CN103472231A - Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof - Google Patents
Indirect competition enzyme linked immunoreagent kit for detecting mercury ions and manufacturing method thereof Download PDFInfo
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Abstract
The invention relates to an indirect competition enzyme linked immunoreagent kit for detecting mercury ions and a manufacturing method of the kit. An elisa plate enveloped by mercury ion envelope antigens, mercury-ion-resisting monoclonal antibodies, elisa second antibodies, a substrate color developing solution, a stop solution, a mercury ion standard solution, a washing liquor concentrated solution and a sample treating liquid are arranged in the kit. The kit can detect the mercury irons in a trace mode, is used for detecting the mercury irons in the environment, the soil, water, foods, medicines, cosmetics and the like, and has the advantages of being rapid, easy and convenient to use, sensitive, peculiar, economical and the like. The kit is few in detection step and high in timeliness, saves detection time, reduces operation errors and can perform field detection. The kit is low in requirement for pretreating samples, simple in treatment process, not only can be used for screening the samples in large batch, but also can perform rapid detection on the samples in small batch, and not only provides technical support for environment and food safety, but also provides effective technological means for food import and export inspection, food inspection, monitoring and evaluation of environmental pollution and the like.
Description
Technical field
The present invention relates to enzyme linked immunological and ion detection, particularly relate to a kind of kit of the indirect competitive enzyme-linked immunosorbent for detection of mercury ion and preparation method thereof.
Background technology
Mercury, be commonly called as mercury, is unique metal existed with liquid state under normal temperature, normal pressure, and stable chemical nature, can evaporate under mercury normal temperature, and the compound of mercuryvapour and mercury has severe toxicity more.Mercury is at the occurring in nature ubiquity, China be mercury production, use and discharge big country, the 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, the infringement human health, and mercury pollution has become global great environmental problem, also causes the great attention of the Chinese government, and the mercury pollution control is listed in " 12 " and plans great special project.Mercury poisoning be take chronic as common, is mainly long-term the suction due to mercuryvapour and mercury compound dust in production or use procedure.Mercuryvapour more easily sees through the alveolus wall cell membrane, with the lipid binding in blood, is distributed to very soon body tissue.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.The chronic mercury poisoning cardinal symptom shows as insanity, gingivitis, tremble and ephritis etc.; Acute mercury poisoning main manifestations is acute corrosivity stomatitis and gastroenteritis, but acute renal failure, liver damage when serious.The skin contact mercury and mercuric compounds can cause contact dermatitis, has allergic reaction character.
Because pollution and the harmfulness of mercury are larger, China has formulated soil, food, water body, cosmetics etc. and has related to the national limit standard in the mercury field, in food, cow's milk mercury allowance standard (GB2762-94) is≤0.01mg/kg, regulation in groundwater quality standard (GB7468-2012), limit the quantity of≤0.001mg/L of the mercury ion that mainly is applicable to 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 littlely, but its sensitivity is not high; Atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry, ICP-AES, atomic fluorescence spectrometry have the advantages such as selectivity is good, the mensuration precision is high, simple, quick, but instrument is more expensive, can only be detected in laboratory, and comparatively strict to operating personnel's technical requirement, limited it and be widely used.(2) immune analysis method: comprise enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography.Enzyme linked immunosorbent assay (ELISA) be take competitiveness enzyme-linked immune response as detecting principle, measures light absorption value (OD) value by microplate reader after reaction solution and carries out judgement as a result, and the method has shortened detection time, can carry out qualitative and quantitative detection to heavy metal.But the ELISA method needs supporting microplate reader and matched reagent, and operating process is more complicated, and the domestic development that now is in, the external valuable product of import, thereby the application of ELISA method has been subject to larger restriction.And commercial rapid detection apparatus, its sensitivity low (under detection, being limited to 0.1mg/L), do not reach the limit value (sewage 0.05mg/L, potable water 0.001mg/L) of GB regulation, and poor selectivity, can't meet sensitive, testing requirement accurately.
Therefore, study a kind of can be easy, responsive, special, economy, mercury ion fast detecting product that the screening amount is large, be current technical matters in the urgent need to address, for environmental contamination reduction, ensure food safety significant.
