CN103792365A - Preparation and application method of fusarium moniliforme colloidal gold test strip - Google Patents
Preparation and application method of fusarium moniliforme colloidal gold test strip Download PDFInfo
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- CN103792365A CN103792365A CN201210435631.7A CN201210435631A CN103792365A CN 103792365 A CN103792365 A CN 103792365A CN 201210435631 A CN201210435631 A CN 201210435631A CN 103792365 A CN103792365 A CN 103792365A
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- Prior art keywords
- moniliformin
- detection
- pad
- fusarium moniliforme
- colloidal gold
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
Abstract
The invention belongs to the field of biological detection, and relates to a preparation and application method of a fusarium moniliforme colloidal gold test strip. The fusarium moniliforme colloidal gold test strip is characterized by comprising a paperboard, wherein a water absorption pad, a detection pad, a gold mark pad and a sample pad are pasted on one side of the paperboard sequentially from top to bottom; the adjacent pads are overlapped and connected at the joint; in the detection pad, a nitrocellulose membrane is used as a base pad; the nitrocellulose membrane is provided with a transverse reference line and a detection line from top to bottom; the detection line coats a fusarium moniliforme-bovine serum albumin conjugate, and the reference line coats a rabbit antimouse polyclonal antibody; the gold mark pad is transversely sprayed with a nanogold-marked monoclonal antibody against fusarium moniliforme, which is generated by a hybridoma cell strain. The test strip is used for detecting the fusarium moniliforme and has the characteristics of quick detection, simplicity in operation and high sensitivity.
Description
Technical field
The invention belongs to food safety detection field, be specifically related to the detection method of residuals in food, particularly a kind of method of preparation and use of moniliformin colloidal gold strip.
Background technology
Moniliformin is the metabolic product of some sickle-like bacteria, because being to be extracted in the culture of fusarium moniliforme in 1973 by people such as Cole at first, and is named as moniliformin moniliformin.This rhzomorph is water-soluble, to the each organ of animal, has an extremely strong toxic action especially to cardiovascular.Chemistry 3,4-diketone-1-hydroxyl cyclobutane by name of fusarium moniliforme, occurring in nature exists in the mode of sodium salt or sylvite.It is faint yellow acicular crystal, has water-soluble.The toxicity of moniliformin is very strong, chicken per os LD
50for 4.0mg/kg.The rat of acute poisoning can cause carrying out property muscle weakness.Expiratory dyspnea, cyanosis, stupor and dead.Someone thinks that some disease is relevant with the corn of ingesting.The toxicological effect of this toxin is that selectivity suppresses ɑ-oxoglutarate dehydrogenasa and acetonate dehydrogenasa system.Wilson etc. have found the hepatotoxicity wind agitation of moniliformin and have caused liver cancer.In food and feed, the detection method of moniliformin has thin-layered chromatography, gas chromatography, online and the high performance liquid chromatography of gas-matter, due to above all analytic approach sample pre-treatments more complicated, when operation, need special technician, testing cost costliness, be unfavorable for applying.
Summary of the invention
The object of the invention is, in order to overcome the deficiencies in the prior art, provides a kind of high specificity, highly sensitive, and simple to operation, is the quick test paper semi-quantitative detection method of detectable moniliformin to sample through simple process.
Object of the present invention can be achieved through the following technical solutions:
Its technical essential of moniliformin colloidal gold strip detection method is: on the backing of test paper, post successively sample pad, gold mark pad, nitrocellulose filter and thieving paper, the material of the upper mark of gold mark pad is that the second kind animal protein and moniliformin detect with the potpourri of antigen or the potpourri of the second kind animal protein and moniliformin antibody; On nitrocellulose filter, be coated with 1 of moniliformin antibody as detection line, 1 of the IgG that is simultaneously also coated with anti-the second kind animal protein is as reference line or be coated with moniliformin and detect with antigen 1 bar as detection line, is also coated with 1 of the work IgG of anti-the second kind animal protein as reference line simultaneously.Time prepared by test paper, by regulating the concentration of detection line and reference line encrusting substance, control the colour developing depth of detection line and reference line while detection, and the developed the color depth and standard substance concentration is mapped, reaches half-quantitative detection.
