CN103675256A - Chloramphenicol immunoaffinity gel detection column - Google Patents
Chloramphenicol immunoaffinity gel detection column Download PDFInfo
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- CN103675256A CN103675256A CN201210335125.0A CN201210335125A CN103675256A CN 103675256 A CN103675256 A CN 103675256A CN 201210335125 A CN201210335125 A CN 201210335125A CN 103675256 A CN103675256 A CN 103675256A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
The invention relates to an immunoaffinity gel detection column for visually fast detecting residual chloramphenicol in food. The chloramphenicol immunoaffinity gel detection column belongs to the field of immunology, enzymology and analytic chemistry. Cyanogen bromide-activated agarose gel is respectively coupled with a chloramphenicol antibody and a hydrogen peroxidase (HRP) antibody to prepare chloramphenicol antibody glue and HRP antibody glue; the cyanogen bromide-activated agarose gel is confined by confining liquid to prepare confining glue. The confining glue and the chloramphenicol antibody glue are mixed to prepare a detection layer, the confining glue and the HRP antibody glue are mixed to prepare a quality control layer, the quality control layer is filled in a 1ml solid phase extraction column, the working conditions of all steps in the detection process can be determined, and a novel immunoaffinity gel detection column for fast qualitatively and semi-quantitatively detecting residual chloramphenicol in food can be researched, with the detection limit of 1mug/L. When the product detects the residual chloramphenicol in food samples, no organic solvents and complicated pretreatment, as well as the assistance of large instruments are not required, the chloramphenicol immunoaffinity gel detection column has good usability and accuracy, and can meet the requirement for visual fast detection.
Description
Technical field
The invention belongs to immunology, zymetology and analytical chemistry field, relate in particular to the preparation of chloromycetin immunity affinity gel test column.
Background technology
Chloromycetin is a kind of broad-spectrum antibiotic, and gram-positive bacteria and Gram-negative bacteria are all had to good inhibiting effect.It is combined by the A unit on ribosomal 50s subunit, and the skin that turns that hinders skin phthalidyl transferase reacts, and stops skin chain extension, thereby reaches the synthetic object of anti-bacteria protein.High inexpensive because of its effect, once in animal husbandry, be widely used in the treatment of the various communicable diseases of animal.But it has stronger toxic and side effect, research institution finds both at home and abroad, residual chloromycetin in food exceeds standard, and easily causes the multiple harm such as the purple hall of human body platelet minimizing property, agranulocytosis, alpastic anemia, particularly premature and neonate is produced to comparatively serious toxicity.The developed country such as European Union, the U.S. forbids that chloromycetin is for animal derived food in succession in recent years, and clearly regulation residual chloromycetin is limited the quantity of as detecting.China Ministry of Agriculture has banned use of this medicine, domestic or livestock products of outlet no matter, and residual chloromycetin is all essential items for inspection.Yet due to CAP good antimicrobial effect and cheap, China still has part cultivation chief commander CAP to be used for the treatment of bird, the disease of domestic animals at present.For ensureing that people ' s health and expansion and countries in the world food trade come and go, must set up highly sensitive, high specificity, simple and quick assay method is monitored residual chloromycetin.
The chloromycetin detection method of reporting for work at present mainly contains: microbial method, enzyme immunoassay, radioimmunology, high performance liquid chromatography and vapor-phase chromatography.The sense cycle of microbial method is longer.Though vapor-phase chromatography sensitivity is higher, also come with some shortcomings, as sample pretreatment process is complicated, analysis speed is slow, instrumentation degree is high and expensive, be not suitable for promoting the use of in basic unit.Though high performance liquid chromatography is simple to operate, detection limit is higher, cannot meet the current requirement that chloromycetin low-residual is limited the quantity of.The immunological assay method that reaction is set up based on antigen and antibody specific, it is the study hotspot of residue of veterinary drug fast detecting and sample sifter choosing method always, the plurality of advantages such as it has simply, quick, processing sample amount is large, sensitivity is higher, high specificity, have broad application prospects.Especially visual tachysynthesis detection technique, as test strips, does not need any instrument and equipment, the youngster minute experimental result that just can detect by an unaided eye, and the field screening and the basic unit that are very suitable for a large amount of samples promote.Yet ELISA test strip is also subject to the impact of sample substrate, in different food products sample detection, be subject to certain limitation.Residual chloromycetin in the sample that the gel column immune detection product that the present invention sets forth is dark for some color and matrix impact is larger detects to analyze provides a kind of effective visual qualitative sxemiquantitative quick easy detection method, for the strong supervision of food security provides reliable technical guarantee.
