CN103412126A - Gold label test paper for rapidly detecting molybdic ions, well as preparation method and application thereof - Google Patents
Gold label test paper for rapidly detecting molybdic ions, well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses gold label test paper for rapidly detecting molybdic ions, as well as a preparation method and an application thereof. The bottom layer of the gold label test paper is a supporting layer, the middle layer of the gold label test paper is an adsorption layer, a protective film is fixedly arranged on the absorption layer, the absorption layer comprises a sample cushion, a gold label antibody combined cushion, a cellulose film layer and a handle-end absorbent cushion from a test end in sequence, and detection imprints printed by a carrier protein solution of coupling molybdic ions and contrast imprints printed by a rabbit anti-mouse IgG antibody solution are arranged on the absorbent cushion; a molybdic ion monoclonal antibody marked by colloidal gold is wrapped in the gold label antibody combined cushion. The gold label test paper can realize the rapid detection of molybdenum ion residual contamination in oil, water and food, and has the advantages of being specific, sensitive, rapid, easy and convenient, vivid and visual in result, economic, and the like; the gold label test paper can be used for screening of samples in large batch, also can be used for the rapid detection on samples in small batch, and is wide in application range.
Description
Technical field
The invention belongs to immunology and the sanitary inspection field that learns a skill, particularly relate to a kind of gold test strip for fast detecting sexavalence molybdenum ion and preparation method thereof and application.
Background technology
China has abundant molybdenum ore resource, 8,400,000 tons of total reservess, and molybdenum is as a kind of transiting state metal element, its compound presents five kinds of valence states, wherein the sexavalence molybdenum is the most stable, also close with the mankind's vital movement relation, lacks and the excessive quality that all can affect life.Molybdenum is one of essential trace element of organism, and various the organizing of human body all contains molybdenum, and becoming total amount in human body is 9mg, and liver, kidney content are the highest.Itself does not have biologically active molybdenum element, after only having the sexavalence molybdenum ion to be combined with purine to form coenzyme, just has biologically active, its physiological function mainly contains and maintains normal metabolism, improve the elasticity of arterial wall, reduce angiocardiopathy, by antioxidation, improve human body immune function, and can suppress the synthetic of nitrosamines carcinogen in vivo, have antitumaous effect; Sexavalence molybdenum shortage can cause the diseases such as carious tooth, kidney stone, Keshan disease, Kaschin-Beck disease, cancer of the esophagus.But the sexavalence molybdenum is excessive simultaneously, can cause that body is poisoning, when human intake's amount surpasses 10～15mg, can cause growth retardation, the illnesss such as gout sample syndrome, arthralgia and deformity, lung's sex change, even occur in Body weight loss.The similar copper deficiency disease of animal molybdenosis performance, Ferguson reported first in 1938 molybdenum too high levels in herbage, can make Grazing Cattle produce continuation diarrhoea, be the poisoning disease of feature by the hair decolouring.The farm cattle " red Pi Baimao disease " that " calf diarrhea " of Britain, Zelanian " ox peat rushes down " and Australia's " ox endemic hematuria disease " and China area, south, Jiangxi in 1981 occurs etc. are all the typical events of sexavalence molybdenosis.The sexavalence molybdenum is safe from harm to biosome under state of nature, but due to molybdenum, be widely used in the numerous areas such as special steel, machinery, oil, chemical industry, national defence, Aero-Space, electronics, nuclear industry, molybdenum ore is exploited in a large number, the sexavalence molybdenum ion enters nature with various forms, environmental pollution sharply increases the weight of, cumulative effect and bioconcentration in addition, environmental pollution and human health have formed serious threat.Therefore, great to the Clinical significance of detecting of sexavalence molybdenum content in ambient soil, water source, feed, food.
At present, testing environment sexavalence molybdenum ion mainly adopts physico-chemical analysis method and immune analysis method, and the physico-chemical analysis method comprises 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 is sensitive, easy etc., 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 detect in laboratory, and comparatively strict to operating personnel's technical requirement, limited it and be widely used; By Naa is a kind ofly to take nuclear reaction and be basic analytical approach, has advantages of trace, fast, accurately and non-destructive and can analyze multielement simultaneously, but required equipment is difficult for being possessed by common laboratory, is difficult to be used widely.
Therefore, develop quick, easy, responsive, special, economy, sexavalence molybdenum 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: the deficiency for existing sexavalence molybdenum ion detection technique provides a kind of gold test strip for fast detecting sexavalence molybdenum ion that can be quick, easy, responsive, special;
Preparation method and the application thereof of sexavalence molybdenum ion gold test strip also are provided.
Technical scheme of the present invention:
A kind of gold test strip for fast detecting sexavalence molybdenum ion, bottom is supporting layer, middle layer is adsorbed layer, diaphragm is fixed on adsorbed layer, adsorbed layer is followed successively by the adsorptive pads of sample pad, golden labeling antibody pad, cellulose rete and handle end from test lead, wherein on the cellulose rete, be provided with the detection trace that the carrier protein solution of coupling sexavalence molybdenum ion prints and the contrast trace of printing with the anti-mouse IgG antibody solution of rabbit; In described golden labeling antibody pad, be coated with the specificity sexavalence molybdenum ion monoclonal antibody of colloid gold label.
