CN103472232B - Zinc ion colloidal gold chromatographic Rapid detection test strip or test card and preparation method - Google Patents
Zinc ion colloidal gold chromatographic Rapid detection test strip or test card and preparation method Download PDFInfo
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- CN103472232B CN103472232B CN201310449633.6A CN201310449633A CN103472232B CN 103472232 B CN103472232 B CN 103472232B CN 201310449633 A CN201310449633 A CN 201310449633A CN 103472232 B CN103472232 B CN 103472232B
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Abstract
The present invention discloses a kind of zinc ion colloidal gold chromatographic Rapid detection test strip or test card and preparation method, test strips structure comprises back up pad, sample pad, golden labeling antibody pad, coated film and adsorptive pads, test strips two ends are provided with diaphragm, and described sample pad, golden labeling antibody pad, coated film, adsorptive pads are pasted in back up pad successively; Gold labeling antibody pad is coated with the monoclonal antibody that can identify zinc ion or the polyclonal antibody of colloid gold label; The stealth that the carrier protein solution that coated film is provided with coupling zinc ion is printed detects trace, and contrasts trace with the stealth that goat anti-mouse igg solution is printed.Colloidal gold chromatographic test strip of the present invention or test card high specificity, susceptibility are high, easy to use, quick, by force ageing; Result display image, directly perceived, accurate, cost saving, applied widely, easy to utilize.
Description
Technical field
The present invention relates to a kind of utensil detecting trace zinc ion, particularly relating to a kind of gold label test strip/card for detecting zinc ion fast and preparation method, belong to immunology and sanitary inspection and to learn a skill field.
Background technology
Heavy metal in soil and agricultural product and human health closely related.Heavy metal pollution mainly refers to the significant mercury of bio-toxicity, chromium, cadmium, lead and metalloid arsenic, also comprises the metal pollutants such as the virose heavy metal copper of tool, cobalt, nickel, tin.Heavy metal pollution is different from other types and pollutes, and has the features such as disguise, chronicity and irreversibility.Along with expansion and the large-scale industry development in city, after plurality of heavy metal entered environment, also may work the mischief even if concentration is very low, finally threaten health by potable water or by the mode such as biological concentration and food chain.
Zinc participates in synthesis and the activation of more than 200 kind of enzyme in body, is the metabolic important substance of body, is animal growth and the necessary trace element of maintenance normal physiological function, participates in the synthesis of protein, promote cell division, growth and regeneration; But the abuse of environmental pollution and feed, medicated premix, causes the residual of animal products zinc to exceed standard in various degree.Excess zinc is that one acts on nervous centralis toxin rapidly, and affect brain function by the direct infringement on neurocyte and the antagonism on material various in body, excess intake can cause organism metabolic disorder; Excessive zinc also can obviously suppress erythrocytic immunologic function, cause chick liver, spleen 26S Proteasome Structure and Function impaired.Many experiments and epidemiology survey confirm, if zinc too high levels in vivo, will suppress cytophagous activity and sterilizing power, and reduce immune function of human body, resistibility weakens, and increases disease susceptibility.
Meanwhile, zinc is mainly derived from mining, plating and the discharge of smelting industry pollutant, and the zinc pollution in water and soil has caused the extensive concern of environmental science worker.ZINC IN SOIL may work the mischief to plant growth more than during 200mg/kg, excess zinc can directly cause plant generation zinc pollution poisoning, also possibility remote effect plant is for the absorption of important nutrient Fe, and then makes plant cause growth disorder, even death because of scarce Fe.In the prevention of heavy metal pollution with in administering, the monitoring of heavy metal contaminants and identify most important.
Therefore, the qualitative and quantitative analysis of trace heavy metal is extremely important in food and environment measuring etc.Traditional detection of heavy metal ion method has flameless atomic absorption spectrometry, inductively coupled plasma mass spectrometry, flame atomic absorption spectrometry, voltammetry and the chromatography of ions, and electric atomizing atomic absorption spectrography (AAS) etc., detection must be carried out in the key lab possessing large-sized analytic instrument, Site Detection cannot be used for, and be subject to costly, the restriction such as treatment capacity finite sum detection time is long, be unfavorable for applying aborning, be difficult to conform and the spot check of market product and product import and export the requirement speeded passage through customs.
Therefore, set up the zinc immunoassay technology of screening quick, easy, economic, in enormous quantities, for minimizing environmental pollution, improve food quality and ensure food safety significant.
Summary of the invention
The technical problem to be solved in the present invention: provide a kind of detection zinc ion that can be quick, easy, responsive, special to pollute residual zinc ion gold label test strip or test card; The preparation method of zinc ion gold label test strip is also provided.
technical scheme of the present invention:
A kind of zinc ion colloidal gold chromatographic Rapid detection test strip, comprise back up pad, sample pad, golden labeling antibody pad, coated film and adsorptive pads, test strips two ends are provided with diaphragm, and described sample pad, golden labeling antibody pad, coated film, adsorptive pads are pasted in back up pad successively; Gold labeling antibody pad is coated with the monoclonal antibody that can identify zinc ion or the polyclonal antibody of colloid gold label; The stealth that the carrier protein solution that coated film is provided with coupling zinc ion is printed detects trace, and contrasts trace with the stealth that goat anti-mouse igg solution is printed.
