CN103499688A - Gold-labeled test strip/card for quickly detecting mercury ions - Google Patents
Gold-labeled test strip/card for quickly detecting mercury ions Download PDFInfo
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Abstract
The invention discloses a gold-labeled test strip/card for quickly detecting mercury ions. The gold-labeled test strip/card comprises a bottom support plate layer (1), a sample pad (2), a gold-labeled antibody binding pad (3), a cellulose membrane (4) and an absorbent pad (5). The gold-labeled test strip/card can quickly detect the residual contamination of the mercury ions in environments, soil, water, food, drugs and cosmetics.
Description
Technical field:
What the present invention relates to is a kind of gold label test strip for the fast detecting mercury ion/card and preparation method thereof and application, belongs to immunology and the sanitary inspection field that learns a skill.
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 mercury vapour and mercury has severe toxicity more.It is with a long history that mercury is used, and the abundant business people in ancient times in period, just uses natural mercuric sulphide.Mercury of many uses, be usually used in manufacturing science surveying instrument, medicine, catalyzer, mercury vapor lamp, electrode, mercury fulminate, cosmetics etc.Mercury, at the occurring in nature ubiquity, all contains micro-mercury in general animals and plants.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, has also caused the great attention of the Chinese government, and the mercury pollution control is listed in " 12 planning " great special project.Mercury poisoning be take chronic as common, mainly, in production or use activity, sucks for a long time due to mercury vapour and mercury compound dust.Mercury vapour more easily sees through the alveolus wall cell membrane, with the lipid binding in blood, is distributed to very soon each tissue of whole body.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, trembles, 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.Fash is the erythema papule, can merge in flakes or form blister, and rear the something lost has pigmentation.
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 for testing environment mercury ion mainly contains:
(1) physics and chemistry analytical approach: comprise 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 own 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) immunological 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.Shorten 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 still 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.Colloidal gold immunity chromatography (GICA), it detects principle is when sample to be checked and after being adsorbed on colloid gold label thing (antibody or the monoclonal antibody) combination on film, pass through chromatography, with be fixed on specific antigen on film or antibody generation specific binding and be trapped, be gathered in to detect and be with, can be by being observed visually the colour developing result of colloid gold label thing.The colloidal gold immunochromatographimethod technology is easy to carry because having, easy and simple to handle, quick, high specificity, susceptibility is high, influence factor is few, be difficult for the advantage such as pollution without specific apparatus and sample, be applicable to batch samples is carried out to on-the-spot primary dcreening operation, thereby be widely used in the various fields such as immunology, histology, pathology and cell biology.
At present mostly ripe mercury detection technique is by large-sized analytic instrument, can't meet cheapness, Site Detection demand fast.And commercial rapid detection apparatus and mercury test strips, 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) that GB is stipulated, and poor selectivity, can't meet sensitive, testing requirement accurately.Therefore, study a kind of novel mercury ion fast detecting gold test strip product, can be quick, easy, responsive, special, economy, Site Detection heavy metal Hg that the screening amount is large
2+, be current in the urgent need to, for environmental contamination reduction, improve food quality, ensure food safety significant.
Summary of the invention:
Purpose of the present invention provides a kind of detection mercury ion that can be quick, easy, responsive, special to pollute residual fast detecting mercury ion gold label test strip and test card.In order to realize purpose of the present invention, intend adopting following technical scheme:
One aspect of the present invention relates to a kind of test strips for the fast detecting mercury ion, described base layer support plate (1), sample pad (2), golden labeling antibody pad (3), cellulose membrane (4), adsorptive pads (5), coated film (6) and the wild card (7) of comprising, it is characterized in that: described test strips is to take the base layer support plate as bottom support, and sample pad, golden labeling antibody pad, cellulose membrane, adsorptive pads and coated film are pasted on the base layer support plate successively.Described test card is to take test strips as basis, adds that wild card makes.Described base layer support plate and wild card are made by rigid plastic, and described sample pad and golden labeling antibody pad are made by glass fibre cotton; Described golden labeling antibody pad is coated with the monoclonal antibody that can identify mercury ion of colloid gold label; Stealthy detection line (T line) and nature controlling line (C line) are shown in described cellulose membrane middle part parallel successively, detection line is coated with the conjugate of mercury ion and carrier protein formation, and nature controlling line is coated with rabbit anti-mouse igg (RaMIgG) (or sheep anti-mouse igg (GaMIgG)).
