CN102288751A - Colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda - Google Patents
Colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda Download PDFInfo
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- CN102288751A CN102288751A CN2011101132127A CN201110113212A CN102288751A CN 102288751 A CN102288751 A CN 102288751A CN 2011101132127 A CN2011101132127 A CN 2011101132127A CN 201110113212 A CN201110113212 A CN 201110113212A CN 102288751 A CN102288751 A CN 102288751A
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Abstract
The invention relates to a colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda, which is prepared by adopting a nano colloidal gold particle labeled antibody technique and a mode of antigen detection through double-antibody sandwich immunoassay, and provides the colloidal gold immunochromatography testing strip for rapid detection of slow Edwardsiella tarda, a preparation method of the same and application of the same. The testing strip comprises a sample pad, a gold labeling pad, a nitrocellulose membrane with a detecting line antibody and a quality control line antibody, an absorption pad, a bottom plate and a bottom plate, and the sample pad, the gold labeling pad, the nitrocellulose membrane and the absorption pad are sequentially overlapped, connected and bonded on the bottom plate. The testing strip can be simply operated in detection and application and has good specificity, high sensitivity and can be used conveniently and rapidly, no special-speed instrument or equipment is needed, no special training is required, results are clean and are easy to distinguish, and the testing strip can be easily promoted and is suitable for detection of infection and contamination of the slow Edwardsiella tarda.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of fast detecting Edwardsiella tarda colloidal gold immuno-chromatography test paper strip and preparation thereof and the application on Edwardsiella tarda detects.
Background technology
The Chi Huanaidehuajunshi disease is to culture a fulminating bacterioid disease in the fishery, is popular in economic cultured fishes such as fork-tail silver xenocypris section, Anguillidae and lefteye flounder, carp, mainly causes liver, the ephrosis of fish.This disease is mainly in the contour water temperature phase of change, autumn of spring and summer, and peak summer, and its popular area is wide, and mortality ratio is higher, endangers very seriously, has caused the great attention of culture fishery.The detection of Chi Huanaidehuajunshi disease is identified mainly be confined to bacteriology checking or pathological observation at present, bacterium isolation identification diagnosis of complex routinely is time-consuming, easily incured loss through delay control opportunity.In addition, also there is the ELISA detection method of utilizing the anti-tarda polyclonal antibody of rabbit, anti-tarda monoclonal antibody to set up to be applied to the immunology detection of tarda.Though ELISA detects and also can diagnose out the tarda disease in several hours, has higher specificity and sensitivity, exist operative technique to require relatively shortcoming such as costliness of high, equipment, be not easy to basic unit's production application.Based on popular situation and the control requirement of Wdwardsiella tarda in aquatic livestock, convenient quick, the sensitive specific detection technology of development research has become a kind of exigence.The present invention is based on immunology detection principle and monoclonal antibody, polyclonal antibody technology of preparing and colloidal gold-labeled method, developed a kind of colloidal gold immunochromatographydetection detection test paper bar of Edwardsiella tarda.
Colloidal gold immunochromatographydetection detection test paper bar technology is a novel vitro diagnostic techniques that has grown up since the nineties in 20th century, closely during the last ten years, colloid gold label has developed into a later important immunolabelling technique of the isotope labeling that continues, enzyme labeling and fluorescence labeling.Because it is quick, convenient, do not need specific installation, the result judges advantages such as directly perceived, this method is rapidly developed in recent years, has particularly obtained widespread use in the medical test at biomedical sector, but it is less to be used for the product of Animal diseases prevention and control detection range.This research has been developed the immune colloidal gold detection test paper strip of aquatic products disease detection at Edwardsiella tarda, develops corresponding quick diagnosis reagent kit with the effective control control of strong promotion culture fishery to bacterial disease.The collaurum detection method sensitivity that research is set up, accurate, high specificity, after the method is set up, simultaneously can with classical conventional method complementation, expectation will have wider application prospect at aquatic livestock diagnosis and treatment mechanism, aquaculture of aquatic animal enterprise, inspection and quarantine department etc., and certain economic and social benefit are arranged.
Summary of the invention
The object of the present invention is to provide a kind of can be used for the detecting sensitivity of Edwardsiella tarda, colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof and the application in the Chi Huanaidehuajunshi detection accurately and rapidly.
The invention provides Edwardsiella tarda colloidal gold immunochromatographydetection detection test paper bar, it is characterized in that: this test strips comprises sample pad, gold mark pad, nitrocellulose filter, absorption pad, base plate, on base plate, be initiating terminal with the sample pad, overlapping successively stickup fixed sample pad, gold mark pad, nitrocellulose filter, absorption pad.
