti-Hg of hybridoma secretion
2+ion monoclonal antibody (Hg
2+-EDTAmAb) stability.
(4) preparation of monoclonal antibody.Adopt in body and induce ascites legal system for monoclonal antibody, get healthy Balb/c female mice in 8 week age, lumbar injection FIA0.5mL/ only, uses after 10 ~ 15 days.By the centrifugal 10min of positive hybridoma cell 1000r/min cultivated, abandon supernatant, collecting cell precipitates.Cell precipitation is suspended with the PBS of sterilizing, mixes, cell number is adjusted to 10
6individual/mL, lumbar injection Balb/C mouse 0.5mL/ only.Inoculating cell produced ascites after 7 ~ 10 days, collected, in 37 DEG C of water-bath 30min, 4 DEG C of placements are spent the night, the centrifugal 5min of 12000r/min, discard the precipitation of the fat on upper strata, IFA and lower floor, saturated ammonium sulfate salting out method measures IgG content after purifying and tires, and-20 DEG C save backup.
the qualification of embodiment four, anti-mercury ion monoclonal antibody
(1) titer of ascites measures.To the mouse peritoneal injection cloning cell line 10 after lumbar injection whiteruss 10d
7individual cell, extracts ascites after 7d, and saturated ammonium sulfate salting out method is purified, and indirect ELISA measures and tires.Measurement result, the titer of ascites of monoclonal antibody is 1: 6.9 × 10
5.
(2) affinity qualification.Saturated ELISA mensuration affinity costant (
ka), the Hg of 3.4 μ g/mL and 1.7 μ g/mL is respectively by concentration
2+-ITCBE-OVA bag quilt, adds the Hg of doubling dilution
2+-ITCBEmAb, then add GaMIgG-HRP, TMB develop the color survey
a 450nmvalue, with Hg
2+-ITCBEmAb concentration is horizontal ordinate, with
a 450nmvalue for ordinate, draws corresponding 2 response curves, with every bar curve upper planar section
a 450nmvalue, as 100%, curve calculates 50%
a 450nmhg corresponding during value
2+-ITCBEmAb concentration, according to formula
kaff=(n-1)/2(n [Ab'] t-[Ab] t) calculates
ka.Monoclonal antibody
kabe 1.52 × 10
10l/mol.
(3) susceptibility qualification.Hg is measured with stop band restrain
2+-EDTAmAb is to variable concentrations Hg
2+the inhibiting rate of-EDTA, with inhibiting rate B/B
0for ordinate, with variable concentrations Hg
2+the logarithm value of-EDTA is horizontal ordinate, and drawing standard suppresses curve, carries out correlation regression analysis, calculates Hg
2+-EDTAmAb is to Hg
2+-EDTA's
iC 50.Qualification result,
iC 50for 10.31ng/mL.
(4) specificity identification.Adopt its specificity of cross reaction test for identification.Cross reaction test selects the huge legendary turtle compound (huge legendary turtle conjunction method is the same) of mercury, chromium, lead, zinc, copper, caesium, cobalt, molybdenum, iron and EDTA and EDTA solution as inhibitor, measures each mortifier with stop band restrain
iC 50 , with Hg
2+-EDTAmAb is to Hg
2+-EDTA's
iC 50and Hg
2+-EDTAmAb is to each competitor
iC 50the percentage of ratio be its cross reacting rate (CR%).Qualification result, is less than 1% with other heavy metal ion cross reaction.
the preparation of embodiment five, mercury ion enzyme linked immunological kit
(1) determination of ELIAS secondary antibody and monoclonal antibody working concentration: adopt the screening of square formation method to determine the working concentration of ELIAS secondary antibody (RaRIgG-HRP) and monoclonal antibody, be respectively 1: 1000 and 1: 10000.