Described mercury ion gold label test strip/card, described sequestrant comprises isothiocyanic acid benzyl ethylenediamine tetraacetic acid (ITCBE), ethylenediamine tetraacetic acid (EDTA), diethylene triamine pentacetic acid (DTPA) (DTPA), reduced glutathione (GSH); Described carrier protein comprises chicken ovalbumin (OVA), bovine serum albumin(BSA) BSA, keyhole limpet hemocyanin (KLH).
Described mercury ion gold label test strip/card is characterized in that: described detectable antigens mercury ion-sequestrant-carrier protein, and with Hg
2+-ITCBE-OVA is example, and its preparation process is as follows:
(1) take the carrier protein solution that 20mg OVA is dissolved in the middle 20mg/mL of formation of HEPES damping fluid (10mM/L) of 1mL pH9.0.
(2) take 10mg 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 OVA carrier protein solution, after mixing, adds 100ul tri-n-butylamine (1.5M), shaking table room temperature reaction 24h, make OVA-ITCBE solution.
(4) get 11.25mg mercuric nitrate (Hg (NO
3)
2) be dissolved in 100ul distilled water, form uniform Hg
2+solution;
(5) 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 into PBS dialysis 10d in bag filter, forms Hg
2+-ITCBE-OVA, collect packing, and-20 ℃ frozen.
Described metal mercury ions gold label test strip/card, it is characterized in that: prepared by following steps by described gold test strip:
(1) colloidal gold solution preparation: adopt the preparation of citrate reducing process, get the 1000mL Erlenmeyer flask, add the 975mL distilled water to boil, add the 15mL trisodium citrate to continue heating 5min, add the 10mL1% gold chloride, continue to be heated to solution and present Chinese red, after being cooled to room temperature, 4 ℃ save backup.
(2) monoclonal antibody preparation: with mercury ion-sequestrant-carrier protein immune mouse, with Hg
2+-ITCBE-OVA is example, is described as follows: use Hg
2+5 of 6 weeks female BALB/C mice of-ITCBE-OVA immunity, dosage be 50 μ g/0.2mL/ only, the subcutaneous branch injection in back.Select the standby mouse of Fusion of Cells, indirect ELISA detects Hg in antiserum
2+chelate pAb tires, and blocking-up ELISA detects Hg
2+-ITCBE pAb is to Hg
2+the IC of-ITCBE
50, select the highest, the IC that tires
50minimum mouse, super exempting from for Fusion of Cells.Adopt the aseptic spleen of getting of cell-fusion techniques to prepare splenocyte, merged with NS0 myeloma cell under 50%PEG (pH8.0) effect; Carry out the screening of positive hybridoma cell strain with indirect ELISA and blocking-up ELISA, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is high, 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; Adopt afterwards in body and induce the standby Hg of ascites legal system
2+-ITCBE-OVA mAb, get healthy Balb/c female mice in 8 week age, and lumbar injection FIA0.5mL/ only, is used in 10～15 days afterwards.The centrifugal 10min of positive hybridoma cell 1000rpm 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, collected, in 37 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, and the centrifugal 5min of 12000rpm discards 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.