The preparation that the invention provides Edwardsiella tarda colloidal gold immunochromatographydetection detection test paper bar may further comprise the steps:
(1) anti-Edwardsiella tarda monoclonal antibody of preparation or polyclonal antibody;
(2) preparation of nano-colloid gold grain;
(3) the colloid gold label thing of anti-Edwardsiella tarda monoclonal antibody of preparation or polyclonal antibody forms golden labeling antibody;
(4) golden labeling antibody is evenly sprayed on the plain film of glass fibre, drying forms gold mark pad;
(5) will resist Edwardsiella tarda monoclonal antibody or anti-Edwardsiella tarda polyclonal antibody to spray to detection line position on the nitrocellulose filter as the detection line capture antibody, two anti-or staphylococcal protein As of anti-golden labeling antibody spray to nature controlling line position on the nitrocellulose filter as the nature controlling line capture antibody, seal blank site, fully dry;
(6) absorption pad, the nitrocellulose filter that has detection line and nature controlling line, gold mark pad, sample pad, base plate are assembled into the colloidal gold immunochromatographydetection detection test paper bar.
Description of drawings
Fig. 1 is the assembling assumption diagram of Edwardsiella tarda colloidal gold immunochromatographydetection detection test paper bar of the present invention, wherein: the 1st, sample pad; The 2nd, gold mark pad; The 3rd, detection line colour developing position; The 4th, nature controlling line colour developing position; The 5th, nitrocellulose filter; The 6th, absorption pad; The 7th, base plate.
Fig. 2 is the appearance assumption diagram of Edwardsiella tarda colloidal gold immunochromatographydetection detection test paper bar of the present invention, wherein: the 8th, plastic casing; The 9th, the result reads window; The 10th, the test sample sample application zone.
Embodiment
The specific embodiment of the present invention is elaborated with reference to the following examples, but is not limited thereto.
The preparation of this test strip may further comprise the steps: the spraying of preparation, the detection line on the nitrocellulose filter and the nature controlling line antibody of the selection of the preparation of the foundation of anti-Edwardsiella tarda cell strain of monoclonal antibody, the anti-Edwardsiella tarda polyclonal antibody of rabbit serum, pairing antibody, the preparation of nano-colloid gold grain, the optimization of nano colloid gold mark pH value, the optimization of nano colloid gold labelled protein concentration, gold mark pad and sealing, the assembling of test strips, detection method.
The foundation of embodiment 1, anti-Edwardsiella tarda cell strain of monoclonal antibody
Preparation Edwardsiella tarda inactivation antigen, immune Balb/c mouse prepares splenocyte suspension after the immunity; Prepare myeloma cell's suspension simultaneously; Splenocyte and myeloma cell are merged screening positive monoclonal cell hole through cell hydridization; Through further positive colonyization and screening thereof, set up anti-Edwardsiella tarda monoclonal antibody hybridoma cell strain 2 strains, respectively called after ETMcAb3A7, ETMcAb4F11.This 2 strain cell line on June 6th, 2010 China's typical culture collection center preservation (China. Wuhan. Wuhan University), deposit number is respectively CCTCC NO:C201061 and CCTCC NO:C201062.
The preparation of embodiment 2, anti-Edwardsiella tarda monoclonal antibody ascites
Cultivate anti-Edwardsiella tarda cell strain of monoclonal antibody ETMcAb3A7, ETMcAb4F11, collect the monoclonal anti body cell respectively, difference lumbar injection Balb/c mouse, clean environment is raised.Observe mouse web portion behind about 7-10d, obvious ascites person is arranged with 12
#Syringe needle reclaims ascites, 3000rmin by the puncture of abdominal cavity downside
-1Centrifugal 5min, precipitation red blood cell and haemocyte are collected the flaxen ascites in upper strata, preserve standby.
The preparation of embodiment 3, the anti-Edwardsiella tarda polyclonal antibody of rabbit serum
Preparation Edwardsiella tarda inactivation antigen, head exempts to add the complete Freund's adjuvant of equivalent, the injection new zealand rabbit, 2-3 carries out two and exempts from week, and antigen adds the equivalent incomplete Freund's adjuvant, and three exempt from after one month.Immunity extracts blood by arteria carotis, through 5000rmin after finishing
-1Serum is collected in centrifugal back, and aseptic subpackaged ,-80 ℃ of preservations are standby.