The cardboard bar that described back up pad is hard plastic bar or does not absorb water; Described sample pad and golden labeling antibody pad are made up of glass fibre cotton; Described adsorptive pads is made up of absorbent filter; Described coated film is made up of nitrocellulose filter; Described carrier protein is chicken egg white (OVA), hemocyanin (KLH) or bovine serum albumin (BSA).
Described diaphragm covers on sample pad, golden labeling antibody pad and adsorptive pads, diaphragm is printed with sample mark line, this sample mark line deflection 0.3-0.5cm place, sample pad side; The stealthy package amount detecting the carrier protein solution of coupling sexavalence zinc ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On stealthy contrast trace, the package amount of goat anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; The arrangement mode that described stealth detects trace and stealthy contrast trace be "
︱ ︱", "
++" or " ● ● ".
The preparation method of zinc ion colloidal gold chromatographic Rapid detection test strip, described preparation process is as follows:
(1) preparation of coupling zinc ionophoric protein solution:
Take that 20mg carrier protein is dissolved in 1mL, concentration is 10mmoL/L, pH value is in the damping fluid of N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid of 9.0, form carrier protein solution; Taking 10mg p-amino phenyl-diethylenetriamine pentaacetic acid is dissolved in during 1mL dimethyl Asia soughs, and forms metal cheating agents solution; Get 100uL DTPA solution to be under agitation dropwise added in 0.5mL carrier protein solution, regulate its pH to 9.0 with the KOH of 10mol/L, room temperature reaction 24h, form DTPA-carrier protein solution; Take 100mg zinc chloride to be dissolved in 100 μ L red fuming nitric acid (RFNA)s, add pure water and dissolve and obtain that final volume is 1mL, concentration is the Zn of 733.7mmoL/L
2+solution;
By Zn
2+solution joins in DTPA-carrier protein solution, regulates its pH value to 7.4, incubated at room 4h, with PBS damping fluid dialysis 10d, forms Zn
2+-DTPA-carrier protein artificial antigen, collect packing ,-20 DEG C are frozen;
(2) preparation of monoclonal antibody: use Zn
2+-DTPA-carrier protein immunity BALB/C mice in 6 week age, adopts cell-fusion techniques, merges under PEG-1500 effect with NS0 myeloma cell; Carrying out the screening of positive hybridoma cell strain by ELISA method, cultivating through expanding, frozen and identify, prepare hybridoma cell strain, adopt in body afterwards and induce ascites legal system for zinc ion monoclonal antibody;
(3) golden labeling antibody preparation:
By the centrifugal 30min of zinc ion monoclonal antibody solution 10000r/min to be marked, get colloidal gold solution 10mL, with 0.1 mol/L K
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution; Get equivalent zinc ion monoclonal antibody solution, mix under magnetic agitation with colloidal gold solution, the centrifugal 30min of incubated at room 15min, 10000r/min, abandons supernatant, is the Na of 0.02 mol/L by gained precipitation with often liter containing 10 g BSA, 0.5 g Sodium azide, concentration
2b
4o
7solution dilution, namely obtains the zinc ion monoclonal antibody of colloid gold label, and 4 DEG C save backup;
(4) golden labeling antibody pad preparation: cut glass fibre cotton by specification, be evenly sprayed on glass fibre cotton by unidirectional for golden labeling antibody X-only specking instrument, 37 DEG C of dry 1h, sealing, 4 DEG C save backup;
(5) the stealthy preparation detecting trace and stealthy contrast trace: by Zn
2+-DTPA-carrier protein artificial antigen is put in X-only unidirectional specking instrument storage pool A, and goat anti-mouse igg solution is put in storage pool B, and start respectively fixed fire is central in film, the stealth forming spacing 0.5cm detects trace and stealthy contrast trace, natural drying, sealing, 4 DEG C save backup;
(6) test strips assembling: sample pad, golden labeling antibody pad, coated film and adsorptive pads are pasted in back up pad successively in order, and the surface mount diaphragm at test strips two ends, slot-cutting machine cuts into test strips.
Wherein zinc ion monoclonal antibody is 1: 5.2 × 10 with the mark ratio of colloidal gold solution
4, in gained colloidal gold solution, the particle diameter of gold grain is 30 nm; The concentration of contained goat anti-mouse igg solution is 0.34 μ g/mL.
A kind of zinc ion colloidal gold chromatographic quick detection test paper card, comprise housing and the test paper core being positioned at housing, test paper core comprises base layer support plate, sample pad, golden labeling antibody pad, coated film and adsorptive pads, and described sample pad, golden labeling antibody pad, coated film and adsorptive pads are pasted on base layer support plate successively; Gold labeling antibody pad is coated with the monoclonal antibody that can identify zinc ion of colloid gold label; The stealth that the carrier protein solution that described coated film is provided with coupling zinc ion is printed detects trace, and contrasts trace with the stealth that goat anti-mouse igg solution is printed.
Described housing is made up of base and cover, base and cover are connected together by chimeric, and base is provided with the groove placing test paper core, and cover is provided with view window, well, view window is corresponding with the coated film of test paper core, and well is corresponding with the sample pad of test paper core.