Described cellulose rete is made with nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane; Described sample pad is made with PVDF membrane, nylon membrane, glass fibre cotton or polyester film; Adsorptive pads is made with absorbent filter, and supporting layer is made with the toughness material do not absorbed water; Gold labeling antibody pad is made with glass fibre cotton.
Described
The carrier protein of coupling sexavalence molybdenum ion is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin; The package amount that detects the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On the contrast trace, the package amount of rabbit anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; Described detection trace and the contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace or " 10 " font arrangement trace.
Described diaphragm covers on sample pad, golden labeling antibody pad and adsorptive pads, and deflection sample pad one side 0.3-0.6 cm place is printed with the sample mark line on the sample pad diaphragm corresponding with golden labeling antibody pad intersection.
By following methods, prepared by the carrier protein solution of described coupling sexavalence molybdenum ion:
Get 20 mL molybdic acids and 1mL concentration and be 10 mmol/L, pH value and be 8.0 HEPES damping fluid and mix, obtain Mo
6+Solution; Take 10 mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid ITCBE and be dissolved in 1mL DMSO, form the metal-chelating agent solution; By Mo
6+Solution and metal-chelating agent solution mix, the pH value to 7.4 of regulator solution, and 25 ℃ are stirred 24 h, and rotating speed is 1000 r/min, forms Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA and be dissolved in the HEPES damping fluid that 1mL concentration is 10 mmol/L, pH 8.0, form carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens mother liquor joins in 1mL carrier protein solution, regulates pH value to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves into dialysis 7 d in bag filter, collects dislysate, prepares carrier protein Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 ℃ frozen standby.
Prepared by the following method by the specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label:
Adopt the preparation of citrate reducing process, in Erlenmeyer flask, add 975 mL distilled waters to boil, add the 15mL trisodium citrate, continue heating 5 min, adding 10 mL mass concentrations is 1% gold chloride, continue to be heated to solution and be Chinese red, obtain colloidal gold solution after being cooled to room temperature, 4 ℃ save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2CO
3Solution is regulated the pH value to 8.2 of colloidal gold solution, under magnetic agitation, dropwise adding concentration is the sexavalence molybdenum ion monoclonal antibody solution of 0.2 mg/mL, room temperature incubation 20 min, centrifugal 30 min of 10000 r/min, abandon supernatant, sediment with every liter contain 100 g BSA, concentration is the Na of 0.02 mol/L
2B
4O
7It is the Na of 0.02 mol/L that solution dilution, centrifugal 30 min of 10000 r/min then, the sediment obtained contain 10 g BSA, 0.5 g Sodium azide, concentration with every liter again
2B
4O
7Solution dilution, namely obtain the sexavalence molybdenum ion monoclonal antibody of colloid gold label, and 4 ℃ save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, in every 1mL collaurum, contains 3 ng sexavalence molybdenum ion monoclonal antibodies.
The preparation method of described gold test strip comprises the following steps:
(1) preparation of sexavalence molybdenum ion carrier protein couplet thing:
The HEPES damping fluid that to get 20 mL molybdic acids and 1mL concentration be 10 mmol/L, pH 8.0 mixes, and obtains Mo
6+Solution; Take 10 mg ITCBE and be dissolved in 1mL DMSO, form the metal-chelating agent solution; By Mo
6+Solution and metal-chelating agent solution mix, the pH value to 7.4 of regulator solution, and 25 ℃ are stirred 24 h, and rotating speed is 1000 r/min, forms Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA, being dissolved in 1mL concentration is in the HEPES damping fluid of 10 mmol/L, pH 8.0, forms carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens mother liquor joins in 1mL carrier protein solution, regulates pH value to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves into dialysis 7 d in bag filter, collects dislysate, prepares carrier protein couplet thing Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 ℃ are frozen standby;
(2) preparation of sexavalence molybdenum ion gold labeling antibody;
(3) preparation of golden labeling antibody pad;
(4) detect the preparation of trace and contrast trace;
With the sexavalence molybdenum ion carrier protein couplet thing of gained, on the cellulose rete, make the detection trace, on the cellulose rete, make the contrast trace by the anti-mouse IgG antibody of rabbit; The package amount that detects the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and the concentration of carrier protein is 1mg/mL; On described contrast trace, the package amount of rabbit anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL;
Sexavalence molybdenum ion gold labeling antibody, for the preparation of golden labeling antibody pad, then is assembled into gold test strip by supporting layer, adsorbed layer and diaphragm successively.