Summary of the invention
The technical problem to be solved in the present invention: for the existing deficiency that detects the mercury ion technology, prepare hypersensitivity, the high-affinity of anti-mercury ion, the monoclonal antibody of 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 that detects mercury ion, comprise box body, be provided with monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, mercury ion standard solution, washing lotion concentrate and the sample preparation liquid of the ELISA Plate be coated with the mercury ion envelope antigen, anti-mercury ion in box body.
By following methods, prepared by described mercury ion envelope antigen:
(1) taking 20 mg ovalbumins (OVA), to be dissolved in 1 mL concentration be in 0.01 mol/L, the pH value HEPES damping fluid that is 9.0, forms carrier protein solution;
(2) take 10mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid intercalating agent (ITCBE) and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO);
(3) 0.5mL metal-chelating agent solution dropwise is added in carrier protein solution, adds the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, make OVA-ITCBE solution;
(4) get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in OVA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms Hg
2+-ITCBE-OVA envelope antigen, collect packing, and-20 ℃ frozen.
Described ELISA Plate is coated as follows: envelope antigen is Hg
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, the carbonate buffer solution that the pH that coated solution is 0.05 moL/L is 9.6, coated dosage is 100 μ L/ holes, and 37 ℃ of incubation 2 h or room temperature 8 h, with washing lotion concentrate PBST washing 3 times, then with 5% pig serum, seal, every hole 250 μ L, 37 ℃ of incubation 1 h, then wash 3 times with PBST.
Prepared by the following method by described anti-mercury ion monoclonal antibody:
(1) artificial immunity antigen is synthetic: taking 20mg bovine serum albumin(BSA) (BSA), to be dissolved in 1mL, concentration be in 10mmol/L, the pH HEPES damping fluid that is 9.0, forms BSA carrier protein solution;
Take 10mg ITCBE and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO); 0.5mL metal-chelating agent solution dropwise is added in BSA carrier protein solution, after mixing, adds the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms immunizing antigen Hg
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age 5 of female Balb/c mouse as immune animal, the subcutaneous branch injection in back, immunizing dose is every each protein content 50 μ g, volume 0.2mL, head exempts to use Freund's complete adjuvant, and the booster immunization incomplete Freunds adjuvant carries out immunity for the second time in 3 weeks after head exempts from, once, immunity is 5 times altogether in interval immunity in 2 weeks later;
(3) select the standby mouse of Fusion of Cells: indirect ELISA detects Hg in antiserum
2+polyclonal antibody (the Hg of-EDTA chelate
2+-EDTA pAb) tire, blocking-up ELISA 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, super exempting from for Fusion of Cells;
(4) prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG, carry out the screening of positive hybridoma cell strain by indirect elisa method and blocking-up ELISA method, the cell screened is carried out to limited dilution cloning 3 times; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA is measured antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Hg of ascites legal system
2+-EDTA monoclonal antibody (Hg
2+-EDTA mAb), get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freunds adjuvant (FIA) 0.5mL/ only, is used in 10～15 days afterwards; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned to supernatant, the collecting cell precipitation; With the PBS of sterilizing, cell precipitation is suspended, mixes, cell number is adjusted to 10
6/ mL, lumbar injection 0.5mL/ only, produce ascites after inoculating cell 7-10 days, collected ascites, in 37 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, the centrifugal 5min of 12000r/min, discard upper strata fat, FIA and lower sediment thing, with the saturated ammonium sulfate salting out method, carries out purifying, measure IgG content and tire ,-20 ℃ save backup.
A kind of construction method that detects the indirect competitive enzyme-linked immunosorbent kit of mercury ion, kit comprises Hg
2+-ITCBE-OVA is coated with and uses the ELISA Plate of 5% pig serum sealing; C1 liquid: the monoclonal antibody of the anti-mercury ion that working concentration is 1: 10000; C2 liquid: the sheep anti mouse ELIAS secondary antibody GaMIgG-HRP that working concentration is 1: 1000 or the anti-mouse ELIAS secondary antibody of rabbit RaMIgG-HRP, 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; The mercury ion standard solution is that mercuric nitrate dilutes the solution of the series concentration obtained with 2% nitric acid; The phosphate buffer that washing lotion concentrate PBST is 0.1mol/L, pH7.4, wherein contain 0.05% Tween-20; The EDTA solution that sample preparation liquid is 0.1 mol/L.
