CN103412126B - 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 sanitary inspection to learn a skill field, particularly relating to a kind of gold test strip for detecting sexavalence molybdenum ion fast and preparation method thereof and application.
Background technology
China has abundant molybdenum ore resource, total reserves 8,400,000 tons, and molybdenum is as a kind of transiting state metal element, its compound presents five kinds of valence states, wherein sexavalence molybdenum is the most stable, also close with the vital movement relation of the mankind, lacks and the excessive quality that all can affect life.Molybdenum is one of required trace element of organism, and various the organizing of human body all contains molybdenum, and in adult body, total amount is 9mg, and liver, kidney content are the highest.Molybdenum element itself does not have biologically active, just there is biologically active after only having sexavalence molybdenum ion to be combined with purine to form coenzyme, its physiological function mainly contains and maintains normal metabolism, improve the elasticity of arterial wall, reduce angiocardiopathy, improve human body immune function by antioxidation, and the synthesis of nitrosamines carcinogen in vivo can be suppressed, there is 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 excessive body that can cause of sexavalence molybdenum is poisoning simultaneously, when human intake's amount is more than 10 ~ 15mg, can growth retardation be caused, Body weight loss, gout sample syndrome, arthralgia and the illness such as deformity, lung's sex change even occur.Animal molybdenosis shows similar copper deficiency disease, and in Ferguson reported first in 1938 herbage, molybdenum content is too high, can make Grazing Cattle produce persistent diarrhea, the poisoning disease that is feature by hair decolouring.The farm cattle " red Pi Baimao disease " etc. that " calf diarrhea ", Zelanian " ox peat rushes down " of Britain and Australia's " ox endemic hematuria disease " and China area, south, Jiangxi in 1981 occurs are all the typical events of sexavalence molybdenosis.Sexavalence molybdenum is safe from harm to biosome in its natural state, but the numerous areas such as special steel, machinery, oil, chemical industry, national defence, Aero-Space, electronics, nuclear industry are widely used in due to molybdenum, molybdenum ore is exploited in a large number, sexavalence molybdenum ion enters nature in a variety of manners, environmental pollution sharp deterioration, in addition cumulative effect and bioconcentration, environmental pollution and human health constitute 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 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 little, but its sensitivity are not high; Electrochemical methods is 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, and comparatively strict to operating personnel's technical requirement, limit it and widely use; 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 required equipment is not easily by common laboratory is possessed, and is difficult to be used widely.
Therefore, develop quick, easy, responsive, special, the quick testing product of economy, screening amount are large sexavalence molybdenum ion, 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 sexavalence molybdenum ion detection technique, provides a kind of gold test strip for detecting sexavalence molybdenum ion fast that can be quick, easy, responsive, special;
Additionally provide preparation method and the application thereof of sexavalence molybdenum ion gold test strip.
Technical scheme of the present invention:
A kind of gold test strip for detecting sexavalence molybdenum ion fast, 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, the detection trace that the carrier protein solution being wherein provided with coupling sexavalence molybdenum ion on cellulose rete is printed and the contrast trace printed with rabbit anti-mouse IgG antibody solution; The specificity sexavalence molybdenum ion monoclonal antibody of colloid gold label is coated with in described golden labeling antibody pad.
Described cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made; Described sample pad PVDF membrane, nylon membrane, glass fibre cotton or polyester film are made; Adsorptive pads absorbent filter is made, and the supporting layer toughness material do not absorbed water is made; Gold labeling antibody pad glass fibre cotton is made.
Described
the carrier protein of coupling sexavalence molybdenum ion is bovine serum albumin(BSA), chicken ovalbumin or hemocyanin; The package amount detecting the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On 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 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, 0.3-0.6 cm place, sample pad side is partial to by the diaphragm that sample pad is corresponding with golden labeling antibody pad intersection and is printed with sample mark line.
