CN103472230B - Detect indirect competitive enzyme-linked immunosorbent kit of lead ion and preparation method thereof - Google Patents
Detect indirect competitive enzyme-linked immunosorbent kit of lead 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 lead ion and preparation method thereof, in box body, be provided with the ELISA Plate with lead ion envelope antigen bag quilt, lead ion monoclonal antibody, ELIAS secondary antibody, substrate nitrite ion, stop buffer, lead ion standard solution, washing lotion concentrate and sample treatment liquid.Lead ion indirect competitive enzyme-linked immunosorbent kit of the present invention, energy trace detection lead ion, the lead ion that can be used in environment, soil, water, food and implements pollutes residual detection, there is quick, easy, responsive, special, economic dispatch characteristic, detecting step is few, save detection time, reduce operate miss; By force ageing, can Site Detection be carried out; Both can be used for gross sample screening, can be used for again the quick detection of little batch sample, for food import and export inspection, Food Inspection, environmental pollution monitoring evaluation etc. provide effective 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 lead ion and preparation method thereof.
Background technology
Plumbous (Pb) is a kind of cinerous heavy metal, is one of environmental poisonous substance known by us, 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, but lead contamination can not be ignored in air, soil, food, tableware, toy.Lead is Systematic toxin, has high accumulative and polyaffinity, is absorbed by approach such as digestion, breathings by body.Acute lead poisoning show as more have a stomach-ache, have a headache, tremble, nerve is irritated, go into a coma time serious, until dead.Chronic lead poisoning shows as neurotoxicity mostly, causes certain infringement to hematological system, immune system, kidney etc.Lead threatens one of maximum environmental contaminants to children, and children to be comparatively grown up sensitivity to lead element.Because blood-brain barrier is not grown complete, the Pb-B of infant 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 national limit standard in plumbous field, limited amount≤0.5mg/kg is had to lead content in agricultural product security quality safety requirements for non-environmental pollution aquatic products (GB18406.4-2001), regulation in groundwater quality standard (GB/T14848-93), mainly be applicable to the lead ion limitation≤0.05mg/L 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 little, but its sensitivity are not high; Electrochemical methods has the features such as sensitive, easy, high to the technical requirement of operating personnel; 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, estimating precision is high, simple, quick, but instrument costly, can only detect in laboratory, comparatively strict to operating personnel's technical requirement; By Naa is a kind of analytical approach based on nuclear reaction, have trace, fast, accurately and non-destructive and simultaneously can analyze the advantage of multielement, but equipment needed thereby is not easily by common laboratory is possessed, and is difficult to be used widely.(2) immunological analysis method: comprise enzyme linked immunosorbent assay (ELISA).With competitiveness enzyme-linked immune response for Cleaning Principle, measure light absorption value (OD) value by microplate reader after reaction solution and carry out result judgement, shorten detection time, qualitative and quantitative detection can be carried out to left drug.But ELISA method needs supporting microplate reader and matched reagent, and operating process is more complicated.
Therefore, develop quick, easy, responsive, special, economy, the quick testing product of lead ion that screening amount is large, for minimizing environmental pollution, 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 the monoclonal antibody of the hypersensitivity of lead 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 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 detecting lead ion, comprise box body, be provided with in box body with lead ion envelope antigen bag by ELISA Plate, the monoclonal antibody of lead ion, ELIAS secondary antibody, substrate nitrite ion, stop buffer, lead ion standard solution, washing lotion concentrate and the sample treatment liquid crossed.
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 lead ion standard solution is the series concentration solution that phosphate buffer (PBS) that lead 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 lead 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 plumbi nitras to be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen.
Described ELISA Plate wraps quilt as follows: envelope antigen is Pb
2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, wrap by solution to be 0.05moL/L, pH be 9.6 carbonate buffer solution, 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 lead 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 of 100uL, 1.5moL/L, shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg plumbi nitras to be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 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 Pb in antiserum
2+polyclonal antibody (the Pb of-EDTA chelate
2+-EDTApAb) to tire, stop band restrain 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, 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 hybridoma cell strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Pb
2+-EDTA monoclonal antibody (Pb
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 lead ion, kit comprises Pb
2+the ELISA Plate that-ITCBE-OVA bag is closed by the Swine serum also with 5%; C1 liquid: working concentration is the lead ion monoclonal antibody 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; Lead ion standard solution: the nitric acid with 2% dilutes the plumbi nitras series concentration solution 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 Pb
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 incubation at 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 lead 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 plumbi nitras to be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 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 Pb in antiserum
2+polyclonal antibody (the Pb of-EDTA chelate
2+-EDTApAb) to tire, stop band restrain 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, 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 hybridoma cell strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Pb
2+-EDTA monoclonal antibody (Pb
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 lead ion, obtains appropriate molecule in conjunction with the artificial immunity antigen of ratio and envelope antigen, solves the immunogene of lead ion
propertyproblem, obtains high-titer, sensitivity, special polyvalent antibody after immune animal, for the establishment of kit is laid a good foundation.
