CN101692091A - Gold labeled immunochromatographic strip assay semiquantitative detection test paper for detecting dienestrol - Google Patents
Gold labeled immunochromatographic strip assay semiquantitative detection test paper for detecting dienestrol Download PDFInfo
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- CN101692091A CN101692091A CN200910177917A CN200910177917A CN101692091A CN 101692091 A CN101692091 A CN 101692091A CN 200910177917 A CN200910177917 A CN 200910177917A CN 200910177917 A CN200910177917 A CN 200910177917A CN 101692091 A CN101692091 A CN 101692091A
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Abstract
The invention provides gold labeled immunochromatographic strip assay semiquantitative detection test paper for detecting dienestrol. The test paper is characterized in that a sample absorption pad, a colloid gold label pad, a chromatography pad and a water absorption pad are pasted on the back lining of the test paper; and a detection reaction pad is covered by dienestrol antigen serving as a detection line and second antibody serving as a quality control line at the same time. The semiquantitative detection test paper has the advantages that the test paper has strong specificity and can realize semiquantitative detection; the test paper can be used at a temperature of between 4 and 40 DEG C and can obtain a result after 5 minutes; and the test paper is suitable for quickly detecting dienestrol drugs in animal-derived foodstuff samples by sanitation departments, quality inspection departments, customs and livestock farms and foodstuff enterprises.
Description
Technical field:
Immune detection of the present invention and detection of veterinary drugs in food technical field.The present invention relates to the residual fast detecting instrument of a kind of estrogen dienestrol, particularly, the present invention relates to a kind of dienestrol gold-marking immunity chromatography half-quantitative detection test paper and preparation method thereof and using method.
Background technology:
(dienestrol DE) is a kind of nonsteroidal estrogen of synthetic to dienestrol, its chemistry by name 3, two (p-hydroxybenzene)-2 of 4-, the 4-hexadiene contains phenolic hydroxyl group, be a kind of faintly acid compound, be mainly used in the growth that improves milk production of cow and livestock and poultry animal and fatten.With respect to natural estrogen, synthetic estrogen is more stable usually, in vivo the hold-up time long, easily residual in the fat of humans and animals and hetero-organization thereof, therefore, can pass through food chain harm humans health.Be embodied in women child and grow in advance, the male children mammogenesis becomes womanlike; Male genetic dysplasia and pathology and women with breast cancer and endometriosis incidence rise, and have carcinogenesis simultaneously.At present, many in the world countries forbid its application.
Existing dienestrol detection method is a chromatography, but these methods need perfect laboratory, experienced technician and expensive instrument and equipment, and testing process is loaded down with trivial details, the examination of incompatible on-the-spot great amount of samples.Provide a new approach based on the immunoreactive immunology detection technology of antigen-antibody for the analyzing and testing of micromolecule residue.It is convenient that the enzyme linked immunosorbent assay (ELISA) technology of setting up based on this principle has, fast, and characteristics such as sensitivity, but this technical requirement testing staff has higher technical level, also need simple equipment such as microplate reader, general 1-4 hour analysis time, its limitation is arranged still.Therefore, need us to develop more simple and fast testing tool easily.
Summary of the invention:
The object of the present invention is to provide a kind of easy, quick, sensitive, cheap dienestrol immunochromatography half-quantitative detection test paper and using method thereof.
Dienestrol gold-marking immunity chromatography half-quantitative detection test paper provided by the invention is that (polyvinyl chloride PVC) posts absorption of sample pad, colloid gold label pad, detection reaction pad and adsorptive pads successively on the backing scribbling the Polyvinylchloride that adhesive sticker do not absorb water.
Described colloid gold label pad is glass fibre or acetate fiber or nylon membrane, and material of mark is the dienestrol polyclonal antibody on it.
Be coated with the dienestrol artificial antigen as detection line above the described detection reaction pad, also be coated with second antibody simultaneously as nature controlling line.The dienestrol artificial antigen is the conjugate of dienestrol and carrier protein among the present invention, and carrier protein is generally used bovine serum albumin(BSA) (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin (KLH).
Second antibody is the antibody with the not homozoic anti-dienestrol antibody of dienestrol antibody.