Summary of the invention
The object of the present invention is to provide a kind of chloromycetin in can qualitative semiquantitative detection food simple, save time, sensitive visual immune affinity gel test column product, gel detection post be in the 1mL of standard solid-phase extraction column, be provided with two-layer: to be chloramphenicol antibody glue mix with sealing glue the detection layers that adds formation on upper strata, the color of this layer can be eliminated or weaken to the chloromycetin containing in testing sample, thereby reach according to colloid shade the object that detects the chloromycetin in sample; Lower floor is that HRP antibody glue mixes with sealing glue the Quality Control layer that adds formation, as long as testing process adds enzyme labeling thing, this layer all can develop the color.If this layer colour developing, detects normally, if this layer colourless, detect inefficacy, thereby reach the object of Quality Control.The technical scheme that the present invention takes is:
The preparation of sealing glue
(1) swelling: take 1.00g cyanogen bromide-activated Ago-Gel, join in the HCl solution preparing of 80mL, stir swelling 10min (after swelling, the final volume of glue is about 3.5mL).
(2) drip washing: the good Ago-Gel of swelling is transferred in tool sand plate layer chromatography post, then added 200mL HCl drip washing, then rinse pillar with coupling buffer, regulate pillar pH to neutral.
(3) sealing: add 9mL glycocoll confining liquid, sealing pillar, concussion reaction 2h under room temperature.
(4) clean: first add the coupling buffer drip washing of 10mL once, then with the sodium acetate buffer solution of 10mL and the coupling buffer of 10mL, rinse respectively 3 times.
(5) store: liquid to be cleaned is found time, and the sealing glue preparing hangs with PBS, and about 10mL PBS (ablastins that contain 0.3ml/L) is positioned over 4 ℃ of preservations stand-by.
The preparation of antibody glue
(1) swelling: accurately take 0.50g cyanogen bromide-activated Ago-Gel, join in the HCL solution preparing of 40mL, stir swelling 10min (after swelling, the final volume of glue is about 1.8mL).
(2) drip washing: the good Ago-Gel of swelling is transferred in tool sand plate layer chromatography post, then added 200mL HCL drip washing, then rinse pillar with coupling buffer, regulate pillar pH to neutral, leacheate has all been drenched.
(3) coupling antibody: get 0.50mg/mL antibody, the coupling buffer dilution with 1mL, then joins in tool sand plate layer chromatography post, sealing pillar, concussion reaction 2h under room temperature.
(4) sealing: add the coupling buffer drip washing pillar of 10mL, then add the confining liquid of 10mL, again seal pillar, room temperature concussion reaction 2h.
(5) clean: first add the coupling buffer drip washing of 10mL once, then with the sodium acetate buffer solution of 10mL and the coupling buffer of 10mL, rinse respectively 3 times;
(6) store: liquid to be cleaned is found time, and the antibody glue preparing hangs with PBS, and about 5mL PBS (ablastins that contain 0.3ml/L) is positioned over 4 ℃ of preservations stand-by.
The preparation of test column
(1) prepare Quality Control layer and detection layers:
Quality Control layer is, after being mixed with certain proportion by HRP antibody glue and sealing glue, to get 150 μ L epoxy glues and join in the SPE plastic column of 1mL, with syringe piston, PBS is extruded.
Detection layers is, after being mixed in certain proportion by the antibody glue of having optimized and sealing glue, to get 150 μ L and add wherein, with syringe piston, PBS is extruded.