The carrier protein solution of described coupling sexavalence molybdenum ion is prepared by following methods:
Get the HEPES damping fluid that 20 mL molybdic acids are 10 mmol/L with 1mL concentration, pH value is 8.0 to mix, obtain Mo
6+solution; Taking 10 mg isothiocycmatobenzyl ethylenediamine tetraacetic acid ITCBE is dissolved in 1mL DMSO, forms metal-chelating agent solution; By Mo
6+solution and the mixing of metal-chelating agent solution, regulate the pH value to 7.4 of solution, and 25 DEG C are stirred 24 h, and rotating speed is 1000 r/min, form Mo
6+-ITCBE chelate haptens mother liquor;
Taking 20 mg carrier protein BSA or OVA, to be dissolved in 1mL concentration be 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, adjust ph to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves in bag filter 7 d that dialyse, collects dislysate, prepare carrier protein Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 DEG C frozen for subsequent use.
The specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label is prepared by the following method:
Adopt citrate reduction legal system standby, in Erlenmeyer flask, add 975 mL distilled waters boil, add 15mL trisodium citrate, continue heating 5 min, adding 10 mL mass concentrations is the gold chloride of 1%, continuing to be heated to solution is Chinese red, obtains colloidal gold solution after being cooled to room temperature, and 4 DEG C save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution, the sexavalence molybdenum ion monoclonal antibody solution that concentration is 0.2 mg/mL is dropwise added under magnetic agitation, incubation at room temperature 20 min, centrifugal 30 min of 10000 r/min, abandon supernatant, the Na that sediment uses often liter to contain 100 g BSA, concentration is 0.02 mol/L
2b
4o
7solution dilution, then centrifugal 30 min of 10000 r/min, the sediment obtained is the Na of 0.02 mol/L again containing 10 g BSA, 0.5 g Sodium azide, concentration with often liter
2b
4o
7solution dilution, namely obtain the sexavalence molybdenum ion monoclonal antibody of colloid gold label, 4 DEG C save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, containing 3 ng sexavalence molybdenum ion monoclonal antibodies in every 1mL collaurum.
The preparation method of described gold test strip, comprises the following steps:
(1) preparation of sexavalence molybdenum ion carrier protein couplet thing:
Get the HEPES damping fluid that 20 mL molybdic acids and 1mL concentration are 10 mmol/L, pH 8.0 to mix, obtain Mo
6+solution; Taking 10 mg ITCBE is dissolved in 1mL DMSO, forms metal-chelating agent solution; By Mo
6+solution and the mixing of metal-chelating agent solution, regulate the pH value to 7.4 of solution, and 25 DEG C are stirred 24 h, and rotating speed is 1000 r/min, form 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, adjust ph to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves in bag filter 7 d that dialyse, collects dislysate, prepare carrier protein couplet thing Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 DEG C are frozen for subsequent use;
(2) preparation of sexavalence molybdenum ion gold labeling antibody;
(3) preparation of golden labeling antibody pad;
(4) preparation of trace and contrast trace is detected;
On cellulose rete, make detection trace with the sexavalence molybdenum ion carrier protein couplet thing of gained, on cellulose rete, make contrast trace by rabbit anti-mouse IgG antibody; The package amount detecting 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;
Supporting layer, adsorbed layer and diaphragm, for the preparation of golden labeling antibody pad, are then assembled into gold test strip by sexavalence molybdenum ion gold labeling antibody successively.
Described sexavalence molybdenum ion monoclonal antibody specific is prepared by following methods:
Immune animal: with Mo
6+-ITCBE-BSA is immunizing antigen, using Balb/c mouse as immune animal, dorsal sc multi-point injection, immunizing dose is every only each 50 ~ 100 μ g, 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;
Select Fusion of Cells mouse for subsequent use: indirect ELISA detects Mo in antiserum
6+chelate polyclonal antibody (pAb) is tired, and stop band restrain detects Mo
6+-EDTA pAb is to Mo
6+the half-inhibition concentration of-EDTA (
iC 50), select tire the highest,
iC 50minimum mouse, surpasses and exempts from for Fusion of Cells;
Prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell PEG solution carries out Fusion of Cells, 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 multiparity female mouse lumbar injection hybridoma cell strain, gather the monoclonal antibody that ascites purifying obtains anti-sexavalence molybdenum ion.