By following methods, prepared by described sexavalence molybdenum ion monoclonal antibody specific:
Immune animal: with Mo
6+-ITCBE-BSA is immunizing antigen, using the Balb/c mouse as immune animal, the subcutaneous multi-point injection in back, immunizing dose is every each 50～100 μ g, head exempts to use Freund's complete adjuvant, and booster immunization incomplete Freunds adjuvant, head carry out immunity for the second time 3 weeks after exempting from, once, immunity is 5 times altogether in all immunity in interval 2 later;
Select the standby mouse of Fusion of Cells: indirect ELISA detects Mo in antiserum
6+Chelate polyclonal antibody (pAb) is tired, and blocking-up ELISA detects Mo
6+-EDTA pAb is to Mo
6+The half-inhibition concentration of-EDTA (
IC 50), select tire the highest,
IC 50Minimum mouse, super exempting from for Fusion of Cells;
Prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG solution, through 4 limiting dilution assay subclones, obtain the cell strain of monoclonal antibody of the anti-sexavalence molybdenum ion of stably excreting;
Preparation monoclonal antibody: to the female mouse lumbar injection of multiparity hybridoma cell strain, gather the monoclonal antibody that the ascites purifying obtains anti-sexavalence molybdenum ion.
The application of described gold test strip in fast detecting sexavalence molybdenum ion.
Positive beneficial effect of the present invention:
(1) gold test strip of the present invention, detect sexavalence molybdenum ion speed fast, in 5min, goes out testing result, greatly saved detection time than physics and chemistry detection method (3 d);
(2) gold test strip of the present invention, detection method is easy, and without any need for auxiliary instrumentation and reagent, the people can operate per capita, easy to carry, but Site Detection;
(3) gold test strip of the present invention, susceptibility is high, and detection sensitivity is 5ng/mL, meets national limit standard requirement, suitable with the sensitivity of physics and chemistry detection method; High specificity, with other heavy metal ion no cross reaction; Economy, compare with the physics and chemistry detection method, detects a sample and can save at least testing cost more than 300 yuan.
(4) gold test strip convenient storage of the present invention, long shelf-life, and less demanding to storing temperature, the term of validity under 2-8 ℃ is 1 year, the drying at room temperature lower shelf-life of condition is six months.
(5) preparation method of gold test strip of the present invention, prepared sexavalence molybdenum ion carrier protein couplet thing, obtained suitable molecule in conjunction with than artificial immunity antigen and envelope antigen, obtained high-titer, sensitivity, special antiserum; Adopt the sodium citrate reducing process to prepare colloidal gold solution, course of reaction is gentleer, can under normal temperature and condition of neutral pH, carry out, and is easy to operate and control.
(6) preparation method of gold test strip of the present invention, prepared the monoclonal antibody of anti-sexavalence molybdenum ion high-titer, high-affinity, high specificity, and the antibody titer of ascites is 1: 6.4 * 10
5, affinity costant
KaBe 1.65 * 10
10L/moL, cross reacting rate is less than 0.01%, for the trace fast detecting of its sexavalence molybdenum ion provides guarantee.
(7) sexavalence molybdenum ion gold test strip of the present invention can be applicable to fast detecting soil, water, in food, the sexavalence molybdenum ion pollutes residual, have fast, easy, responsive, special, the economic dispatch characteristic, 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, also be the food import and export check, the Food Inspection law enforcement, 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, and have significant economic benefit and social benefit.
The accompanying drawing explanation
Fig. 1 is the plan structure schematic diagram of test strips of the present invention.
Fig. 2 is the side structure schematic diagram of Fig. 1 test strips.
Fig. 3 is the synthetic Technology Roadmap of sexavalence molybdenum ion carrier protein couplet thing.
In figure, 1 is supporting layer, and 2 is sample pad, and 3 is golden labeling antibody pad, and 4 is the cellulose rete, and 5 is adsorptive pads, and 6 for detecting trace, and 7 are the contrast trace, and 8-1 is the test lead diaphragm, and 8-2 is the handle end diaphragm, and 9 is the sample mark line.
Embodiment
Below with embodiment, illustrate the present invention, but do not mean any limitation of the invention, as special instruction not, percentage composition wherein is weight percentage.
Prepare the gold test strip that detects the sexavalence molybdenum ion, at first will prepare the artificial immunity antigen of sexavalence molybdenum ion, immune Balb/c mouse afterwards, prepare the monoclonal antibody of anti-sexavalence molybdenum ion, assembles on this basis the gold test strip of sexavalence molybdenum ion.
The preparation of embodiment mono-, sexavalence molybdenum ion artificial immunity antigen
Adopt different sulphur hydrocyanic ester method.Getting the 20mL molybdic acid, is that 10mmol/L pH value is that 8.0 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid mixes and forms Mo with concentration
6+Solution; Take 10mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid (ITCBE) and be dissolved in 1mL dimethyl sulfoxide (DMSO) (DMSO), form the metal-chelating agent solution; By Mo
6+After solution and metal-chelating agent solution mix, the pH value to 7.4 of regulator solution, 25 ℃ are stirred 24 h, and rotating speed is 1000 r/min, forms Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA, be dissolved in 1mL concentration and be 10 mmol/L, pH value and be in 8.0 HEPES damping fluid, form carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens solution joins in 1mL carrier protein solution, regulates pH value to 9.0, and at room temperature 1000 r/min stir 24 h, then moves into dialysis 7 d in bag filter, collects dislysate, prepares sexavalence molybdenum ion artificial immunity antigen
(The carrier protein couplet thing) Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 ℃ frozen standby.Referring to Fig. 3.