Described ELISA Plate is coated as follows: envelope antigen is Hg
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, the carbonate buffer solution that the pH that coated solution is 0.05 moL/L is 9.6, coated dosage is 100 μ L/ holes, and 37 ℃ of incubation 2 h or room temperature 8 h, with washing lotion concentrate PBST washing 3 times, then with 5% pig serum, seal, every hole 250 μ L, 37 ℃ of incubation 1 h, then wash 3 times with PBST.
Prepared by the following method by described anti-mercury ion monoclonal antibody:
(1) artificial immunity antigen is synthetic: taking 20mg bovine serum albumin(BSA) (BSA), to be dissolved in 1mL, concentration be in 10mmol/L, the pH HEPES damping fluid that is 9.0, forms BSA carrier protein solution;
Take 10mg ITCBE and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO); 0.5mL metal-chelating agent solution dropwise is added in BSA carrier protein solution, after mixing, adds the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms immunizing antigen Hg
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age 5 of female Balb/c mouse as immune animal, the subcutaneous branch injection in back, immunizing dose is every each 50 μ g, head exempts to use Freund's complete adjuvant, and the booster immunization incomplete Freunds adjuvant carries out immunity for the second time in 3 weeks after head exempts from, once, immunity is 5 times altogether in interval immunity in 2 weeks later;
(3) select the standby mouse of Fusion of Cells: indirect ELISA detects Hg in antiserum
2+polyclonal antibody (the Hg of-EDTA chelate
2+-EDTA pAb) tire, blocking-up ELISA 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, super exempting from for Fusion of Cells;
(4) prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG, carry out the screening of positive hybridoma cell strain by indirect elisa method and blocking-up ELISA method, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is higher, Growth of Cells is vigorous 3 times; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA is measured antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Hg of ascites legal system
2+-EDTA monoclonal antibody (Hg
2+-EDTA mAb), get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freunds adjuvant (FIA) 0.5mL/ only, is used in 10～15 days afterwards; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned to supernatant, the collecting cell precipitation; With the PBS of sterilizing, cell precipitation is suspended, 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 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, the centrifugal 5min of 12000r/min, discard upper strata fat, FIA and lower sediment thing, with the saturated ammonium sulfate salting out method, carries out purifying, measure IgG content and tire ,-20 ℃ save backup.
Positive beneficial effect of the present invention:
1. the construction method of kit of the present invention, the artificial antigen that has prepared mercury ion, obtained suitable molecule in conjunction with than artificial immunity antigen and envelope antigen, and obtained high-titer, sensitivity and special antiserum, for the establishment of kit provides necessary condition.
2. the construction method of kit of the present invention, 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, be 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, for the trace fast detecting of mercury ion provides guarantee.
3. mercury ion indirect competitive enzyme-linked immunosorbent kit of the present invention, can the trace detection mercury ion, has quick, easy, responsive, special and economic dispatch characteristic, and detecting step is few, saves detection time, the reduction operate miss.Fast, 45～50 min go out result, than physics and chemistry detection method (3 d), greatly save time; Easy, without any need for auxiliary instrumentation and reagent, the people can operate per capita; Sensitivity, detection sensitivity is 0.5 ng/mL, meets national limit standard requirement, suitable with the sensitivity of physics and chemistry detection method; Special, with other heavy metal ion no cross reaction; Economy, compare with the physics and chemistry detection method, and testing cost is less than 1/20 of the physico-chemical analysis method; That detects is ageing strong, 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., to the pre-treatment of sample, require low and processing procedure is simple, both can be used for the screening of gross sample, can carry out the fast detecting of little batch sample again, it is not only environment, food security provides technical support, it is also the food import and export check, Food Inspection, environmental pollution monitoring evaluation etc. provides effective technological means and detection method, for improving food security, ensure people's physical and mental health, keep environmental friendliness and sustainable development to have important practical significance, the popularization of this technology will have significant economic benefit and social benefit.