The each several part preparation method of dienestrol immunochromatography half-quantitative detection test paper of the present invention with and function as follows:
1, backing:,, play the fixing function of supporting other ingredients of test paper as the PVC plate for simultaneously scribbling the toughness material of adhesive sticker.
2, the preparation of absorption of sample pad: filter paper or all-glass paper were immersed in the PBS solution of pH7.4 2 minutes, and took out 80 ℃ and dry or other mode dryings,, be convenient to sample solution and move up as the absorption of sample pad.
3, the preparation of colloid gold label thing pad: this part plays carrying colloid gold label antibody.Comprise the preparation of aurosol, the golden mark of dienestrol antibody, the processing of colloid gold label thing.
(1) preparation of collaurum colloidal sol: with HAuCl
4Be mixed with 0.01% aqueous solution earlier, get 100ml and be heated to and boil.Stir the 1% trisodium citrate (Na that accurately adds 1.2ml down
3C
6H
5O
72H
2O) aqueous solution.Continue heated and boiled 15min.Can be observed flaxen aqueous solution of chloraurate and gray very soon after sodium citrate adds this moment, continuous and change into black, stablizes gradually subsequently to become red.About 2～the 3min of overall process.Return to original volume with distilled water after being cooled to room temperature.The uniformity coefficient of electron microscope observation aurosol particle, prepared after measured collaurum sol particle diameter is 40nm.
(2) colloid gold label dienestrol antibody: get two test tubes, the 40nm collaurum colloidal sol of preparation adds an amount of 25mM K in wherein respectively adding 1.2ml step (1)
2CO
3PH is adjusted into 90, and adding 10 μ l concentration respectively is 1mg/ml dienestrol antibody, mixing, room temperature is placed 10min, adds 12 μ l 2%PEG20000 respectively, and room temperature is placed 5min, the centrifugal 20min of 10000rpm, absorb supernatant gently,, and focus in the new pipe with the loose collaurum precipitation of 20 μ l PBS solution weight suspension, add 20 μ l glycerine respectively, fully mixing is colloid gold label dienestrol antibody, and-20 ℃ of preservations are standby.
(3) preparation of colloid gold label pad: with Biodot point film machine with colloid gold label dienestrol antibody specking on the collaurum pad, dienestrol-BSA conjugate and goat anti-rabbit igg are coated on the detection zone and the Quality Control district of nitrocellulose filter, fully dry.
4. the preparation of adsorptive pads: after all-glass paper or filter paper or thieving paper drying at room temperature, be adsorptive pads.The main effect of adsorptive pads is to absorb the unnecessary sample solution that moves up.
The assembling of dienestrol immunochromatography half-quantitative detection test paper of the present invention: on backing, post absorption of sample pad, colloid gold label pad, detection reaction pad and adsorptive pads successively, promptly get dienestrol immunochromatography half-quantitative detection test paper of the present invention.During detection, can as required described test paper be cut into the reagent strip of suitable width.
On the other hand, the invention provides a kind of residual method of dienestrol that detects, this method is preferably used detection test paper of the present invention.Described detection method comprises: randomly at room temperature place frozen sample to be checked and reagent strip a period of time earlier before detecting (1), is generally more than 1 hour, makes it restore to room temperature; (2) test strips is immersed sample solution 4-6 second, liquid level must not surpass the MARK line on the test strips, lies on the testing table observations after 5 minutes.If agent plate drips 3 sample solutions in well, get final product observations after 5 minutes.The result judges: if there is dienestrol to exist in the sample, red nature controlling line band then only occurs, this moment, the result was positive.If red stripes all appears in detection line and nature controlling line, then the result is negative.Reagent did not lose efficacy if red stripes appears in nature controlling line.
Embodiment:
Detection line is coated with two rare female phenol monoclonal antibody or two rare female phenol detection the antigen 1 bar, wide 1mm that concentration is 0.2-20 μ g/ml; Article two, to be coated with concentration be 2-6mm to line.When the detection line comparison was shallow according to line color, then sample was positive.Control line develops the color all the time, if control line does not develop the color, shows the test paper failure.