The selection of embodiment 4, pairing antibody
The selection of pairing antibody is to select anti-Edwardsiella tarda monoclonal antibody, the polyclonal antibody that has prepared for use, after all associated antibodies do pairing experiments, select sensitivity, specificity one group of antibody preferably, be respectively applied for the spraying of colloid gold label and nitrocellulose filter detection line capture antibody.
We have selected anti-Edwardsiella tarda monoclonal antibody ETMcAb3A7, ETMcAb4F11 and the anti-Edwardsiella tarda polyclonal antibody of rabbit of prepared in laboratory, through sandwich method experiment and golden labeling antibody immunochromatography experiment, select a pair of best results antibody at last in twos.Experimental result shows, anti-Edwardsiella tarda monoclonal antibody ETMcAb3A7 and ETMcAb4F11 are best of breed as colloid gold label antibody, detection line antibody respectively, this scheme implementation can reduce the appearance of false negative signal in the sample detection, improve the colloid gold label effect of antibody, and can obtain reliable result.
The preparation of embodiment 5, nano-colloid gold grain
Get one of 250mL triangular flask, prepare 1% chlorauride 100mL, ebuillition of heated; 1% sodium citrate of getting 1mL adds in the above-mentioned solution, mixes rapidly, keeps boiling 15min again, and distilled water returns to the 100mL volume behind the natural cooling; Colloidal gold solution is with 4 ℃ of preservations of reagent bottle of silication, and is standby.
After collaurum is fired, observe after leaving standstill 4h,, then abandon or adopt, fire again if there is floating thing on the colloidal gold solution surface; The colloidal gold solution surface of 4 ℃ of preservations also should be abandoned or adopted if floating thing or flocculent deposit are arranged, and fires again.
Embodiment 6, nano colloid gold mark pH value are optimized
Get several 5mL test tubes, add 1.5mL 20nm collaurum respectively; Use 0.05moLL
-1K
2CO
3And 0.1moLL
-1HCl is adjusted to 6～10 with the pH of colloidal gold solution; Get the 1.5ml centrifuge tube, respectively above-mentioned collaurum is got 1mL from low to high respectively by pH and added in the pipe triplicate; Every hole adds monoclonal antibody 200 μ L respectively, and concentration is 1mgmL
-1, mixing mixes rapidly, places 30min under the room temperature; Every hole adds 5%KCl 50 μ L respectively, leaves standstill 4h behind the mixing; Observing colloid gold change color, the less group of record change color, preferred PH is 8.0-9.8; Adjust in preferred pH scope, repeat above-mentioned steps, optimal pH is 8.2.
Test findings shows: keeping the stable optimal pH scope of colloidal gold solution is 8.2-9.0, and testing optimum pH value is 8.2.
Embodiment 7, nano colloid gold labelled protein concentration are optimized
The present invention also is to provide a kind of acquisition to keep the method for the stable antibody optimum mark dosage of collaurum, comprising:
Get the 1.5mL centrifuge tube of 10 cleanings, add the colloidal gold solution 1mL of optimum pH scope respectively; Add dilution good antibody to be marked 20 μ g, 18 μ g, 16 μ g, 14 μ g, 12 μ g, 10 μ g, 8 μ g, 6 μ g, 4 μ g, 0 μ g respectively successively in 1～No. 10 pipe, mixing leaves standstill 2min; Add 10%KCl solution 50 μ L in 10 pipes respectively, room temperature leaves standstill 2h behind the mixing; Observe change color.
Do not add liquid color in the test tube that antibody and addition be not enough to the stable colloid gold and present variation, add the test tube that the antibody amount meets or exceeds minimum consistent dose and then keep red constant by red stain indigo plant.Compare with control tube (No. 1 pipe), color is the most approaching, contain the contained antibody dosage of the minimum test tube of antibody dosage, be the necessary antibody consistent dose of 1mL collaurum, add 20% antibody on this basis again, be the necessary antibody optimum mark of 1mL collaurum dosage.