The accompanying drawing explanation
Fig. 1 is artificial immunizing antigen Hg
2+the Technology Roadmap that-ITCBE-Protein is synthetic;
Fig. 2 is for detecting the canonical plotting of mercury ion enzyme linked immunological kit.
Embodiment
Below illustrate the present invention with embodiment, but do not mean any limitation of the invention, as special instruction not, percentage composition wherein is weight percentage.
the preparation of embodiment mono-, mercury ion artificial immunity antigen
Adopt the standby artificial complete antigen of different sulphur hydrocyanic ester legal system, 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) taking 20 mg ovalbumin OVA, to be dissolved in 1 mL concentration be in 0.01 mol/L, the pH value HEPES damping fluid that is 9.0, forms carrier protein solution;
(2) take 10mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid intercalating agent (ITCBE) and be dissolved in formation metal-chelating agent solution in 1mL dimethyl sulfoxide (DMSO) (DMSO);
(3) 0.5mL metal-chelating agent solution dropwise is added in carrier protein solution, adds the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, make OVA-ITCBE solution;
(4) get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in OVA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms Hg
2+-ITCBE-OVA envelope antigen, collect packing, and-20 ℃ frozen.
With the standby immunizing antigen Hg of legal system
2+-ITCBE-BSA.
embodiment bis-, mercury ion artificial immunity antigen are identified
Adopt the bicinchoninic acid method 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, by the bicinchoninic acid method, build concentration examination criteria curve.The linear equation of BSA concentration standard curve is: y=0.0035x+0.0065, R
2=0.9981, wherein y is the absorbance that sample is the 562nm place at wavelength, and x is sample protein concentration.
Adopt the ICP-AES method to measure Hg
2+hg in-ITCBE-BSA
2+concentration.Hg by 100 μ g/mL
2+hg in-ITCBE-BSA
2+standard reserving solution 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 with 2% nitric acid, the automatic drawing standard curve of instrument software, and draw equation of linear regression Y=0.0174X+0.0019, related coefficient 0.9989; Measured instrument software automatic analysis result under the optimum optimization experiment condition of 253.7nm wavelength.
the preparation of embodiment tri-, anti-mercury ion monoclonal antibody
(1) immune animal.Use Hg
2+5 of-ITCBE-BSA immunity Balb/c mouse, immunizing dose is 50 μ g0.2 mL/, the subcutaneous multi-point injection in back.Head exempts from, with Plondrel phthalate buffer (PBS) dilution Hg
2+-ITCBE-BSA, with equivalent Freund's complete adjuvant (CFA) mixing and emulsifying; Booster immunization, with sterilizing PBS dilution Hg
2+-ITCBE-BSA, with equivalent incomplete Freunds adjuvant (IFA) mixing and emulsifying, after exempting from, head carries out immunity for the second time in 3 weeks, interval immunity in 3 weeks later once, is exempted from 5 times altogether, and blood is got in docking in immune latter 10 days for the third time, 37 ℃ of water-bath 30 min, 4 ℃ of placements are spent the night, centrifugal 5 min of 800 4 ℃ of r/min, and after getting supernatant ,-20 ℃ save backup.
(2) the standby mouse of Fusion of Cells is selected.Indirect ELISA detects Hg in antiserum
2+with ethylenediamine tetraacetic acid (EDTA) chelate Hg
2+the polyclonal antibody of-EDTA (pAb) is tired, and blocking-up ELISA detects Hg
2+-EDTA pAb is to Hg
2+the half-inhibition concentration of-EDTA (
iC 50), select tire the highest,
iC 50minimum mouse, super exempting 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 ℃, the splenocyte for preparing and NS0 myeloma cell are mixed in 50 mL centrifuge tubes in the ratio of 10: 1, add GNK solution to 40 mL, centrifugal 10 min of 1000 r/min, abandon supernatant, break up cell mass, this fusion pipe is moved in 40 ℃ of water-baths.50% PEG(pH 8.0 with 1 mL suction pipe by preheating) be added drop-wise in fusion pipe, the limit edged shakes fusion pipe gently, adds in 1 min, and continue slowly to shake fusion pipe 1.5 min in water-bath; Then slowly add GNK solution to 40 mL, 37 ℃ of standing 5 min of water-bath, centrifugal 10 min of 1000 r/min, abandon supernatant; Break up cell mass, add 40 mL HAT piping and druming and mix, be added on the 96 porocyte culture plates containing feeder cells, every hole 100 μ L, put 37 ℃, 5% CO
2in incubator, cultivate.