The evaluation of embodiment bis-, sexavalence molybdenum ion artificial immunity antigen
Adopt the bicinchoninic acid method to measure Mo
6+The concentration of carrier protein BSA in-ITCBE-BSA.Using BSA as standard protein, build concentration examination criteria curve by the bicinchoninic acid method.The linear equation of BSA concentration standard curve is: y=0.0004x+0.0072, 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 Mo
6+Mo in-ITCBE-BSA
6+Concentration.Mo by 100 μ g/mL
6+Standard reserving solution, the nitric acid with 2% are diluted to the concentration gradient of 0 μ g/mL, 0.25 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 4 μ g/mL, the automatic drawing standard curve of instrument software, and draw equation of linear regression; 50 times of dilutions of sample solution, measure under the optimum optimization experiment condition of 226 nm wavelength, instrument software automatic analysis result.
The preparation of embodiment tri-, anti-sexavalence molybdenum ion monoclonal antibody
(1) animal immune.Immunity Balb/c mouse, use Mo
6+5 of-ITCBE-BSA immunity female Balb/C mouse in 6 age in week, 50 μ g0.2 mL/, the subcutaneous multi-point injection in back.Head exempts from, with sterilizing PBS dilution Mo
6+-ITCBE-BSA, with equivalent CFA mixing and emulsifying; Booster immunization, with sterilizing PBS dilution Mo
6+-ITCBE-BSA, with equivalent IFA mixing and emulsifying, after head exempts from, carry out immunity for the second time 3 weeks, once, immunity is 5 times altogether for all immunity in later interval 2, after the immunity, blood is got in 10 d dockings for the third time, 37 ℃ of water-bath 30 min, 4 ℃ of placements are spent the night, centrifugal 5 min of 4,000 4 ℃ of r/min, get supernatant ,-20 ℃ save backup.
(2) the standby mouse of Fusion of Cells is selected.Indirect ELISA detects Mo in antiserum
6+Chelate pAb tires, and blocking-up ELISA detects Mo
6+-EDTA pAb is to Mo
6+-EDTA's
IC 50, select tire the highest,
IC 50Minimum mouse, after super exempting from for Fusion of Cells.
(3) positive hybridoma cell strain screening.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.With 50% the PEG(pH 8.0 of 1mL suction pipe by preheating) be added drop-wise in fusion pipe, the limit edged shakes fusion pipe gently, in 1min, adds, 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 Tissue Culture Plate in 96 holes, every hole 100 μ L, be placed in the CO of 37 ℃ 5%
2In incubator, cultivate.
The screening of positive hybridoma cell, carry out the screening of positive hybridoma cell strain with indirect ELISA and blocking-up ELISA.Selection strong positive, the hole that inhibition is good, Growth of Cells is vigorous, carry out limited dilution cloning 3 times, respectively at recovery hybridoma after frozen 15 d, 30 d and 60 d, after cultivation, get supernatant, indirect ELISA is measured antibody titer, investigates the stability of hybridoma secrete monoclonal antibody.
(4) preparation of monoclonal antibody.Adopt in body and induce the standby monoclonal antibody of ascites legal system.Get 8 age in week healthy Balb/c female mice, lumbar injection IFA 0.5 mL/ only, is used after 10～15 d.Centrifugal 10 min of positive hybridoma cell 1000 r/min by cultivating, abandon supernatant, the collecting cell precipitation.With sterilizing PBS, cell precipitation is suspended, mixes, cell number is adjusted to 10
6Individual/mL, lumbar injection Balb/C mouse 0.5 mL/ are only.After inoculating cell 7-10 d, produce ascites, collect, in 37 ℃ of water-bath 30 min, 4 ℃ of placements are spent the night, and the centrifugal 5min of 12000 r/min 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.
The evaluation of embodiment tetra-, anti-sexavalence molybdenum ion monoclonal antibody
(1) hypotype is identified.Take out the hybridoma supernatant of cultivating, after dilution in 1: 50, operate with reference to mouse monoclonal antibody subtype identifying test paper bar instructions.Get dilution 0.15 mL and add in the kit tubule, room temperature is placed 1 min.After it dissolves naturally, mix gently, then test strips is inserted into to the tubule bottom, after 5 min, when band foremost occurs in the middle of to "+" two, i.e. the secreted Subclass of antibody of visible and hybridoma cell strain and the position at the corresponding place of light chain type, thereby definite antibody subtype.Qualification result: the hypotype of monoclonal antibody is IgG
1/ λ.
(2) binding capacity is measured.Indirect ELISA is measured its binding capacity.Measurement result, monoclonal antibody titer of ascites are 1: 6.4 * 10
5.