Described detection refers to antigen the conjugates that moniliformin and carrier mass gelatin form, and immunity refers to antigen the conjugates that moniliformin and carrier mass keyhole limpet hemocyanin (KLH) form.
Described carrier mass also can be selected other macromolecular substances, as: lipoprotein, polyamino acid, glucosan, oralbumin etc., but require to detect with the carrier of antigen and immunity antigen without cross-immune reaction.
The second described kind animal protein refers to that non-antibody source belongs to the albumen of animal, and for example, antibody is rabbit source property, and the second kind animal protein can be the chicken such as oralbumin, porcine hemoglobin, duck or other non-rabbit source property animal proteins.
The each several part of test paper described in the invention is processed with function as follows:
Backing: for simultaneously scribbling the toughness material not absorbing water of adhesive sticker, as PVC plate, play fixing other ingredients of test paper of supporting;
Sample pad preparation: by about 2 min in the PBS of filter paper or all-glass paper immersion pH 7.0-8.4, take out, 80 ℃ of oven dry or other modes are dry, as sample pad, play a part to absorb sample solution when detection, are convenient to sample solution and move up;
The preparation of colloid gold label part: this part plays antigen or the antibody of fixing colloid gold label; Preparation process comprises preparation, colloid gold label moniliformin antigen or the moniliformin antibody of colloidal gold solution;
Colloid gold label section processes:
(1) preparation of colloidal gold solution: by gold chloride (HAuCl
4) be mixed with 1% mother liquor with ultrapure water, get the mother liquor of 1 mL, be settled to 100 mL with ultrapure water, be made into 0.01% solution, be heated to boiling, add the trisodium citrate aqueous solution of 1 – 5 mL 1%, continue to be heated to occur transparent orange red till, be colloidal gold solution;
(2) colloid gold label moniliformin antigen: moniliformin detection is used respectively to PBS(0.01mo1/L with antigen and the second kind animal protein, pH 7.0-7.5) dissolved dilution is to 2-4 mg/mL, every 100 mL colloidal gold solutions add the detection antigen of 1-3 mL 2-4 mg/mL and 1-1. 5 mL 2-4 mg/mL the second kind animal proteins to shake 2 min, with the K of 0. 2mol/L
2cO
3:, regulate pH to 8.4, concussion 5 min add 11% PEG-10000 2 mL, concussion 5 min, centrifugal 15 min of 8000-15000r/min, remove supernatant, will precipitate the mol/L with PBS(0. 0l, pH 7.0-7.5) redissolve, with centrifugal 15 min of 6000-13000r/min, remove supernatant, will precipitate and use PBS(0.0l mol/L, pH 7.0-7.5) dilute 500-2000 times of product as golden mark moniliformin antigen;
(3) colloid gold label moniliformin antibody: moniliformin monoclonal antibody or polyclonal antibody and the second kind albumen are used respectively to PBS(0.01 mo1/L, pH 7.0-7.5) dissolved dilution is to 2-4 mg/mL, every 100 mL colloidal gold solutions add moniliformin antibody and 1-1.5 mL 2-4 mg/mL the second kind animal protein of 1-3 mL 2-4 mg/mL, shake 2 min, with the K of 0. 2 mol/L
2cO
3, regulate pH to 8.4, concussion 5 min, add 11% PEG-10000 2 mL, concussion 5 min, centrifugal 15 min of 8000-15000r/min, except supernatant, to precipitate mo1/L with PBS(0.01, pH 7.0-7.5) redissolve, centrifugal 15 min of 6000-13000 r/min, remove supernatant, to precipitate and use PBS(0.01 mol/L, pH 7.0-7.5) dilute 500-2000 doubly, product is as gold mark moniliformin antibody;
(4) gold mark pad is processed: moniliformin antigen or moniliformin antibody are poured in a groove, glass fibre or filter paper are immersed to 1 min, take out, after drying at room temperature, as gold mark pad;
(5) nitrocellulose filter divides preparation: the glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane or cellulose acetate film or nylon membrane 30 min, take out, 37 ℃ of oven dry, the moniliformin of coated 1 variable concentrations in top detects with antigen line or moniliformin antibody line as detection line, and the work IgG line of coated 1 anti-the second kind animal protein is as with reference to line simultaneously.In the time that colloid gold label part tagged object is moniliformin detection antigen and the second kind albumen, detection line is coated moniliformin antibody.This is detection reaction part, and this part Main Function is by reaction result with macroscopic characterization out;
(6) thieving paper preparation: by after all-glass paper or filter paper or thieving paper drying at room temperature, as water suction part.This part Main Function is the mobile unnecessary sample solution coming up to absorb;
(7) test paper assembling: on backing, be pasted with successively sample pad, gold mark pad, nitrocellulose filter and thieving paper, moniliformin Test paper.
