CN102426239A - Colloidal gold chromatographic test strip for rapidly testing lead ion - Google Patents
Colloidal gold chromatographic test strip for rapidly testing lead ion Download PDFInfo
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Abstract
A colloidal gold chromatographic test strip for rapidly testing lead ion belongs to the field of immunological technique. The test strip provided by the invention is composed of a liner plate and a sample pad, a colloidal-gold combination pad, a coating membrane and a water absorption pad which are successively connected on the liner plate. The colloidal-gold combination pad is glass fiber cotton for adsorbing lead ion colloidal-gold antibody. A linear invisible detection line T printed by a lead ion and carrier protein coupled solution and a linear contrast line C printed by a goat-anti-mouse IgG solution are successively arranged on the coating membrane, wherein the two lines are parallelly arranged. The colloidal gold chromatographic test strip for rapidly testing lead ion in the invention has strong specificity and high sensitivity, and can be used to more rapidly, sensitively and simply test lead ion residues. By the adoption of the test strip, the determination result is vivid, visual, accurate, simple and clear, and false positive and false negative human misjudgments are not easy to occur. The test strip provided by the invention can be used to save cost, has a wide application range, is convenient for popularization, and has an extensive market prospect as well as obvious economic and social benefits.
Description
Technical field
What the present invention relates to is a kind of colloid gold chromatographic test paper strip of fast detecting lead ion, belongs to the immunological technique field.
Background technology
Lead is a kind of primary raw material in the current industrial production, is very big to the harm of human body.Saturnism can cause neurasthenia and indigestion, and serious taken place lead encephalopathy forms insane carbuncle, dementia or spiritual irritated entanglement, hypopsia even blind.Nineteen twenty-three begins in gasoline, to add plumbous as after the octane promoter, has more quickened the pollution of global lead.Plumbous pollution is the extend over the entire globe scope, the existing increase at double of lead tolerance in the ice in the Greenland and the South Pole.Therefore we can say and nowadays difficultly in the world find the soil lead content not receive a slice " pure land " of the effect of human activity.
Regulation in China's groundwater quality standard (GB/T14848-93) is applicable to mainly that the lead ion of centralized Drinking Water water source and worker, agricultural water is limited the quantity of≤0.05mg/L.
At present, the method for detection lead ion mainly contains instrumental method and enzyme-linked immunosorbent assay (ELISA).Heavy metal instrumental analysis detection method commonly used at present comprises: ICP-AES, graphite oven atomic absorption, UV-VIS spectrophotometry, atomic fluorometry etc.Though more than the accurate single total amount of planting metal in the measuring samples of these detection methods; But detect effort, time-consuming, expensive relatively; Need carry out a large amount of sample pretreatments, and the detection of sample needs to carry out having the indoor of large-scale analytical equipment, can not be used for on-the-spot the detection.Because the restriction of above-mentioned shortcoming, the sample number of detection is restricted, causes having bigger uncertainty in the evaluation work of heavy metal pollution degree of carrying out subsequently and risk.The detection that is established as this pollutant of heavy metal immunological detection method provides another kind of approach.This method has that detection speed is very fast, the sensitivity of cost, simple portable, height and characteristics optionally.Can be applied to produce any polluter of suitable antibodies on this theoretical method.(Reardan etc. since people such as Reardan produce through metal-intercalating agent antigen first and isolate monoclonal antibody; 1985); Metal-chelate mixture compound antibody such as external increasing anti-Hg, In, Cd, Pb are developed out (Johnson etc., 2003), can be used for the field quick detection and the conventional sense of heavy metal contaminants; This has very big meaning for remedying and resuming work of heavy metal pollution area, thereby development and popularization and application potentiality are very big.The foreign scholar further prepares the metal specific monoclonal antibody through selecting or synthesizing difunctional intercalating agent huge legendary turtle and close heavy metal ion and prepare comlete antigen with carrier protein couplet.Heavy metal ion in the applied immunology detection method testing environment also is in breadboard experimental stage at present.
