CN101169414A - Water body Chlamydomonas reinhaidtii toxin detection method - Google Patents
Water body Chlamydomonas reinhaidtii toxin detection method Download PDFInfo
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- CN101169414A CN101169414A CNA2007100702748A CN200710070274A CN101169414A CN 101169414 A CN101169414 A CN 101169414A CN A2007100702748 A CNA2007100702748 A CN A2007100702748A CN 200710070274 A CN200710070274 A CN 200710070274A CN 101169414 A CN101169414 A CN 101169414A
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Abstract
The invention relates to a measuring method of micro-capsule alga toxin in water. The method includes the steps as follows: firstly, compounding and appraising of the micro-capsule alga toxin; secondly, preparing and purifying of polyclonal antibody antibody: immunizing the New-Zealand rabbit by making the micro-capsule alga toxin holoantigen MC-LR-KLH as the immunity resource, and preparing the polyclonal antibody and purifying by ammonium sulphate according to the normal method; thirdly, preparing immune bead: coupling the micro-capsule alga toxin holoantigen MC-LR-BSA and the nanometer bead, and preparing the immune bead containing MC-LR-BSA; fourthly, embedding the antibody to the pyroxylin film; fifthly, producing testing board, combing the coupling mat of the magnetic scale MC-LR-BSA, the pyroxylin film embedding the polyclonal antibody, the sample mat, the sopping mat, the covering film, the testing board out card into a testing board; sixthly, sample testing, respectively adding standard goods and testing samples with different concentrations into the sampling holds on the testing board, the samples flow on the test paper through the chromatography effect, after 3 to 5 minutes of the reaction under the room temperature, the testing board is put into a magnetic single testing apparatus to be tested, and the testing apparatus can further output the biology reaction signals being transferred to the magnetic field signals by the way of the electric signals; after the standard curve being drawn, the specific value of the micro-capsule alga toxin content in the sample to be tested is counted based on the standard curve.
Description
Technical field
The present invention relates to the detection method that a kind of energy rapid sensitive detects Microcystin (MC) in the water body, be used for the detection of Microcystin content in physical environment water body, the potable water.
Background technology
The body eutrophication that is on the rise makes water pollution become global environment problem, and blue-green algae is the advantage algae kind that forms wawter bloom in the most freshwater lakes of China, and these algae can produce has obvious hepatotoxic peptide matters, is called Microcystin.Its toxicity is number two in the known toxin of nature, is only second to dioxin.Microcystin has multiple different isomeride, and wherein MC-LR is present known a kind of fresh water cyanophycean toxin that toxicity is the strongest, acute hazard is maximum.
The detection method of Microcystin mainly contains bioanalysis, chemical analysis, biochemical analysis method and immunoassay etc. in the water body.Bioanalysis is to utilize mouse peritoneal injection or oral cavity to irritate to feed the toxicity of estimating Microcystin, and this method is simple to operate, but sensitivity is relatively poor, and can't determine toxin type and structure; Chemical analysis is maximum method of using at present, mainly comprise gas chromatography (GC), thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), liquid chromatography/mass spectrometry analysis (LC/MS) and Capillary Electrophoresis (CE) etc., these class methods have good sensitivity and selectivity, but instrument is bulky, cost an arm and a leg, need the higher indoor environment of environmental baseline, and special technician operation, the testing cost costliness; The biochemical analysis method comprises that mainly enzymatic activity suppresses detection technique, and its principle is to utilize Microcystin that the inhibition degree of protein phosphatase enzymatic activity is detected by colourimetry, and this method sensitivity is too low, and has the interference of endogenous enzyme.Immunoassay is the method for the detection Microcystin preferentially elected of American National Environmental Protection Agency, and the most frequently used is competitive non-even phase enzyme linked immunosorbent detection method, and this method is simple to operate, but the reaction time is long, can not be quantitative, and sensitivity is on the low side, can not on-the-spot testing result.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned each method, provide a kind of short based on nanometer magnetic immunological technique, highly sensitive, reaction time, instrument and equipment is cheap, can be used for the on-the-spot anatoxic detection method of Microcystis aeruginosa in the water body that detects.