Beneficial effect of the present invention:
1. high specificity.This test paper detecting method specificity is extremely strong, does not have cross reaction substantially with medicines such as stilbestrol, estradiol, has avoided the interference of other medicines to detecting.
2. highly sensitive.This test paper lowest detection is limited to 10 μ g/kg.
3. can realize half-quantitative detection.This detection method not only can qualitative detection, the more important thing is according to detection line color and control line color contrast to reach the detection by quantitative purpose.
4. simple to operation.Can directly detect milk, animals urine, can detect after the tissue sample simple process such as meat.This test paper does not need any professional training, does not need any instrument and equipment, and the ordinary person can operate, as long as test paper is inserted in the detection liquid interpretation with the naked eye.Therefore, this method is suitable for face width, and health detects superintendent office, animal-breeding unit, slaughterhouse and consumer and can use per capita.
5. speed is fast, and flux is big, colour stable.Test paper inserts in the sample liquid, but 5min is just observations later on, people can be simultaneously, continuous detecting, detect flux and be higher than the HPLC method far away, then faster more than 50 times than two rare female phenol ELTSA kit detection speeds, and its color can forever be preserved, and can not resemble variable color soon the ELISA substrate.
6. the temperature optimum range is wide.All can use at 4-35 ℃, the result is normal, does not need to carry out at low temperatures, need not take the insulation measure, and indoor field all can be used.
7. test paper long shelf-life.According to the accelerated aging test result, the test paper shelf-life can reach 2 years under the drying condition.
8. with low cost.This method detects cost well below two rare female phenol ELISA kit detection methods, and after large-scale production, its cost also can reduce significantly.
It is in order further to understand the present invention better that following embodiment is provided, and never content of the present invention and protection domain is constituted any restriction.
Embodiment
The embodiment 1 immunogenic synthetic preparation that reaches immune serum
1.1 the preparation of two rare female phenol artificial antigens:
Two rare female naphtholic coupler antigens are divided into immunity and use antigen with antigen and detection, and the former is used for immune animal, and the latter is used for colloid gold label or the detection line bag quilt in screening of Antibody Preparation detection of antibodies and the test paper preparation.
The immunity antigen preparation:
Take by weighing 26.6 milligrams of (0.1mmol) dienestrols and be dissolved among the 1mlDMSO, fully stirring is dissolved it fully, adds 11.7 milligrams of sodium chloroacetates, stirring at room 1 hour;
Take by weighing 50 milligrams of KLH, PBS dissolving with 0.1mol/L, slowly dripping DMSO fully stirs, make it account for 50% of total solution, the dienestrol of sodium chloroacetate activation is joined in the KLH solution, transfer pH value of solution value to 11, add EDC 0.2-0.3M (about 100mg) with 10M NaOH, stirring reaction spends the night, and promptly gets dienestrol-KLH conjugate.
Then, conjugate is crossed the SephadexG-25M gel chromatography,, regulate flow velocity to 3ml/min with three times of bed volume balances of 0.01M pH 7.4PBS, sample concentration joins chromatography purification conjugate in the good chromatographic column of balance to 5ml, eluent pH 7.4 0.01M PBS.
After the immunogene packing after the purification-20 ℃ frozen.
1.2 the bag preparation of antigen:
The dienestrol that takes by weighing 26.6 milligrams (0.1mmol) is dissolved in the 1ml anhydrous pyridine, adds 10 milligrams of succinic anhydrides, stirs 4 hours N under the room temperature
2Dry up, redissolve with certain volume DMSO.
Take by weighing 40 milligrams of ovalbumins (OVA), PBS dissolving with 0.1mol/L, slowly dripping DMSO fully stirs, make it account for 50% of total solution, the dienestrol of succinic anhydride activation is joined in the OVA solution, transfer pH value of solution value to 11, add EDC 0.2-0.3M (about 100-150mg) with 10M NaOH, stirring reaction spends the night, and promptly gets dienestrol-ovalbumin conjugate (DE-OVA).