(3) affinity is identified.Saturated ELISA mensuration affinity costant (
Ka), by concentration, be respectively the Mo of 3.4 μ g/mL and 1.7 μ g/mL
6+-ITCBE-BSA is coated, adds the Mo of doubling dilution
6+-EDTA mAb, then add GaMIgG-HRP, the TMB survey that develops the color
A 450nmValue, with Mo
6+-EDTA 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 Mo during value
6+-EDTA mAb concentration, according to formula
Kaff=(n-1)/2(n[Ab'] t-[Ab] t) calculate
Ka.The monoclonal antibody of calculating
KaBe 1.65 * 10
10L/mol.
(4) susceptibility is identified.ELISA measures Mo with blocking-up
6+-EDTA mAb is to variable concentrations Mo
6+The inhibiting rate of-EDTA, with inhibiting rate B/B
0For ordinate, with variable concentrations Mo
6+The logarithm value of-EDTA is horizontal ordinate, and drawing standard suppresses curve, carries out the correlation regression analysis, calculates Mo
6+-EDTA mAb is to Mo
6+-EDTA's
IC 50.Qualification result,
IC 50Be 11.35 ng/mL.
(5) specificity identification.Adopt its specificity of cross reaction test for identification.Qualification result, be less than 0.01% with other heavy metal ion cross reaction.
The preparation of embodiment five, sexavalence molybdenum ion gold test strip
(1) colloidal gold solution preparation.Adopt the preparation of citrate reducing process, get 1000 mL Erlenmeyer flasks, add the 975mL distilled water to boil, add 15 mL trisodium citrates to continue heating 5 min, add the gold chloride of 10 mL 1%, 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 centrifugal 30 min of monoclonal antibody solution 10000r/min be marked, with 0.1 mol/L K
2CO
3Solution is regulated the pH value to 8.2 of colloidal gold solution, and under magnetic agitation, dropwise adding concentration is 0.2 mg/mL monoclonal antibody solution, room temperature incubation 20 min, and centrifugal 30 min of 10000r/min, abandon supernatant, and sediment is with 0.02 mol/L Na
2B
4O
7Solution (containing 100 g/L BSA) dilution, centrifugal 30 min of 10000 r/min, sediment is with 0.02 mol/L Na
2B
4O
7Solution (containing 10 g/L BSA, 0.5 g/L Sodium azide) dilution, 4 ℃ save backup;
(3) golden labeling antibody pad preparation.Cut the glass fibre cotton that specification is 20 * 4 mm, golden labeling antibody evenly is sprayed on glass fibre cotton with the unidirectional specking instrument of X-only, 37 ℃ of drying 1 h, seal 4 ℃ and save backup;
(4) preparation of detection line, nature controlling line on the cellulose rete.By the concentration made, be the Mo of 1mg/ml
6+-ITCBE-BSA is put in the unidirectional specking instrument of X-only storage pool A, and concentration is that the RaMIgG of 1mg/ml is put in storage pool B, and start fixed fire respectively, in the central authorities of film, forms detection line and the nature controlling line of spacing 0.5 cm, natural drying, sealing, and 4 ℃ save backup.
(5) gold test strip assembling.Sample pad, golden labeling antibody pad, cellulose membrane, adsorptive pads are pasted on back up pad in order successively, and paste the MAX line in sample pad and golden labeling antibody pad surface, on slot-cutting machine, cut into the gold test strip of width 4 mm.
Embodiment six: detect the test strips structure of micro-sexavalence molybdenum ion, referring to Fig. 1, Fig. 2.In figure, supporting layer 1 use plastic slice bar is made, sample pad 2 use glass fibre cottons are made, on gold labeling antibody pad 3, absorption has the monoclonal antibody of anti-sexavalence molybdenum ion, cellulose rete 4 adopts nitrocellulose filter, adsorptive pads 5 use absorbent filters are made, to number 2,3,4,5 each layers and be pasted and fixed on successively from right to left on supporting layer 1, the intersection fiber infiltration that crosses one another each other.On cellulose rete 4, be provided with and detect trace 6 and contrast trace 7, detect trace and print with bovine serum albumin(BSA) (BSA) solution of coupling sexavalence molybdenum ion, the contrast trace is printed with the anti-mouse IgG antibody solution of rabbit, two traces be arranged in parallel into "
︱ ︱".8-1 covers sample pad 2 and the test lead diaphragm (white) above golden labeling antibody pad 3; 8-2 is the handle end diaphragm (as yellow) covered above adsorptive pads 5; in sample pad 2 and golden labeling antibody pad 3 intersections, corresponding white diaphragm deflection sample pad 2 one sides, approximately be printed with sample mark line 9 in 0.5 cm place, on the diaphragm on sample mark line right side, be printed on arrow and MAX printed words.
Wherein the preparation of each assembly and golden labeling antibody, artificial immunity antigen, anti-sexavalence molybdenum ion monoclonal antibody etc. is referring to above embodiment.