(3) golden labeling antibody preparation: will the centrifugal 30min of monoclonal antibody solution 10000r/min be marked, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution; According to the coagulation phenomenon, determine mercury ion-sequestrant-carrier protein mAb and the suitableeest label concentration of colloidal gold solution, Hg
2+-ITCBE-OVAmAb is 0.175mg/mL.Get colloidal gold solution 10mL, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution.The concentration of getting equivalent is the 0.175mg/mL monoclonal antibody solution, under magnetic agitation, mixes, and incubated at room 15min, the centrifugal 30min of 10000r/min, abandon supernatant, sediment 0.02mol/LNa
2b
4o
7solution (containing 10g/L OVA, 0.5g/L Sodium azide) dilution, 4 ℃ save backup.
(4) golden labeling antibody pad preparation: cut the glass fibre cotton that specification is 20 * 4mm, golden labeling antibody evenly is sprayed on glass fibre cotton with the unidirectional specking instrument of X-only, 37 ℃ of dry 1h, sealing, 4 ℃ save backup.
(5) detection line, nature controlling line preparation on nitrocellulose filter: the mercury ion-sequestrant that is 1mg/ml by concentration-carrier protein is put in the unidirectional specking instrument of X-only storage pool A, the RaMIgG that concentration is 1mg/ml (GaMIgG) is put in storage pool B, the fixed fire of start difference is in film central authorities, form detection line and the nature controlling line of spacing 0.5cm, natural drying, sealing, 4 ℃ save backup.
(6) gold label test strip assembling: be pasted on successively on back up pad in order by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads, and paste the MAX line in sample pad and golden labeling antibody pad surface, cut into the gold test strip of width 4mm on slot-cutting machine.
(7) gold test strip card assembling: the gold label test strip prepared is added to wild card makes.
On described mercury ion gold label test strip/card detection line, the package amount of mercury ion-sequestrant-carrier protein is 1 μ L/cm, and carrier protein concentration is 1mg/mL.The package amount of RaMIgG on described nature controlling line (or GaMIgG) is 1 μ L/cm, and the concentration of RaMIgG (or GaMIgG) is 1mg/mL.
A kind of gold label test strip of fast detecting mercury ion/card, its detecting step:
(1) sample pretreating method: sample comprises soil, water, food, feed, animal tissue, blood, urine, cosmetics, medicine.Thing to be checked adopts the pressure dispelling tank to clear up pre-service.Get 1.000-3.000g sample (solid thing to be checked grinds and smashs to pieces) or 3ml fluid sample and put into the teflon inner canister, add nitric acid 3ml soaked overnight.Add 30% hydrogen peroxide 3ml, build inner cap, screw stainless steel outer sleeve, put into thermostatic drying chamber, 120-140 ℃, 3h, naturally cool to room temperature, filters digestive juice, proceeds in the 10ml volumetric flask stand-by.According to the 1:1 volume, treatments of the sample liquid and 10% sequestrant (ITCBE, EDTA, DTPA, GSH) are mixed, make the abundant chelating of mercury ion and sequestrant, form sample liquid to be measured.
(2) detecting step: get 100 μ L testing samples, described mercury ion gold label test strip is inserted in testing sample, room temperature reaction 5min, horizontal positioned, observations, after 10min, result is invalid.Or draw testing sample 100 μ L with dropper, and drip on the sample well of test card, reaction 5min, observations, after 10min, result is invalid.
(3) analyzing and testing result: T line, C line all develop the color, and are judged to feminine gender, mean that in sample, ion concentration of mercury is less than in 2ng/mL or sample not containing mercury ion; Only C line colour developing, and the T line does not develop the color, and is judged to the positive, means in sample that ion concentration of mercury is greater than 1ng/mL; If the C line does not develop the color, no matter whether the T line develops the color, and all is judged to gold test strip and loses efficacy.
The detection sensitivity of described mercury ion gold test strip is 1ng/mL; Described mercury ion gold test strip can be applicable to mercury ion in environment, soil, water, food, cosmetics, medicine and pollutes in residual fast detecting.
