CN105301251A - Preparation and using method of quintozene colloidal gold test strip - Google Patents
Preparation and using method of quintozene colloidal gold test strip Download PDFInfo
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- CN105301251A CN105301251A CN201410357558.5A CN201410357558A CN105301251A CN 105301251 A CN105301251 A CN 105301251A CN 201410357558 A CN201410357558 A CN 201410357558A CN 105301251 A CN105301251 A CN 105301251A
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- pentachloronitrobenzene
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Abstract
The invention relates to a preparation and using method of a quintozene colloidal gold test strip. According to the method, a sample pad, a gold labeling pad, a nitrocellulose film and a sample absorption pad are sequentially pasted onto the backing of test paper; the nitrocellulose film is coated with an antigen 1 strip for detection, and the antigen 1 strip serves as a detection line; meanwhile, the nitrocellulose film is coated with an IgG 1 strip, and the IgG 1 strip resists the second generic-specific animal protein and serves as a reference line. The quick detection test strip is high in specificity, can be utilized for semi-quantitative detection, and is suitable for quick detection of remaining quintozene in soil, seed, fruit and ginseng; the method provided by the invention has the benefits of being high in specificity and sensitivity, and simple and convenient to operate, and realizing semi-quantitative detection.
Description
Technical field
The invention belongs to field of detection of food safety, be specifically related to the method for preparation and use of a kind of quick colloidal gold strip of pentachloronitrobenzene medicament residue in food.
Background technology
Pentachloronitrobenzene has another name called grogs and falls apart, water insoluble, is dissolved in organic solvent, stable chemical nature, and not volatile, oxidation Sum decomposition, is not subject to the shadow of sunlight and soda acid yet, but understands explosive decomposition under the condition of high temperature drying, reduce drug effect.To people, animal, fish low toxicity, long in the soil longevity of residure.
Pentachloronitrobenzene is eaten and too much may be damaged cardiovascular system, central nervous system, liver and kidney.During small white mouse acute poisoning, there is accelerated breathing, cyanosis, tremble, spasmodic tic, incoordination, even dead.Under chronic effect, initial stage RBC number and content of hemoglobin increase, and suppress hematopoiesis function subsequently, exhaustion, tic, Some Animals can be lethal.In food, the detection method gas chromatography of pentachloronitrobenzene, due to above analytic approach sample pre-treatments more complicated, needs special technician, testing cost costliness, is unfavorable for applying during operation.
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 pentachloronitrobenzene.
Object of the present invention realizes by following technical scheme
Its technical essential of pentachloronitrobenzene colloidal gold strip detection method is: the backing of test paper posts successively sample pad and gold mark pad and nitrocellulose filter and water suction thieving paper, and the material that gold mark pad is labeled is the potpourri of the second kind animal protein and pentachloronitrobenzene detection antigen or the potpourri of the second kind animal protein and pentachloronitrobenzene antibody; Nitrocellulose filter is coated with pentachloronitrobenzene 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 pentachloronitrobenzene 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 pentachloronitrobenzene and carrier mass gelatin are formed, immunity antigen refer to the conjugates that pentachloronitrobenzene 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 gold mark pad: this part plays antigen or the antibody of fixing colloid gold label.
Preparation process comprises the preparation of colloidal gold solution, colloid gold label pentachloronitrobenzene antigen or pentachloronitrobenzene antibody.
(1) 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 pentachloronitrobenzene antigen: pentachloronitrobenzene 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 the detection antigen 1-1.5mL2-4mg/mL second kind animal protein concussion 2min of the 2-4mg/mL of 1-3mL, with the K of 0.2mol/L
2cO
3regulate pH to 8.4, concussion 5min adds 11%PEG-100002mL, concussion 5min, the centrifugal 15min of 6000-13000r/min, removing supernatant, by precipitation PBS(0.0lmol/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 8000-15000r/min, removing supernatant, using precipitation PBS(0.0lmol/L, pH7.0-7.5) release 500-2000 times of product as gold mark pentachloronitrobenzene antigen.
(3) colloid gold label pentachloronitrobenzene antibody: pentachloronitrobenzene 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 pentachloronitrobenzene 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, 6000-13000r/min, removing supernatant, by precipitation PBS(0.01mo1/L, pH7.0-7.5) redissolve, with the centrifugal 15min of 8000-15000r/min, removing supernatant, by precipitation PBS(0.0lmol/L, pH7.0-7.5) dilute 500-2000 doubly, product is as gold mark pentachloronitrobenzene antibody.