Then, above-mentioned DE-OVA is crossed the SephadexG-25M gel chromatography,, regulate flow velocity to 3ml/min with three times of bed volume balances of 0.01M pH 7.4PBS, sample concentration joins chromatography purification conjugate in the good chromatographic column of balance to 5ml, eluent pH 7.4 0.01M PBS.
-20 ℃ of preservations after the envelope antigen packing after the purification.
1.3 two rare female phenol Monoclonal Antibody:
Get 5 of health female secondary Balb/c mouse in 4 age in week and carry out immunity.Fundamental immunity uses antigen and equivalent complete Freund's adjuvant with the abundant mixing emulsification of stirrer the two rare female phenol immunity of 0.1mL for the first time, carries out lumbar injection, and injection volume 0.2mL/ only.Begin after 2 weeks to carry out booster immunization, adjuvant is changed to incomplete Freund's adjuvant, every two all booster immunizations once, gets spleen behind the last immunity 3d and merges.
With HAT nutrient solution, the incomplete nutrient solution of TMDM, IMDM complete culture solution and 50%PEG-2000 solution pre-temperature in 37 ℃ of water-baths, the beaker that another is filled with water is put into the pre-temperature of 37 ℃ of water-baths simultaneously earlier.The above-mentioned splenocyte for preparing and bone marrow cell and splenocyte are mixed in 1: 4 ratio, and the full nutrient solution that toos many or too much for use in the plastic centrifuge tube of 50mL is washed 1 time, and the centrifugal 8min of 1000r/min abandons supernatant, with dripping an exhaustion residual liquid.Put in 37 ℃ of water-baths, at the bottom of the suction pipe tubular stinger, in 60s, add 1Ml, the 50%PEG-2000 of preheating while stirring.After leaving standstill 60s, in 37 ℃ of water-baths, add the complete culture solution of 10mL preheating in the 60s.Light gently the suspension once is with the centrifugal 6min supernatant discarded of 1000r/min, earlier with 6mL left and right sides complete culture solution.Mixing gently, in 96 orifice plates that feeder cells are arranged, every hole 0.1mL once inoculates 8 blocks of plates with the mixing suspension inoculation.Culture plate is placed 37 ℃, 5%C0
2Cultivate in the incubator.Merge back 7d, change liquid 1 time, behind 13d, use the complete culture solution of 20%NBS instead according to the increment situation with HT nutrient solution half amount.Occur the hybridoma colony behind about 7d, cell is big, round and bright.Treat that colony grows at the bottom of the hole at 1/3 o'clock, clonal growth mark on the cover plate of culture plate, be that desirable supernatant detects corresponding specific antibody this moment.After the complete cloning of cell, cell is injected mouse peritoneal, when ascites such as certain interval of time are abundant, get ascites, behind ascites usefulness albumin A immunoaffinity chromatography purifying, promptly obtain two rare female phenol monoclonal antibodies.
1.4 monoclonal antibody is tired and specific assay
1.4.1 indirect ELISA detects antibody titer
DE-BSA with 10 μ g/ml presses every hole 100 μ l coated elisa plates, and 4 ℃ of bags are washed 5 times by 24h, pat dry, and every hole adds the bovine serum albumin(BSA) phosphate buffer (confining liquid) of 200 μ l 2%, and 4 ℃ are sealed 12h down, wash 3 times, pat dry.The monoclonal antibody 100 μ l that add different extension rates, hatch 2h for 37 ℃, wash three times, add 100 μ l enzymes mark sheep anti-mouse antibody immediately, hatch 30min for 37 ℃, wash three times, add 50 μ l developer A liquid and 50 μ l developer B liquid, room temperature lucifuge effect 15min adds 50 μ l stop buffer cessation reactions, and microplate reader detects OD value (450nm).Negative serum control wells and parallel repeating hole are set simultaneously.Antibody is with 1000 times, 2000 times, 4000 times, 8000 times, 16000 times, 32000 times dilutions.With the antibody dilution that doubles negative serum OD value is antibody titer.Testing result sees Table-1
Table-1 antibody titer testing result (average OD value)
From table-1 as can be known, antibody titer is more than 32000, to satisfy requirement of experiment.