Detect principle: detect principle because of the object difference of colloid gold label, and difference slightly.
In the time that colloid gold label part tagged object is moniliformin detection antigen and the second kind animal protein, if contain moniliformin in sample, sample solution is by the absorption of the sample pad of test paper and by moving in capillary action, the little translational speed of free moniliformin molecular weight in sample is fast, first arrive detection line, first coated moniliformin antibody is combined on detection line, because moniliformin antibody coated on detection line only has specific binding site, and in sample, free moniliformin binding ability detects strong with antigen than the moniliformin of parataxic, so antigen moniliformin antibody capture on tested survey line again for the detection of colloid gold label, so detection line is colourless, this is positive, if, without moniliformin, the moniliformin of colloid gold label detects and arrives after detection line with regard to moniliformin antibody capture coated on tested survey line with antigen, forms macroscopic redness in sample, this is negative.No matter in sample, whether contain moniliformin, the work IgG that moves on to while reaching reference line anti-the second kind animal protein coated on can referenced line on the second kind animal protein of colloid gold label catches and forms macroscopic redness, and this is reference line.
In the time that colloid gold label part tagged object is moniliformin antibody and the second kind animal protein, if contain moniliformin in sample, sample solution is absorbed and reaches colloid gold label part by moving on in capillary action by the sample pad of test paper, the moniliformin antibody response of the moniliformin in sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because the moniliformin antibody of colloid gold label only has specific binding site, moniliformin in sample solution with it in conjunction with after, detection on detection line just can not be combined with the moniliformin antibody of colloid gold label with antigen again, so detection line is colourless, this is positive, in the time there is no moniliformin in sample, detected with antigen capture when the moniliformin antibody of colloid gold label arrives detection line, form macroscopic redness.No matter in sample, whether contain moniliformin, the work IgG that moves on to while reaching reference line anti-the second kind animal protein coated on can referenced line on the second kind albumen of colloid gold label catches and forms macroscopic redness, and this is reference line.
The test paper of above two kinds of modes, time prepared by test paper, pass through to regulate detection line and reference line encrusting substance concentration, control the colour developing depth of detection line and reference line while detection, and the developed the color depth and standard substance concentration is mapped, can reaches half-quantitative detection object.
Accompanying drawing explanation
The structural representation that accompanying drawing 1 is test strip of the present invention:
In figure, 1 is that sample liquid absorption portion 2 is that colloid gold label part 3 is that detection reaction part 4 is that detection line 5 is that reference line 6 is the part that absorbs water.
embodiment
Moniliformin semi-quantitative rapid detection test paper is prepared as described in technique scheme.
In embodiment 1 corn, moniliformin detects
Pre-treatment: get 3 g and pulverize samples, add 30 mL sample extracting solutions (methyl alcohol is diluted by 7:3 volume ratio with deionized water, 7 parts of methyl alcohol add 3 parts of deionized waters, for extracting the moniliformin of sample); Thermal agitation 5 min; Centrifugal 5 min of 4000 r/min, or use common Filter paper filtering; Supernatant or filtrate are pressed 1:19 dilution proportion with deionized water; After getting dilution, liquid is to be measured.
The using method of moniliformin semi-quantitative rapid detection test paper product: sample thief filtrate 100 μ g slowly drip on the glass fibre membrane of test strips, the colour developing situation of observing detection line in test strips (T line), reference line (C line) after 15 minutes.Limit the quantity and judge the content of moniliformin in sample extracting solution according to the detection principle of test strips: if only have a red ribbon to occur judging that sample liquid is positive, moniliformin exceeds standard; If occur, two red ribbons judge that sample liquid is negative, and category-B Trichothecenes toxin does not exceed standard; If colourless band occurs that this test strips lost efficacy.