Described gold test strip is detecting the application in sexavalence molybdenum ion fast.
Positive beneficial effect of the present invention:
(1) gold test strip of the present invention, detect sexavalence molybdenum ion speed fast, go out testing result in 5min, than Physico-chemical tests method, (3 d) save detection time greatly;
(2) gold test strip of the present invention, detection method is easy, and without any need for auxiliary instrumentation and reagent, people can operate per capita, easy to carry, can 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 Physico-chemical tests method; High specificity, with other heavy metal ion no cross reaction; Economy, compared with Physico-chemical tests method, detects a sample and at least can save 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 at 2-8 DEG C is 1 year, and the drying at room temperature condition lower shelf-life is six months.
(5) preparation method of gold test strip of the present invention, has prepared sexavalence molybdenum ion carrier protein couplet thing, obtains appropriate molecule in conjunction with the artificial immunity antigen of ratio and envelope antigen, obtains high-titer, sensitivity, special antiserum; Adopt reduction of sodium citrate legal system for colloidal gold solution, course of reaction is gentleer, can carry out, be easy to operate and control under normal temperature and condition of neutral pH.
(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, 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%, and the trace for its sexavalence molybdenum ion detects fast and provides guarantee.
(7) sexavalence molybdenum ion gold test strip of the present invention can be applicable to detect soil fast, water, in food, sexavalence molybdenum ion pollutes residual, have fast, easy, responsive, special, economic dispatch characteristic, 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 is enforced the law, 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, and there is significant economic benefit and social benefit.
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 Technology Roadmap of sexavalence molybdenum ion carrier protein couplet thing synthesis.
In figure, 1 is supporting layer, and 2 is sample pad, and 3 is golden labeling antibody pad, and 4 is cellulose rete, and 5 is adsorptive pads, and 6 for detecting trace, and 7 is contrast trace, and 8-1 is test lead diaphragm, and 8-2 is handle end diaphragm, and 9 is sample mark line.
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.
Prepare the gold test strip detecting sexavalence molybdenum ion, first will prepare the artificial immunity antigen of sexavalence molybdenum ion, afterwards immune Balb/c mouse, prepare the monoclonal antibody of anti-sexavalence molybdenum ion, assemble the gold test strip of sexavalence molybdenum ion on this basis.
the preparation of embodiment one, sexavalence molybdenum ion artificial immunity antigen
Adopt different sulphur hydrocyanic ester method.Get 20mL molybdic acid, with concentration to be 10mmol/L pH value be 8.0 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid mix and form Mo
6+solution; Take 10mg isothiocycmatobenzyl ethylenediamine tetraacetic acid (ITCBE) and be dissolved in 1mL dimethyl sulfoxide (DMSO) (DMSO), form metal-chelating agent solution; By Mo
6+after solution and metal-chelating agent solution mix, regulate the pH value to 7.4 of solution, 25 DEG C are stirred 24 h, and rotating speed is 1000 r/min, form Mo
6+-ITCBE chelate haptens mother liquor;
Take 20 mg carrier protein BSA or OVA, be dissolved in that 1mL concentration is 10 mmol/L, pH value is in the HEPES damping fluid of 8.0, form carrier protein solution; Get 1mL Mo
6+-ITCBE chelate haptens solution joins in 1mL carrier protein solution, adjust ph to 9.0, and at room temperature 1000 r/min stir 24 h, then moves in bag filter 7 d that dialyse, collects dislysate, prepare sexavalence molybdenum ion artificial immunity antigen
(carrier protein couplet thing) Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 DEG C frozen for subsequent use.See Fig. 3.
the qualification of embodiment two, sexavalence molybdenum ion artificial immunity antigen
Bicinchoninic acid method is adopted to measure Mo
6+the concentration of carrier protein BSA in-ITCBE-BSA.Using BSA as standard protein, build Concentration Testing typical curve by bicinchoninic acid method.The linear equation of BSA concentration standard curve is: y=0.0004x+0.0072, 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 Mo
6+mo in-ITCBE-BSA
6+concentration.By the Mo of 100 μ g/mL
6+standard reserving solution, the nitric acid with 2% is 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 draws equation of linear regression; Sample solution 50 times dilution, measures under the optimum optimization experiment condition of 226 nm wavelength, instrument software automatic analysis result.