The accompanying drawing explanation:
The structural representation that Fig. 1 is mercury ion fast detecting gold label test strip: wherein 1, sample pad, 2, golden labeling antibody pad, 3, nature controlling line (C line), 4, detection line (T line), 5, adsorptive pads, 6, nitrocellulose filter, 7, back up pad, 8, coated film;
The colour developing result schematic diagram that Fig. 2 is mercury ion fast detecting gold test strip;
Fig. 3 is mercury ion fast detecting gold test strip card structure schematic diagram: wherein 1, handle, and 2, nature controlling line, 3, detection line, 4, the loading end.
Embodiment:
Embodiment mono-, the preparation of mercury ion artificial immunity antigen
Adopt preparation artificial immunity antigen mercury ion-sequestrant-carrier protein, with Hg
2+-ITCBE-OVA is example, takes the carrier protein solution that 20mg ovalbumin (OVA) is dissolved in the middle 20mg/mL of formation of HEPES damping fluid (10mM/L) of 1mL pH9.0.Take 10mg1-(the different thiocyanate phenyl of 4-)-ethylenediamine tetraacetic acid (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 OVA carrier protein solution, after mixing, adds 100ul tri-n-butylamine (1.5M), shaking table room temperature reaction 24h, make OVA-ITCBE solution.Get 11.25mg mercuric nitrate (Hg (NO
3)
2) 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 into PBS dialysis 10d in bag filter, forms Hg
2+-ITCBE-OVA, collect packing, and-20 ℃ frozen.Prepare artificial immunity antigen Hg
2+-ITCBE-OVA.
Ultraviolet spectrophotometry scans the maximum absorption band of visible OVA at the 280nm place, and conjugate is in the situation that shown the superposition character of spectrogram separately in the concentration ultraviolet scanning spectrum identical with OVA concentration; The mobility speed of the visible OVA of SDS-PAGE electrophoresis result is greater than conjugate, and conjugate Hg is described
2+the molecular weight of-ITCBE-OVA is greater than OVA, proves the coupling success.
So the SM aqueous solution does not have characteristic absorption peak under ultraviolet wavelength,
Embodiment bis-, mercury ion artificial immunity antigen are identified
Adopt the bicinchoninic acid method to measure Hg
2+carrier protein OVA concentration in-ITCBE-OVA.Using OVA 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 OVA concentration standard curve is: y=0.0035x+0.0065, R
2=0.9981, wherein y is that sample is 562nm place absorbance at wavelength, and x is sample protein concentration.
Adopt the ICP-AES method to measure Hg
2+-ITCBE-OVA Hg
2+in concentration.Hg by 100 μ g/mL
2+hg in-ITCBE-OVA
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.
Embodiment tri-, the preparation of anti-mercury ion monoclonal antibody
Use Hg
2+5 of 6 weeks female BALB/C mice of-ITCBE-OVA immunity, dosage be 50 μ g/0.2mL/ only, the subcutaneous branch injection in back.Select the standby mouse of Fusion of Cells, indirect ELISA detects Hg in antiserum
2+chelate pAb tires, and blocking-up ELISA detects Hg
2+-ITCBE pAb is to Hg
2+the IC of-ITCBE
50, select the highest, the IC that tires
50minimum mouse, super exempting from for Fusion of Cells.Adopt the aseptic spleen of getting of cell-fusion techniques to prepare splenocyte, merged with NS0 myeloma cell under 50%PEG (pH8.0) effect; Carry out the screening of positive hybridoma cell strain with indirect ELISA and blocking-up ELISA, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is high, 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; Adopt afterwards in body and induce the standby Hg of ascites legal system
2+-ITCBE-OVA mAb, get healthy Balb/c female mice in 8 week age, and lumbar injection FIA0.5mL/ only, is used in 10～15 days afterwards.The centrifugal 10min of positive hybridoma cell 1000rpm 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, collected, in 37 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, and the centrifugal 5min of 12000rpm discards 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.