Purpose of the present invention is finished by following technical solution:
1) Microcystin holoantigen synthetic and identifying: with glutaraldehyde as difunctional cross-linking reagent, respectively BSA, KLH and algae phycotoxin MC-LR are carried out coupling according to a conventional method, obtain the holoantigen MC-LR-BSA and the MC-LR-KLH of Microcystin, and utilize ultraviolet spectrophotometer to identify;
2) Polyclonal Antibody Preparation and purifying: as immunogene, immune new zealand rabbit prepares polyclonal antibody and according to a conventional method with the ammonium sulfate purifying with MC-LR-KLH; Obtain the IgG of the anti-MC-LR-KLH of rabbit.
3) preparation of immunomagnetic beads: with MC-LR-BSA and nanometer magnetic bead coupling, preparation contains the immunomagnetic beads of MC-LR-BSA; Concrete grammar is: draw an amount of magnetic bead to small test tube, place magnetic field that magnetic bead is separated with stock solution, the MES cleaning buffer solution cleans and resuspended magnetic bead, with carbodiimides of small size (EDC) and N-hydroxy-succinamide (NHS) add in the magnetic bead solution as far as possible, the molecular proportion that makes EDC and NHS is 1: 1～1: 3, and to make it molecular amounts be 5～19 times of magnetic bead surfaces carboxyl, 37 ℃ of concussions were hatched 1 hour, clean magnetic bead with MES cleaning buffer solution and boric acid cleaning buffer solution respectively, subsequently an amount of MC-LR-BSA is added in the magnetic bead suspension, make protein in magnetic bead, reach finite concentration, 37 ℃ of concussions were hatched 1 hour, clean magnetic bead with the boric acid cleaning buffer solution, magnetic bead is transferred to new pipe, discard the boric acid cleaning buffer solution, and use the coupling buffer resuspension, the magnetic bead that will be marked with MC-LR-BSA antigen with quantitative sample adding device adds magnetic bead coupling pad;
4) embedded antibody is to nitrocellulose filter: the purpose antibody that will prepare the bag quilt with spray film instrument is stated from the nitrocellulose filter, makes purpose antibody reach a certain amount of, and the reaction between the magnetic mark antigen reaches the best, drying at room temperature.With 3% bovine serum albumin-phosphate buffer (BSA-PBS), room temperature sealing 60 minutes; With 0.01% lauryl sodium sulfate-phosphate buffer (SDS-PBS) washing 15 minutes, totally three times, after the drying at room temperature, be stored in 4-20 ℃.
5) making of detection test board: coupling pad, the nitrocellulose filter of embedding polyclonal antibody, sample pad, adsorptive pads, coverlay, the test board wild card of respectively magnetic being marked MC-LR-BSA are formed test board (this device is similar to golden device for mark).
6) sample detection: the standard items and the test sample of variable concentrations are added sample hole on the test board, sample flows through from test strips by the chromatography effect, after reacting 3～5 minutes under the room temperature, test board is put into the magnetic signal detector detect, and this detecting instrument can further be exported the biologically signal that changes into field signal in the mode of electric signal; Behind the drawing standard curve, the establishing criteria curve is obtained the occurrence of Microcystin content in the testing sample.
The present invention be based on a kind of nanometer magnetic immunological technique carry out the anatoxic detection method of Microcystis aeruginosa in the water body, it have highly sensitive, reaction time short, instrument and equipment is cheap, can be used for characteristics such as on-the-spot detection.
Description of drawings
Fig. 1 is a detection methodologies FB(flow block) of the present invention.
Fig. 2 is a test board used for it structural representation of the present invention.
Fig. 3 is that the concentration of MC-LR of the present invention is 0.001ppb test result figure.