The cardboard bar that described back up pad is hard plastic bar or does not absorb water; Described sample pad and golden labeling antibody pad are made up of glass fibre cotton; Described adsorptive pads is made up of absorbent filter; Described coated film is made up of nitrocellulose filter; Described carrier protein is chicken egg white, hemocyanin or bovine serum albumin; The package amount that described stealth detects the carrier protein of coupling sexavalence zinc ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On stealthy contrast trace, the package amount of goat anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; The arrangement mode that described stealth detects trace and stealthy contrast trace be "
︱ ︱", "
++" or " ● ● ".
The carrier protein solution of described coupling zinc ion is prepared by following steps:
(1) take that 20mg carrier protein is dissolved in 1mL, concentration is 10mmoL/L, pH value is in the damping fluid of N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid of 9.0, form carrier protein solution;
(2) taking 10mg metal cheating agents p-amino phenyl-diethylenetriamine pentaacetic acid is dissolved in during 1mL dimethyl Asia soughs, and forms metal cheating agents solution;
(3) get 100uL DTPA solution to stir down gently and be dropwise added drop-wise in 0.5mL carrier protein solution, regulate pH to 9.0 with 10mol/L KOH, under room temperature, react 24h, form DTPA-carrier protein solution;
(4) take 100mg zinc chloride to be dissolved in 100 μ L red fuming nitric acid (RFNA)s, add pure water and dissolve and obtain that final volume is 1mL, concentration is the Zn of 733.7mmoL/L
2+solution;
(5) by Zn
2+solution joins in DTPA-carrier protein solution, adjust ph to 7.4, incubated at room 4h, to dialyse 10d, namely form Zn with PBS
2+-DTPA-carrier protein artificial antigen, collect packing ,-20 DEG C frozen.
Described colloid gold label can identify that the monoclonal antibody of zinc ion is prepared by following methods:
(1) preparation of monoclonal antibody: use Zn
2+-DTPA-KLH immunity female BAl BIc/C mouse in 6 week age, dosage is 20 μ g/0.2mL/, and dorsal sc branch is injected; Adopt cell-fusion techniques, merge with NS0 myeloma cell under PEG-1500 effect; The screening of positive hybridoma cell strain is carried out by indirect ELISA and stop band restrain method, limited dilution cloning is carried out after screening, then expand cultivation, frozen and identify, obtain hybridoma cell strain, adopt in body afterwards and induce ascites legal system for zinc ion monoclonal antibody;
(2) golden labeling antibody preparation: by the centrifugal 30min of zinc ion monoclonal antibody solution 10000r/min to be marked, get colloidal gold solution 10mL, with the K of 0.1 mol/L
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution; Get equivalent zinc ion monoclonal antibody solution, mix with colloidal gold solution under magnetic agitation, incubated at room 15min, the centrifugal 30min of 10000r/min, abandon supernatant, be the Na2B4O7 solution dilution of 0.02 mol/L containing 10 g BSA, 0.5 g Sodium azide, concentration with often liter by the sediment obtained, namely obtain the zinc ion monoclonal antibody of colloid gold label, 4 DEG C save backup; In gained colloidal gold solution, the particle diameter of gold grain is 30 nm, the monoclonal antibody containing 5 ng zinc ions in every milliliter of collaurum.
zinc ion test strip/jig of the present invention has following advantages:
high specificity, susceptibility is high.Zn
2+-Strip is prepared from based on the monoclonal antibody of colloid gold label high-affinity or polyclonal antibody, formed without covalent bond between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, colloid gold label on the specificity of monoclonal antibody or polyclonal antibody and affinity impact very little, and there is higher mark rate.Therefore, this test strips or jig have stronger specificity and higher susceptibility, and lowest detection is limited to 1 μ g/L, less with other metallic ion cross reaction.
easy, quick, by force ageing.Use Zn
2+-Strip, without the need to other reagent any and instrument, can execute-in-place, only test strips need be inserted measuring samples or measuring samples be dripped 10-20 second on test card, in 5 minutes, can testing result be judged.
result intuitive display, image, accurately.Zn
2+-Strip with show reddish brown colo(u)r streak "
︱" and "
︱ ︱" the positive and negative marker as testing result, namely show on coated film a reddish brown colo(u)r streak "
︱" time, represent in test sample containing Zn
2+, show two reddish brown colo(u)r streaks "
︱ ︱" time, represent in test sample not containing Zn
2+.It is vivid, directly perceived, accurate, simple and clear that result judges, not easily occurs the artificially erroneous judgement such as false positive and false negative.
the preparation method of gold label test strip of the present invention, has prepared zinc ionophoric protein conjugate, 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.
5. the preparation method of gold test strip of the present invention, prepared the monoclonal antibody of anti-zinc ion high-titer, high-affinity, high specificity, antibody titer of ascites is 1: 1.2 × 10
6, cross reacting rate is less than 0.01%, and the trace for zinc ion detects fast and provides guarantee.
6. applied widely, cost saving, is convenient to promote.Use Zn
2+-Strip is lower than instrumental analysis expense, applied widely, the needs of different levels personnel can be met, comprise professional inspection, customs quarantine control, health quarantine, quality monitoring and processing enterprise and plant family etc., easy to utilize, there are wide market outlook and obvious economical, societal benefits.
Accompanying drawing explanation
Fig. 1, zinc ion test strip plan structure schematic diagram.
Fig. 2, zinc ion test strip cross-sectional view.
Fig. 3, zinc ion Test paper card plan structure schematic diagram.
Fig. 4, zinc ion Test paper card cross-sectional view.