Embodiment tetra-, anti-mercury ion Identification of Monoclonal Antibodies
Titer of ascites is measured.To the injection of the mouse peritoneal after lumbar injection whiteruss 10d cloning cell line 10
7individual cell, extract ascites after 7d, the saturated ammonium sulfate salting out method is purified, and indirect ELISA is measured and tired.Measurement result, the monoclonal antibody titer of ascites is 1:6.9 * 10
5.
Affinity is identified.Saturated ELISA measures affinity costant (Ka), is 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, A is surveyed in the TMB colour developing
450nmvalue, with Hg
2+-ITCBE mAb concentration is horizontal ordinate, with A
450nmvalue, for ordinate, is drawn corresponding 2 response curves, with the A of every curve upper planar section
450nmvalue, as 100%, is calculated 50%A on curve
450nmcorresponding Hg during value
2+-ITCBE mAb concentration, according to formula Kaff=(n-1)/2 (n[Ab '] t-[Ab] t) calculating K a.The Ka of monoclonal antibody is 1.52 * 10
10l/mol.
Susceptibility is identified.With blocking-up, ELISA measures Hg
2+-ITCBE mAb is to variable concentrations Hg
2+the inhibiting rate of-ITCBE, with inhibiting rate B/B
0for ordinate, with variable concentrations Hg
2+the logarithm value of-ITCBE is horizontal ordinate, and drawing standard suppresses curve, carries out the correlation regression analysis, calculates Hg
2+-ITCBE mAb is to Hg
2+the IC of-ITCBE
50.Qualification result, IC
50be 28.14 μ g/L.
Specificity identification.Adopt its specificity of cross reaction test for identification.The huge legendary turtle compound (it is the same that huge legendary turtle is closed method) of cross reaction test selection mercury, chromium, lead, zinc, copper, caesium, cobalt, molybdenum, iron and ITCBE and ITCBE solution, as inhibitor, are measured the IC of each mortifier with blocking-up ELISA
50, with Hg
2+-ITCBE mAb is to Hg
2+the IC of-ITCBE
50and Hg
2+the IC of-ITCBE mAb to each competition thing
50the percentage of ratio be its cross reacting rate (cross-reactivity, CR%).Qualification result, be less than 0.01% with other heavy metal ion cross reaction.
Embodiment five, mercury ion gold label test strip/blocking are standby
(1) colloidal gold solution preparation: adopt the preparation of citrate reducing process, get the 1000mL Erlenmeyer flask, add the 975mL distilled water to boil, add the 15mL trisodium citrate to continue heating 5min, add the 10mL1% gold chloride, continue to be heated to solution and present Chinese red, after being cooled to room temperature, 4 ℃ save backup.
(2) golden labeling antibody preparation: will the centrifugal 30min of monoclonal antibody solution 10000r/min be marked, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution; According to the coagulation phenomenon, determine mercury ion-sequestrant-carrier protein mAb and the suitableeest label concentration of colloidal gold solution, Hg
2+-ITCBE-OVAmAb is 0.175mg/mL.Get colloidal gold solution 10mL, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution.The concentration of getting equivalent is the 0.175mg/mL monoclonal antibody solution, under magnetic agitation, mixes, and incubated at room 15min, the centrifugal 30min of 10000r/min, abandon supernatant, sediment 0.02mol/LNa
2b
4o
7solution (containing 10g/L OVA, 0.5g/L Sodium azide) dilution, 4 ℃ save backup.
(3) golden labeling antibody pad preparation: cut the glass fibre cotton that specification is 20 * 4mm, golden labeling antibody evenly is sprayed on glass fibre cotton with the unidirectional specking instrument of X-only, 37 ℃ of dry 1h, sealing, 4 ℃ save backup.
(4) detection line, nature controlling line preparation on nitrocellulose filter: the mercury ion-sequestrant that is 1mg/ml by concentration-carrier protein is put in the unidirectional specking instrument of X-only storage pool A, the RaMIgG that concentration is 1mg/ml (GaMIgG) is put in storage pool B, the fixed fire of start difference is in film central authorities, form detection line and the nature controlling line of spacing 0.5cm, natural drying, sealing, 4 ℃ save backup.