(4) gold mark pad process: pour in a groove by pentachloronitrobenzene antigen or pentachloronitrobenzene antibody, immerses 1min by glass fibre or filter paper, takes out, and after drying at room temperature, namely makes gold mark pad.
Cellulose nitrate film 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 pentachloronitrobenzene detection of 1 variable concentrations with antigen line or pentachloronitrobenzene 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 pentachloronitrobenzene detection antigen and the second kind albumen time, detection line then wraps by pentachloronitrobenzene antibody.This is nitrocellulose filter, 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 absorption of sample pad successively, pentachloronitrobenzene 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 pentachloronitrobenzene detection antigen and the second kind animal protein time, if containing pentachloronitrobenzene 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 pentachloronitrobenzene molecular weight in sample is fast, first arrive detection line, the pentachloronitrobenzene antibody first wrapping quilt on detection line is combined, because pentachloronitrobenzene antibody detection line wrapping quilt only has specific binding site, and the pentachloronitrobenzene binding ability of dissociating in sample is stronger than the pentachloronitrobenzene detection antigen of parataxic, so the detection antigen of colloid gold label can not pentachloronitrobenzene antibody capture again on tested survey line, so detection line is colourless, this is the positive, if without pentachloronitrobenzene in sample, then just tested survey line wraps the pentachloronitrobenzene antibody capture of quilt after the pentachloronitrobenzene detection antigen of colloid gold label arrives detection line, form macroscopic redness, this is feminine gender.No matter in sample whether containing pentachloronitrobenzene, 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 gold mark pad tagged object be pentachloronitrobenzene antibody and the second kind animal protein time, if containing pentachloronitrobenzene 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 pentachloronitrobenzene antibody response of the pentachloronitrobenzene in sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because the pentachloronitrobenzene antibody of colloid gold label only has specific binding site, after pentachloronitrobenzene in sample solution combines with it, detection antigen on detection line just can not be combined with the pentachloronitrobenzene antibody of colloid gold label again, so detection line is colourless, this is the positive, when not having pentachloronitrobenzene in sample, detected antigen capture when the pentachloronitrobenzene antibody of colloid gold label arrives detection line, then form macroscopic redness.No matter in sample whether containing pentachloronitrobenzene, 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
Fig. 1 is pentachloronitrobenzene colloidal-gold detecting-card shell and test strip arrangement figure in the enclosure, and 1 is test card shell, and wherein 2 is test strips, and 3 is detection window, and 4 is well.
Fig. 2 is pentachloronitrobenzene test card test strip structural representation, and wherein 5 is supporting backboard, and 6 is nitrocellulose filter, and 7 is gold mark pad, and 8 is sample pad, and 9 is thieving paper.
Fig. 3 test strip nitrocellulose filter shows trace band schematic diagram, and wherein 10 is detection line, and 11 is line of reference.
Embodiment
Pentachloronitrobenzene test card arranges test strip 2 in test card shell 1, structure and the arranging of test strip 2 of test card shell 1 see accompanying drawing 1, test card shell 1 is rectangular flat shell shape shell, long 70mm, wide 20mm, thick 5mm, shell wall thickness 1mm, be made up of engineering plastics, test card shell 1 is formed by upper cover and lower cover two semi join, test card shell 1 has covered and has detected fenestra 3 and well 4.Test strip 2 is placed in the lower cover of test card shell 1.
Test strip 2 is fillet thin slices of sandwich construction, length of a film 60mm, and the wide about 4mm of sheet, thickness is less than 2.5mm.Test strip 2 is sandwich constructions: bottom is supporting backboard 5, and be polyethylene sheets, thickness is about 0.5mm, long 60mm, wide 4mm; Nitrocellulose filter 6 is pasted, cellulose nitrate thickness 0.5mm, long 20mm, wide 4mm in the middle part of supporting backboard 5.Nitrocellulose filter 6 has two isolated laterally display trace bands, see accompanying drawing 3, in two display traces, one is detection line 10, and containing pentachloronitrobenzene conjugate, another is line of reference 11, containing the IgG of the second kind animal protein.Thieving paper 9 is pasted in backboard 5 one end in supporting, the other end pastes sample pad 8, and thieving paper 9 is inner to be overlapped with nitrocellulose filter 6, then has one section of gold to mark pad 7 between sample pad 8 and nitrocellulose filter 6, sample pad 8 and nitrocellulose filter 6 are linked together by gold mark pad 7, lap width is at 1-2mm.Sample pad 8 material is water adsorption glass fiber or polyester film.
