CN105486857A - Diclazuril colloidal gold test paper strip preparation method and usage method thereof - Google Patents
Diclazuril colloidal gold test paper strip preparation method and usage method thereof Download PDFInfo
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- CN105486857A CN105486857A CN201410531018.4A CN201410531018A CN105486857A CN 105486857 A CN105486857 A CN 105486857A CN 201410531018 A CN201410531018 A CN 201410531018A CN 105486857 A CN105486857 A CN 105486857A
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- diclazuril
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Abstract
The invention belongs to the field of biological detection, and relates to a diclazuril colloidal gold test paper strip preparation method and a usage method thereof. The test paper strip comprises a paperboard, an absorbent pad, a detection pad, a colloidal gold pad and a sample pad are sequentially pasted on one side of the paperboard from up to down, and adjacent pads overlappingly connect at a joint; the detection pad treats a cellulose nitrate membrane as a base pad, a transverse reference wire and a detection wire are arranged on the cellulose nitrate membrane in a top-down manner, the detection wire is coated with a diclazuril-bovine serum albumin conjugate, and the reference wire is coated with a rabbit-anti-mouse polyclonal antibody; and the colloidal gold pad is transversely sprayed with a nanogold labeled anti-diclazuril monoclonal antibody which is generated by a hybridoma cell strain. The test paper strip which is used for diclazuril detection has the characteristics of rapid detection, simple operation and high sensitivity.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to the detection method of residuals in food, particularly a kind of method of preparation and use of diclazuril colloidal gold strip.
Background technology
Diclazuril diclazuril belongs to triazine benzene acetonitrile compound, is novel, efficient, low toxicity anticoccidial drug, is widely used in chicken coccidiasis.To the coccidia Main Function peak phase, different with the different genera of coccidia, as to the sexual cycle of Eimeria tenella Main Function point 2nd generation schizont coccidia.But to huge, E.brunetti schizont is invalid.To the zygote stage of Eimeria maxima application point coccidia; Have efficiently the E.brunetti microgametophyte stage.A ground gram ball profit also has inhibiting effect to formation Sporulated Oocysts.In food and feed, the detection method of diclazuril 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, need special technician, testing cost costliness during operation, be unfavorable for applying.
Summary of the invention
The object of the invention is, in order to overcome the deficiencies in the prior art, to provide a kind of high specificity, highly sensitive, and simple to operation, to sample through simple process and the quick test paper semi-quantitative detection method of detectable diclazuril.
Object of the present invention realizes by following technical scheme:
Its technical essential of diclazuril colloidal gold strip detection method is: the backing of test paper posts 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 diclazuril detection antigen or the potpourri of the second kind animal protein and diclazuril antibody; Nitrocellulose filter is coated with diclazuril antibody 1 as detection line, the IgG1 bar being simultaneously also coated with anti-second kind animal protein is as reference line or be coated with diclazuril detection antigen 1 bar as detection line, is also coated with the work IgG1 bar of anti-second kind animal protein as reference line simultaneously.When prepared by test paper by regulating the concentration of detection line and reference line encrusting substance, the colour developing depth of detection line and reference line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration, reaches half-quantitative detection.
Described detection antigen refers to that the conjugates that diclazuril and carrier mass gelatin are formed, immunity antigen refer to the conjugates that diclazuril and carrier mass keyhole limpet hemocyanin (KLH) are formed.
Described carrier mass also can select other macromolecular substances, as: lipoprotein, polyamino acid, glucosan, oralbumin etc., but require that the carrier of detection antigen and immunity antigen is without cross-immune reaction.
The second described kind animal protein refers to that non-antibody source belongs to the albumen of animal, and such as, antibody is rabbit source property, then 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 process of test paper described in the invention and function as follows:
Backing: for one side scribbles the toughness material do not absorbed water of adhesive sticker, as PVC board, plays fixing other ingredients of support test paper.
Sample pad preparation: filter paper or all-glass paper are immersed about 2min in the PBS of pH7.0-8.4, take out, dries or other modes dryings, namely as sample pad, plays a part to absorb sample solution, be convenient to sample solution and move up during detection for 80 DEG C.
The preparation of colloid gold label part: this part plays antigen or the antibody of fixing colloid gold label.
Preparation process comprises the preparation of colloidal gold solution, colloid gold label diclazuril antigen or diclazuril antibody.
The process of colloid gold label part.
(1) the preparation of colloidal gold solution: by gold chloride (HAuCl
4) to be mixed with ultrapure water 1% mother liquor, get the mother liquor of 1mL, be settled to 100mL with ultrapure water, be made into the solution of 0.01%, be heated to boiling, add the trisodium citrate aqueous solution of 1 – 5mL1%, continue to be heated to occur transparent orange red till, be colloidal gold solution.