1.4.2 indirect competitive ELISA detects antibody specificity
The ELISA method of operating is with 1.Free dienestrol and encrusting substance competition that every hole adds 100 μ l variable concentrations add antibody subsequently, draw different OD values.According to the result of 1.4.1, used polyclonal antibody optium concentration is 1: 2000.Putting in order of dienestrol series concentration and test hole sees Table-2, and parallel repeating hole and blank hole are set simultaneously.With the 0 OD value that suppresses the hole is maximal value B
0, other inhibition concentration hole OD value is B, B/B
0The dienestrol concentration of=50% o'clock correspondence is the IC of antibody for this reason
50Value.Testing result sees Table-2
Table-2 antibody specificity testing results (OD mean value)
By table-2 data as can be known, dienestrol polyclonal antibody IC
50Value about 9 μ g/L, the IC of other several analogs
50Value is not seen obvious inhibition in the concentration range of being tested, in this scope, almost do not have cross reactivity, shows that the antibody specificity that the present invention prepares meets the requirements.
The preparation of embodiment 2 dienestrol immunochromatography half-quantitative detection test paper
2.1 sample liquid absorption pad is handled:
Filter paper or glass fibre are immersed 2min among the PBS of 0.2mol/L pH7.4, take out, 80 ℃ of oven dry or other mode dryings, sample liquid absorption pad.
2.2 the preparation of colloid gold label pad and processing:
The preparation of colloid gold label pad and processing comprise the preparation of collaurum colloidal sol, the two rare female phenol antibody of collaurum mark, the processing of colloid gold label pad.
Collaurum colloidal sol preparation: with HAuCl
4Be mixed with 0.01% aqueous solution earlier, get 100ml and be heated to and boil.Stir the 1% trisodium citrate (Na that accurately adds 1.2ml down
3C
6H
5O
72H
2O) aqueous solution.Continue heated and boiled 15min.Can be observed flaxen aqueous solution of chloraurate and gray very soon after sodium citrate adds this moment, continuous and change into black, stablizes gradually subsequently to become red.About 2～the 3min of overall process.Return to original volume with distilled water after being cooled to room temperature.The uniformity coefficient of electron microscope observation aurosol particle, prepared after measured collaurum sol particle diameter is 40nm.
The two rare female phenol Monoclonal Antibody of colloid gold label: get two test tubes, the 40nm collaurum colloidal sol of preparation adds an amount of 25mM K in wherein respectively adding 1.2ml step (1)
2CO
3PH is adjusted into 9.0, and adding 10 μ l concentration respectively is 1mg/ml dienestrol antibody, mixing, room temperature is placed 10min, adds 12 μ l 2%PEG20000 respectively, and room temperature is placed 5min, the centrifugal 20min of 10000rpm, absorb supernatant gently,, and focus in the new pipe with the loose collaurum precipitation of 20 μ l PBS solution weight suspension, add 20 μ l glycerine respectively, fully mixing is colloid gold label dienestrol antibody, and-20 ℃ of preservations are standby.
The two rare female phenol monoclonal antibodies of gold mark are poured in the groove, and glass fibre or filter paper are immersed 1min, take out, and drying at room temperature or vacuum freeze drying promptly become the colloid gold label pad.
2.3 the assembling of dienestrol immunochromatography half-quantitative detection test paper
On backing, post absorption of sample pad, colloid gold label pad, detection reaction pad and adsorptive pads successively, promptly get dienestrol immunochromatography half-quantitative detection test paper of the present invention.During detection, can as required described test paper be cut into the reagent strip of suitable width.
Embodiment 3 dienestrol gold-marking immunity chromatography half-quantitative detection test paper sensitivity experiments
Get the standard solution that two rare female phenol concentration are respectively 5ng/ml, 10ng/ml, 15ng/ml, each concentration is prepared 10 parts of test strips that prepare, and on request test strips is inserted in the above-mentioned solution observations after 5 minutes.
The result shows: the detection line on the test strips in the standard solution of insertion 10ng/ml and 15ng/ml is invisible, and the detection line color and the control line difference of the appearance on the test strips of insertion 5ng/ml standard solution are not obvious.