In figure, 1: back up pad, 2: sample pad; 3: golden labeling antibody pad, 4: coated film, 5: stealthy detection trace; 6: stealthy contrast trace, 7: adsorptive pads, 8-1: sample end diaphragm; 8-2: handle end diaphragm, 9: sample mark line, 10: well; 11: view window, 12: panel, 13: pickup groove; 14: base, 15: groove.
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 and detect zinc ion gold label test strip or test card, first will prepare zinc ion artificial immunity antigen, immune Balb/c mouse, prepares the monoclonal antibody of anti-zinc ion afterwards, assembles zinc ion gold label test strip or test card on this basis.
embodiment 1, Zn
2+
the synthesis of artificial antigen
Take 20mg KLH, be dissolved in N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd damping fluid (HBS) that 1mL concentration is 10mmoL/L, pH9.0, form KLH solution.
Take 10mg metal cheating agents p-amino phenyl-diethylenetriamine pentaacetic acid (P-NH2-Bn-DTPA), be dissolved in 1mL dimethyl Asia and sough in (DMSO), form metal cheating agents solution; Get 160uL metal cheating agents solution dropwise joins in the KLH solution of 1mL under stirring gently, regulates pH to 9.0, react 24h under room temperature by the NaOH solution of 10mol/L; The super filter tube purifying of reaction product 30Kd, reaction product ultrafiltration is added in super filter tube, super filter tube first rinses 3 times with 10 mmoL/L, pH9.0 HBS, then washes 2 times with the HBS damping fluid of 10 mmoL/L, pH7.4, removing wherein unreacted Small molecular metal cheating agents.
Getting 16 μ L concentration is that 733.7mmoL/L zinc solution is added drop-wise in reacted carrier protein solution, and regulate its pH value to 9.0 by NaOH solution, room temperature shaker reacts 24 hours, then moves in bag filter and dialyses 10 days.Products therefrom is the artificial antigen Zn after the zinc ion huge legendary turtle conjunction of purifying
2+-DTPA-KLH.The protein concentration in artificial antigen is measured at a wavelength of 280 nm with protein nucleic acid analyser.
Envelope antigen Zn is obtained with legal system
2+-DTPA-BSA or Zn
2+-DTPA-OVA.Reaction principle is shown in following reaction equation.
embodiment 2, Zn
2+
the preparation of monoclonal antibody
(1) animal immune.Immunity Balb/c mouse, uses Zn
2+-DTPA-KLH immunity female Balb/C mouse 5 in 6 weeks age, dosage be 20 μ g/0.2mL/ only, the injection of dorsal sc branch.Head exempts from, and dilutes Zn with sterilizing PBS
2+-DTPA-KLH, with equivalent CFA mixing and emulsifying; Booster immunization, dilutes Zn with sterilizing PBS
2+-DTPA-KLH, 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 Zn in antiserum
2+chelate pAb tires, and stop band restrain detects Zn
2+-DTPA pAb is to Zn
2+-DTPA'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-1500 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-1500(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 96 porocyte culture plates, every hole 100 μ L, be placed in 37 DEG C, the CO of 5%
2cultivate in incubator.
The screening of positive hybridoma cell, carries out the screening of positive hybridoma cell strain 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.Ascites is produced 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, measure the IgG content of obtained antibody with protein nucleic acid analyser at a wavelength of 280 nm, calculate the affinity constant of this monoclonal antibody;-20 DEG C save backup.
embodiment 3, anti-zn
2+mAb
qualification
(1) titer of ascites measures.To the injected in mice cloning cell line 10 after lumbar injection whiteruss 10 d
7individual cell, extracts ascites after 10 d, and saturated ammonium sulfate salting out method is purified, and indirect ELISA measures Zn
2+the titer of ascites of mAb is 1: 1.2 × 10
6;
(2) susceptibility qualification.Zn is measured with stop band restrain
2+mAb is to variable concentrations Zn
2+the inhibiting rate of-EDTA titer, with inhibiting rate B/B
0for ordinate, with variable concentrations Zn
2+the logarithm value of-EDTA is horizontal ordinate, and drawing standard suppresses curve, carries out correlation regression analysis, calculates Zn
2+mAb is to Zn
2+-EDTA's
iC 50.Zn
2+the equation of linear regression of mAb is y=-33.827
x+ 99.512, R
2=0.9891,
iC 50 be 29.08 μ g/L.
(3) specificity identification.Employing cross reaction is tested, select the chelate of the metallic ion such as mercury, lead, cadmium, copper, chromium, cobalt, iron and EDTA, EDTA, ITCBE, DOTA, DTPA be mortifier, measures each mortifier with stop band restrain
iC 50, with Zn
2+mAb is to Zn
2+'s
iC 50with to other each competitor
iC 50percent be its cross reacting rate (CR%).Qualification result, is less than 0.01% with other heavy metal ion cross reacting rate.
embodiment 4,zn
2+ the preparation of gold label test strip/test card
(1) colloidal gold solution preparation.Adopt citrate reduction legal system standby, get 1000mL Erlenmeyer flask, add 975mL distilled water and boil, add 15mL trisodium citrate and continue heating 5min, add the gold chloride of 10mL 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 mAb solution 10000r/min will be treated, with the K of 0.1 mol/L
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution; According to coagulation phenomenon, determine Zn
2+-DTPA-BSA mAb marks ratio, according to 1: 5.2 × 10 with the optimum of colloidal gold solution
4ratio dilute, contained IgG concentration is 0.34 μ g/mL.
