CN105301238A - Preparation of test paper for detecting excessive caffeine - Google Patents
Preparation of test paper for detecting excessive caffeine Download PDFInfo
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- CN105301238A CN105301238A CN201410354444.5A CN201410354444A CN105301238A CN 105301238 A CN105301238 A CN 105301238A CN 201410354444 A CN201410354444 A CN 201410354444A CN 105301238 A CN105301238 A CN 105301238A
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- caffeine
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Abstract
The invention relates to test paper for detecting excessive caffeine and preparation of the test paper. A liquid sample absorption part, a colloid gold marking part, a detection and reaction part and a water absorption part are sequentially glued on a lining of the test paper, and the detection and reaction part is coated with a detecting antigen as a detection line and simultaneously coated with an IgG1 strip resistant to second species animal protein as a reference line. The quick detection test strip is strong in specificity, can realize semi-quantitative detection, is suitable for quickly detecting excessive caffeine in canned drink manufacturing plants, food hygiene quality inspection departments, personal families and the like, and has the characteristics of being quick in detection, simple to operate and high in sensitivity when being used for detecting caffeine.
Description
Technical field
Belong to field of detection of food safety, be specifically related to the detection method having the material that also exceeds standard in food, particularly a kind of caffeine test paper detecting method.
Background technology
Caffeine is a kind of alkaloid extract from tealeaves, coffee berry, and moderately using has the effect of dispelling fatigue, excitor nerve, is used for the treatment of neurasthenia and stupor recovery clinically.But, heavy dose of or Long-Time Service also can cause damage to human body, particularly it also has additive, once stop using there will be down in spirits, the various withrawal symptom such as tired weak from head to foot, although it is additive more weak, withrawal symptom is very not serious yet. but when causing dosage constantly to increase due to the tolerance of medicine, caffeine just not only acts on cerebral cortex, can also direct excited oblongata, paroxysmal convulsions and bone is caused to tremble, infringement liver, stomach, the important internal organs such as kidney, bring out respiratory inflammation, the diseases such as mammary glands in women knurl, even cause smoker's mentally disabled of future generation, cacomelia.Therefore the psychotropic substances scope by national regulatory is also put into.Abuse caffeine usually also has and sucks and inject two kinds of forms, its excited spread effect and toxicity, symptom, drug-dependent close with amphetamine.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of special fast and simple, with low cost, result detection method is accurately provided, and simple to operation, to sample through simple process and the quick test paper quantitative detecting method of detectable caffeine.
Object of the present invention realizes by following technical scheme:
Its technical essential of caffeine immune chromatography test paper detecting method is: being lining with of test paper posts sample liquid absorption portion and colloid gold label part and detection reaction part and water absorbent portion successively, and the material that colloid gold label part is labeled is caffeine detection antibody; Be coated with caffeine antigen above detection reaction part as detection line, be also coated with anti-anti-as reference line with two of kind animal protein 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 caffeine and carrier mass gelatin are formed, immunity antigen refer to the conjugates that caffeine and carrier mass bovine serum albumin(BSA) (BSA) 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.
Two anti-finger antibody sources of described same kind animal protein belong to the albumen of animal, and such as, antibody is rabbit source property, then resisting with two of kind animal protein is also rabbit source property animal protein.
The each several part process of test paper described in the invention and function as follows:
Served as a contrast: for one side scribbles the toughness material do not absorbed water of adhesive sticker, as PVC board, played fixing other compositions of support test paper
The effect of part.
Prepared by sample liquid absorption portion: polyester film or all-glass paper are immersed about 2min in the PBS of pH7.4, takes out, about 45 DEG C oven dry, namely as sample liquid absorption portion, plays a part to absorb sample solution, be convenient to sample solution and move up during detection.
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 caffeine antigen or caffeine antibody.The process of colloid gold label part.