(5) gold label test strip assembling: be pasted on successively on back up pad in order by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads, and paste the MAX line in sample pad and golden labeling antibody pad surface, cut into the gold test strip of width 4mm on slot-cutting machine.
(6) gold test strip card assembling: the gold label test strip prepared is added to wild card makes.
Embodiment six, mercury ion gold label test strip/card performance measurement
(1) sensitivity testing of mercury ion gold label test strip of the present invention/card
Measure with the mercury ion gold test strip that concentration is respectively 0,1,2,4,8,16,32,64,128, the mercury ion standard items of 256ng/mL, each concentration is established 6 repetitions, according to the color of detection line and control line, carries out the naked eyes judgement or with reading bar instrument value read.Two lines appear in the colour developing district, and the T line color obviously is shallower than the C line color; A C line only appears in the district that perhaps develops the color, and the T line does not develop the color, and is judged to the positive.Positive findings means content mercury ion >=1ng/mL; Detection line (T line) and two lines of control line (C line) appear in the colour developing district, and the T line color is not shallower than the C line color, is judged to feminine gender.Invalid: colour developing two, district line (T line and C line) does not all occur, shows to operate wrong or test paper inefficacy, and it is invalid to be judged to.The results are shown in Table 1, as shown in Table 1, the range estimation of mercury ion gold test strip detects and is limited to 1ng/mL.
Table 1 is measured gold test strip sensitivity testing (n=6)
| Ion concentration of mercury (ng/mL) | 0 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 |
| Result | - | + | + | + | + | + | + | + | + | + |
(2) specific test of mercury ion gold label test strip of the present invention/card
The cross reacting rate of take is judged the specificity of this test paper as index.Employing cross reaction test, selecting chelate, the ITCBE of the metallic ion such as lead, molybdenum, cadmium, copper, zinc, chromium, cobalt, iron and ITCBE is mortifier, measure with the mercury ion gold test strip that final concentration is respectively 0,1,2,4,8,16,32,64,128,256ng/mL heavy metal ion standard items, each concentration is established 6 repetitions, with this, judges its specificity.The results are shown in Table 2, mercury ion gold test strip and mercury ion specific binding, with other heavy metal ion no cross reaction, cross reaction is 2ng/mL.
Table 2 is measured gold test strip specific test (n=6)
(3) replica test of mercury ion gold label test strip of the present invention/card
Get 6 batches of mercury ion gold test strips of different batches, respectively 6d to ion concentration of mercury be 0,1,2, the cosmetics of 4ng/mL prepare sample, water sample, cow's milk prepare sample and detected, each concentration is established 6 repetitions, checks its repeatability.The results are shown in Table 3, testing result is in full accord, proves that the gold test strip testing result of different batches production is reliable and stable, has good repeatability.
Table 3CAP-Strip replica test (n=6)
(4) storage life of mercury ion gold label test strip of the present invention/card test
Retention period check: the mercury ion gold test strip of different batches is stored in respectively to general refrigerator (2～8 ℃) and within 12 months, preserves 6 months with drying at room temperature, the testing result of different holding times of research, determine its storage life.Result shows, mercury ion gold test strip its outward appearance, accuracy, susceptibility, specificity etc. in storage life all do not change, identical with the test result of the standby test paper of new system, its term of validity be 2～8 ℃ 12 months, lower 6 months of drying at room temperature condition.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (10)
1. the gold label test strip for the fast detecting mercury ion, comprise base layer support plate (1), sample pad (2), gold labeling antibody pad (3), cellulose membrane (4), adsorptive pads (5), it is characterized in that: described test strips is to take the base layer support plate as bottom support, by sample pad, gold labeling antibody pad, cellulose membrane, adsorptive pads and coated film are pasted on the above base layer support plate of base layer support plate successively and wild card is made by rigid plastic, described sample pad and golden labeling antibody pad be take glass fibre cotton as matrix, described golden labeling antibody pad is coated with the monoclonal antibody that can identify mercury ion of colloid gold label, stealthy detection line (T line) and nature controlling line (C line) are shown in described cellulose membrane middle part parallel successively, detection line is coated with the conjugate that mercury ion and carrier protein form by sequestrant, and nature controlling line is coated with rabbit anti-mouse igg (RaMIgG) or sheep anti-mouse igg (GaMIgG)).