(2) mercury ion enzyme linked immunological kit assembling:
Kit comprises: Hg
2+-ITCBE-OVA bag quilt is also with 8 × 12(96 hole that 5% Swine serum is closed) or 4 × 12(48 hole) ELISA Plate; C1 liquid: working concentration is the monoclonal antibody of the anti-mercury ion of 1: 10000; C2 liquid: working concentration is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or the rabbit against murine ELIAS secondary antibody RaMIgG-HRP of 1: 1000, and wherein enzyme is horseradish peroxidase; C3 liquid: developer A is urea peroxide; C4 liquid: developer B, is tetramethyl benzidine (TMB); C5 liquid: stop buffer is the sulfuric acid solution of 2moL/L; Mercury ion standard solution be mercuric nitrate with 2% nitric acid be diluted to the series concentration solution that 0ng/mL, 1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL, 32ng/mL, 64ng/mL, 128ng/mL, 256ng/mL obtain; Washing lotion concentrate PBST is the phosphate buffer of 0.1mol/L, pH7.4, wherein contains the Tween-20 of 0.05%; Sample treatment liquid is the EDTA solution of 0.1mol/L.
(3) typical curve of mercury ion enzyme linked immunological kit: stop band restrain measures monoclonal antibody to variable concentrations Hg
2+the inhibiting rate of standard items, with inhibiting rate B/B
0%(B is Hg
2+various criterion concentration
a 450value, B
0hg
2+0 normal concentration
a 450value) be ordinate, with the logarithm value of various criterion product concentration for horizontal ordinate, drawing standard curve on semilogarithmic paper, derivation regression equation, carries out regretional analysis.Typical curve is shown in accompanying drawing 2.Equation of linear regression is y=-35.16x+85.632, R
2=0.9928,
iC 50be 10.31 μ g/L.
the performance measurement of embodiment six, mercury ion enzyme linked immunological kit
(1) sensitivity measures.B/B is limited to according to stop band restrain lowest detection
0the method of=120%, calculates the sensitivity of kit, determines detectability according to Regression Equations.Measurement result, detects and is limited to 0.5ng/mL.
(2) sensing range measures.Be B/B according to stop band restrain sensing range
0the method of=20% ~ 80%, calculates the sensitivity of kit, determines sensing range according to Regression Equations, sensing range is 0.5 ~ 73.62ng/mL.
(3) accuracy measures.By Hg
2+standard items are respectively 1 with final concentration, 2,4,8,10ng/mL adds in feed, cow's milk, if 6 repetitions, determines its accuracy with the recovery and the coefficient of variation (CV).The recovery of feed sample 91.2% ~ 98.41%, average 96.1%, the coefficient of variation (CV) 10.9% ~ 14.8%, average 13.09%; The recovery of cow's milk is 89.7% ~ 98.1%, and average 95.43%, CV 11.2% ~ 13.9%, and average 13.46%; The average CV of feed and cow's milk is less than 15%, shows that kit has high accuracy.In table 2.
The interpolation recovery test of table 2 mercury ion enzyme linked immunological kit (
n=6)
(4) specific assay.Employing cross reaction is tested, select the chelate of the metallic ion such as molybdenum, lead, cadmium, copper, zinc, chromium, cobalt, iron and EDTA, EDTA is mortifier, measures each mortifier with stop band restrain
iC 50, with Hg
2+mAb is to Hg
2+'s
iC 50with to other each competitor
iC 50percent be its cross reacting rate (CR%).In table 3.
Table 3Hg
2+the cross reaction of-EDTAmAb and other metallo-chelate
(5) stability: the kit getting same batch is stored in 4 DEG C, mensuration 6 each months in the middle of the month of preservation
a 450value,
iC 50, R
2situation of change, determines its stability.Result shows, along with kit storage life extends, and the A of each standard items
450nmvalue reduces to some extent, but its IC
50, R
2change is little, and curve is good, and this kit steady quality in 4 DEG C, 6 months storage lives is described.See the following form 4.