(1) preparation of aqueous solution of chloraurate: 10g gold chloride 1000m1 distilled water is dissolved, is made into the aqueous solution of 1%, is placed in
4 DEG C for subsequent use, and the term of validity is 3 days.
(2) preparation of colloidal gold solution: 1% gold chloride is diluted to 0.01% with distilled water, put electric furnace to boil, add 4ml1% citrate three sodium by every 100ml0.01% gold chloride, continue to boil, namely stop heating until liquid is shiny red, after being cooled to room temperature, supply dehydration.Outward appearance should be pure, bright, without precipitation and floating thing.
(3) configuration of golden labeling antibody conserving liquid: l0gBSA, 5g skimmed milk power, 0.5gNaN3 and 1mlTween-20 are dissolved in the PBS of 1000ml0.01M, pH7.0, by 0.22u membrane filtration mistake, be placed in 40 DEG C for subsequent use, the term of validity is 15 days.
(4) colloid gold label caffeine antibody: adjust collaurum pH value to 7.6, by 20 μ g antibody with 0.1M wet chemical
/ ml collaurum adds the monoclonal antibody of anti-caffeine, mixing, leave standstill 30 minutes, with 12000rpm centrifugal 30 minutes, abandoning supernatant, precipitation mark cleansing solution washes twice, last abandoning supernatant, will the precipitation golden labeling antibody conserving liquid dissolving of 1/10th initial colloid gold volumes, be placed in 4 DEG C for subsequent use, the term of validity is 7 days, and product is as gold mark caffeine antibody.
(5) colloid gold label part process: the gold mark caffeine antigen after dilution or gold mark caffeine antibody are poured in a groove, glass fibre or filter paper is immersed 1min, takes out, after 37 DEG C of dryings, namely as colloid gold label part.
Prepared by detection reaction part: the glutaraldehyde solution with 0.8% or 0.2% Carbodiimide solution immersion nitrocellulose membrane 30min, take out, 37 DEG C of oven dry, top bag by the caffeine detection of 1 variable concentrations with antigen line or caffeine antibody line as detection line, wrap by 1 anti-two anti-line with kind animal protein as with reference to line simultaneously.When colloid gold label portion markings object is caffeine detection antigen, detection line then wraps by caffeine antibody.This is detection reaction part, and this part Main Function is by reaction result with macroscopic characterization out.
Prepared by water absorbent portion: 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 assemble: on backing, paste sample liquid absorption portion, colloid gold label part, detection reaction part and water absorbent portion successively, caffeine Test paper.
Cleaning Principle: Cleaning Principle because of the object of colloid gold label different, and slightly difference.
Because colloid gold label portion markings object is caffeine antibody, if so containing caffeine in sample, sample solution is absorbed by the sample liquid absorption portion of test paper and reaches colloid gold label part by capillary action moves on to, the caffeine antibody response of the caffeine in sample solution and colloid gold label forms bond, bond moves on to detection line on continuing, because the caffeine antibody of colloid gold label only has specific binding site, after caffeine in sample solution combines with it, detection antigen on detection line just can not be combined with the caffeine antibody of colloid gold label again, so detection line is colourless, this is the positive, when not having caffeine in sample, detected antigen capture when the caffeine antibody of colloid gold label arrives detection line, then form macroscopic redness.No matter in sample whether containing caffeine, the caffeine antibody of colloid gold label and foreign protein wherein to move on to when reaching reference line all referenced line can wrap quilt anti-and anti-ly catch the macroscopic redness of formation with two of kind animal protein, this is reference line.
When prepared by test paper by regulating detection line and reference line encrusting substance concentration, thus 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 caffeine test card test strip structural representation.
Embodiment
End liner 1, sample pad 2 and thieving paper 5 are the general parts in this area.Above-mentioned nitrocellulose filter 4, the glass fibre membrane 3 covering golden labeling antibody, end liner 1, sample pad 2 and thieving paper 5 are pasted successively, obtains test paper plate, finally this test paper plate is cut into the test strips of different in width.