In embodiment 2 feeds, moniliformin detects
Pre-treatment: get 3 g and pulverize samples, add 30 mL sample extracting solutions (methyl alcohol is diluted by 7:3 volume ratio with deionized water, 7 parts of methyl alcohol add 3 parts of deionized waters, for extracting the moniliformin of sample); Thermal agitation 5min; Centrifugal 5 min of 4000 r/min, or use common Filter paper filtering; Supernatant or filtrate are pressed 1:49 dilution proportion with deionized water; After getting dilution, liquid is to be measured.
The using method of moniliformin semi-quantitative rapid detection test paper product: sample thief filtrate 100 μ L slowly drip on the glass fibre membrane of test strips, the colour developing situation of observing detection line in test strips (T line), reference line (C line) after 15 minutes.Limit the quantity and judge the content of moniliformin in sample extracting solution according to the detection principle of test strips: if only have a red ribbon to occur judging that sample liquid is positive, moniliformin exceeds standard; If occur, two red ribbons occur judging that sample liquid is negative, and moniliformin does not exceed standard; If colourless band occurs that this test strips lost efficacy.
Claims (4)
1. moniliformin colloidal gold strip, it is characterized in that: on the backing of test paper, post successively sample pad, gold mark pad, nitrocellulose filter and thieving paper, the material of the upper mark of gold mark pad is the potpourri of the second kind animal protein and moniliformin antibody; Nitrocellulose filter is coated with and detects with moniliformin antigen 1 bar as detection line, is also coated with 1 of the IgG of anti-the second kind animal protein as line of reference simultaneously.
2. moniliformin colloidal gold strip according to claim 1, is characterized in that: it is the conjugates of moniliformin and carrier mass formation that moniliformin detects with antigen.
3. moniliformin colloidal gold strip according to claim 2, is characterized in that: described carrier mass is protein, protein fragments, synthetic polypeptide, semi-synthetic polypeptide or polysaccharide.
4. moniliformin colloidal gold strip according to claim 1, is characterized in that: the second described kind animal protein is the protein of non-antibody source animal.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210435631.7A CN103792365A (en) | 2012-11-05 | 2012-11-05 | Preparation and application method of fusarium moniliforme colloidal gold test strip |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210435631.7A CN103792365A (en) | 2012-11-05 | 2012-11-05 | Preparation and application method of fusarium moniliforme colloidal gold test strip |
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| CN103792365A true CN103792365A (en) | 2014-05-14 |
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| CN201210435631.7A Pending CN103792365A (en) | 2012-11-05 | 2012-11-05 | Preparation and application method of fusarium moniliforme colloidal gold test strip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021093884A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting diacetoxyscirpenol pollution, and preparation method therefor and application thereof |
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| US5770458A (en) * | 1995-02-10 | 1998-06-23 | Roche Diagnostics Systems, Inc. | Apparatus and method for conducting a binding assay on an absorbant carrier material |
| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
| CN101482566A (en) * | 2009-01-19 | 2009-07-15 | 南昌博恒生物制品有限公司 | Production and use of test paper for fast detecting deoxynivalenol in cereal |
| WO2009102420A1 (en) * | 2008-02-11 | 2009-08-20 | Biotechpharma Corporation | Integrated device for analyte testing, confirmation, and donor identity verification |
-
2012
- 2012-11-05 CN CN201210435631.7A patent/CN103792365A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5770458A (en) * | 1995-02-10 | 1998-06-23 | Roche Diagnostics Systems, Inc. | Apparatus and method for conducting a binding assay on an absorbant carrier material |
| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
| WO2009102420A1 (en) * | 2008-02-11 | 2009-08-20 | Biotechpharma Corporation | Integrated device for analyte testing, confirmation, and donor identity verification |
| CN101482566A (en) * | 2009-01-19 | 2009-07-15 | 南昌博恒生物制品有限公司 | Production and use of test paper for fast detecting deoxynivalenol in cereal |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021093884A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting diacetoxyscirpenol pollution, and preparation method therefor and application thereof |
| US11635429B2 (en) | 2019-11-15 | 2023-04-25 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Immunochromatography test strip for detecting pollution of diacetoxyscirpenol, preparation method therefor, and application thereof |
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Application publication date: 20140514 |