2. fast detecting mercury ion gold label test strip according to claim 1 is characterized in that: described sequestrant is selected from a kind of in isothiocyanic acid benzyl ethylenediamine tetraacetic acid (ITCBE), ethylenediamine tetraacetic acid (EDTA), diethylene triamine pentacetic acid (DTPA) (DTPA), reduced glutathione (GSH); Described carrier protein is selected from a kind of in chicken ovalbumin (OVA), bovine serum albumin(BSA) (BSA), keyhole limpet hemocyanin (KLH); Preferably, described conjugate is Hg
2+-ITCBE-OVA.
3. fast detecting mercury ion gold label test strip according to claim 1 and 2, is characterized in that described monoclonal antibody is that the conjugate formed by sequestrant by antigen mercury ion and carrier protein adopts monoclonal antibody technique to prepare.
4. according to fast detecting mercury ion gold label test strip claimed in claim 3, it is characterized in that described conjugate is Hg
2+-ITCBE-OVA, described gold label test strip prepares in interior method by comprising the steps:
(1) take the carrier protein solution that 20mg OVA is dissolved in the middle 20mg/mL of formation of HEPES damping fluid (10mM/L) of 1mL pH9.0;
(2) take 10mg 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 OVA carrier protein solution, after mixing, adds 100ul tri-n-butylamine (1.5M), shaking table room temperature reaction 24h, make OVA-ITCBE solution;
(4) get 11.25mg mercuric nitrate (Hg (NO
3)
2) be dissolved in 100ul distilled water, form uniform Hg
2+solution;
(5) 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 into PBS dialysis 10d in bag filter, forms Hg
2+-ITCBE-OVA, collect packing, and-20 ℃ frozen;
(6) colloidal gold solution preparation: adopt the preparation of citrate reducing process, get the 1000mL Erlenmeyer flask, add the 975mL distilled water to boil, add the 15mL trisodium citrate to continue heating 5min, add the 10mL1% gold chloride, continue to be heated to solution and present Chinese red, after being cooled to room temperature, 4 ℃ save backup.
(7) monoclonal antibody preparation: with mercury ion-sequestrant-carrier protein immune mouse, with Hg
2+-ITCBE-OVA is example, is described as follows: use Hg
2+5 of 6 weeks female BALB/C mice of-ITCBE-OVA immunity, dosage be 50 μ g/0.2mL/ only, the subcutaneous branch injection in back.Select the standby mouse of Fusion of Cells, indirect ELISA detects Hg in antiserum
2+chelate pAb tires, and blocking-up ELISA detects Hg
2+-ITCBE pAb is to Hg
2+the IC of-ITCBE
50, select the highest, the IC that tires
50minimum mouse, super exempting from for Fusion of Cells.Adopt the aseptic spleen of getting of cell-fusion techniques to prepare splenocyte, merged with NS0 myeloma cell under 50%PEG (pH8.0) effect; Carry out the screening of positive hybridoma cell strain with indirect ELISA and blocking-up ELISA, limited dilution cloning is carried out in selection strong positive, the hole that inhibiting rate is high, 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; Adopt afterwards in body and induce the standby Hg of ascites legal system
2+iTCBE-OVA mAb, get healthy Balb/c female mice in 8 week age, and lumbar injection FIA0.5mL/ only, is used in 10～15 days afterwards.The centrifugal 10min of positive hybridoma cell 1000rpm 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/ is only; Produce ascites after inoculating cell 7-10 days, collected, in 37 ℃ of water-bath 30min, 4 ℃ of placements are spent the night, and the centrifugal 5min of 12000rpm discards 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;
(8) golden labeling antibody preparation: will the centrifugal 30min of monoclonal antibody solution 10000r/min be marked, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution; According to the coagulation phenomenon, determine mercury ion-sequestrant-carrier protein mAb and the suitableeest label concentration of colloidal gold solution, Hg