(1) detection reaction principle
After sexavalence molybdenum ion test strips test lead inserted testing sample solution, solution to be measured drives sexavalence molybdenum ion to be measured by syphonic effect and golden labeling antibody spreads to the cellulose rete together, and infiltrate the adsorptive pads of handle end.In diffusion process, sexavalence molybdenum ion to be measured can combine with golden labeling antibody, and then seal the antigen-combining site of sexavalence molybdenum ion on golden labeling antibody, stop the detection trace of golden labeling antibody coupling sexavalence molybdenum ion carrier protein on the cellulose rete to be combined, can not show the detection trace, the anti-mouse IgG antibody of rabbit can be combined with golden labeling antibody, formation brownish red contrast trace band "
︱", namely reddish brown colour band "
︱" the positive expression of trace; Otherwise, in sample solution, without the sexavalence molybdenum ion, can not stop the carrier protein detection trace of golden labeling antibody coupling sexavalence molybdenum ion on cellulose membrane to be combined, demonstration brownish red detection trace "
︱", the same anti-mouse IgG antibody of rabbit also is combined with golden labeling antibody, demonstration brownish red contrast trace band "
︱", form two reddish brown colour bands "
︱ ︱" negative expression.If on cellulose membrane, do not have reddish brown colour band to show, show that test strips lost efficacy.
(2) testing sample preparation and detecting step:
For detection of soil: take 1.0 g pedotheques in 50 mL microwave tubes, add mixed acid solution (2 mL HNO after adding the 1mL distilled water to infiltrate
3With 6 mL HCl), room temperature reaction 12 h, be placed in Hyperfrequency waves eliminating stove, and 180 ℃ keep 15 min, are cooled to room temperature, and NaOH solution is regulated pH to 7.4, with 50 mL volumetric flask constant volumes.In water sample digestion solution after constant volume, adding volumetric molar concentration according to the volume ratio of 9: 1 is the EDTA intercalating agent of 100mmoL/L, mixes, and makes the sexavalence molybdenum from the abundant huge legendary turtle of EDTA, closing, and obtains pedotheque and detects solution.
Method of operating: test strips is inserted in testing sample, and insertion depth is no more than mark line, approximately takes out test strip 10～20 seconds, and horizontal positioned is observed and judged testing result in 5 min.
Embodiment seven: test strips structure and embodiment six are basic identical; difference is: golden labeling antibody pad absorption has the polyclonal antibody of anti-sexavalence molybdenum ion; sample pad is made with nylon membrane; the cellulose rete adopts the pure cellulose film; detect trace and be " ten " with the contrast trace, the diaphragm that covers the handle end above adsorptive pads is blue look.
For detection of the pork sample: take pork sample 1.0g, in the teflon crucible, add nitric acid 2mL, room temperature reaction 4h, then with nitric acid 4 mL, the meat sample is all transferred in digest tube, added 1.5 mL 30% superoxol reaction 1 h, be placed in afterwards Hyperfrequency waves eliminating stove, 180 ℃ keep 15 min, be cooled to room temperature, NaOH solution is regulated pH to 7.4, with 50 mL volumetric flask constant volumes.In water sample digestion solution after constant volume, adding volumetric molar concentration according to the volume ratio of 9: 1 is the EDTA intercalating agent of 100 mmoL/L, mixes, and makes the sexavalence molybdenum from the abundant huge legendary turtle of EDTA, closing, and obtains pork sample detection solution.
Embodiment eight: test strips structure and embodiment six are basic identical; difference is: sample pad is made with pvdf membrane; the carrier protein solution of coupling sexavalence molybdenum ion is chicken ovalbumin (OVA); the cellulose rete adopts the carboxylation cellulose membrane, and the handle end diaphragm covered above adsorptive pads is green.
For detection of water sample: in water sample, adding volumetric molar concentration according to the volume ratio of 9: 1 is the EDTA intercalating agent of 100 mmoL/L, mixes, and makes the sexavalence molybdenum from the abundant huge legendary turtle of EDTA, closing, and obtains water sample detection solution.
Embodiment nine: test strips structure and embodiment six are basic identical, and difference is: sample pad is made with polyester film, and the cellulose rete adopts the carboxylation cellulose membrane, and coupling sexavalence molybdenum ion carrier protein solution is hemocyanin (KLH).
The performance measurement of embodiment ten, sexavalence molybdenum ion gold test strip
(1) sensitivity testing.With the sexavalence molybdenum ion gold test strip in embodiment, measure the sexavalence molybdenum ion standard items that concentration is respectively 0 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 160 ng/mL, 320 ng/mL, 640 ng/mL, each concentration is established 6 repetitions, judges its susceptibility according to test findings.The results are shown in Table 1, as shown in Table 1, the range estimation of sexavalence molybdenum ion gold test strip detects and is limited to 5 ng/mL.