the preparation of embodiment three, anti-sexavalence molybdenum ion monoclonal antibody
(1) animal immune.Immunity Balb/c mouse, uses Mo
6+-ITCBE-BSA immunity female Balb/C mouse 5 in 6 weeks age, 50 μ g0.2 mL/, dorsal sc multi-point injection.Head exempts from, and dilutes Mo with sterilizing PBS
6+-ITCBE-BSA, with equivalent CFA mixing and emulsifying; Booster immunization, dilutes Mo with sterilizing PBS
6+-ITCBE-BSA, with equivalent IFA mixing and emulsifying, within 3 weeks, carry out second time immunity after head exempts from, the immunity in 2 weeks of later interval once, is total to immunity 5 times, after third time immunity, blood is got in 10 d dockings, 37 DEG C of water-bath 30 min, 4 DEG C of placements are spent the night, centrifugal 5 min of 4000 r/min 4 DEG C, get supernatant ,-20 DEG C save backup.
(2) Fusion of Cells mouse for subsequent use is selected.Indirect ELISA detects Mo in antiserum
6+chelate pAb tires, and stop band restrain detects Mo
6+-EDTA pAb is to Mo
6+-EDTA's
iC 50, select tire the highest,
iC 50minimum mouse, surpasses after exempting from for Fusion of Cells.
(3) positive hybridoma cell strain screening.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 50 mL centrifuge tubes, 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 DEG C of water-baths.With 1mL suction pipe by the PEG(pH 8.0 of 50% of preheating) be added drop-wise in fusion pipe, limit edged shakes fusion pipe gently, adds in 1min, and continues slowly to shake fusion pipe 1.5 min in a water bath; Then slowly add GNK solution to 40 mL, 37 DEG C of water-baths leave standstill centrifugal 10 min of 5 min, 1000 r/min, abandon supernatant.Break up cell mass, add 40 mL HAT and blow and beat mixing, be added on the Tissue Culture Plate in 96 holes, every hole 100 μ L, is placed in the CO of 37 DEG C 5%
2cultivate in incubator.
The screening of positive hybridoma cell, carries out the screening of positive hybridoma cell strain with indirect ELISA and stop band restrain.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 15 d, 30 d and 60 d, supernatant is got after cultivation, indirect ELISA measures antibody titer, investigates the stability of hybridoma secrete monoclonal antibody.
(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 IFA 0.5 mL/ only, uses after 10 ~ 15 d.By centrifugal 10 min of positive hybridoma cell 1000 r/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 0.5 mL/ only.Produce ascites after inoculating cell 7-10 d, collect, in 37 DEG C of water-bath 30 min, 4 DEG C of placements are spent the night, the centrifugal 5min of 12000 r/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, anti-sexavalence molybdenum ion monoclonal antibody
(1) hypotype qualification.Take out the hybridoma supernatant cultivated, after 1: 50 dilution, with reference to the operation of mouse monoclonal antibody subtype identifying test paper bar instructions.Getting dilution 0.15 mL adds in kit tubule, and room temperature places 1 min.After it dissolves naturally, mix gently, then test strips is inserted into bottom tubule, after 5 min, when band foremost two to when occurring in the middle of "+", namely as seen with the position at the Subclass of antibody secreted by hybridoma cell strain and the corresponding place of light chain type, thus determine antibody subtype.Qualification result: the hypotype of monoclonal antibody is IgG
1/ λ.
(2) binding capacity measures.Indirect ELISA measures its binding capacity.Measurement result, monoclonal antibody titer of ascites is 1: 6.4 × 10
5.
(3) affinity qualification.Saturated ELISA mensuration affinity costant (
ka), the Mo of 3.4 μ g/mL and 1.7 μ g/mL is respectively by concentration
6+-ITCBE-BSA bag quilt, adds the Mo of doubling dilution
6+-EDTA mAb, then add GaMIgG-HRP, TMB develop the color survey
a 450nmvalue, with Mo
6+-EDTA mAb 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 450nmmo corresponding during value
6+-EDTA mAb concentration, according to formula
kaff=(n-1)/2(n [Ab'] t-[Ab] t) calculates
ka.The monoclonal antibody calculated
kabe 1.65 × 10
10l/mol.