(2) prepare test column:
Test column from down to up, is respectively Quality Control layer and detection layers, separates the air layer of a 3mm between Quality Control layer and detection layers, can prevent from adding the rear reagent of substrate colour developing and color to move to detection layers from contrast layer.
First tygon pad is added in the plastic column of 1mL, then add Quality Control layer epoxy glue, add second layer tygon pad, above Quality Control layer, about 3mm is except adding three-layer polyethylene pad, then the epoxy glue that adds its detection layers, finally adds the 4th layer of pad at top.
Accompanying drawing explanation
Gel detection post forms schematic diagram and sees Figure of description.
Advantage of the present invention and good effect are:
(1) chloromycetin provided by the invention immunity affinity gel test column, can single-minded identification chloromycetin, has very high selectivity.
(2) chloromycetin immunity affinity gel test column of the present invention is a kind of visual qualitative half-quantitative detection product.Detection to target determinand, test column can provide testing result with having or not of detection layers color, with the visual determining signal of Yes/No, replys, easy to operate, and result judgement is simple.
(3) the chloromycetin immunity affinity gel test column that the present invention makes both can make it to connect with decontaminating column, eliminated sample substrate impact; Cleaning buffer solution and the sample solution that can hold again larger volume, improved method sensitivity.Testing product is without any need for organic solvent and complicated pre-treatment, and large-scale instrument is auxiliary, has good ease for use and accuracy, meets the requirement of field quick detection.
Embodiment
Below in conjunction with embodiment, the present invention is further described.
The present invention is solidificated in the antibody of chloromycetin on Ago-Gel, then fills post and forms.Utilize the high degree of specificity of antigen-antibody reaction, when sample solution passes through immune affinity gel test column, chloromycetin is attached on post specifically, adds after enzyme mark, and chloromycetin and the competition of enzyme mark are attached on chloramphenicol antibody, then add substrate colour developing.Its specific embodiment is:
(1) prepare chloromycetin immunity affinity gel test column Quality Control layer and detection layers:
1. chloromycetin immunity affinity gel test column Quality Control layer is, after being mixed with 1: 100 ratio by HRP antibody glue and sealing glue, to get 150 μ L epoxy glues and join in the solid-phase extraction column of 1mL, with syringe piston, PBS is extruded;
2. chloromycetin immunity affinity gel test column detection layers is, after being mixed with 1: 20 ratio by the chloramphenicol antibody glue of having optimized and sealing glue, to get 150 μ L and add wherein, with syringe piston, PBS is extruded;
(2) prepare chloromycetin immunity affinity gel test column:
1. test column from down to up, is respectively Quality Control layer and detection layers, separates the air layer of a 3mm between Quality Control layer and detection layers, can prevent from adding the rear reagent of substrate colour developing and color to move to detection layers from Quality Control layer.
2. first tygon pad is added in the plastic column of 1mL, then add Quality Control layer epoxy glue, add second layer tygon pad, above Quality Control layer, about 3mm place adds three-layer polyethylene pad, then the epoxy glue that adds its detection layers, finally at top, add the 4th layer of pad, test column can not be reused through experimental verification.
(3) when chloromycetin immunity affinity gel test column detects in food samples residual chloromycetin, without any need for organic solvent and complicated pre-treatment, and large-scale instrument is auxiliary, and the detection of chloromycetin immunity affinity gel test column chlorine detection mycin is limited to 1 μ g/L.
Claims (4)
1. the composition of chloromycetin immunity affinity gel test column, it is characterized in that: gel detection post be in the 1mL of standard solid-phase extraction column, be provided with two-layer, upper strata is that chloramphenicol antibody glue mixes with sealing glue the detection layers that adds formation, lower floor is that HRP antibody glue mixes with sealing glue the Quality Control layer that adds formation, separates the air layer of a 3mm between Quality Control layer and detection layers.