Summary of the invention
The object of the invention: to existing deficiency and the defective that detects the technology of lead ion, a kind of preparation method of gold mark chromatograph test strip of fast detecting lead ion is provided, it is residual to make it detect lead ion quick more, sensitive, easily.
Technical scheme of the present invention: a kind of lead ion colloidal gold chromatographic test strip; Be made up of liner plate (1) and the sample pad (2), gold-marking binding pad (3), coated film (4) and the adsorptive pads (5) that on liner plate, are connected successively: gold-marking binding pad is the glass fibre cotton of the golden labeling antibody of adsorpting lead ion; The stealthy detection line T line (6) of the orthoscopic that the solution of useful successively lead ion coupling carrier albumen is printed on coated film; With the stealthy control line C line (7) of orthoscopic that sheep anti-mouse igg solution is printed, two line parallels are arranged.
The hard plastic bar of described liner plate for not absorbing water, or the cardboard bar that do not absorb water; Described sample pad is glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Described coated film is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane; Described adsorptive pads is absorbent filter or filter paper for oil.
Described lead ion gold labeling antibody is the lead ion monoclonal antibody of colloid gold label; The carrier protein of described coupling lead ion is keyhole limpet hemocyanin KLH, or is bovine serum albumin(BSA) BSA, and the sequestrant of coupling lead ion and carrier protein is to sulphur cyanobenzyl-disodium salt ITCBE.
Beneficial effect of the present invention: test strip of the present invention has advantage:
1. high specificity, susceptibility is high.This colloidal gold chromatographic test strip is that the basis is prepared from the monoclonal antibody of colloid gold label high-affinity; No covalent bond formation between gold grain and the antibody molecule in the gold labeling antibody; The two combines through the Van der Waals force between the charges of different polarity; The specificity of colloid gold label antagonist and affinity influence are very little, and have higher mark rate.Therefore, test strips has stronger specificity and higher susceptibility, detects minimum limiting the quantity of and can reach 5ppb.
2. easy, quick, ageing strong.Use the colloidal gold chromatographic test strip, need not any other reagent and instrument, but execute-in-place, and test strips is the decidable testing result in 10min after adding test sample liquid.
3. the result shows image, directly perceived, accurate.Test strip all with exhibit red line T line and C line as the testing result positive and negative marker; When promptly on coated film, showing a red line C line; Be illustrated in and contain checking matter in the test sample, when showing two red line T lines and C line, be illustrated in and do not contain checking matter in the test sample.The result judges image, directly perceived, accurate, simple and clear, is not prone to artificial erroneous judgement such as false positive and false negative.
4. cost saving, applied widely, be convenient to promote.Use test strip, decline to a great extent than expense with instrumental analysis and ELISA kit.In addition, test strips applied widely can satisfy different levels personnel needs, comprises professional inspection; Customs quarantine control, health quarantine, quality monitoring; Processing enterprise and plant family etc., easy to utilize, have vast market prospect and tangible economical, societal benefits.
Description of drawings
Fig. 1, lead ion colloidal gold chromatographic test strip cross-sectional view.1, liner plate, 2, sample pad, 3, gold-marking binding pad, 4, coated film, 5, adsorptive pads, 6, detection line T line, 7, control line C line.
Fig. 2, lead ion colloidal gold chromatographic test strip plan structure synoptic diagram.
Embodiment
The preparation of embodiment 1, lead ion colloidal gold chromatographic test strip
Process the lead ion test strip, at first need prepare the carrier protein of coupling lead ion, be used to prepare relevant detection line (T line) and antibody; And need the plumbous golden labeling antibody of preparation, be used to prepare corresponding golden labeling antibody cellucotton; Need prepare sheep anti-mouse igg antibody in addition, be used to prepare control line (C line).
1, lead and carrier protein couplet
Immunizing antigen is plumbous-to the preparation of sulphur cyanobenzyl-disodium salt-keyhole limpet hemocyanin (Pb-ITCBE-KLH).