2. the construction method of kit of the present invention, has prepared lead ion high-titer, sensitivity, special monoclonal antibody, and antibody titer of ascites is 1: 7.2 × 10
5, affinity costant
kabe 1.87 × 10
10l/mol, is less than 1% with the cross reacting rate of other heavy metal ion, and the trace for lead ion detects fast and provides guarantee.
3. lead ion indirect competitive enzyme-linked immunosorbent kit of the present invention, energy trace detection lead ion, have quick, easy, responsive, special, 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 0.2ng/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. 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, 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 illustrates:
Fig. 1 is artificial immunizing antigen Pb
2+the Technology Roadmap of-ITCBE-Protein synthesis;
Fig. 2 is the canonical plotting detecting lead 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, lead ion artificial immunity antigen
Adopt different sulphur hydrocyanic ester legal system for artificial complete antigen, 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) 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 plumbi nitras to be dissolved in 100uL distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen.
With legal system for immunizing antigen Pb
2+-ITCBE-BSA.
embodiment two, lead ion artificial immunity Antigen Identification
Bicinchoninic acid method is adopted 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, Concentration Testing typical curve is built by bicinchoninic acid method, the linear equation of BSA concentration standard curve is: y=0.0022x+0.008, R
2=0.9987, wherein y is sample is 562nm place absorbance at wavelength, and x is sample protein concentration.
ICP-AES method is adopted to measure Pb
2+pb in-ITCBE-BSA
2+concentration.By the Pb of 100 μ g/mL
2+pb 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, 0.3 μ g/mL, 0.4 μ g/mL, 0.5 μ g/mL, the automatic drawing standard curve of instrument software, and draw equation of linear regression Y=0.27X+0.0045, related coefficient 0.9998, measure under the optimum optimization experiment condition of 283nm wavelength, instrument software automatic analysis result.
the preparation of embodiment three, lead ion monoclonal antibody
(1) immune animal.Use Pb
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 Pb with sterile phosphate damping fluid (PBS)
2+-ITCBE-BSA, with equivalent Freund's complete adjuvant (CFA) mixing and emulsifying; Booster immunization, dilutes Pb 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 Pb in antiserum
2+with ethylenediamine tetraacetic acid (EDTA) chelate Pb
2+the polyclonal antibody (pAb) of-EDTA is tired, and stop band restrain detects Pb
2+-EDTApAb is to Pb
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-Pb of hybridoma secretion
2+ion monoclonal antibody (Pb
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, lead 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: 7.2 × 10
5.
(2) affinity qualification.Saturated ELISA mensuration affinity costant (
ka), the Pb of 3.4 μ g/mL and 1.7 μ g/mL is respectively by concentration
2+-ITCBE-OVA bag quilt, adds the Pb of doubling dilution
2+-ITCBEmAb, then add GaMIgG-HRP, TMB develop the color survey
a 450nmvalue, with Pb
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 450nmpb 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.Pb is measured with stop band restrain
2+-EDTAmAb 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 correlation regression analysis, calculates Pb
2+-EDTAmAb 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 (huge legendary turtle conjunction method is the same) of lead, mercury, chromium, zinc, copper, caesium, cobalt, molybdenum, iron and ITCBE and ITCBE solution as inhibitor, measures each mortifier with stop band restrain
iC 50 , with Pb
2+-ITCBEmAb is to Pb
2+-ITCBE's
iC 50 and Pb
2+-ITCBEmAb is to each competitor
iC 50
the percentage of ratio be its cross reacting rate (CR%).Qualification result, is less than 0.01% with other heavy metal ion cross reaction.
the preparation of embodiment five, lead 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) assembling of lead ion enzyme linked immunological kit:
Kit comprises Pb
2+-ITCBE-OVA bag quilt is also by the ELISA Plate that 5% Swine serum is closed; C1 liquid: working concentration is the monoclonal antibody of the lead 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; 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; 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 lead ion enzyme linked immunological kit: stop band restrain 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, 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=-35.074x+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 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 0.208ng/mL.
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 2.87 ~ 147.57ng/mL.
Accuracy measures.By Pb
2+standard items are respectively 2 with final concentration, 4,8,16,32ng/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 90.8% ~ 98.62%, average 96.21%, the coefficient of variation (CV) 10.6% ~ 13.7%, average 12.16%; The recovery of cow's milk is 93.12% ~ 98.82%, and average 96.68%, CV 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 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 Pb
2+mAb is to Pb
2+'s
iC 50with to other each competitor
iC 50percent be its cross reacting rate (CR%).In table 3.
Table 3Pb
2+the cross reaction of-EDTAmAb and other metallo-chelate
(4) 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.In table 4.