(2) colloid gold label diclazuril antigen: diclazuril detection antigen and the second kind animal protein are used PBS(0.01mo1/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds detection antigen and the 1-1.5mL2-4mg/mL second kind animal protein concussion 2min of 1-3mL2-4mg/mL, with the K of 0.2mol/L
2cO
3:, regulate pH to 8.4, concussion 5min adds 11%PEG-100002mL, concussion 5min, the centrifugal 15min of 8000-15000r/min, removing supernatant, by precipitation PBS(0.0lmol/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, removing supernatant, using precipitation PBS(0.0lmol/L, pH7.0-7.5) dilute 500-2000 times of product as gold mark diclazuril antigen.
(3) colloid gold label diclazuril antibody: diclazuril monoclonal antibody or polyclonal antibody and the second kind albumen are used PBS(0.01mo1/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds diclazuril antibody and the 1-1.5mL2-4mg/mL second kind animal protein of 1-3mL2-4mg/mL, concussion 2min, with the K of 0.2mol/L
2cO
3, regulate pH to 8.4, concussion 5min, add 11%PEG-100002mL, the centrifugal 15min of concussion 5min, 8000-15000r/min, except supernatant, by precipitation PBS(0.01mo1/L, pH7.0-7.5) redissolve, the centrifugal 15min of 6000-13000r/min, removing supernatant, by precipitation PBS(0.01mol/L, pH7.0-7.5) dilute 500-2000 doubly, product is as gold mark diclazuril antibody.
(4) gold mark pad process: pour in a groove by diclazuril antigen or diclazuril antibody, immerses 1min by glass fibre or filter paper, takes out, after drying at room temperature, namely as gold mark pad.
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 30min, take out, 37 DEG C of oven dry, top bag by the diclazuril detection of 1 variable concentrations with antigen line or diclazuril antibody line as detection line, wrap by the work IgG line of 1 anti-second kind animal protein as with reference to line simultaneously.When colloid gold label portion markings object be diclazuril detection antigen and the second kind albumen time, detection line then wraps by diclazuril antibody.This is detection reaction part, and this part Main Function is by reaction result with macroscopic characterization out.
Prepared by thieving paper: after all-glass paper or filter paper or thieving paper drying at room temperature, namely as water absorbent portion.This part Main Function is the mobile unnecessary sample solution come up to absorb.
Test paper is assembled: on backing, be pasted with sample pad, gold mark pad, nitrocellulose filter and thieving paper successively, diclazuril Test paper.
Cleaning Principle: Cleaning Principle because of the object of colloid gold label different, and slightly difference.
When colloid gold label portion markings object be diclazuril detection antigen and the second kind animal protein time, if containing diclazuril in sample, sample solution is by the absorption of the sample pad of test paper and by capillary action is moved, the little translational speed of free diclazuril molecular weight in sample is fast, first arrive detection line, the diclazuril antibody first wrapping quilt on detection line is combined, because diclazuril antibody detection line wrapping quilt only has specific binding site, and the diclazuril binding ability of dissociating in sample is stronger than the diclazuril detection antigen of parataxic, so the detection antigen of colloid gold label can not diclazuril antibody capture again on tested survey line, so detection line is colourless, this is the positive, if without diclazuril in sample, then just tested survey line wraps the diclazuril antibody capture of quilt after the diclazuril detection antigen of colloid gold label arrives detection line, form macroscopic redness, this is feminine gender.No matter in sample whether containing diclazuril, the second kind animal protein of colloid gold label to move on to when reaching reference line the work IgG that all referenced line can wrap the anti-second kind animal protein of quilt and catch and form macroscopic redness, this is reference line.
When colloid gold label portion markings object be diclazuril antibody and the second kind animal protein time, if containing diclazuril in sample, sample solution is absorbed by the sample pad of test paper and reaches colloid gold label part by capillary action moves on to, the diclazuril antibody response of the diclazuril in sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because the diclazuril antibody of colloid gold label only has specific binding site, after diclazuril in sample solution combines with it, detection antigen on detection line just can not be combined with the diclazuril antibody of colloid gold label again, so detection line is colourless, this is the positive, when not having diclazuril in sample, detected antigen capture when the diclazuril antibody of colloid gold label arrives detection line, then form macroscopic redness.No matter in sample whether containing diclazuril, the second kind albumen of colloid gold label to move on to when reaching reference line the work IgG that all referenced line can wrap the anti-second kind animal protein of quilt and catch and form macroscopic redness, this is reference line.
The test paper of above two kinds of modes, when prepared by test paper by regulating detection line and reference line encrusting substance concentration, the colour developing depth of detection line and reference line when controlling to detect, and the depth that developed the color is mapped with standard substance concentration, can reach half-quantitative detection object.