2. the composition of chloromycetin claimed in claim 1 immunity affinity gel test column, it is characterized in that: first tygon pad is added in the solid-phase extraction column of 1mL, then the epoxy glue making by 1: 100 blending ratio with HRP antibody glue and sealing glue that adds 150 μ L, add second layer tygon pad, with syringe piston, PBS is extruded, above the Quality Control layer assembling, about 3mm place adds three-layer polyethylene pad, then the epoxy glue making by 1: 20 blending ratio with chloramphenicol antibody glue and sealing glue that adds 150 μ L, finally at top, add the 4th layer of pad, with syringe piston, PBS is extruded.
3. chloromycetin immunity affinity gel test column claimed in claim 1, is characterized in that: the detection of described chloromycetin immunity affinity gel test column chlorine detection mycin is limited to 1 μ g/L.
4. chloromycetin claimed in claim 1 immunity affinity gel test column, is characterized in that:
(1) preparation of sealing glue is used cyanogen bromide-activated Ago-Gel to add after HCl solution swelling to be transferred in special tool sand plate layer chromatography post and to carry out drip washing with HCl solution, after rinsing pillar with coupling buffer again, add glycocoll confining liquid room temperature sealing 2h, with coupling buffer and sodium acetate buffer solution, repeatedly rinse gel again, liquid to be cleaned is found time, the sealing glue preparing hangs with PBS for 1: 3 by volume, the ablastins that add again 0.3ml/L, are positioned over 4 ℃ of preservations stand-by;
(2) preparation method of antibody glue is with sealing glue, and difference was only that before sealing the chloromycetin specific antibody of 0.5mg/mL is added into room temperature coupling 2h in the middle of gel.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210335125.0A CN103675256A (en) | 2012-09-12 | 2012-09-12 | Chloramphenicol immunoaffinity gel detection column |
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| CN201210335125.0A CN103675256A (en) | 2012-09-12 | 2012-09-12 | Chloramphenicol immunoaffinity gel detection column |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104190387A (en) * | 2014-09-11 | 2014-12-10 | 福州新北生化工业有限公司 | Gel for eliminating pyrogen in liquid as well as preparation method and application gel for eliminating pyrogen in liquid |
| CN106370870A (en) * | 2016-09-21 | 2017-02-01 | 中国农业大学 | Kit for detecting clenbuterol |
| CN107389958A (en) * | 2017-08-30 | 2017-11-24 | 深圳大学 | The detection gel column of lincomycin and the detection method of lincomycin |
| CN112505026A (en) * | 2020-11-23 | 2021-03-16 | 深圳大学 | Visual gel detection device and method for soybean allergen |
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| RU2374649C1 (en) * | 2008-05-04 | 2009-11-27 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Test system for immunoenzymometric determination of toxicants |
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2012
- 2012-09-12 CN CN201210335125.0A patent/CN103675256A/en active Pending
Patent Citations (2)
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| RU2374649C1 (en) * | 2008-05-04 | 2009-11-27 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Test system for immunoenzymometric determination of toxicants |
| CN102466731A (en) * | 2010-11-19 | 2012-05-23 | 南京神州英诺华医疗科技有限公司 | Novel detecting method for blood type and blood matching test |
Non-Patent Citations (2)
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| IRINA YU. GORYACHEVA ET AL: "Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk", 《FOOD CONTROL》 * |
| 乔好等: "氯霉素免疫亲和柱的制备及在牛奶中痕量氯霉素残留分析中的应用", 《食品研究与开发》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104190387A (en) * | 2014-09-11 | 2014-12-10 | 福州新北生化工业有限公司 | Gel for eliminating pyrogen in liquid as well as preparation method and application gel for eliminating pyrogen in liquid |
| CN106370870A (en) * | 2016-09-21 | 2017-02-01 | 中国农业大学 | Kit for detecting clenbuterol |
| CN107389958A (en) * | 2017-08-30 | 2017-11-24 | 深圳大学 | The detection gel column of lincomycin and the detection method of lincomycin |
| CN112505026A (en) * | 2020-11-23 | 2021-03-16 | 深圳大学 | Visual gel detection device and method for soybean allergen |
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Application publication date: 20140326 |