CN103472230A - Indirect competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof - Google Patents
Indirect competition enzyme linked immunoreagent kit for detecting lead 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 lead ions and a manufacturing method of the kit. An elisa plate enveloped by lead ion envelope antigens, lead-ion monoclonal antibodies, elisa second antibodies, a substrate color developing solution, a stop solution, a lead ion standard solution, a washing liquor concentrated solution and a sample treating liquid are arranged in the kit. The indirect competition enzyme linked immunoreagent kit can detect the lead irons in a trace mode, is used for detecting the lead iron contamination residual in the environment, the soil, water, foods and containers, 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 not only can be used for screening samples in large batch, but also can perform rapid detection on samples in small batch, and provides effective 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 lead ion and preparation method thereof.
Background technology
Plumbous (Pb) is a kind of cinerous heavy metal, is one of our known environmental poisonous substance, widely distributed at occurring in nature.Along with the development of industrial or agricultural, plumbous purposes is more and more extensive, can look for its trace in air, soil, food, tableware, toy, but lead contamination can not be ignored.Lead is the general poisonous substance, has the high property accumulated and polyaffinity, can by body, be absorbed by approach such as digestion, breathings.More acute lead poisoning shows as and has a stomach-ache, has a headache, trembles, the nerve agitation, and stupor when serious, until dead.Chronic lead poisoning shows as neurotoxicity mostly, and hematological system, immune system, kidney etc. are caused to certain infringement.Lead is children to be threatened to one of maximum environmental contaminants, and children are to the lead element sensitivity of being grown up.Due to blood-brain barrier, do not grow completely, infant's Pb-B just can cause cognition dysfunction or development delay when reaching 100 μ g/L.
Due to lead contamination and harmfulness larger, China has formulated soil, food, water body, toy, apparatus etc. and has related to the national limit standard in plumbous field, in agricultural product security quality safety requirements for non-environmental pollution aquatic products (GB18406.4-2001), lead content is had to limited amount≤0.5mg/kg, regulation in groundwater quality standard (GB/T14848-93), mainly be applicable to the limit the quantity of≤0.05mg/L of lead ion of centralized Drinking Water water source and industrial or agricultural water, cooked meat product (GB2726-2005) requires lead content≤0.5mg/kg.
At present, the method for testing environment lead ion mainly contains (1) physics and chemistry analytic approach: comprise ultraviolet spectrophotometry (UV), electrochemical methods (EC), atomic absorption spectrography (AAS) (AAS), inductively coupled plasma mass spectrometry (ICP-MS), ICP-AES (ICP-AES), By Hydride Generation-atomic Fluorescence Spectrometry (HG-AFS), By Naa (NAA) etc.These methods respectively have relative merits, and ultraviolet spectrophotometry is simple to operate, fast, disturb littlely, but its sensitivity is not high; Electrochemical methods has the characteristics such as sensitive, easy, high to operating personnel's technical requirement; Atomic absorption spectrography (AAS), inductively coupled plasma mass spectrometry, ICP-AES, By Hydride Generation-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, comparatively strict to operating personnel's technical requirement; By Naa is a kind ofly to take nuclear reaction as basic analytical approach, has advantages of trace, fast, accurately and non-destructive and can analyze multielement, but equipment needed thereby is difficult for being possessed by common laboratory, is difficult to be used widely simultaneously.(2) immunological analysis method: comprise enzyme linked immunosorbent assay (ELISA).Take competitiveness enzyme-linked immune response as detecting principle, measure light absorption value (OD) value by microplate reader after reaction solution and carry out judgement as a result, shortened detection time, can carry out qualitative and quantitative detection to left drug.But the ELISA method needs supporting microplate reader and matched reagent, and operating process is more complicated.
Therefore, develop quick, easy, responsive, special, economy, lead ion fast detecting product that the screening amount is large, for environmental contamination reduction, improve food quality, ensure food safety significant.
Summary of the invention
The technical problem to be solved in the present invention: for the deficiency of existing lead ion detection technique, prepare hypersensitivity, the high-affinity of lead 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 lead ion, the lead ion in the testing environment 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 lead ion, comprise box body, be provided with the ELISA Plate be coated with the lead ion envelope antigen, monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, lead ion standard solution, washing lotion concentrate and the sample preparation liquid of lead ion in box body.
By following methods, prepared by described lead ion envelope antigen:
(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 plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-OVA envelope antigen, collect packing, and-20 ℃ frozen.