Get colloidal gold solution 10mL, with 0.1 mol/L K
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution.The concentration of getting equivalent is 0.34mg/mL mAb solution, mixes under magnetic agitation, and the centrifugal 30min of incubated at room 15min, 10000r/min, abandons supernatant, the sediment Na of 0.02 mol/L
2b
4o
7solution (containing 10 g/L BSA, 0.5 g/L Sodium azide) dilutes, and 4 DEG C save backup; Wherein in colloidal gold solution, the particle diameter of gold grain is 30nm, and labelled amount is 1mL collaurum: 5ng Zn
2+mAb.
(3) preparation of golden labeling antibody pad.Cut the glass fibre cotton that specification is 20 × 4mm, be evenly sprayed on glass fibre cotton by unidirectional for golden labeling antibody X-only specking instrument, 37 DEG C of dry 1h, sealing, 4 DEG C save backup.
(4) preparation of detection line, nature controlling line.Be the Zn of 1mg/mL by concentration
2+-DTPA-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 film central authorities respectively in start, and form detection line and nature controlling line that spacing is 0.5cm, natural drying, sealing, 4 DEG C save backup.
(5) assembling of test strips.Sample pad, golden labeling antibody pad, coated film, adsorptive pads are pasted in back up pad in order successively, and the surface mount diaphragm at test strips two ends, slot-cutting machine cuts into the gold label test strip of width 4mm.
embodiment 5: the structure of zinc ion test strips, see Fig. 1, Fig. 2.In figure, back up pad 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-zinc ion, coated film 4 adopts nitrocellulose filter, adsorptive pads 7 is made with absorbent filter, be pasted and fixed on successively from right to left in back up pad 1 by each layer of numbering 2,3,4,7, intersection fiber crosses one another infiltration each other.Coated film 4 is provided with and stealthy detects trace 5 and stealthy contrast trace 6, stealthy trace bovine serum albumin(BSA) (BSA) solution of coupling zinc ion that detects is printed, stealthy contrast trace goat anti-mouse igg antibody solution printing, two trace one-tenth arranged in parallel "
︱ ︱".8-1 covers the sample end 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 7; 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-zinc ion monoclonal antibody etc. is see above embodiment.
(1) detection reaction principle.After zinc ion test strips test lead inserts testing sample solution, solution to be measured drives zinc ion to be measured and golden labeling antibody together to coated film diffusion by syphonic effect, and infiltrates the adsorptive pads of handle end.In diffusion process, zinc ion to be measured can combine with golden labeling antibody, and then close the antigen-combining site of zinc ion on golden labeling antibody, the detection trace of golden labeling antibody coupling zinc ionophoric albumen on coated film is stoped to be combined, detection trace can not be shown, goat anti-mouse igg antibody then can be combined with golden labeling antibody, formation brownish red contrast trace "
︱", namely a brownish red trace is positive expression; Otherwise, without zinc ion in sample solution, the carrier protein of golden labeling antibody coupling zinc ion on coated film then can not be stoped to detect trace be combined, display brownish red detects trace, same goat anti-mouse igg antibody is also combined with golden labeling antibody, display brownish red contrast trace, forming two reddish brown colour bands is negative expression.If cellulose membrane does not have brownish red trace show, then show that test strips lost efficacy.
(2) detecting step.Sample pretreatment, sample comprises soil, water, food, feed, animal tissue, blood, urine etc., and the grinding of solid thing to be checked is smashed to pieces, and it is nitrated to carry out nitric acid-perchloric acid, and liquid thing to be checked directly adds nitric acid nitrating.
1. solid sample is nitrated: get 1g sample respectively and put into glass digest tube, add 15mL red fuming nitric acid (RFNA), be placed in vent window and spend the night; Then add the dense perchloric acid of 5mL, be slowly heated to perchloric acid white smoke ease under constant temperature to the greatest extent, simultaneously till solution clear, transfer in 25mL volumetric flask stand-by after cooling.
2. fluid sample is nitrated: liquid thing to be checked directly adds 15mL nitric acid, and room temperature leaves standstill 2h, in thermostatic electrothermal plate, during volume concentration to 2 ~ 3mL, with 2.5% nitric acid dilution after cooling, stand-by in constant volume to 25mL volumetric flask.
According to 1:1 volume ratio, treatments of the sample liquid and 10% EDTA sequestrant are mixed, make Zn
2+chelating abundant with EDTA, forms sample liquid to be measured.
Detecting step: test strips is directly inserted in testing sample, horizontal positioned, observations in 5min, after 10min, result is invalid; When detecting with test card, draw testing sample 100 μ L or two droplet with dropper and drip in the well of test card, observations in 5min.