After earlier the KLH albumen of the ITCBE of 2mg and 4.5mg being reacted 24h in the HEPES of 3mL, pH9.4 (hydroxyethyl croak piperazine second thiosulfonic acid) damping fluid; Under 4 ℃, the condition of 6000r/min; Ultrafiltration 10min removes the ITCBE in the not coupling; Wash with the HEPES damping fluid of pH7.4 again and get trapped substance, dropwise add the Pb of 1 mol/L then
2+Behind the standard solution 100 μ L reaction 1h, the Pb that huge legendary turtle is not closed is removed in ultrafiltration
2+,, finally obtain the solution of 2mL again with HEPES damping fluid dilution trapped substance.Before the use, with the HEPES damping fluid of 100mmol/L EDTA and 0.1mol/L pH7.4 to the ultra-filtration centrifuge tube pre-service.
Detection antigen lead-to the preparation of sulphur cyanobenzyl-disodium salt-bovine serum albumin(BSA) (Pb-ITCBE-BSA), replace KLH with BSA, method is the same.
No Pb
2+Detect the preparation to sulphur cyanobenzyl-disodium salt-bovine serum albumin(BSA) (ITCBE-BSA) of antigen, substitute Pb with tri-distilled water
2+Standard solution get final product, substitute KLH with BSA, all the other steps are the same.
2, anti-lead ion MONOCLONAL ANTIBODIES SPECIFIC FOR
The monoclonal antibody preparation: with immunizing antigen (Pb-ITCBE-KLH) the female BALB/c mouse in immune 6-8 age in week that the lead ion carrier protein combines, immunizing dose is 100 μ g/ time, adopts quick adjuvant, the leg muscle injection.Head exempts from, and quick adjuvant 1:1 mixes with lead ion carrier protein immunizing antigen and equal-volume, fully mixing; Head exempts to carry out booster immunization after 3 weeks, and method is the same, continuous immunity 3-4 time, each 3 weeks at interval, last immunity back 10～15 days.Comlete antigen metallic ion-sequestrant-carrier protein and sequestrant-carrier protein is coated elisa plate simultaneously; Detecting mice serum with indirect ELISA tires; The result shows that mice serum is tired and can reach 1 ︰, 8000～1 ︰ 128000; And the OD value of metallic ion-sequestrant-carrier protein explains that much larger than sequestrant-carrier protein detected value the antigen of preparation has better immunogenicity.Tail vein booster immunization is carried out to mouse in 3-4 back of immunity.
3, MONOCLONAL ANTIBODIES SPECIFIC FOR and ascites purifying
Get immune mouse spleen cell and SP2/0 myeloma cell and merge, filter out the positive cell strain and the enlarged culture of the monoclonal antibody of anti-lead ion of stably excreting and ITCBE sequestrant complex compound, the injection cell advances in the mouse body to induce ascites; With sad ammonium sulfate method purifying.
4, the preparation of lead ion gold labeling antibody and gold mark thing pad
Trisodium citrate reduction method prepares collaurum: because very easily moisture absorption of chlorauride when therefore using the chlorauride of low dose of encapsulation, must once have been joined.The chlorauride of 1g once is dissolved in is made into 1% the WS in the distilled water.Be placed in 4 ℃ of refrigerators and preserve, the holding time reaches about some months to 1 year.Get 1% chlorauric acid solution 10mL again, be mixed with the chlorauric acid solution that concentration is 0.1g/L.Get 0.1g/L chlorauric acid solution 50 mL and put into conical flask; Be heated to boiling and lasting 2min with the constant temperature magnetic stirrer; Under the 100r/min magnetic agitation, add 1% trisodium citrate aqueous solution 2mL, keep temperature and stirring rate constant; Continue agitating heating 6min, be bright claret until solution.The room temperature cooling, 4 ℃ of preservations are subsequent use.Obtaining diameter is the colloidal gold solution of 20nm.