The screening of positive hybridoma cell, carry out the screening of positive hybridoma cell strain by indirect elisa method and blocking-up ELISA method.Limited dilution cloning is carried out in selection strong positive, the hole that inhibition is good, Growth of Cells is vigorous 3 times, respectively at recovery hybridoma after frozen 15 d, 30 d and 60 d, get supernatant after cultivation, indirect elisa method is measured antibody titer, investigates the anti-Hg of hybridoma secretion
2+ion monoclonal antibody (Hg
2+-EDTA mAb) stability.
(4) preparation of monoclonal antibody.Adopt in body and induce the standby monoclonal antibody of ascites legal system, get healthy Balb/c female mice in 8 week age, lumbar injection FIA 0.5 mL/ only, is used in 10～15 days afterwards.The centrifugal 10min of positive hybridoma cell 1000 r/min by cultivating, abandon supernatant, the collecting cell precipitation.With the PBS of sterilizing, cell precipitation is suspended, mixes, cell number is adjusted to 10
6individual/mL, lumbar injection Balb/C mouse 0.5 mL/ only.Inoculating cell produced ascites after 7～10 days, was collected, in 37 ℃ of water-bath 30 min, 4 ℃ of placements are spent the night, and centrifugal 5 min of 12000 r/min discard the precipitation of fat, IFA and the lower floor on upper strata, the saturated ammonium sulfate salting out method is measured IgG content and tires after purifying, and-20 ℃ save backup.
the evaluation of embodiment tetra-, anti-mercury ion monoclonal antibody
(1) titer of ascites is measured.To the injection of the mouse peritoneal after lumbar injection whiteruss 10 d cloning cell line 10
7individual cell, extract ascites after 7 d, and the saturated ammonium sulfate salting out method is purified, and indirect ELISA is measured and tired.Measurement result, the titer of ascites of monoclonal antibody is 1: 6.9 * 10
5.
(2) affinity is identified.Saturated ELISA mensuration affinity costant (
ka), be respectively the Hg of 3.4 μ g/mL and 1.7 μ g/mL by concentration
2+-ITCBE-OVA is coated, adds the Hg of doubling dilution
2+-ITCBE mAb, then add GaMIgG-HRP, the TMB survey that develops the color
a 450nmvalue, with Hg
2+-ITCBE mAb concentration is horizontal ordinate, with
a 450nmvalue for ordinate, is drawn corresponding 2 response curves, with every curve upper planar section
a 450nmvalue, as 100%, calculates 50% on curve
a 450nmcorresponding Hg during value
2+-ITCBE mAb concentration, according to formula
kaff=(n-1)/2(n[Ab'] t-[Ab] t) calculate
ka.Monoclonal antibody
kabe 1.52 * 10
10l/mol.
(3) susceptibility is identified.With blocking-up, ELISA measures Hg
2+-EDTA mAb 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 the correlation regression analysis, calculates Hg
2+-EDTA mAb 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 (it is the same that huge legendary turtle is closed method) of mercury, chromium, lead, zinc, copper, caesium, cobalt, molybdenum, iron and EDTA and EDTA solution as inhibitor, with blocking ELISA, measures each mortifier
iC 50 , with Hg
2+-EDTA mAb is to Hg
2+-EDTA's
iC 50and Hg
2+-EDTA mAb is to each competition thing
iC 50the percentage of ratio be its cross reacting rate (CR%).Qualification result, be less than 1% with other heavy metal ion cross reaction.