After measured, the range estimation sensitivity of this test strips/card is 5 μ g/L, and machine-readable sensitivity is 1 μ g/L; Can be applicable to Zn in environment, soil, water, food
2+pollute residual quick detection.
embodiment 6: test strips structure is substantially identical with embodiment 6; difference is: golden labeling antibody pad is adsorbed with the polyclonal antibody of anti-zinc ion; sample pad nylon membrane is made; coated film adopts pure cellulose film; detect trace and contrast trace and be " ten ", the handle end diaphragm covered above adsorptive pads is blue look.
embodiment 7: test strips structure is substantially identical with embodiment 6; difference is: sample pad pvdf membrane is made; the carrier protein solution of coupling zinc ion is chicken ovalbumin (OVA), and coated film adopts carboxylated cellulose film, and the handle end diaphragm covered above adsorptive pads is green.
embodiment 8: test strips structure is substantially identical with embodiment 6, difference is: sample pad polyester film is made, coated film adopts carboxylated cellulose film, and the carrier protein solution of coupling zinc ion is hemocyanin (KLH), detection trace and contrast trace are arranged as " ● ● ".
embodiment 9:zinc ion Test paper card, see Fig. 3, Fig. 4.Test card comprises housing and is positioned at the test paper core of housing, and test paper core comprises back up pad, sample pad, golden labeling antibody pad, coated film and adsorptive pads, and the structure of test paper core is identical with example 6, and difference is:
Test card case material is plastics, and housing base 14 is provided with the pickup groove 13 of fixing test paper core; Well 10 on panel 12 is corresponding with the sample pad 2 of test paper core, and well 10 is the positions dripping measuring samples; Panel 12 is provided with the groove 15 combined with base, view window 11 on panel 12 is corresponding with the coated film 4 of test paper core, be observe the window of result of determination, its side is printed on T and C, and detects trace 5 and stealth respectively to contrast trace 6 corresponding with the stealth on coated film 4.
During detection, test card is kept flat, drip analyte sample fluid from well, in 5 minutes, judge testing result from view window.
Result judge: the position display as T corresponding on coated film have a reddish brown colo(u)r streak "
︱" time, represent that testing result is positive, illustrate in testing sample containing zinc ion; Position display as corresponding on coated film T, C have two reddish brown colo(u)r streaks "
︱ ︱" time, represent that testing result is negative, testing sample is described not containing zinc ion; As not having reddish brown colo(u)r streak to show on coated film, then show that test card lost efficacy.
embodiment 10:test card structure is substantially identical with embodiment 10, difference is: substitute zinc ion monoclonal antibody with anti-zinc ion polyclonal antibody, the carrier protein of coupling zinc ion is chicken ovalbumin OVA, substitutes goat anti-mouse igg on coated film, prepare contrast trace with rabbit anti-mouse IgG.
embodiment 11,zn
2+ the performance measurement of gold label test strip/paper slip card
(1) Zn
2+the sensitivity testing of gold label test strip/card.Use Zn
2+-Strip measures the Zn that concentration is respectively 0,1.25,2.5,5,10,20,40,80,160,320,640 μ g/L
2+standard items, each concentration establishes 6 repetitions.With the relative optical density number (G/D × A-ROD (pixel) reading bar instrument reading T line sweep area optical density, in formula, G represents chart, D represents optical density, A represents scan area, D × A is the optical density of scan area, ROD represents relative optical density number), with the percent (G/D × A-ROD (pixel) %) of variable concentrations standard items and zero standard product relative optical density number for ordinate, with the common logarithm value of various criterion product concentration for horizontal ordinate, semilogarithmic paper is drawn the typical curve of Test paper, add regression equation, carry out correlation regression analysis.According to the colour developing result of Test paper, get 80% for machine-readable susceptibility, get 50% for range estimation susceptibility, determine Zn
2+the detectability of-Strip.Zn
2+the range estimation sensitivity of gold test strip is 5 μ g/L, and machine-readable is sensitivity 1 μ g/L.
Table 1 measures Zn
2+-Strip sensitivity testing (μ g/L, n=6)
| Zn 2+Concentration | 0 | 1.25 | 2.5 | 5 | 10 | 16 | 20 | 40 | 80 | 160 | 320 | 640 |
| Result | － | － | － | + | + | + | + | + | + | + | + | + |
(2) Zn
2+the specific test of gold label test strip/card.Employing cross reaction is tested, select mercury, molybdenum, cadmium, copper, lead, etc. the chelate of metallic ion and EDTA, take ITCBE as mortifier, use Zn
2+-Strip measures final concentration and is respectively 0,1.25,2.5,5,10,20,40,80,160,320,640 μ g/L heavy metal ion standard items, and each concentration establishes 6 repetitions, judges its specificity with this.The results are shown in Table 2, Zn
2+-Strip and Zn
2+specific binding, with other heavy metal ion no cross reaction.
Table 2 measures Zn
2+-Strip specific test (n=6)
(3) Zn
2+the replica test of gold label test strip/card.Get 6 crowdes of Zn of different batches
2+gold test strip, respectively at the 1st, 7,14,28,56,112 day to Zn
2+concentration is that the sample such as soil, tap water, pork of 0,2,4,8 μ g/L detects, and 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 Zn
2+-Strip replica test (n=6)
（4）
(5) Zn
2+the storage life test of gold label test strip/test card.Retention period check: by the Zn of different batches
2+gold test strip is stored in general refrigerator (2 ~ 8 DEG C) 12 months respectively and drying at room temperature preserves 6 months, and the testing result of research different holding time, determines its storage life.Result shows, Zn
2+gold test strip its outward appearance, accuracy, susceptibility, specificity etc. in storage life all do not change, and identical for the test result of test paper with brand-new, its term of validity is lower 12 months of 2 ~ 8 DEG C of conditions, lower 6 months of drying at room temperature condition.