With 0.1 mol/L K
2CO
3Or 0.1 mol/L HCl to regulate colloidal gold solution be pH 9.0,10 mL colloidal gold solutions are added in the 50 mL beakers, with magnetic stirrer 250 rpm stirring, dropwise add 150 μ L antibody-solutions, equilibrate overnight.Dropwise add 5% BSA, the final concentration that makes BSA is 1%, continues to stir 5 min, in order to saturated free collaurum.With centrifugal 15 min of golden labeling antibody solution normal temperature low speed (3000 rpm), discard the deposition that forms by the gold grain that condenses again.Get red supernatant solution in 4 ℃ with centrifugal 40 min of 11000 rpm.Solution is divided into two layers: transparent supernatant, the flowable kermesinus deposition in the pipe end.The light suction and abandoning supernatant, flowable kermesinus deposition is with resuspended liquid suspendible, centrifugal again behind the recovery original volume, the CBS damping fluid that used resuspended liquid is 0.05M.So repeat 2～4 times, thoroughly to remove unconjugated protein.It is subsequent use as 1/10,4 ℃ of preservation of original volume to use resuspended liquid will precipitate suspendible at last, obtains lead ion colloid gold label antibody.
Be adsorbed in the processed glass cellucotton with the CBS damping fluid of the 0.05M colloid gold label antibody with 1:100～500 dilutions, 37 ℃ of dryings make the gold-marking binding pad of lead ion.
4, lead ion colloid gold chromatographic test paper strip detection reaction principle
Plumbous (Pb) atomic weight is merely 207, belongs to small-molecule substance.Antibody can not be caught free lead, needs to detect the Pb-ITCBE complex compound.The present invention adopts competition law, i.e. Pb-ITCBE complex compound in the sample and the anti-Pb-ITCBE monoclonal antibody that is fixed on the envelope antigen Pb-ITCBE-BSA competition colloid gold label on the coated film.
After test strips is with the terminal immersion of sample pad sample; Through capillary action swimming from the bottom up, dissolve gold mark pad is gone up dry golden labeling antibody to sample solution along test strips, if lead content is less than 10ng/mL in the testing sample; Immune response can direct swimming take place to the Pb-ITCBE-BSA on detection line (T) and the nitrocellulose membrane in then golden labeling antibody; Thereby colloid gold particle is assembled, and forms red lines, and other unconjugated golden labeling antibodies continue through capillarity swimming forward then; Sheep anti mouse two anti-generation immune responses for the second time with control line (C); The red lines of same formation just have two red lines like this on the coated film, the expression sample is negative.If lead content is greater than 10ng/mL in the testing sample, golden labeling antibody all with sample in Pb-ITCBE immunity takes place combines, just do not have again antibody to combine, thereby the T line representes that with regard to not having red lines appearance sample is positive with T line coating antigen.Whether the C line is effectively set for check gold-marking immunity chromatography method itself, so no matter whether have Pb in the sample, control line all should develop the color.If control line does not develop the color, explain that then test strips lost efficacy.
Be sprayed on the envelope antigen and the sheep anti-mouse igg of selected concentration on the coated film, respectively as detection line (T) and control line (C), at 37 ℃ of oven drying 10 min.In kind, certain density golden labeling antibody is coated on the pad.Test strips consists of a liner plate, is stained with sample pad, gold-marking binding pad, coated film and adsorptive pads above that in order.The plate that posts is cut into the wide bar of 3mm, then test strips is preserved with the drying agent sealing of packing in the aluminium foil bag.
The test sample pre-treatment becomes digestive juice: get the even quality sample of 1.0g pork, in the teflon crucible, add nitric acid 2mL, room temperature is placed 4h.Sample transfer in digest tube, with 2mL nitric acid rinse crucible repeatedly, is all shifted digest tube, add 30% hydrogen peroxide of 1.5mL again.Put into the microwave digestion appearance, microwave heating digests 15 min.Taking-up is left standstill to cooling, regulates pH to 7.2, and digestive juice is transferred to the 20mL volumetric flask, and constant volume is to be measured.