The storage life of table 4 kit
Claims (4)
1. one kind is detected the indirect competitive enzyme-linked immunosorbent kit of mercury ion, comprise box body, it is characterized in that: be provided with in box body with mercury ion envelope antigen bag by the monoclonal antibody of the ELISA Plate crossed, anti-mercury ion, ELIAS secondary antibody, substrate nitrite ion, stop buffer, mercury ion standard solution, washing lotion concentrate and sample treatment liquid;
Described ELIAS secondary antibody is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or rabbit against murine ELIAS secondary antibody RaMIgG-HRP, and concentration is 100ng/mL; Described substrate nitrite ion is made up of developer A and developer B, and developer A is hydrogen peroxide or urea peroxide, and developer B is tetramethyl benzidine (TMB); Described stop buffer is the sulfuric acid solution of 2mol/L; Described mercury ion standard solution is the series concentration solution that phosphate buffer (PBS) that mercury ion is dissolved in 0.01mol/L, pH7.4 obtains; Described washing lotion concentrate is the phosphate buffer of 0.1mol/L, pH7.4, wherein contains the Tween-20 of 0.05%; Described sample treatment liquid is ethylenediamine tetraacetic acid (EDTA) solution of 0.1mol/L; Solid phase material for the preparation of described ELISA Plate is polystyrene, tygon or polypropylene;
Described mercury ion envelope antigen is prepared by following methods:
(1) take 20mg ovalbumin OVA and be dissolved in that 1mL concentration is 0.01mol/L, pH value is in the HEPES damping fluid of 9.0, form carrier protein solution;
(2) take 10mg isothiocycmatobenzyl ethylenediamine tetraacetic acid intercalating agent (ITCBE) and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO);
(3) be added in carrier protein solution by 0.5mL metal-chelator dropwise, add the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, makes OVA-ITCBE solution;
(4) get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg2+ solution; Joined by Hg2+ solution in OVA-ITCBE solution, adjust ph to 7.4, at room temperature shaking table hatches 4h, and then move in bag filter and to dialyse 10d with PBS, namely form Hg2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen;
Described anti-mercury ion monoclonal antibody is prepared by the following method:
(1) artificial antigen synthesis: take that 20mg bovine serum albumin(BSA) (BSA) is dissolved in 1mL, concentration be 10mmol/L, pH is in the HEPES damping fluid of 9.0, form BSA carrier protein solution;
Take 10mgITCBE and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO); Be added in BSA carrier protein solution by 0.5mL metal-chelator dropwise, after mixing, add the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg mercuric nitrate to be dissolved in 100uL distilled water, form uniform Hg2+ solution; Joined by Hg2+ solution in BSA-ITCBE solution, adjust ph to 7.4, at room temperature shaking table hatches 4h, and then move in bag filter and to dialyse 10d with PBS, namely form immunizing antigen Hg2+-ITCBE-BSA, collect packing ,-20 DEG C frozen;
(2) immune animal: take Hg2+-ITCBE-BSA as immunizing antigen, using 6 week age female Balb/c mouse 5 only as immune animal, dorsal sc branch is injected, immunizing dose is every only each 50 μ g, head exempts from Freund's complete adjuvant, booster immunization incomplete Freund's adjuvant, within 3 weeks, carries out second time immunity after head exempts from, the immunity in 2 weeks of later interval once, is total to immunity 5 times;
(3) Fusion of Cells mouse for subsequent use is selected: the polyclonal antibody (Hg2+-EDTApAb) that indirect ELISA detects Hg2+-EDTA chelate in antiserum is tired, stop band restrain method detects Hg2+-EDTA to the half-inhibition concentration IC50 of Hg2+-ITCBE-BSA, select the highest, that IC50 is minimum mouse of tiring, surpass and exempt from for Fusion of Cells;
(4) positive hybridoma cell is prepared: get NS0 myeloma cell and immune mouse spleen cell PEG carries out Fusion of Cells, carry out the screening of positive hybridoma cell strain by indirect elisa method and stop band restrain method, selection strong positive, the hole that inhibiting rate is higher, Growth of Cells is vigorous carry out 3 limited dilution clonings; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA measures antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Hg2+-EDTA monoclonal antibody (Hg2+-EDTAmAb), get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freund's adjuvant (FIA) 0.5mL/ only, uses after 10 ~ 15 days; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned supernatant, and collecting cell precipitates; Cell precipitation is suspended with the PBS of sterilizing, mixes, cell number is adjusted to 106/mL, and lumbar injection Balb/C mouse 0.5mL/ only, produces ascites after inoculating cell 7-10 days, carry out collection ascites, in 37 DEG C of water-bath 30min, 4 DEG C of placements are spent the night, the centrifugal 5min of 12000r/min, discard upper-layer fat, FIA and lower sediment thing, carry out purifying with saturated ammonium sulfate salting out method, measure IgG content and tire ,-20 DEG C save backup.