From then on as seen, dienestrol gold-marking immunity chromatography half-quantitative detection test paper of the present invention sensitivity is very high, detects to be limited to 10ng/ml.
The test of experimental example 4 test strips storage lives
The test strips preservation condition is 2-8 ℃, and through 12 months mensuration, the quality of test strips there was not variation, control line, detection line colour developing normally, and detection sensitivity does not change.Consideration has improper preservation condition and occurs in transportation and use, and test strips was placed for two weeks under 37 ℃ of preservation conditions, carries out breaking test, and the result shows that the every index of this test strips is normal fully.Can draw test strips from above result can preserve more than 12 months at 2-8 ℃.
Dienestrol adds the detection test in embodiment 5 samples
5.1, sample pre-treatments
Accurately take by weighing 1g flesh of fish sample in the 5ml centrifuge tube, grind to form the congee shape, add two rare female phenol standard items 100ng, mixing, added 10ml methyl alcohol sonic oscillation subsequently 1 hour, the centrifuging and taking supernatant, nitrogen dries up, use the 1mlDMSO sample dissolution, with 10 times of sample diluting liquid dilutions, the preparation sample solution.
5.2, detect with test strips
Test strips is inserted in the above-mentioned solution for preparing, and the positive of a band appears in observations after 5 minutes, the negative of two bands occur.The result shows, adds sample and has only band colour developing, and sample is judged to be feminine gender.
Claims (8)
1. a kind of gold-marking immunity chromatography half-quantitative detection test paper that detects dienestrol that detects dienestrol, comprising: dienestrol monoclonal antibody specific, dienestrol-carrier protein couplet thing.
2. the gold-marking immunity chromatography half-quantitative detection test paper of dienestrol according to claim 1, it is characterized in that: described detection test paper also comprises absorption of sample pad, colloid gold label thing pad, chromatography pad and the adsorptive pads on backing, the backing.
3. detection test paper according to claim 1 is characterized in that: described dienestrol specific antibody is a monoclonal antibody.
4. detection test paper according to claim 1 is characterized in that: described carrier protein is the blue albumen of bovine serum albumin(BSA), ovalbumin or key hole copper.
5. detection test paper according to claim 2 is characterized in that: be coated with dienestrol-carrier protein couplet thing and second antibody on the described chromatography pad.
6. detection test paper according to claim 5 is characterized in that: described dienestrol-carrier protein couplet thing bag is a detection line by line.
7. detection test paper according to claim 5 is characterized in that: described second antibody bag is a nature controlling line by line.
8. the method for dienestrol content in the test sample comprises step:
(1) sample pre-treatments, methods such as employing pulverizing, homogenate are utilized the two rare female phenol in the organic solvent extraction sample.
(2) detect with the arbitrary described detection test paper of claim 1-7, will detect test paper and insert in the sample solution observations after five minutes.
(3) analyzing and testing result, it is positive a red zone sample only to occur, and it is negative two bands to occur.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103308699A (en) * | 2013-06-17 | 2013-09-18 | 杭州南开日新生物技术有限公司 | Colloidal gold reagent plate for quickly detecting methyltestosterone in aquatic product |
| CN105246404A (en) * | 2013-03-14 | 2016-01-13 | 聚合物技术系统公司 | Direct temperature measurement of a test strip |
| CN106404783A (en) * | 2016-09-29 | 2017-02-15 | 网易（杭州）网络有限公司 | Test paper detection method and device |
-
2009
- 2009-10-19 CN CN200910177917A patent/CN101692091A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105246404A (en) * | 2013-03-14 | 2016-01-13 | 聚合物技术系统公司 | Direct temperature measurement of a test strip |
| CN103308699A (en) * | 2013-06-17 | 2013-09-18 | 杭州南开日新生物技术有限公司 | Colloidal gold reagent plate for quickly detecting methyltestosterone in aquatic product |
| CN106404783A (en) * | 2016-09-29 | 2017-02-15 | 网易（杭州）网络有限公司 | Test paper detection method and device |
| CN106404783B (en) * | 2016-09-29 | 2019-07-30 | 网易（杭州）网络有限公司 | Test paper detecting method and device |
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Application publication date: 20100407 |