Fig. 4 is that the concentration of MC-LR of the present invention is 0.1ppb test result figure.
Fig. 5 is that the concentration of MC-LR of the present invention is 1ppb test result figure.
Fig. 6 is that the concentration of MC-LR of the present invention is 50ppb test result figure.
Fig. 7 is the canonical plotting of concentration that the present invention works as MC-LR magnetic signal intensity when being 0.001-50ppb.
Embodiment
The present invention will be described in detail below in conjunction with the accompanying drawings and the specific embodiments: as shown in Figure 1, the anatoxic detection method of Microcystis aeruginosa is in the water body of the present invention:
1) Microcystin holoantigen synthetic and identifying: with glutaraldehyde as difunctional cross-linking reagent, respectively BSA, KLH and algae phycotoxin MC-LR are carried out coupling according to a conventional method, obtain the holoantigen MC-LR-BSA and the MC-LR-KLH of Microcystin, and utilize ultraviolet spectrophotometer to identify; Wave spectrum scanning shows that MC-LR-BSA compares with 0.2%BSA, and absorption peak generation blue shift phenomenon illustrates in our the existing material MC-LR and BSA coupling.
2) Polyclonal Antibody Preparation and purifying: as immunogene, immune new zealand rabbit prepares polyclonal antibody and according to a conventional method with the ammonium sulfate purifying with MC-LR-KLH; The SDS-PAGE electrophoresis result shows to possess the antibody specificity band.
3) preparation of immunomagnetic beads is with MC-LR-BSA and nanometer magnetic bead coupling, and preparation contains the immunomagnetic beads of MC-LR-BSA.Concrete grammar: draw 20 microlitre magnetic beads to small test tube, place magnetic field that magnetic bead is separated with stock solution, clean magnetic bead twice with 40 microlitre MES cleaning buffer solutions, the resuspension magnetic bead is in 20 microlitre MES cleaning buffer solutions.Respectively the carbodiimides about 60 micrograms (EDC) and 80 microgram left and right sides N-hydroxy-succinamide (NHS) are added in the magnetic bead solution, 37 ℃ of concussions were hatched 1 hour.Clean magnetic bead twice with 40 microlitre MES cleaning buffer solutions respectively subsequently, behind twice of 20 microlitre boric acid cleaning buffer solutions cleaning magnetic bead, the resuspension magnetic bead is in 20 microlitre boric acid cleaning buffer solutions.MC-LR-BSA about 5 microlitres (8 mg/ml) is added in the magnetic bead suspension, and 37 ℃ of concussions were hatched 3 hours; The 10% bovine serum albumin(BSA) confining liquid that adds 3 microlitres again, 37 ℃ of concussions were hatched 1 hour; Clean magnetic bead twice with 20 microlitre boric acid cleaning buffer solutions, and magnetic bead is transferred in the new pipe; Discard the boric acid cleaning buffer solution, with 15 microlitre coupling buffer resuspension magnetic beads; The magnetic bead that will be marked with MC-LR-BSA antigen with quantitative sample adding device adds magnetic bead coupling pad.
4) embedded antibody is to nitrocellulose filter: the purpose antibody that will prepare the bag quilt with spray film instrument is stated from the nitrocellulose filter, makes purpose antibody reach a certain amount of, drying under the room temperature.With 3% bovine serum albumin-phosphate buffer (BSA-PBS), room temperature sealing 60 minutes; With 0.01% lauryl sodium sulfate-phosphate buffer (SDS-PBS) washing 15 minutes, totally three times, after the drying at room temperature, be stored in 4-20 ℃.
5) making of detection test board: coupling pad, the nitrocellulose filter of embedding polyclonal antibody, sample pad, adsorptive pads, coverlay, the test board wild card of magnetic being marked MC-LR-BSA are formed test board (this device is similar to golden device for mark).