the preparation of embodiment five, mercury ion enzyme linked immunological kit
(1) determining 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 is coated with and uses 8 * 12(96 hole of 5% pig serum sealing) or 4 * 12(48 hole) ELISA Plate; C1 liquid: the monoclonal antibody of the anti-mercury ion that working concentration is 1: 10000; C2 liquid: the sheep anti mouse ELIAS secondary antibody GaMIgG-HRP that working concentration is 1: 1000 or the anti-mouse ELIAS secondary antibody of rabbit RaMIgG-HRP, 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; To be mercuric nitrate be diluted to 2% nitric acid 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 to the mercury ion standard solution; The phosphate buffer that washing lotion concentrate PBST is 0.1mol/L, pH7.4, wherein contain 0.05% Tween-20; The EDTA solution that sample preparation liquid is 0.1 mol/L.
(3) typical curve of mercury ion enzyme linked immunological kit: blocking-up ELISA 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, the logarithm value of various criterion product concentration of take is horizontal ordinate, drawing standard curve on semilogarithmic paper, and the derivation regression equation, carry out regretional analysis.Typical curve is shown in accompanying drawing 2.Equation of linear regression be 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 is measured.Be limited to B/B according to blocking-up ELISA lowest detection
0=120% method, calculate the sensitivity of kit according to Regression Equations, determine detectability.Measurement result, detect and be limited to 0.5 ng/mL.
(2) sensing range is measured.According to blocking-up ELISA sensing range, be B/B
0=20%～80% method, calculate the sensitivity of kit according to Regression Equations, determine sensing range, and sensing range is 0.5～73.62 ng/mL.
(3) accuracy is measured.By Hg
2+standard items are respectively 1,2,4,8,10 ng/mL with final concentration to be added in feed, cow's milk, establishes 6 repetitions, with the recovery and the coefficient of variation (CV), determines its accuracy.The recovery of feed sample is 91.2%～98.41%, average 96.1%, and the coefficient of variation (CV) is 10.9%～14.8%, and average 13.09%; The recovery of cow's milk is 89.7%～98.1%, average 95.43%, and CV is 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 test, to select the metallic ions such as molybdenum, lead, cadmium, copper, zinc, chromium, cobalt, iron and chelate, the EDTA of EDTA be mortifier, with blocking ELISA, measures each mortifier
iC 50, with Hg
2+mAb is to Hg
2+'s
iC 50with other is respectively competed to thing
iC 50percent be its cross reacting rate (CR%).In Table 3.
Table 3 Hg
2+the cross reaction of-EDTA mAb and other metallo-chelate
(5) stability: the kit of getting same batch is stored in 4 ℃, mensuration 6 each months in the middle of the month of preservation
a 450value,
iC 50, R
2situation of change, determine its stability.Result shows, along with the prolongation of kit storage life, the A of each standard items
450nmvalue reduces to some extent, but its IC
50, R
2change not quite, curve is good, and this kit steady quality in 4 ℃, 6 months storage lives is described.See the following form 4.
The storage life of table 4 kit
Claims (8)
1. an indirect competitive enzyme-linked immunosorbent kit that detects mercury ion, comprise box body, it is characterized in that: the monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, mercury ion standard solution, washing lotion concentrate and the sample preparation liquid that are provided with the ELISA Plate be coated with the mercury ion envelope antigen, anti-mercury ion in box body.
2. indirect competitive enzyme-linked immunosorbent kit according to claim 1 is characterized in that:
described ELIAS secondary antibody is sheep anti mouse ELIAS secondary antibody GaMIgG-HRP or the anti-mouse ELIAS secondary antibody of rabbit RaMIgG-HRP, and concentration is 100 ng/mL; Described substrate nitrite ion is comprised of developer A and developer B, and developer A is urea peroxide, and developer B is tetramethyl benzidine; The sulfuric acid solution that described stop buffer is 2 mol/L; Described mercury ion standard solution is that mercury ion is dissolved in the series concentration solution that the phosphate buffer of 0.01 mol/L, pH7.4 obtains; The phosphate buffer that described washing lotion concentrate is 0.1 mol/L, pH7.4, wherein contain 0.05% Tween-20; The edta solution that described sample preparation liquid is 0.1 mol/L; Solid phase material for the preparation of described ELISA Plate is polystyrene, tygon or polypropylene.