Described ELISA Plate is coated as follows: envelope antigen is Pb
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, coated solution is the carbonate buffer solution that 0.05 moL/L, pH are 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 lead 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 of 100uL, 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age 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 Pb in antiserum
2+polyclonal antibody (the Pb of-EDTA chelate
2+-EDTA pAb) tire, blocking-up ELISA method detects Pb
2+-EDTA is to Pb
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 hybridoma cell strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Pb of ascites legal system
2+-EDTA monoclonal antibody (Pb
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 lead ion, kit comprises Pb
2+-ITCBE-OVA is coated with and uses the ELISA Plate of 5% pig serum sealing; C1 liquid: the lead ion monoclonal antibody 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; Lead ion standard solution: the plumbi nitras series concentration solution that the nitric acid dilution with 2% obtains; Washing lotion concentrate: PBST, be the phosphate buffer of 0.1mol/L, pH7.4, wherein contains 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 Pb
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 incubation 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 lead 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 plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age 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 Pb in antiserum
2+polyclonal antibody (the Pb of-EDTA chelate
2+-EDTA pAb) tire, blocking-up ELISA method detects Pb
2+-EDTA is to Pb
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 hybridoma cell strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Pb of ascites legal system
2+-EDTA monoclonal antibody (Pb
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 lead ion, obtained suitable molecule in conjunction with than artificial immunity antigen and envelope antigen, solved the immunogene of lead ion
the propertyproblem, obtained high-titer, sensitivity, special polyvalent antibody, for the establishment of kit is laid a good foundation after immune animal.
2. the construction method of kit of the present invention, prepared lead ion high-titer, sensitivity, special monoclonal antibody, and the antibody titer of ascites is 1: 7.2 * 10
5, affinity costant
kabe 1.87 * 10
10l/mol, be less than 1% with the cross reacting rate of other heavy metal ion, for the trace fast detecting of lead ion provides guarantee.
3. lead ion indirect competitive enzyme-linked immunosorbent kit of the present invention, can the trace detection lead ion, has quick, easy, responsive, special, 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 0.2 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. lead ion indirect competitive enzyme-linked immunosorbent kit of the present invention, mainly for detection of environment, soil, water, food, in implements, lead ion pollutes residual, 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 Pb
2+the Technology Roadmap that-ITCBE-Protein is synthetic;
Fig. 2 is for detecting the canonical plotting of lead 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-, lead ion artificial immunity antigen
Adopt the standby artificial complete antigen of different sulphur hydrocyanic ester legal system, comprise immunizing antigen Pb
2+-ITCBE-BSA and envelope antigen Pb
2+-ITCBE-OVA.With envelope antigen Pb
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 plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-OVA envelope antigen, collect packing, and-20 ℃ frozen.
With the standby immunizing antigen Pb of legal system
2+-ITCBE-BSA.
embodiment bis-, lead ion artificial immunity antigen are identified
Adopt the bicinchoninic acid method to measure Pb
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 examination criteria curve by the bicinchoninic acid method, the linear equation of BSA concentration standard curve is: y=0.0022x+0.008, R
2=0.9987, wherein y is that sample is 562nm place absorbance at wavelength, and x is sample protein concentration.
Adopt the ICP-AES method to measure Pb
2+pb in-ITCBE-BSA
2+concentration.Pb by 100 μ g/mL
2+pb 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, 0.3 μ g/mL, 0.4 μ g/mL, 0.5 μ g/mL with 2% nitric acid, the automatic drawing standard curve of instrument software, and draw equation of linear regression Y=0.27X+0.0045, related coefficient 0.9998, measured instrument software automatic analysis result under the optimum optimization experiment condition of 283nm wavelength.
the preparation of embodiment tri-, lead ion monoclonal antibody
(1) immune animal.Use Pb
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 Pb
2+-ITCBE-BSA, with equivalent Freund's complete adjuvant (CFA) mixing and emulsifying; Booster immunization, with sterilizing PBS dilution Pb
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 Pb in antiserum
2+with ethylenediamine tetraacetic acid (EDTA) chelate Pb
2+the polyclonal antibody of-EDTA (pAb) is tired, and blocking-up ELISA detects Pb
2+-EDTA pAb is to Pb
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-Pb of hybridoma secretion
2+ion monoclonal antibody (Pb
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-, lead 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: 7.2 * 10
5.