6) sample detection: the standard items and the test sample of variable concentrations are added sample hole on the test board, sample flows through from test strips by the chromatography effect, reaction is after 3-5 minute under the room temperature, test board is put into the magnetic signal detector detect, and this detecting instrument can further be exported the biologically signal that changes into field signal in the electric signal mode; Behind the drawing standard curve, the establishing criteria curve is obtained the occurrence of testing sample.
The pure water sample detection: its concrete testing result and 0.001ppb basically identical, specifically as shown in Figure 3.
Variable concentrations MC-LR canonical reference product detect: concrete as Fig. 3,4,5, shown in 6.
Testing sample detects: the positive sample with clear and definite negative sample and variable concentrations is tested, and the peak value height of p-wire presents certain correlativity with the concentration of positive, and is concrete as Fig. 7.Get the water body sample in different waters, Taihu Lake respectively and test, obviously there is serious pollution in the Taihu Lake water sample 5-6 month in 2007 as can be seen.And the water quality of water delivering orifice is better than water inlet slightly.
Other embodiments of the invention can be selected arbitrarily in above-mentioned scope, and those skilled in the art is understanding on the basis of content of the present invention, in conjunction with existing disclosed technology, as patent gazette etc., can fully implement the present invention.
Claims (2)
1. the detection method of Microcystin in the water body, this method is: 1) Microcystin holoantigen synthetic and identifying: with glutaraldehyde as difunctional cross-linking reagent, respectively BSA, KLH and algae phycotoxin MC-LR are carried out coupling according to a conventional method, obtain the holoantigen MC-LR-BSA and the MC-LR-KLH of Microcystin, and utilize ultraviolet spectrophotometer to identify; 2) Polyclonal Antibody Preparation and purifying: as immunogene, immune new zealand rabbit prepares polyclonal antibody and according to a conventional method with the ammonium sulfate purifying with MC-LR-KLH; 3) preparation of immunomagnetic beads: with MC-LR-BSA and nanometer magnetic bead coupling, preparation contains the immunomagnetic beads of MC-LR-BSA; 4) embedded antibody is to nitrocellulose filter: the purpose antibody that will prepare the bag quilt with spray film instrument is stated from the nitrocellulose filter, makes purpose antibody reach a certain amount of, drying under the room temperature; With 3% bovine serum albumin-phosphate buffer (BSA-PBS), room temperature sealing 60 minutes; With 0.01% lauryl sodium sulfate-phosphate buffer (SDS-PBS) washing 15 minutes, totally three times, after the drying at room temperature, be stored in 4～20 ℃; 5) making of detection test board: coupling pad, the nitrocellulose filter of embedding polyclonal antibody, sample pad, adsorptive pads, coverlay, the test board wild card of magnetic being marked MC-LR-BSA are formed test board; 6) sample detection: the standard items and the test sample of variable concentrations are added sample hole on the test board, sample flows through from test strips by the chromatography effect, after reacting 3～5 minutes under the room temperature, test board is put into the magnetic signal detector detect, and this detecting instrument can further be exported the biologically signal that changes into field signal in the electric signal mode; Behind the drawing standard curve, the establishing criteria curve is obtained the occurrence of testing sample.