(4) susceptibility qualification.Mo is measured with stop band restrain
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 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, is 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 citrate reduction legal system standby, get 1000 mL Erlenmeyer flasks, add 975mL distilled water and boil, add 15 mL trisodium citrates and 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 DEG C save backup;
(2) golden labeling antibody preparation.Centrifugal 30 min of mark monoclonal antibody solution 10000r/min will be treated, with 0.1 mol/L K
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution, and dropwise adding concentration is under magnetic stirring 0.2 mg/mL monoclonal antibody solution, and centrifugal 30 min of incubation at room temperature 20 min, 10000r/min, abandon supernatant, sediment 0.02 mol/L Na
2b
4o
7solution (containing 100 g/L BSA) dilutes, centrifugal 30 min of 10000 r/min, sediment 0.02 mol/L Na
2b
4o
7solution (containing 10 g/L BSA, 0.5 g/L Sodium azide) dilutes, and 4 DEG C save backup;
(3) golden labeling antibody pad preparation.Cut the glass fibre cotton that specification is 20 × 4 mm, unidirectional for golden labeling antibody X-only specking instrument is evenly sprayed on glass fibre cotton, 37 DEG C of drying 1 h, seal 4 DEG C and save backup;
(4) preparation of detection line, nature controlling line on cellulose rete.Be the Mo of 1mg/ml by obtained concentration
6+-ITCBE-BSA is put in X-only unidirectional specking instrument storage pool A, and concentration is that the RaMIgG of 1mg/ml is put in storage pool B, and fixed fire is in the central authorities of film respectively in start, and form detection line and the nature controlling line of spacing 0.5 cm, natural drying, sealing, 4 DEG C save backup.
(5) gold test strip assembling.Sample pad, golden labeling antibody pad, cellulose membrane, adsorptive pads are pasted in back up pad in order successively, and at sample pad and golden labeling antibody pad surface mount MAX line, slot-cutting machine cut into the gold test strip of width 4 mm.
embodiment six: the test strips structure detecting micro-sexavalence molybdenum ion, see Fig. 1, Fig. 2.In figure, supporting layer 1 plastic slice bar is made, sample pad 2 is made with glass fibre cotton, gold labeling antibody pad 3 is adsorbed with the monoclonal antibody of anti-sexavalence molybdenum ion, cellulose rete 4 adopts nitrocellulose filter, adsorptive pads 5 is made with absorbent filter, be pasted and fixed on successively from right to left on supporting layer 1 by each layer of numbering 2,3,4,5, intersection fiber crosses one another infiltration each other.Cellulose rete 4 is provided with and detects trace 6 and contrast trace 7, detect trace bovine serum albumin(BSA) (BSA) solution of coupling sexavalence molybdenum ion and prints, the rabbit anti-mouse IgG antibody solution printing of contrast trace, two trace one-tenth arranged in parallel "
︱ ︱".8-1 covers the test lead diaphragm (white) above sample pad 2 and golden labeling antibody pad 3; 8-2 is the handle end diaphragm (as yellow) covered above adsorptive pads 5; be partial to cm place, sample pad 2 side about 0.5 at sample pad 2 and golden labeling antibody pad 3 intersection, corresponding white diaphragm and be printed with sample mark line 9, the diaphragm on the right side of sample mark line is 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 see above embodiment.