2. indirect competitive enzyme-linked immunosorbent kit according to claim 1, it is characterized in that: described ELISA Plate wraps quilt as follows: envelope antigen is Hg2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, and to wrap by solution be 0.05moL/L, and pH is the carbonate buffer solution of 9.6, and coating agent amount is 100 μ L/ holes, 37 DEG C of incubation 2h or room temperature 8h, wash 3 times with washing lotion concentrate PBST, then close with the Swine serum of 5%, every hole 250 μ L, 37 DEG C of incubation 1h, then wash 3 times with PBST.
3. the construction method detecting the indirect competitive enzyme-linked immunosorbent kit of mercury ion as claimed in claim 1, is characterized in that: described kit comprises the ELISA Plate that Hg2+-ITCBE-OVA bag is closed by the Swine serum also with 5%; C1 liquid: working concentration is the monoclonal antibody of the anti-mercury ion of 1: 10000; C2 liquid: working concentration is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or the rabbit against murine ELIAS secondary antibody RaMIgG-HRP of 1: 1000, and wherein enzyme is horseradish peroxidase; C3 liquid: developer A is urea peroxide; C4 liquid: developer B, is tetramethyl benzidine (TMB); C5 liquid: stop buffer is the sulfuric acid solution of 2moL/L; Mercury ion standard solution be mercuric nitrate with 2% nitric acid dilute the solution of the series concentration obtained; Washing lotion concentrate PBST is the phosphate buffer of 0.1mol/L, pH7.4, wherein contains the Tween-20 of 0.05%; Sample treatment liquid is the EDTA solution of 0.1mol/L.
4. the construction method of enzyme linked immunological kit according to claim 3, it is characterized in that: described ELISA Plate wraps quilt as follows: envelope antigen is Hg2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, and to wrap by solution be 0.05moL/L, and pH is the carbonate buffer solution of 9.6, and coating agent amount is 100 μ L/ holes, 37 DEG C of incubation 2h or room temperature 8h, wash 3 times with washing lotion concentrate PBST, then close with the Swine serum of 5%, every hole 250 μ L, 37 DEG C of incubation 1h, then wash 3 times with PBST.
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| CN105044006B (en) * | 2015-07-14 | 2018-04-03 | 上海拜豪生物科技有限公司 | Mercury-IgG chelate and preparation method and application thereof |
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| CN104987400A (en) * | 2015-07-14 | 2015-10-21 | 上海拜豪生物科技有限公司 | Mercury-hemoglobin chelate as well as preparation method and application thereof |
| CN105388280A (en) * | 2015-11-12 | 2016-03-09 | 江苏省检验检疫科学技术研究院 | Indirect competitive ELISA kit and detection method for detecting content of mercury in textiles based on mercury monoclonal antibody |
| CN106872692B (en) * | 2017-02-23 | 2019-06-07 | 河南科技学院 | Competitive ELISA detection kit and its application in mercury ion food pollution |
| CN109187948B (en) * | 2018-08-17 | 2021-10-29 | 郑州大学 | Paroxarsone and nitrophenylarsonic acid duplex detection test paper |
| CN110646606A (en) * | 2019-10-12 | 2020-01-03 | 江南大学 | Fluorescent quantitative rapid detection test strip for heavy metal mercury ions and preparation method and application thereof |
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