3. indirect competitive enzyme-linked immunosorbent kit according to claim 1 and 2, it is characterized in that: prepared by following methods by described mercury ion envelope antigen:
(1) taking 20 mg ovalbumins, to be dissolved in 1 mL concentration be in 0.01 mol/L, the pH value HEPES damping fluid that is 9.0, forms carrier protein solution;
(2) take 10mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid intercalating agent and be dissolved in formation metal-chelating agent solution in the 1mL dimethyl sulfoxide (DMSO);
(3) 0.5mL metal-chelating agent solution dropwise is added in carrier protein solution, adds the tri-n-butylamine of 100uL, 1.5moL/L after mixing, shaking table room temperature reaction 24h, make OVA-ITCBE solution;
(4) get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in OVA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms Hg
2+-ITCBE-OVA envelope antigen, collect packing, and-20 ℃ frozen.
4. indirect competitive enzyme-linked immunosorbent kit according to claim 3 is characterized in that: described ELISA Plate is coated as follows: envelope antigen is Hg
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, the carbonate buffer solution that the pH that coated solution is 0.05 moL/L is 9.6, coated dosage is 100 μ L/ holes, and 37 ℃ of incubation 2 h or room temperature 8 h, with washing lotion concentrate PBST washing 3 times, then with 5% pig serum, seal, every hole 250 μ L, 37 ℃ of incubation 1 h, then wash 3 times with PBST.
5. according to the described indirect competitive enzyme-linked immunosorbent kit of claim 1-4 any one, it is characterized in that: prepared by described anti-mercury ion monoclonal antibody by the following method:
(1) artificial immunity antigen is synthetic: taking the 20mg bovine serum albumin(BSA), to be dissolved in 1mL, concentration be in 10mmol/L, the pH HEPES damping fluid that is 9.0, forms BSA carrier protein solution;
Take 10mg ITCBE and be dissolved in formation metal-chelating agent solution in the 1mL dimethyl sulfoxide (DMSO); 0.5mL metal-chelating agent solution dropwise is added in BSA carrier protein solution, after mixing, adds the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms immunizing antigen Hg
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age 5 of female Balb/c mouse as immune animal, the subcutaneous branch injection in back, immunizing dose is every each protein content 50 μ g, volume 0.2mL, head exempts to use Freund's complete adjuvant, and the booster immunization incomplete Freunds adjuvant carries out immunity for the second time in 3 weeks after head exempts from, once, immunity is 5 times altogether in interval immunity in 2 weeks later;
(3) select the standby mouse of Fusion of Cells: indirect ELISA detects Hg in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and blocking-up ELISA 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, super exempting from for Fusion of Cells;
(4) prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG, carry out the screening of positive hybridoma cell strain by indirect elisa method and blocking-up ELISA method, the cell screened is carried out to limited dilution cloning 3 times; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA is measured antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Hg of ascites legal system
2+-EDTA monoclonal antibody, get healthy Balb/c female mice in 8 week age, and lumbar injection incomplete Freunds adjuvant 0.5mL/ only, is used in 10～15 days afterwards; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned to supernatant, the collecting cell precipitation; With the PBS of sterilizing, cell precipitation is suspended, mixes, cell number is adjusted to 10
6/ mL, lumbar injection 0.5mL/ only, produce ascites after inoculating cell 7-10 days, collected ascites, in 37 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, the centrifugal 5min of 12000r/min, discard upper strata fat, FIA and lower sediment thing, with the saturated ammonium sulfate salting out method, carries out purifying, measure IgG content and tire ,-20 ℃ save backup.