(1) detection reaction principle
After sexavalence molybdenum ion test strips test lead inserts testing sample solution, solution to be measured drives sexavalence molybdenum ion to be measured and golden labeling antibody together to the diffusion of cellulose rete by syphonic effect, and infiltrates the adsorptive pads of handle end.In diffusion process, sexavalence molybdenum ion to be measured can combine with golden labeling antibody, and then close the antigen-combining site of sexavalence molybdenum ion on golden labeling antibody, the detection trace of golden labeling antibody coupling sexavalence molybdenum ion carrier protein on cellulose rete is stoped to be combined, detection trace can not be shown, rabbit anti-mouse IgG antibody then can be combined with golden labeling antibody, formation brownish red contrast trace band "
︱", namely reddish brown colour band "
︱" trace is positive expression; Otherwise, without sexavalence molybdenum ion in sample solution, then the carrier protein of golden labeling antibody coupling sexavalence molybdenum ion on cellulose membrane can not be stoped to detect trace and to be combined, display brownish red detection trace "
︱", same rabbit anti-mouse IgG antibody is also combined with golden labeling antibody, display brownish red contrast trace band "
︱", formed two reddish brown colour bands "
︱ ︱" be negative expression.If cellulose membrane does not have reddish brown colour band show, then show that test strips lost efficacy.
(2) testing sample preparation and detecting step:
For detecting soil: take 1.0 g pedotheques in 50 mL microwave tubes, adding after 1mL distilled water infiltrates and adding mixed acid solution (2 mL HNO
3with 6 mL HCl), room temperature reaction 12 h, is placed in Hyperfrequency waves eliminating stove, and 180 DEG C keep 15 min, are cooled to room temperature, and NaOH solution regulates pH to 7.4, with 50 mL volumetric flask constant volumes.Add according to the volume ratio of 9: 1 the EDTA intercalating agent that volumetric molar concentration is 100mmoL/L in water sample digestion solution after constant volume, mix, sexavalence molybdenum is closed from the abundant huge legendary turtle of EDTA, obtain pedotheque and detect solution.
Method of operating: test strips inserted in testing sample, insertion depth is no more than mark line, about 10 ~ 20 seconds took out test strip, horizontal positioned, observe and judge testing result in 5 min.
embodiment seven: test strips structure is substantially identical with embodiment six; difference is: golden labeling antibody pad is adsorbed with the polyclonal antibody of anti-sexavalence molybdenum ion; sample pad nylon membrane is made; cellulose rete adopts pure cellulose film; detect trace and contrast trace and be " ten ", the diaphragm covering the handle end above adsorptive pads is blue look.
For detecting pork sample: take pork sample 1.0g, nitric acid 2mL is added in teflon crucible, room temperature reaction 4h, then with nitric acid 4 mL, meat sample is all transferred in digest tube, adds 1.5 mL 30% superoxols and react 1 h, be placed in Hyperfrequency waves eliminating stove, 180 DEG C keep 15 min, be cooled to room temperature, NaOH solution regulates pH to 7.4, with 50 mL volumetric flask constant volumes.Add according to the volume ratio of 9: 1 the EDTA intercalating agent that volumetric molar concentration is 100 mmoL/L in water sample digestion solution after constant volume, mix, sexavalence molybdenum is closed from the abundant huge legendary turtle of EDTA, obtains pork sample detection solution.
embodiment eight: test strips structure is substantially identical with embodiment six; difference is: sample pad pvdf membrane is made; the carrier protein solution of coupling sexavalence molybdenum ion is chicken ovalbumin (OVA); cellulose rete adopts carboxylated cellulose film, and the handle end diaphragm covered above adsorptive pads is green.
For detecting water sample: add according to the volume ratio of 9: 1 the EDTA intercalating agent that volumetric molar concentration is 100 mmoL/L in water sample, mix, sexavalence molybdenum being closed from the abundant huge legendary turtle of EDTA, obtains water sample detection solution.
embodiment nine: test strips structure is substantially identical with embodiment six, and difference is: sample pad polyester film is made, and cellulose rete adopts carboxylated cellulose film, 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.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 are measured with the sexavalence molybdenum ion gold test strip in embodiment, each concentration establishes 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 measures 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 is tested, the chelate, the EDTA that select the metallic ions such as mercury, lead, cadmium, copper, zinc, chromium, cobalt, iron and EDTA are mortifier, measure final concentration with sexavalence molybdenum ion gold test strip 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 establishes 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 (n=6) of table 2 gold test strip
(3) repeatability measures.Get 6 batches of sexavalence molybdenum ion gold test strips of different batches, respectively the 6th d to sexavalence molybdenum ion concentration be 0n g/mL, 5 ng/mL, the soil of 10 ng/mL, 20ng/mL, water sample, pork prepares sample and detects, each concentration establishes 6 repetitions, checks its repeatability.The results are shown in Table 3, testing result is completely the same, proves that the gold test strip testing result that different batches is produced 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 general refrigerator (2 ~ 8 DEG C) and preserves 6 months with drying at room temperature in 12 months, the testing result of research different holding time, determines 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 for the test result of test paper with brand-new, and its term of validity is at 2 ~ 8 DEG C 12 months, lower 6 months of drying at room temperature condition.