Above-mentioned colloidal gold strip is detecting the application in caffeine
This test strips can be used for detecting the fluid sample containing caffeine in beverage, food, during operation, sample liquid to be detected (after the extract dilution of medicine, be directly used in detection) directly drip in the sample pad 2 of this test strips, due to rainbow action principle, sample liquid to be detected and the caffeine monoclonal antibody colloid gold label thing contained by colloidal gold film 3 is driven to spread to nitrocellulose filter 4 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 caffeine protein conjugate, and line of reference reacts containing the IgG of the second kind animal protein, formation aubergine band.If corresponding caffeine 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 caffeine protein conjugate react, thus not develop the color.
The result occurred is three kinds of results:
1., when respectively there is an aubergine band on the detection line 6 and control line 7 position of the antibody of the caffeine of test strips, result is positive findings;
2., when an aubergine band appears in control line 7 position of only test strips, be negative findings;
3. be null result when there is not aubergine band on control line 8 position of test strips;
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (5)
1. detect caffeine test card, it is characterized in that being made up of, in one end of PVC offset plate by the gold conjugation pad of caffeine monoclonal antibody colloid gold label thing, the nitrocellulose filter wrapping the IgG1 bar of quilt caffeine protein conjugate and anti-second kind animal protein, sample pad, absorbent wool, PVC offset plate and mould of plastics bag; Adhere to sample pad, pad successively, middle subsides nitrocellulose filter, the other end adheres to absorbent wool.
2. the test strips of caffeine in beverage according to claim 1, is characterized in that: caffeine detection antigen is the conjugates that caffeine and carrier mass are formed.
3. the test strips of caffeine in beverage according to claim 2, it is characterized in that: described carrier mass, is protein, protein fragments, improvement on synthesis, semi-synthetic polypeptide or polysaccharide.
4. the test strips of caffeine in beverage according to claim 1, is characterized in that: the IgG of described anti-second kind animal protein is the protein of non-antibody sources animal.
5. the pre-service of antibody according to claim 1: caffeine monoclonal antibody will be marked at 1000r/min, under 4 DEG C of conditions, centrifugal 20min, gets supernatant, is diluted to 1mg/mL with 0.01mol/LPBS; Or the caffeine monoclonal antibody that will mark with 0.01mol/LPBS is diluted to 1mg/mL, cross 0.22nm filter membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410354444.5A CN105301238A (en) | 2014-07-24 | 2014-07-24 | Preparation of test paper for detecting excessive caffeine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410354444.5A CN105301238A (en) | 2014-07-24 | 2014-07-24 | Preparation of test paper for detecting excessive caffeine |
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| CN105301238A true CN105301238A (en) | 2016-02-03 |
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| CN201410354444.5A Pending CN105301238A (en) | 2014-07-24 | 2014-07-24 | Preparation of test paper for detecting excessive caffeine |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030077222A1 (en) * | 2001-05-07 | 2003-04-24 | Mcgill University | Individualization of therapy with analgesics |
| CN102590521A (en) * | 2012-02-27 | 2012-07-18 | 上海凯创生物技术有限公司 | Colloidal gold detection kit for rapidly detecting caffeine and preparation process of colloidal gold detection kit |
| CN102796102A (en) * | 2012-06-13 | 2012-11-28 | 广州万孚生物技术股份有限公司 | Caffeine hapten, conjugate, applications of caffeine hapten and conjugate, and method for detecting or determining caffeine |
-
2014
- 2014-07-24 CN CN201410354444.5A patent/CN105301238A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030077222A1 (en) * | 2001-05-07 | 2003-04-24 | Mcgill University | Individualization of therapy with analgesics |
| CN102590521A (en) * | 2012-02-27 | 2012-07-18 | 上海凯创生物技术有限公司 | Colloidal gold detection kit for rapidly detecting caffeine and preparation process of colloidal gold detection kit |
| CN102796102A (en) * | 2012-06-13 | 2012-11-28 | 广州万孚生物技术股份有限公司 | Caffeine hapten, conjugate, applications of caffeine hapten and conjugate, and method for detecting or determining caffeine |
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Application publication date: 20160203 |
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