(2) affinity is identified.Saturated ELISA mensuration affinity costant (
ka), be respectively the Pb of 3.4 μ g/mL and 1.7 μ g/mL by concentration
2+-ITCBE-OVA is coated, adds the Pb of doubling dilution
2+-ITCBE mAb, then add GaMIgG-HRP, the TMB survey that develops the color
a 450nmvalue, with Pb
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 Pb 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 Pb
2+-EDTA mAb is to variable concentrations Pb
2+the inhibiting rate of-EDTA, with inhibiting rate B/B
0for ordinate, with variable concentrations Pb
2+the logarithm value of-EDTA is horizontal ordinate, and drawing standard suppresses curve, carries out the correlation regression analysis, calculates Pb
2+-EDTA mAb is to Pb
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 lead, mercury, chromium, zinc, copper, caesium, cobalt, molybdenum, iron and ITCBE and ITCBE solution as inhibitor, with blocking ELISA, measures each mortifier
iC 50 , with Pb
2+-ITCBE mAb is to Pb
2+-ITCBE's
iC 50 and Pb
2+-ITCBE mAb is to each competition thing
iC 50
the percentage of ratio be its cross reacting rate (CR%).Qualification result, be less than 0.01% with other heavy metal ion cross reaction.
the preparation of embodiment five, lead 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) assembling of lead ion enzyme linked immunological kit:
Kit comprises Pb
2+-ITCBE-OVA is coated with and uses the ELISA Plate of 5% pig serum sealing; C1 liquid: the monoclonal antibody of the lead 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; The lead ion standard solution is plumbi nitras (Pb (NO
3)
2) nitric acid with 2% is diluted to the solution of the series concentration that 0ng/mL, 1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL, 32ng/mL, 64ng/mL, 128ng/mL, 256ng/mL obtain; 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 lead ion enzyme linked immunological kit: blocking-up ELISA measures monoclonal antibody to variable concentrations Pb
2+the inhibiting rate of standard items, with inhibiting rate B/B
0%(B is Pb
2+various criterion concentration
a 450value, B
0pb
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=-35.074x+96.108, R
2=0.9918,
iC 50 be 20.41 μ g/L.
the performance measurement of embodiment six, lead ion enzyme linked immunological kit
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 0.208 ng/mL.
Sensing range is measured.According to blocking-up ELISA sensing range, be B/B
0the method of=20%-80%, calculate the sensitivity of kit according to Regression Equations, determine sensing range, sensing range 2.87～147.57 ng/mL.
Accuracy is measured.By Pb
2+standard items are respectively 2,4,8,16,32 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 90.8%～98.62%, average 96.21%, and the coefficient of variation (CV) is 10.6%～13.7%, and average 12.16%; The recovery of cow's milk is 93.12%～98.82%, average 96.68%, and CV is 10.7%～13.9%, and average 12.96%; Average CV is less than 15%, shows that kit has high accuracy.In Table 2.
Table 2 lead ion enzyme linked immunological kit interpolation recovery test (
n=6)
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 Pb
2+mAb is to Pb
2+'s
iC 50with other is respectively competed to thing
iC 50percent be its cross reacting rate (CR%).In Table 3.
Table 3 Pb
2+the cross reaction of-EDTA mAb and other metallo-chelate
(4) 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.In Table 4.
The storage life of table 4 kit
Claims (8)
1. an indirect competitive enzyme-linked immunosorbent kit that detects lead ion, comprise box body, it is characterized in that: be provided with the ELISA Plate be coated with the lead ion envelope antigen, monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, lead ion standard solution, washing lotion concentrate and the sample preparation liquid of lead 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 lead ion standard solution is that lead 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 lead ion envelope antigen:
(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 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 plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
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 Pb
2+-ITCBE-OVA, coated concentration is 2 μ g/mL, coated solution is the carbonate buffer solution that 0.05 moL/L, pH are 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 lead 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 of 100uL, 1.5moL/L, shaking table room temperature reaction 24h, make BSA-ITCBE solution; Get the 11.25mg plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age 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 Pb in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and blocking-up ELISA method detects Pb
2+-EDTA is to Pb
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 hybridoma cell strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Pb 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 lead ion, and it is characterized in that: described kit comprises Pb
2+-ITCBE-OVA is coated with and uses the ELISA Plate of 5% pig serum sealing; C1 liquid: the lead ion monoclonal antibody 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; Lead ion standard solution: the plumbi nitras series concentration solution that the nitric acid dilution with 2% obtains; Washing lotion concentrate: PBST, be the phosphate buffer of 0.1mol/L, pH7.4, wherein contains 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 Pb
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 incubation 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 lead 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 plumbi nitras and be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing, and-20 ℃ frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age 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 Pb in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and blocking-up ELISA method detects Pb
2+-EDTA is to Pb
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 hybridoma cell strain;
(5) prepare monoclonal antibody: adopt in body and induce the standby anti-Pb 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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