Claims (9)
1. a zinc ion colloidal gold chromatographic Rapid detection test strip, comprise back up pad, sample pad, golden labeling antibody pad, coated film and adsorptive pads, test strips two ends are provided with diaphragm, it is characterized in that: described sample pad, golden labeling antibody pad, coated film, adsorptive pads are pasted in back up pad successively; Gold labeling antibody pad is coated with the monoclonal antibody that can identify zinc ion or the polyclonal antibody of colloid gold label; The stealth that the carrier protein solution that coated film is provided with coupling zinc ion is printed detects trace, and contrasts trace with the stealth that goat anti-mouse igg solution is printed;
Described coupling zinc ionophoric protein solution is prepared by following methods:
Take that 20mg carrier protein is dissolved in 1mL, concentration is 10mmoL/L, pH value is in the damping fluid of N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid of 9.0, form carrier protein solution; Taking 10mg p-amino phenyl-diethylenetriamine pentaacetic acid is dissolved in during 1mL dimethyl Asia soughs, and forms metal cheating agents solution; Get 100 μ L DTPA solution to be under agitation dropwise added in 0.5mL carrier protein solution, regulate its pH to 9.0 with the KOH of 10mol/L, room temperature reaction 24h, form DTPA-carrier protein solution; Take 100mg zinc chloride to be dissolved in 100 μ L red fuming nitric acid (RFNA)s, add pure water and dissolve and obtain that final volume is 1mL, concentration is the Zn of 733.7mmoL/L
2+solution;
By Zn
2+solution joins in DTPA-carrier protein solution, regulates its pH value to 7.4, incubated at room 4h, with PBS damping fluid dialysis 10d, forms Zn
2+-DTPA-carrier protein artificial antigen, collect packing ,-20 DEG C frozen.
2. test strips according to claim 1, is characterized in that: the cardboard bar that described back up pad is hard plastic bar or does not absorb water; Described sample pad and golden labeling antibody pad are made up of glass fibre cotton; Described adsorptive pads is made up of absorbent filter; Described coated film is made up of nitrocellulose filter; Described carrier protein is chicken egg white, hemocyanin or bovine serum albumin.
3. test strips according to claim 1 and 2, is characterized in that: described diaphragm covers on sample pad, golden labeling antibody pad and adsorptive pads, diaphragm is printed with sample mark line, this sample mark line deflection 0.3-0.5cm place, sample pad side; The stealthy package amount detecting the carrier protein solution of coupling divalent zinc ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On stealthy contrast trace, the package amount of goat anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; The arrangement mode that described stealth detects trace and stealthy contrast trace be "
︱ ︱", "
++" or " ● ● ".
4. the preparation method of zinc ion colloidal gold chromatographic Rapid detection test strip described in claim 1, is characterized in that: gold label test strip preparation process is as follows:
(1) preparation of coupling zinc ionophoric protein solution:
Take that 20mg carrier protein is dissolved in 1mL, concentration is 10mmoL/L, pH value is in the damping fluid of N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid of 9.0, form carrier protein solution; Taking 10mg p-amino phenyl-diethylenetriamine pentaacetic acid is dissolved in during 1mL dimethyl Asia soughs, and forms metal cheating agents solution; Get 100 μ L DTPA solution to be under agitation dropwise added in 0.5mL carrier protein solution, regulate its pH to 9.0 with the KOH of 10mol/L, room temperature reaction 24h, form DTPA-carrier protein solution; Take 100mg zinc chloride to be dissolved in 100 μ L red fuming nitric acid (RFNA)s, add pure water and dissolve and obtain that final volume is 1mL, concentration is the Zn of 733.7mmoL/L
2+solution;
By Zn
2+solution joins in DTPA-carrier protein solution, regulates its pH value to 7.4, incubated at room 4h, with PBS damping fluid dialysis 10d, forms Zn
2+-DTPA-carrier protein artificial antigen, collect packing ,-20 DEG C are frozen;
(2) preparation of monoclonal antibody: use Zn
2+-DTPA-carrier protein immunity BALB/C mice in 6 week age, adopts cell-fusion techniques, merges under PEG-1500 effect with NS0 myeloma cell; Carrying out the screening of positive hybridoma cell strain by ELISA method, cultivating through expanding, frozen and identify, prepare hybridoma cell strain, adopt in body afterwards and induce ascites legal system for zinc ion monoclonal antibody;
(3) golden labeling antibody preparation:
By the centrifugal 30min of zinc ion monoclonal antibody solution 10000r/min to be marked, get colloidal gold solution 10mL, with 0.1 mol/L K
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution; Get equivalent zinc ion monoclonal antibody solution, mix under magnetic agitation with colloidal gold solution, the centrifugal 30min of incubated at room 15min, 10000r/min, abandons supernatant, is the Na of 0.02 mol/L by gained precipitation with often liter containing 10 g BSA, 0.5 g Sodium azide, concentration
2b
4o
7solution dilution, namely obtains the zinc ion monoclonal antibody of colloid gold label, and 4 DEG C save backup;
(4) golden labeling antibody pad preparation: cut glass fibre cotton by specification, be evenly sprayed on glass fibre cotton by the unidirectional specking instrument of golden labeling antibody, 37 DEG C of dry 1h, sealing, 4 DEG C save backup;
(5) the stealthy preparation detecting trace and stealthy contrast trace: by Zn
2+-DTPA-carrier protein artificial antigen is put in unidirectional specking instrument storage pool A, and goat anti-mouse igg solution is put in storage pool B, and start respectively fixed fire is central in film, the stealth forming spacing 0.5cm detects trace and stealthy contrast trace, natural drying, sealing, 4 DEG C save backup;
(6) test strips assembling: sample pad, golden labeling antibody pad, coated film and adsorptive pads are pasted in back up pad successively in order, and the surface mount diaphragm at test strips two ends, slot-cutting machine cuts into test strips.