Method of operating: according to the volume ratio of 1:1 ITCBE sequestrant mixing, make the abundant chelating of lead ion and ITCBE, obtain the pork sample solution with pork treatments of the sample liquid and 4mmol/L.
Lead ion colloidal gold chromatographic test strip sample pad end is inserted in the 100 μ L analyte sample fluids, and insertion depth is no more than mark line, and about 3-5 min reads the test strips testing result, when reading, and test strips horizontal positioned, top view.
The result judges: if red line colour developing of C line is only arranged on coated film, the expression testing result is positive, explains that plumbum ion concentration is greater than 10ng/mL in testing sample; If two red lines of C line and T line all develop the color on coated film, the expression testing result is negative, explains that plumbum ion concentration is lower than 10ng/mL in testing sample; If C line red line does not develop the color on coated film, show that then test strips lost efficacy.
Claims (3)
1. lead ion colloidal gold chromatographic test strip; It is characterized in that being made up of liner plate (1) and the sample pad (2), gold-marking binding pad (3), coated film (4) and the adsorptive pads (5) that on liner plate, are connected successively: gold-marking binding pad is the glass fibre cotton of the golden labeling antibody of adsorpting lead ion; The stealthy detection line T line (6) of the orthoscopic that the solution of useful successively lead ion coupling carrier albumen is printed on coated film; With the stealthy control line C line (7) of orthoscopic that sheep anti-mouse igg solution is printed, two line parallels are arranged.
2. test strips according to claim 1 is characterized in that: the hard plastic bar of described liner plate for not absorbing water, or the cardboard bar that do not absorb water; Described sample pad is glass fibre cotton, nylon membrane, PVDF membrane or polyester film; Described coated film is nitrocellulose filter, pure cellulose film or carboxylation cellulose membrane; Described adsorptive pads is absorbent filter or filter paper for oil.
3. according to the described test strips of claim l, it is characterized in that: described lead ion gold labeling antibody is the lead ion monoclonal antibody of colloid gold label; The carrier protein of described coupling lead ion is keyhole limpet hemocyanin KLH, or is bovine serum albumin(BSA) BSA, and the sequestrant of coupling lead ion and carrier protein is to sulphur cyanobenzyl-disodium salt ITCBE.
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| CN102643917A (en) * | 2012-04-25 | 2012-08-22 | 宁波大学 | Lead ion detection test paper and preparation method thereof |
| CN102680105A (en) * | 2012-05-26 | 2012-09-19 | 东华大学 | Nanofiber membrane blood lead color sensor and detection method thereof |
| CN103412126A (en) * | 2013-08-25 | 2013-11-27 | 河南科技学院 | Gold label test paper for rapidly detecting molybdic ions, well as preparation method and application thereof |
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| CN103412126A (en) * | 2013-08-25 | 2013-11-27 | 河南科技学院 | Gold label test paper for rapidly detecting molybdic ions, well as preparation method and application thereof |
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| CN103472230A (en) * | 2013-09-28 | 2013-12-25 | 河南科技学院 | Indirect competition enzyme linked immunoreagent kit for detecting lead ions and manufacturing method thereof |
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| CN106546730A (en) * | 2016-10-28 | 2017-03-29 | 华中科技大学 | A kind of lead ion visible detection method |
| CN106546730B (en) * | 2016-10-28 | 2018-07-24 | 华中科技大学 | A kind of lead ion visible detection method |
| CN108226488A (en) * | 2017-11-30 | 2018-06-29 | 上海拜豪生物科技有限公司 | One heavy metal species fluorescence immunoassay detection method and its chromatography kit |
| CN110045108A (en) * | 2019-05-28 | 2019-07-23 | 江南大学 | A kind of gutter oil enzyme-linked immunologic detecting kit and its detection method based on capsaicine index |
| CN112526122A (en) * | 2020-11-13 | 2021-03-19 | 山东美正生物科技有限公司 | Fluorescent quantitative test strip for rapidly detecting heavy metal lead |
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Application publication date: 20120425 |