Table 1 is measured gold test strip sensitivity testing (n=6)
| Sexavalence molybdenum ion concentration (ng/mL) | 0 | 5 | 10 | 20 | 40 | 80 | 160 | 320 | 640 |
| Result | - | + | + | + | + | + | + | + | + |
(2) specific assay.Employing cross reaction test, selecting chelate, the EDTA of the metallic ion such as mercury, lead, cadmium, copper, zinc, chromium, cobalt, iron and EDTA is mortifier, with sexavalence molybdenum ion gold test strip, measure final concentration and be respectively 0 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 40 ng/mL, 80 ng/mL, 160 ng/mL, 320 ng/mL, 640 ng/mL heavy metal ion standard items, each concentration is established 6 repetitions, judges its specificity with this.Result shows, sexavalence molybdenum ion gold test strip can with sexavalence molybdenum ion specific binding, with other heavy metal ion no cross reaction.In Table 2.
The specific test of table 2 gold test strip (n=6)
(3) repeatability is measured.Get 6 batches of sexavalence molybdenum ion gold test strips of different batches, at the 6th d, be that 0n g/mL, 5 ng/mL, the soil of 10 ng/mL, 20ng/mL, water sample, pork prepare sample and detects to sexavalence molybdenum ion concentration respectively, 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 3 CAP-Strip replica test (n=6)
(4) retention period check.The sexavalence molybdenum ion gold test strip of different batches is stored in respectively to general refrigerator (2～8 ℃) and with drying at room temperature, preserved 6 months in 12 months, the testing result of different holding times of research, determine its storage life.Result shows, sexavalence molybdenum 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 ℃ lower 12 months, lower 6 months of drying at room temperature condition.
Claims (10)
1. gold test strip for fast detecting sexavalence molybdenum ion, bottom is supporting layer, middle layer is adsorbed layer, diaphragm is fixed on adsorbed layer, it is characterized in that: adsorbed layer is followed successively by the adsorptive pads of sample pad, golden labeling antibody pad, cellulose rete and handle end from test lead, wherein on the cellulose rete, is provided with the detection trace that the carrier protein solution of coupling sexavalence molybdenum ion prints and the contrast trace of printing with the anti-mouse IgG antibody solution of rabbit; In described golden labeling antibody pad, be coated with the specificity sexavalence molybdenum ion monoclonal antibody of colloid gold label.
2. gold test strip according to claim 1, it is characterized in that: described cellulose rete is made with nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane; Described sample pad is made with PVDF membrane, nylon membrane, glass fibre cotton or polyester film; Adsorptive pads is made with absorbent filter, and supporting layer is made with the toughness material do not absorbed water; Gold labeling antibody pad is made with glass fibre cotton.
3. gold test strip according to claim 1, is characterized in that: described
The carrier protein of coupling sexavalence molybdenum ion is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin; The package amount that detects the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On the contrast trace, the package amount of rabbit anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; Described detection trace and the contrast trace be arranged in parallel "
︱ ︱" orthoscopic trace or " 10 " font arrangement trace.
4. gold test strip according to claim 1; it is characterized in that: described diaphragm covers on sample pad, golden labeling antibody pad and adsorptive pads, and deflection sample pad one side 0.3-0.6 cm place is printed with the sample mark line on the sample pad diaphragm corresponding with golden labeling antibody pad intersection.
5. according to the described gold test strip of claim 1-4 any one, it is characterized in that: prepared by following methods by the carrier protein solution of described coupling sexavalence molybdenum ion:
Get 20 mL molybdic acids and 1mL concentration and be 10 mmol/L, pH value and be 8.0 HEPES damping fluid and mix, obtain Mo
6+Solution; Take 10 mg isothiocyanic acid benzyl ethylenediamine tetraacetic acid ITCBE and be dissolved in 1mL DMSO, form the metal-chelating agent solution; By Mo
6+Solution and metal-chelating agent solution mix, the pH value to 7.4 of regulator solution, and 25 ℃ are stirred 24 h, and rotating speed is 1000 r/min, forms Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA and be dissolved in the HEPES damping fluid that 1mL concentration is 10 mmol/L, pH 8.0, form carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens mother liquor joins in 1mL carrier protein solution, regulates pH value to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves into dialysis 7 d in bag filter, collects dislysate, prepares carrier protein Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 ℃ frozen standby.
6. according to the described gold test strip of claim 1-4 any one, it is characterized in that: prepared by the specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label by the following method:
Adopt the preparation of citrate reducing process, in Erlenmeyer flask, add 975 mL distilled waters to boil, add the 15mL trisodium citrate, continue heating 5 min, adding 10 mL mass concentrations is 1% gold chloride, continue to be heated to solution and be Chinese red, obtain colloidal gold solution after being cooled to room temperature, 4 ℃ save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2CO
3Solution is regulated the pH value to 8.2 of colloidal gold solution, under magnetic agitation, dropwise adding concentration is the sexavalence molybdenum ion monoclonal antibody solution of 0.2 mg/mL, room temperature incubation 20 min, centrifugal 30 min of 10000 r/min, abandon supernatant, sediment with every liter contain 100 g BSA, concentration is the Na of 0.02 mol/L
2B
4O
7It is the Na of 0.02 mol/L that solution dilution, centrifugal 30 min of 10000 r/min then, the sediment obtained contain 10 g BSA, 0.5 g Sodium azide, concentration with every liter again
2B
4O
7Solution dilution, namely obtain the sexavalence molybdenum ion monoclonal antibody of colloid gold label, and 4 ℃ save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, in every 1mL collaurum, contains 3 ng sexavalence molybdenum ion monoclonal antibodies.