2. the anatoxic detection method of Microcystis aeruginosa in the body according to claim 1, the preparation that it is characterized in that described immunomagnetic beads is: draw 20 microlitre magnetic beads to small test tube, place magnetic field that magnetic bead is separated with stock solution, clean magnetic bead twice with 40 microlitre MES cleaning buffer solutions, the resuspension magnetic bead is in 20 microlitre MES cleaning buffer solutions; Respectively carbodiimides about 60 micrograms (EDC) and 81 microgram N-hydroxy-succinamides (NHS) are added in the magnetic bead solution, 37 ℃ of concussions were hatched 1 hour.Clean magnetic bead twice with 40 microlitre MES cleaning buffer solutions respectively subsequently, behind twice of 20 microlitre boric acid cleaning buffer solutions cleaning magnetic bead, the resuspension magnetic bead is in 20 microlitre boric acid cleaning buffer solutions; MC-LR-BSA about 5 microlitres (8 mg/ml) is added in the magnetic bead suspension, and 37 ℃ of concussions were hatched 3 hours; The 10% bovine serum albumin(BSA) confining liquid that adds 3 microlitres again, 37 ℃ of concussions were hatched 1 hour; Clean magnetic bead twice with 20 microlitre boric acid cleaning buffer solutions, and magnetic bead is transferred in the new pipe; Discard the boric acid cleaning buffer solution, with 30 microlitre coupling buffer resuspension magnetic beads; The magnetic bead that will be marked with MC-LR-BSA antigen with quantitative sample adding device adds magnetic bead coupling pad.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100702748A CN101169414A (en) | 2007-08-09 | 2007-08-09 | Water body Chlamydomonas reinhaidtii toxin detection method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102043055A (en) * | 2010-10-27 | 2011-05-04 | 上海海洋大学 | Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products |
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN101382541B (en) * | 2008-06-27 | 2012-07-11 | 江南大学 | Immunofluorescence quenching detecting method for microcystin-LR |
| CN104655837A (en) * | 2015-02-27 | 2015-05-27 | 南京微测生物科技有限公司 | Fluorescent quantitative test paper strip for simultaneously detecting algal toxins MC-LR/RR/YR and preparation method and application of fluorescent quantitative test paper strip |
| CN107167585A (en) * | 2017-04-07 | 2017-09-15 | 华南农业大学 | A kind of new small molecule structure and its application in terms of blue-green alge hepatotoxin is detected |
| CN110186881A (en) * | 2019-05-17 | 2019-08-30 | 华南农业大学 | A kind of biosensor and method detecting Microcystin |
| CN111718412A (en) * | 2020-03-27 | 2020-09-29 | 清华大学 | Broad-spectrum microcystin monoclonal antibody and preparation method thereof |
| CN112345773A (en) * | 2020-09-24 | 2021-02-09 | 北京健乃喜生物技术有限公司 | Immunoassay device for in vitro instant determination of ABO and/or Rh blood type of human |
-
2007
- 2007-08-09 CN CNA2007100702748A patent/CN101169414A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101382541B (en) * | 2008-06-27 | 2012-07-11 | 江南大学 | Immunofluorescence quenching detecting method for microcystin-LR |
| CN101446575B (en) * | 2008-12-29 | 2011-06-22 | 无锡市疾病预防控制中心 | Preparation and use method of microcystin-LR polyclonal antibody immunoaffinity column |
| CN102043055A (en) * | 2010-10-27 | 2011-05-04 | 上海海洋大学 | Rapid immunomagnetic bead chromatographic method for main allergens in aquatic products |
| CN104655837A (en) * | 2015-02-27 | 2015-05-27 | 南京微测生物科技有限公司 | Fluorescent quantitative test paper strip for simultaneously detecting algal toxins MC-LR/RR/YR and preparation method and application of fluorescent quantitative test paper strip |
| CN107167585A (en) * | 2017-04-07 | 2017-09-15 | 华南农业大学 | A kind of new small molecule structure and its application in terms of blue-green alge hepatotoxin is detected |
| CN110186881A (en) * | 2019-05-17 | 2019-08-30 | 华南农业大学 | A kind of biosensor and method detecting Microcystin |
| CN111718412A (en) * | 2020-03-27 | 2020-09-29 | 清华大学 | Broad-spectrum microcystin monoclonal antibody and preparation method thereof |
| CN111718412B (en) * | 2020-03-27 | 2022-09-13 | 清华大学 | Broad-spectrum microcystin monoclonal antibody and preparation method thereof |
| CN112345773A (en) * | 2020-09-24 | 2021-02-09 | 北京健乃喜生物技术有限公司 | Immunoassay device for in vitro instant determination of ABO and/or Rh blood type of human |
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Open date: 20080430 |