2+-ITCBE-OVAmAb is 0.175mg/mL.Get colloidal gold solution 10mL, use 0.1mol/L K
2cO
3solution is regulated the pH value to 8.2 of colloidal gold solution.The concentration of getting equivalent is the 0.175mg/mL monoclonal antibody solution, under magnetic agitation, mixes, and incubated at room 15min, the centrifugal 30min of 10000r/min, abandon supernatant, sediment 0.02mol/LNa
2b
4o
7solution (containing 10g/L OVA, 0.5g/L Sodium azide) dilution, 4 ℃ save backup;
(9) golden labeling antibody pad preparation: cut the glass fibre cotton that specification is 20 * 4mm, golden labeling antibody evenly is sprayed on glass fibre cotton with the unidirectional specking instrument of X-only, 37 ℃ of dry 1h, sealing, 4 ℃ save backup;
(10) detection line, nature controlling line preparation on nitrocellulose filter: the mercury ion-sequestrant that is 1mg/ml by concentration-carrier protein is put in the unidirectional specking instrument of X-only storage pool A, the RaMIgG that concentration is 1mg/ml (GaMIgG) is put in storage pool B, the fixed fire of start difference is in film central authorities, form detection line and the nature controlling line of spacing 0.5cm, natural drying, sealing, 4 ℃ save backup;
(11) gold label test strip assembling: be pasted on successively on back up pad in order by sample pad, golden labeling antibody pad, nitrocellulose filter, adsorptive pads, and paste the MAX line in sample pad and golden labeling antibody pad surface, cut into the gold test strip of width 4mm on slot-cutting machine.
5. according to the described fast detecting mercury ion of claim 1～4 gold label test strip, the package amount of mercury ion-sequestrant on detection line-carrier protein is 1 μ L/cm, and carrier protein concentration is 1mg/mL.
6. according to the described fast detecting mercury ion of claim 1～4 gold label test strip, it is characterized in that: the package amount of RaMIgG on described nature controlling line (or GaMIgG) is 1 μ L/cm, and the concentration of RaMIgG (or GaMIgG) is 1mg/mL.
7. according to the described fast detecting mercury ion of claim 1～4 gold label test strip, it is characterized in that: in described colloidal gold solution, the particle diameter of gold grain is 30nm, and labelled amount is the 1mL collaurum: the 3ng monoclonal antibody.
8. for detection of the gold test strip card of mercury ion, it is characterized in that: described test card is to take the described fast detecting mercury ion of claim 1-7 any one gold label test strip as basis, adds that wild card makes.
9. a fast detecting mercury ion gold label test strip as described as claim 1～7 or gold test strip for detection of mercury ion claimed in claim 8 are stuck in mercury ion in environment, soil, water, food, cosmetic product or medicine and pollute the application in residual fast detecting, preferably, described detection for detection of mercury ion content the sample at 10ng/mL.
10. application according to claim 9 is characterized in that: after sample pretreating method is Specimen eliminating, adopt 10%EDTA as the sequestrant Treatment Solution, the centrifuging and taking supernatant for detection of.
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| CN114518449A (en) * | 2022-03-18 | 2022-05-20 | 福建农林大学 | Latex microsphere immunochromatographic test strip for detecting heavy metal mercury ions and application thereof |
| CN114527270A (en) * | 2022-03-18 | 2022-05-24 | 福建农林大学 | Nanoflower immunochromatographic test strip for detecting heavy metal mercury ions and application thereof |
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