The storage life of table 4 kit
Claims (6)
1. one kind is detected the indirect competitive enzyme-linked immunosorbent kit of lead ion, comprise box body, it is characterized in that: be provided with in box body with lead ion envelope antigen bag by ELISA Plate, the monoclonal antibody of lead ion, ELIAS secondary antibody, substrate nitrite ion, stop buffer, lead ion standard solution, washing lotion concentrate and the sample treatment liquid crossed;
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; Described stop buffer is the sulfuric acid solution of 2mol/L; Described lead ion standard solution is the series concentration solution that phosphate buffer that lead 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 the edta solution of 0.1mol/L; Solid phase material for the preparation of described ELISA Plate is polystyrene, tygon or polypropylene;
Described lead ion envelope antigen is prepared by following methods:
(1) take 20mg oralbumin 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 to be dissolved in 1mL dimethyl sulfoxide (DMSO) and to form metal-chelating agent solution;
(3) be added in carrier protein solution by 0.5mL metal-chelator dropwise, add the tri-n-butylamine of 100 μ L, 1.5mol/L after mixing, shaking table room temperature reaction 24h, makes OVA-ITCBE solution;
(4) get 11.25mg plumbi nitras to be dissolved in 100 μ L distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-OVA envelope antigen, collect packing ,-20 DEG C frozen.
2. indirect competitive enzyme-linked immunosorbent kit according to claim 1, is characterized in that: described ELISA Plate wraps quilt as follows: envelope antigen is Pb
2+-ITCBE-OVA, bag is 2 μ g/mL by concentration, wrap by solution to be 0.05mol/L, pH be 9.6 carbonate buffer solution, 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.
3. the indirect competitive enzyme-linked immunosorbent kit according to any one of claim 1-2, is characterized in that: described lead ion monoclonal antibody is prepared by the following method:
(1) artificial immunity antigen synthesis: take that 20mg bovine serum albumin(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 to be dissolved in 1mL dimethyl sulfoxide (DMSO) and to form metal-chelating agent solution; Be added in BSA carrier protein solution by 0.5mL metal-chelator dropwise, after mixing, add the tri-n-butylamine of 100 μ L, 1.5mol/L, shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg plumbi nitras to be dissolved in 100 μ L distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 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 Pb in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and stop band restrain 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, 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 hybridoma cell strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Pb
2+-EDTA monoclonal antibody, get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freund's adjuvant 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.
4. the construction method detecting the indirect competitive enzyme-linked immunosorbent kit of lead ion as claimed in claim 1, is characterized in that: described kit comprises Pb
2+the ELISA Plate that-ITCBE-OVA bag is closed by the Swine serum also with 5%; C1 liquid: working concentration is the lead ion monoclonal antibody 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; C5 liquid: stop buffer is the sulfuric acid solution of 2mol/L; Described lead ion standard solution is the series concentration solution that phosphate buffer that lead ion is dissolved in 0.01mol/L, pH7.4 obtains; 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.
5. the construction method of enzyme linked immunological kit according to claim 4, is characterized in that: described ELISA Plate wraps quilt as follows: envelope antigen is Pb
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 incubation at 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.
6. the construction method of enzyme linked immunological kit according to claim 5, is characterized in that: described lead ion monoclonal antibody is prepared by the following method:
(1) artificial immunity antigen synthesis: take that 20mg bovine serum albumin(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 to be dissolved in 1mL dimethyl sulfoxide (DMSO) and to form metal-chelating agent solution; Be added in BSA carrier protein solution by 0.5mL metal-chelator dropwise, after mixing, adding 100 μ L concentration is the tri-n-butylamine of 1.5mol/L, and shaking table room temperature reaction 24h, makes BSA-ITCBE solution; Get 11.25mg plumbi nitras to be dissolved in 100 μ L distilled water, form uniform Pb
2+solution; By Pb
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 Pb
2+-ITCBE-BSA, collect packing ,-20 DEG C are frozen;
(2) immune animal: with Pb
2+-ITCBE-BSA is immunizing antigen, using 6 week age female Balb/c mouse 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 Pb in antiserum
2+the polyclonal antibody of-EDTA chelate is tired, and stop band restrain 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, 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 hybridoma cell strain;
(5) monoclonal antibody is prepared: adopt in body and induce ascites legal system for anti-Pb
2+-EDTA monoclonal antibody, get healthy Balb/c female mice in 8 week age, lumbar injection incomplete Freund's adjuvant 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.
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| CN105823881A (en) * | 2016-05-18 | 2016-08-03 | 深圳先进技术研究院 | Immunoassay method and kit for quick detection of lead |
| CN106872708B (en) * | 2017-02-23 | 2019-07-19 | 河南科技学院 | Competitive ELISA detection kit and its application in lead ion environmental pollution |
| CN108264553B (en) * | 2018-02-09 | 2021-03-30 | 浙江工商大学 | Preparation method of heavy metal lead artificial antigen and application of NOTA in preparation of heavy metal lead artificial antigen reagent |
| CN108456253B (en) * | 2018-02-09 | 2021-06-08 | 中国科学院广州生物医药与健康研究院 | Heavy metal ion specific secondary antibody and preparation method and application thereof |
| CN110407931A (en) * | 2018-12-10 | 2019-11-05 | 浙江工商大学 | A kind of lead ion artificial antigen and its application |
| CN111273028B (en) * | 2020-02-25 | 2023-03-28 | 芜湖天明生物技术有限公司 | rhTSG-6 direct competition ELISA quantitative detection kit and use method and application thereof |
| CN112379088B (en) * | 2020-12-04 | 2022-05-31 | 福建农林大学 | Detection test paper for detecting lead residue based on gold nanoflower technology |
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