Claims (9)
1. one kind for detecting the gold test strip of sexavalence molybdenum ion fast, 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, the detection trace that the carrier protein solution being wherein provided with coupling sexavalence molybdenum ion on cellulose rete is printed and the contrast trace printed with rabbit anti-mouse IgG antibody solution; The specificity sexavalence molybdenum ion monoclonal antibody of colloid gold label is coated with in described golden labeling antibody pad;
The package amount detecting the carrier protein of coupling sexavalence molybdenum ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On contrast trace, the package amount of rabbit anti-mouse IgG antibody is 1 μ L/cm, and antibody concentration is 1mg/mL;
The carrier protein solution of described coupling sexavalence molybdenum ion is prepared by following methods:
Get the HEPES damping fluid that 20 mL molybdic acids are 10 mmol/L with 1mL concentration, pH value is 8.0 to mix, obtain Mo
6+solution; Taking 10 mg isothiocycmatobenzyl ethylenediamine tetraacetic acid ITCBE is dissolved in 1mL DMSO, forms metal-chelating agent solution; By Mo
6+solution and the mixing of metal-chelating agent solution, regulate the pH value to 7.4 of solution, and 25 DEG C are stirred 24 h, and rotating speed is 1000 r/min, form Mo
6+-ITCBE chelate haptens mother liquor;
Taking 20 mg carrier protein BSA or OVA, to be dissolved in 1mL concentration be 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, adjust ph to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves in bag filter 7 d that dialyse, collects dislysate, prepare carrier protein Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 DEG C frozen for subsequent use.
2. gold test strip according to claim 1, is characterized in that: described cellulose rete nitrocellulose filter, pure cellulose film or carboxylated cellulose film are made; Described sample pad PVDF membrane, nylon membrane, glass fibre cotton or polyester film are made; Adsorptive pads absorbent filter is made, and the supporting layer toughness material do not absorbed water is made; Gold labeling antibody pad glass fibre cotton is made.
3. gold test strip according to claim 1, is characterized in that: described in
the carrier protein of coupling sexavalence molybdenum ion is bovine serum albumin(BSA) or chicken ovalbumin; Described detection trace and 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, 0.3-0.6 cm place, sample pad side is partial to by the diaphragm that sample pad is corresponding with golden labeling antibody pad intersection and is printed with sample mark line.
5. the gold test strip according to any one of claim 1-4, is characterized in that: the specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label is prepared by the following method:
Adopt citrate reduction legal system standby, in Erlenmeyer flask, add 975 mL distilled waters boil, add 15mL trisodium citrate, continue heating 5 min, adding 10 mL mass concentrations is the gold chloride of 1%, continuing to be heated to solution is Chinese red, obtains colloidal gold solution after being cooled to room temperature, and 4 DEG C save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution, the sexavalence molybdenum ion monoclonal antibody solution that concentration is 0.2 mg/mL is dropwise added under magnetic agitation, incubation at room temperature 20 min, centrifugal 30 min of 10000 r/min, abandon supernatant, the Na that sediment uses often liter to contain 100 g BSA, concentration is 0.02 mol/L
2b
4o
7solution dilution, then centrifugal 30 min of 10000 r/min, the sediment obtained is the Na of 0.02 mol/L again containing 10 g BSA, 0.5 g Sodium azide, concentration with often liter
2b
4o
7solution dilution, namely obtain the sexavalence molybdenum ion monoclonal antibody of colloid gold label, 4 DEG C save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, containing 3 ng sexavalence molybdenum ion monoclonal antibodies in every 1mL collaurum.