Accompanying drawing explanation
Accompanying drawing is the structural representation of test strip of the present invention:
1 be sample liquid absorption portion, 2 be colloid gold label part in figure, 3 be detection reaction part, 4 be detection line, 5 be reference line, 6 for water absorbent portion.
embodiment
The preparation of diclazuril semi-quantitative rapid detection test paper is as described in technique scheme.
In embodiment 1 chicken, diclazuril detects
Pre-treatment: get 2g chicken meat sample, adds 20ml sample extracting solution (diluted by 9:1 volume ratio by methyl alcohol with deionized water, namely 9 parts of methyl alcohol add 1 part of deionized water, for extracting the diclazuril in sample); Thermal agitation 5min; The centrifugal 5min of 4000r/min, or filter with ordinary filter paper; Supernatant or filtrate press 1:19 dilution proportion with deionized water; After getting dilution, liquid is to be measured.
The using method of diclazuril semi-quantitative rapid detection test paper product: sample thief filtrate 100 μ g slowly drips on the glass fibre membrane of test strips, observes the colour developing situation of detection line in test strips (T line), reference line (C line) after 15 minutes.Limit the quantity according to the Cleaning Principle of test strips and judge the content of diclazuril in sample extracting solution: if only have a red ribbon to occur, judge that sample liquid is as the positive, diclazuril exceeds standard; If there are two red ribbons, judge that sample liquid is as feminine gender, diclazuril does not exceed standard; If colourless band occurs, this test strips lost efficacy.
In embodiment 2 feed, diclazuril detects
Pre-treatment: getting 2g and pulverize sample, adding 20mL sample extracting solution (diluted by 9:1 volume ratio by methyl alcohol with deionized water, namely 9 parts of methyl alcohol add 1 part of deionized water, for extracting the diclazuril in sample); Thermal agitation 5min; The centrifugal 5min of 4000r/min, or filter with ordinary filter paper; Supernatant or filtrate press 1:49 dilution proportion with deionized water; After getting dilution, liquid is to be measured.
The using method of diclazuril semi-quantitative rapid detection test paper product: sample thief filtrate 100 μ L slowly drips on the glass fibre membrane of test strips, observes the colour developing situation of detection line in test strips (T line), reference line (C line) after 15 minutes.Limit the quantity according to the Cleaning Principle of test strips and judge the content of diclazuril in sample extracting solution: if only have a red ribbon to occur, judge that sample liquid is as the positive, diclazuril exceeds standard; If occur, two red ribbons occur, judge that sample liquid is as feminine gender, diclazuril does not exceed standard; If colourless band occurs, this test strips lost efficacy.
Claims (4)
1. diclazuril colloidal gold strip, is characterized in that: on the backing of test paper, post sample pad, gold mark pad, nitrocellulose filter and thieving paper successively, and the material of the upper mark of gold mark pad is the potpourri of the second kind animal protein and diclazuril antibody; Nitrocellulose filter is coated with detection diclazuril antigen 1 bar as detection line, is also coated with the IgG1 bar of anti-second kind animal protein as line of reference simultaneously.
2. diclazuril colloidal gold strip according to claim 1, is characterized in that: diclazuril detection antigen is the conjugates that diclazuril and carrier mass are formed.
3. diclazuril colloidal gold strip according to claim 2, is characterized in that: described carrier mass, is protein, protein fragments, improvement on synthesis, semi-synthetic polypeptide or polysaccharide.
4. diclazuril 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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410531018.4A CN105486857A (en) | 2014-10-10 | 2014-10-10 | Diclazuril colloidal gold test paper strip preparation method and usage method thereof |
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| CN201410531018.4A CN105486857A (en) | 2014-10-10 | 2014-10-10 | Diclazuril colloidal gold test paper strip preparation method and usage method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837771A (en) * | 2006-04-19 | 2006-09-27 | 姚家彪 | Analytic sample of animal drug residue in meat powder and preparation process thereof |
| CN103808939A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Spectinomycin colloidal gold detection card |
| CN104090099A (en) * | 2013-04-07 | 2014-10-08 | 江苏维赛科技生物发展有限公司 | Preparation method and use method of apramycin-detection colloidal gold test paper strip |
-
2014
- 2014-10-10 CN CN201410531018.4A patent/CN105486857A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837771A (en) * | 2006-04-19 | 2006-09-27 | 姚家彪 | Analytic sample of animal drug residue in meat powder and preparation process thereof |
| CN103808939A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Spectinomycin colloidal gold detection card |
| CN104090099A (en) * | 2013-04-07 | 2014-10-08 | 江苏维赛科技生物发展有限公司 | Preparation method and use method of apramycin-detection colloidal gold test paper strip |
Non-Patent Citations (1)
| Title |
|---|
| YAHUI WANG ET AL.: "Development of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Analysis of Diclazuril in Chicken Tissues", 《FOOD ANALYTICAL METHOD》 * |
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