When using pentachloronitrobenzene colloidal-gold detecting-card, first by sample liquid to be detected (after the extract dilution of medicine, be directly used in detection) from well 4, instill the sample pad 8 of pentachloronitrobenzene test strip 2, due to rainbow action principle, the anti-pentachloronitrobenzene monoclonal antibody colloid gold label thing contained by sample liquid to be detected and colloidal gold film 7 is driven to spread to nitrocellulose filter 6 together, observations in 5-10 minute.
The key reaction of test card is immunologic antigen and antibody response, the antibody of the colloid gold label that nitrocellulose filter moves, on p-wire with containing pentachloronitrobenzene protein conjugate, and line of reference reacts containing the IgG of the second kind animal protein, formation brownish red band.If corresponding pentachloronitrobenzene to be measured is higher than permissible value in sample, sample adds rear elder generation and marks the antibody response in padding with gold, and can not with detection zone with pentachloronitrobenzene protein conjugate react, thus not develop the color.Testing result has following three kinds:
1. detection line 10 and line of reference 11 manifest brownish red trace simultaneously, represent that testing result be negative, illustrate in sample and do not contain pentachloronitrobenzene or content lower than permissible value.
2. detection line 10 does not develop the color, and line of reference 11 manifests brownish red trace, represents that testing result is positive, illustrates that in sample, pentachloronitrobenzene content is higher than permissible value.
3. detection line 10 manifests brownish red trace, and line of reference 11 does not develop the color, or detection line 10 and line of reference 11 all do not develop the color, and represents that test strip lost efficacy.
Claims (4)
1. pentachloronitrobenzene colloidal gold strip, it is characterized in that: on the backing of test paper, post sample pad, gold mark pad, nitrocellulose filter and thieving paper successively, the material that gold mark pad part is labeled is the potpourri of the second kind animal protein and pentachloronitrobenzene antibody; Cellulose nitrate membrane portions is coated with detection pentachloronitrobenzene 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. pentachloronitrobenzene colloidal gold strip according to claim 1, is characterized in that: pentachloronitrobenzene detection antigen is the conjugates that pentachloronitrobenzene and carrier mass are formed.
3. pentachloronitrobenzene 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. pentachloronitrobenzene 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 |
|---|---|---|---|
| CN201410357558.5A CN105301251A (en) | 2014-07-25 | 2014-07-25 | Preparation and using method of quintozene colloidal gold test strip |
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| CN201410357558.5A CN105301251A (en) | 2014-07-25 | 2014-07-25 | Preparation and using method of quintozene colloidal gold test strip |
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| CN105301251A true CN105301251A (en) | 2016-02-03 |
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| CN201410357558.5A Pending CN105301251A (en) | 2014-07-25 | 2014-07-25 | Preparation and using method of quintozene colloidal gold test strip |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113281501A (en) * | 2021-05-12 | 2021-08-20 | 北京勤邦生物技术有限公司 | Application of pentachloronitrobenzene artificial antigen in enzyme linked immunosorbent assay kit |
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| CN101526527A (en) * | 2008-03-07 | 2009-09-09 | 中国农业大学 | ELISA kit applicable to residue analysis of terrachlor |
| CN102798719A (en) * | 2012-08-09 | 2012-11-28 | 河南省农业科学院 | Test paper strip for rapidly detecting traces of chlorothalonil and preparation method thereof |
| CN103698524A (en) * | 2013-12-19 | 2014-04-02 | 杭州南开日新生物技术有限公司 | Immunity colloid gold reagent plate for quickly detecting sodium pentachlorophenate and preparation method for immunity colloid gold reagent plate |
| 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 |
-
2014
- 2014-07-25 CN CN201410357558.5A patent/CN105301251A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101526527A (en) * | 2008-03-07 | 2009-09-09 | 中国农业大学 | ELISA kit applicable to residue analysis of terrachlor |
| CN102798719A (en) * | 2012-08-09 | 2012-11-28 | 河南省农业科学院 | Test paper strip for rapidly detecting traces of chlorothalonil and preparation method 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 |
| CN103698524A (en) * | 2013-12-19 | 2014-04-02 | 杭州南开日新生物技术有限公司 | Immunity colloid gold reagent plate for quickly detecting sodium pentachlorophenate and preparation method for immunity colloid gold reagent plate |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113281501A (en) * | 2021-05-12 | 2021-08-20 | 北京勤邦生物技术有限公司 | Application of pentachloronitrobenzene artificial antigen in enzyme linked immunosorbent assay kit |
| CN113281501B (en) * | 2021-05-12 | 2023-07-07 | 北京勤邦科技股份有限公司 | Application of pentachloronitrobenzene artificial antigen in ELISA kit |
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