The present invention is through validation trial and analysis, and the result who draws shows: suitable protein antibodies concentration is 10 μ gmL to keep colloidal gold solution
-1
The preparation of embodiment 8, gold mark pad
Get the test tube of two 50mL, add 10mL 25nm collaurum respectively; Add an amount of 0.05moLL
-1K
2CO
3Adjustment pH is an optimum concentration; Under 4 ℃ of conditions, add the antibody response 20min of optimum mark concentration, 4 ℃ of 12000rmin
-1Centrifugal 30min, supernatant discarded; It is the same centrifugal to add the resuspended liquid suspension of 10mL post precipitation in every centrifuge tube, abandons supernatant, and the pipe end obtains bolarious loose shape precipitation, and resuspended liquid is 0.2moLL
-1The PB damping fluid (contains 1%BSA, 0.02%PEG, 0.3%Tween-20,0.1moLL
-1NaCl); Repeated centrifugation 1 time, supernatant discarded; 0.05moLL
-1PB (contains 1%BSA, 0.02%PEG, 0.3%Tween-20,12% sucrose, 1%Na N
3, 0.1moLL
-1NaCl) resuspended precipitation, 4 ℃ of refrigerator storage are standby;
Good immune colloid gold solution is adjusted to finite concentration to get above-mentioned mark, evenly is sprayed on the plain film of glass fibre, and dried overnight in 37 ℃ of incubators packs and places 4 ℃ of refrigerators standby.
The spraying of the detection line on embodiment 9, the nitrocellulose filter, nature controlling line antibody and sealing
With nitrocellulose filter with 1% methanol solution soak waft wash after, softly wash rapidly dried, drying; To resist Edwardsiella tarda monoclonal antibody (2mgmL
-1), the anti-mouse I of rabbit gG antibody (1mgmL
-1) add respectively in the storage bottle of detection line, nature controlling line of bio-dot collaurum specking system, adjust the position of drawing film, spray the film line; Place 37 ℃ of incubators to dry 2h the nitrocellulose filter of drawing good detection line, nature controlling line; Take out nitrocellulose filter and place neutral protein to soak to take out immediately after wetting, place 37 ℃ of incubators sealing 4h after, it is standby that sealing is kept at 4 ℃ of refrigerators.
Test findings prepared and had immunocompetent detection line and nature controlling line, and the immune nitrocellulose filter that blank site has been sealed with confining liquid.
The assembling of embodiment 10, test strips
Absorption pad, gold mark pad, sample pad are cut into strip 2.5cm, 0.5cm, 1.5cm respectively; With immune nitrocellulose filter along the long axis direction of adhesive sticker base plate, paste apart from adhesive sticker base plate upper edge 2.2cm, following 1.8cm position; Absorption pad and the overlapping 0.3cm of immune nitrocellulose filter place are sticked on the top of nitrocellulose filter; The gold mark is filled up the below that sticks on immune nitrocellulose filter with the overlapping 0.1cm of immune nitrocellulose filter place; Sample pad is filled up the below that overlapping 0.1cm sticks on gold mark pad with the gold mark; Cut into wide * long rectangular for 0.35cm * 6cm with being fixed with base plate with absorption pad, immune nitrocellulose filter, gold mark pad, sample pad from top to bottom successively, test-strips, the plastic casing of packing into is packed in the aluminium foil bag with drying agent, sealing is stored.
Through assembling, cut out, steps such as drying, sealing, promptly can be assembled into the Edwardsiella tarda colloidal gold fast detecting test paper strip.
Embodiment 11, detection method
The processing of sample to be checked: solid-state sample is as morbidity animal lesion tissue, aquatic products or bacterial cultures equal samples, adds an amount of distilled water and grinds centrifugal or leave standstill and treat that precipitation gets supernatant liquor; Liquid sample such as blood, ascites can directly extract the detection sample; The less sample of bacteria containing amount can increase through nutrient culture media to be got bacterium liquid behind the bacterium and directly detects; The Edwardsiella tarda colloidal gold fast detecting test paper strip is recovered room temperature; Extract an amount of sample 80-150 μ L that detects, slowly add in the well on the test card; Observe the colour developing situation of test strip behind the 20min, the record result.
Testing result: if contain Edwardsiella tarda, then with test paper on golden labeling antibody form corresponding compound, up be coated on immune nitrocellulose membrane on monoclonal antibody combine, form red lines, promptly locate to form red stripes at " T ".No matter whether test sample contains Edwardsiella tarda, and golden labeling antibody continues upwards migration and forms the red precipitate line with the rabbit anti-mouse igg that is coated on the film, promptly locates to form red stripes at " C ".
So, if nature controlling line colour developing, the detection line negative result that do not develop the color, test sample does not contain Edwardsiella tarda.The positive result if nature controlling line colour developing, detection line develop the color, test sample contains Edwardsiella tarda.Testing result is judged to be the test strips deterioration failure if nature controlling line does not develop the color then.