6. a claim 1-3 any one detects the construction method of the indirect competitive enzyme-linked immunosorbent kit of mercury ion, and it is characterized in that: described kit comprises Hg
2+-ITCBE-OVA is coated with and uses the ELISA Plate of 5% pig serum sealing; C1 liquid: the monoclonal antibody of the anti-mercury ion that working concentration is 1: 10000; C2 liquid: the sheep anti mouse ELIAS secondary antibody GaMIgG-HRP that working concentration is 1: 1000 or the anti-mouse ELIAS secondary antibody of rabbit RaMIgG-HRP, wherein enzyme is horseradish peroxidase; C3 liquid: developer A is urea peroxide; C4 liquid: developer B is tetramethyl benzidine; C5 liquid: stop buffer is the sulfuric acid solution of 2moL/L; The mercury ion standard solution is that mercuric nitrate dilutes the solution of the series concentration obtained with 2% nitric acid; The phosphate buffer that washing lotion concentrate PBST is 0.1mol/L, pH7.4, wherein contain 0.05% Tween-20; The EDTA solution that sample preparation liquid is 0.1 mol/L.
7. the construction method of enzyme linked immunological kit according to claim 6 is characterized in that: described ELISA Plate is coated as follows: envelope antigen is Hg
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, the carbonate buffer solution that the pH that coated solution is 0.05 moL/L is 9.6, coated dosage is 100 μ L/ holes, and 37 ℃ of incubation 2 h or room temperature 8 h, with washing lotion concentrate PBST washing 3 times, then with 5% pig serum, seal, every hole 250 μ L, 37 ℃ of incubation 1 h, then wash 3 times with PBST.
8. the construction method of enzyme linked immunological kit according to claim 7, it is characterized in that: prepared by described anti-mercury ion monoclonal antibody by the following method:
(1) artificial immunity antigen is synthetic: taking the 20mg bovine serum albumin(BSA), to be dissolved in 1mL, concentration be in 10mmol/L, the pH HEPES damping fluid that is 9.0, forms BSA carrier protein solution;
Take 10mg ITCBE and be dissolved in formation metal-chelating agent solution in the 1mL dimethyl sulfoxide (DMSO); 0.5mL metal-chelating agent solution dropwise is added in BSA carrier protein solution, after mixing, adds the tri-n-butylamine that 100uL concentration is 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg mercuric nitrate and be dissolved in 100uL distilled water, form uniform Hg
2+solution; By Hg
2+solution joins in BSA-ITCBE solution, regulates pH value to 7.4, and at room temperature shaking table is hatched 4h, then moves in bag filter the 10d that dialyses with PBS, forms immunizing antigen Hg
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Hg
2+-ITCBE-BSA is immunizing antigen, using 6 week age 5 of female Balb/c mouse as immune animal, the subcutaneous branch injection in back, immunizing dose is every each 50 μ g, head exempts to use Freund's complete adjuvant, and the booster immunization incomplete Freunds adjuvant carries out immunity for the second time in 3 weeks after head exempts from, once, immunity is 5 times altogether in interval immunity in 2 weeks later;
(3) select the standby mouse of Fusion of Cells: indirect ELISA detects Hg in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and blocking-up ELISA 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, super exempting from for Fusion of Cells;
(4) prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG, carry out the screening of positive hybridoma cell strain by indirect elisa method and blocking-up ELISA method, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is higher, Growth of Cells is vigorous 3 times; Respectively at recovery hybridoma after frozen 15d, 30d and 60d, get supernatant after cultivation, indirect ELISA is measured antibody titer, investigates the stability of hybridoma secrete monoclonal antibody, obtains a strain of hybridoma strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Hg of ascites legal system
2+-EDTA monoclonal antibody, get healthy Balb/c female mice in 8 week age, and lumbar injection incomplete Freunds adjuvant 0.5mL/ only, is used in 10～15 days afterwards; The centrifugal 10min of positive hybridoma cell 1000r/min of cultivation is abandoned to supernatant, the collecting cell precipitation; With the PBS of sterilizing, cell precipitation is suspended, 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 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, the centrifugal 5min of 12000r/min, discard upper strata fat, FIA and lower sediment thing, with the saturated ammonium sulfate salting out method, carries out purifying, measure IgG content and tire ,-20 ℃ save backup.
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| CN109187948A (en) * | 2018-08-17 | 2019-01-11 | 郑州大学 | A kind of roxarsone and Nitarsone duplex Test paper |
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| 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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