7. the preparation method of the described gold test strip of claim 1, it is characterized in that: the method comprises the following steps:
(1) preparation of sexavalence molybdenum ion carrier protein couplet thing:
The HEPES damping fluid that to get 20 mL molybdic acids and 1mL concentration be 10 mmol/L, pH 8.0 mixes, and obtains Mo
6+Solution; Take 10 mg ITCBE and be dissolved in 1mL DMSO, form the metal-chelating agent solution; By Mo
6+Solution and metal-chelating agent solution mix, the pH value to 7.4 of regulator solution, and 25 ℃ are stirred 24 h, and rotating speed is 1000 r/min, forms Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA, being dissolved in 1mL concentration is in the HEPES damping fluid of 10 mmol/L, pH 8.0, forms carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens mother liquor joins in 1mL carrier protein solution, regulates pH value to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves into dialysis 7 d in bag filter, collects dislysate, prepares carrier protein couplet thing Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 ℃ are frozen standby;
(2) preparation of sexavalence molybdenum ion gold labeling antibody;
(3) preparation of golden labeling antibody pad;
(4) detect the preparation of trace and contrast trace;
With the sexavalence molybdenum ion carrier protein couplet thing of gained, on the cellulose rete, make the detection trace, on the cellulose rete, make the contrast trace by the anti-mouse IgG antibody of rabbit; The package amount that detects the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and the concentration of carrier protein is 1mg/mL; On described contrast trace, the package amount of rabbit anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL;
Sexavalence molybdenum ion gold labeling antibody, for the preparation of golden labeling antibody pad, then is assembled into gold test strip by supporting layer, adsorbed layer and diaphragm successively.
8. preparation method according to claim 7, it is characterized in that: the preparation method of the specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label is as follows:
Adopt the citrate reducing process, in Erlenmeyer flask, add 975 mL distilled waters, boil, add 15 mL trisodium citrates, continue heating 5 min, adding 10 mL mass concentrations is 1% gold chloride, continues to be heated to solution and is Chinese red, after being cooled to room temperature, obtain colloidal gold solution, 4 ℃ save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2CO
3Solution is regulated the pH 8.2 of colloidal gold solution, under magnetic agitation, dropwise adding concentration is the sexavalence molybdenum ion monoclonal antibody solution of 0.2 mg/mL, room temperature incubation 20 min, centrifugal 30 min of 10000 r/min, abandon supernatant, sediment with every liter contain 100 g BSA, concentration is the Na of 0.02 mol/L
2B
4O
7Solution dilution, centrifugal 30 min of 10000 r/min then, the sediment obtained with every liter, contains 10 g BSA again and 0.5 g Sodium azide, concentration are 0.02 mol/L Na
2B
4O
7Solution dilution, namely obtain the sexavalence molybdenum ion monoclonal antibody of colloid gold label, and 4 ℃ save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, in every 1mL collaurum, contains 3 ng sexavalence molybdenum ion monoclonal antibodies.
9. according to the described preparation method of claim 7 or 8, it is characterized in that: prepared by following methods by described sexavalence molybdenum ion monoclonal antibody specific:
Immune animal: with Mo
6+-ITCBE-BSA is immunizing antigen, using the Balb/c mouse as immune animal, the subcutaneous multi-point injection in back, immunizing dose is every each 50～100 μ g, head exempts to use Freund's complete adjuvant, and booster immunization incomplete Freunds adjuvant, head carry out immunity for the second time 3 weeks after exempting from, once, immunity is 5 times altogether in all immunity in interval 2 later;
Select the standby mouse of Fusion of Cells: indirect ELISA detects Mo in antiserum
6+Chelate polyclonal antibody (pAb) is tired, and blocking-up ELISA detects Mo
6+-EDTA pAb is to Mo
6+The half-inhibition concentration of-EDTA (
IC 50), select tire the highest,
IC 50Minimum mouse, super exempting from for Fusion of Cells;
Prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell and carry out Fusion of Cells with PEG solution, through 4 limiting dilution assay subclones, obtain the cell strain of monoclonal antibody of the anti-sexavalence molybdenum ion of stably excreting;
Preparation monoclonal antibody: to the female mouse lumbar injection of multiparity hybridoma cell strain, gather the monoclonal antibody that the ascites purifying obtains anti-sexavalence molybdenum ion.
10. the application of the described gold test strip of claim 1-6 any one in fast detecting sexavalence molybdenum ion.
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