6. a preparation method for gold test strip described in claim 1, is characterized in that: the method comprises the following steps:
(1) preparation of sexavalence molybdenum ion carrier protein couplet thing:
Get the HEPES damping fluid that 20 mL molybdic acids and 1mL concentration are 10 mmol/L, pH 8.0 to mix, obtain Mo
6+solution; Taking 10 mg ITCBE is dissolved in 1mL DMSO, forms metal-chelating agent solution; By Mo
6+solution and the mixing of metal-chelating agent solution, regulate the pH value to 7.4 of solution, and 25 DEG C are stirred 24 h, and rotating speed is 1000 r/min, form 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, adjust ph to 9.0, and under room temperature, 1000 r/min stir 24 h, then moves in bag filter 7 d that dialyse, collects dislysate, prepare carrier protein couplet thing Mo
6+-ITCBE-BSA or Mo
6+-ITCBE-OVA ,-20 DEG C are frozen for subsequent use;
(2) preparation of sexavalence molybdenum ion gold labeling antibody;
(3) preparation of golden labeling antibody pad;
(4) preparation of trace and contrast trace is detected;
On cellulose rete, make detection trace with the sexavalence molybdenum ion carrier protein couplet thing of gained, on cellulose rete, make contrast trace by rabbit anti-mouse IgG antibody; The package amount detecting 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;
Supporting layer, adsorbed layer and diaphragm, for the preparation of golden labeling antibody pad, are then assembled into gold test strip by sexavalence molybdenum ion gold labeling antibody successively.
7. preparation method according to claim 6, is characterized in that: the preparation method of the specificity sexavalence molybdenum ion monoclonal antibody of described colloid gold label is as follows:
Adopt citrate reduction method, 975 mL distilled waters are added in Erlenmeyer flask, boil, add 15 mL trisodium citrates, continue heating 5 min, adding 10 mL mass concentrations is the gold chloride of 1%, and continuing to be heated to solution is Chinese red, obtain colloidal gold solution after being cooled to room temperature, 4 DEG C save backup;
Get colloidal gold solution 100 mL, with the K of 0.1 mol/L
2cO
3solution regulates the pH 8.2 of colloidal gold solution, the sexavalence molybdenum ion monoclonal antibody solution that concentration is 0.2 mg/mL is dropwise added, centrifugal 30 min of incubation at room temperature 20 min, 10000 r/min under magnetic agitation, abandon supernatant, the Na that sediment uses often liter to contain 100 g BSA, concentration is 0.02 mol/L
2b
4o
7solution dilution, then centrifugal 30 min of 10000 r/min, the sediment obtained contains 10 g BSA with often liter 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, 4 DEG C save backup; In the colloidal gold solution of gained, the particle diameter of gold grain is 25 nm, containing 3 ng sexavalence molybdenum ion monoclonal antibodies in every 1mL collaurum.
8. the preparation method according to claim 6 or 7, is characterized in that: described sexavalence molybdenum ion monoclonal antibody specific is prepared by following methods:
Immune animal: with Mo
6+-ITCBE-BSA is immunizing antigen, using Balb/c mouse as immune animal, dorsal sc multi-point injection, immunizing dose is every only each 50 ~ 100 μ g, 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;
Select Fusion of Cells mouse for subsequent use: indirect ELISA detects Mo in antiserum
6+chelate polyclonal antibody (pAb) is tired, and stop band restrain detects Mo
6+-EDTA pAb is to Mo
6+the half-inhibition concentration of-EDTA (
iC 50), select tire the highest,
iC 50minimum mouse, surpasses and exempts from for Fusion of Cells;
Prepare positive hybridoma cell: get NS0 myeloma cell and immune mouse spleen cell PEG solution carries out Fusion of Cells, 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 multiparity female mouse lumbar injection hybridoma cell strain, gather the monoclonal antibody that ascites purifying obtains anti-sexavalence molybdenum ion.
9. the gold test strip described in an any one of claim 1-5 is detecting the application in sexavalence molybdenum ion fast.
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