5. preparation method according to claim 4, is characterized in that: wherein zinc ion monoclonal antibody is 1: 5.2 × 10 with the mark ratio of colloidal gold solution
4, in gained colloidal gold solution, the particle diameter of gold grain is 30 nm; The concentration of contained goat anti-mouse igg solution is 0.34 μ g/mL.
6. a zinc ion colloidal gold chromatographic quick detection test paper card, comprise housing and the test paper core being positioned at housing, test paper core comprises base layer support plate, sample pad, golden labeling antibody pad, coated film and adsorptive pads, it is characterized in that: described sample pad, golden labeling antibody pad, coated film and adsorptive pads are pasted on base layer support plate successively; Gold labeling antibody pad is coated with the monoclonal antibody that can identify zinc ion of colloid gold label; The stealth that the carrier protein solution that described coated film is provided with coupling zinc ion is printed detects trace, and contrasts trace with the stealth that goat anti-mouse igg solution is printed;
The carrier protein solution of described coupling zinc ion is prepared by following steps:
(1) take that 20mg carrier protein is dissolved in 1mL, concentration is 10mmoL/L, pH value is in the damping fluid of N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid of 9.0, form carrier protein solution;
(2) taking 10mg metal cheating agents p-amino phenyl-diethylenetriamine pentaacetic acid is dissolved in during 1mL dimethyl Asia soughs, and forms metal cheating agents solution;
(3) get 100 μ L DTPA solution to stir down gently and be dropwise added drop-wise in 0.5mL carrier protein solution, regulate pH to 9.0 with 10mol/L KOH, under room temperature, react 24h, form DTPA-carrier protein solution;
(4) take 100mg zinc chloride to be dissolved in 100 μ L red fuming nitric acid (RFNA)s, add pure water and dissolve and obtain that final volume is 1mL, concentration is the Zn of 733.7mmoL/L
2+solution;
(5) by Zn
2+solution joins in DTPA-carrier protein solution, adjust ph to 7.4, incubated at room 4h, to dialyse 10d, namely form Zn with PBS
2+-DTPA-carrier protein artificial antigen, collect packing ,-20 DEG C frozen.
7. test card according to claim 6, it is characterized in that: described housing is made up of base and cover, base and cover are connected together by chimeric, base is provided with the groove placing test paper core, cover is provided with view window, well, view window is corresponding with the coated film of test paper core, and well is corresponding with the sample pad of test paper core.
8. test card according to claim 6, is characterized in that: the cardboard bar that described back up pad is hard plastic bar or does not absorb water; Described sample pad and golden labeling antibody pad are made up of glass fibre cotton; Described adsorptive pads is made up of absorbent filter; Described coated film is made up of nitrocellulose filter; Described carrier protein is chicken egg white, hemocyanin or bovine serum albumin; The package amount that described stealth detects the carrier protein of coupling divalent zinc ion on trace is 1 μ L/cm, and carrier protein concentration is 1mg/mL; On stealthy contrast trace, the package amount of goat anti-mouse igg antibody is 1 μ L/cm, and antibody concentration is 1mg/mL; The arrangement mode that described stealth detects trace and stealthy contrast trace be "
︱ ︱", "
++" or " ● ● ".
9. the test card according to any one of claim 6-8, is characterized in that: described colloid gold label can identify that the monoclonal antibody of zinc ion is prepared by following methods:
(1) preparation of monoclonal antibody: use Zn
2+-DTPA-KLH immunity female BAl BIc/C mouse in 6 week age, dosage is 20 μ g/0.2mL/, and dorsal sc branch is injected; Adopt cell-fusion techniques, merge with NS0 myeloma cell under PEG-1500 effect; The screening of positive hybridoma cell strain is carried out by indirect ELISA and stop band restrain method, limited dilution cloning is carried out after screening, then expand cultivation, frozen and identify, obtain hybridoma cell strain, adopt in body afterwards and induce ascites legal system for zinc ion monoclonal antibody;
(2) golden labeling antibody preparation: by the centrifugal 30min of zinc ion monoclonal antibody solution 10000r/min to be marked, get colloidal gold solution 10mL, with the K of 0.1 mol/L
2cO
3solution regulates the pH value to 8.2 of colloidal gold solution; Get equivalent zinc ion monoclonal antibody solution, mix with colloidal gold solution under magnetic agitation, the centrifugal 30min of incubated at room 15min, 10000r/min, abandoning supernatant, is the Na of 0.02 mol/L containing 10 g BSA, 0.5 g Sodium azide, concentration with often liter by the sediment obtained
2b
4o
7solution dilution, namely obtains the zinc ion monoclonal antibody of colloid gold label, and 4 DEG C save backup; In gained colloidal gold solution, the particle diameter of gold grain is 30 nm, the monoclonal antibody containing 5 ng zinc ions in every milliliter of collaurum.
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