Claims (7)
1. the colloidal gold immunochromatographydetection detection test paper bar of an Edwardsiella tarda is characterized in that: this test strips comprises sample pad, gold mark pad, nitrocellulose filter, absorption pad, base plate.
2. test strips according to claim 1, it is characterized in that: the material of sample pad is selected from glass fibre, filter paper fibre or polyester film, the golden labeling antibody supporting film of gold mark pad is selected from glass fibre, filter paper fibre or polyester film, absorption pad is selected absorbent filter for use, and base plate is selected the PVC plate of band adhesive sticker for use.
3. according to each described test strips of claim 1-2, it is characterized in that: be initiating terminal with the sample pad on the base plate, overlapping successively stickup is fixed with sample pad, gold mark pad, has nitrocellulose filter, the absorption pad of detection line and nature controlling line antibody.
4. according to each described test strips of claim 1-3, it is characterized in that: the antibody of anti-Edwardsiella tarda is monoclonal antibody or polyclonal antibody; Nature controlling line antibody can be selected IgG or the staphylococcal protein A of goat-anti rabbit, mouse-anti rabbit, the anti-sheep of rabbit, sheep anti mouse, the anti-mouse of rabbit etc. according to the source of golden labeling antibody.
5. the preparation method of the colloidal gold immunochromatographydetection detection test paper bar of an Edwardsiella tarda, it is characterized in that: the preparation process of this test strips comprises: (1) anti-Edwardsiella tarda monoclonal antibody of preparation or polyclonal antibody; (2) preparation nano-colloid gold grain; (3) the nano colloid gold label of anti-Edwardsiella tarda monoclonal antibody of preparation or polyclonal antibody forms golden labeling antibody; (4) golden labeling antibody is evenly sprayed on the golden labeling antibody supporting film drying preparation gold mark pad; (5) will resist Edwardsiella tarda monoclonal antibody or anti-Edwardsiella tarda polyclonal antibody to spray to detection line position on the nitrocellulose filter as the detection line capture antibody, two anti-or staphylococcal protein As of anti-golden labeling antibody spray to nature controlling line position on the nitrocellulose filter as the nature controlling line capture antibody, and the blank site on the sealing nitrocellulose filter; (6) absorption pad, the nitrocellulose filter that has detection line and nature controlling line antibody, gold mark pad, sample pad, base plate are assembled into the colloidal gold immunochromatographydetection detection test paper bar.
6. the application of an Edwardsiella tarda colloidal gold immunochromatographydetection detection test paper bar, it is characterized in that: this test strips is applied to detect Edwardsiella tarda.
7. test strips according to claim 6 is characterized in that this detection step is as follows:
(1) gets the sample application zone that 80-150 μ L sample to be checked is added in test strip;
(2) in 20min, observe testing result.
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| CN102759624A (en) * | 2012-07-18 | 2012-10-31 | 吉林大学 | Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer |
| CN103267853A (en) * | 2013-06-04 | 2013-08-28 | 大连海洋大学 | Edwardsiella ictaluri colloidal gold immunochromatographic strip and preparation method thereof |
| CN103675264A (en) * | 2013-12-05 | 2014-03-26 | 美艾利尔（上海）诊断产品有限公司 | Colloidal gold film for detecting antibodies on sensitization nitrocellulose membrane and preparation method and application thereof |
| CN105606812A (en) * | 2015-12-07 | 2016-05-25 | 福建省淡水水产研究所 | Colloidal gold immunochromatographic test strip for quinolone drugs |
| CN106719274A (en) * | 2016-12-23 | 2017-05-31 | 福建省淡水水产研究所 | A kind of pond on a hill drainage arrangement and its application method |
| CN107607718A (en) * | 2017-09-13 | 2018-01-19 | 中国海洋大学 | A kind of lefteye flounder Edwardsiella tarda disease quick diagnosis test paper and preparation method thereof |
| CN110806477A (en) * | 2018-08-06 | 2020-02-18 | 国家纳米科学中心 | Pathogenic bacterium detection test strip, sensor and application thereof |
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Cited By (10)
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| CN102121937A (en) * | 2010-07-14 | 2011-07-13 | 福建省淡水水产研究所 | Colloidal gold immunochromatographic test strip for quickly detecting Aeromonas hydrophila |
| CN102759624A (en) * | 2012-07-18 | 2012-10-31 | 吉林大学 | Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer |
| CN102759624B (en) * | 2012-07-18 | 2015-04-22 | 吉林大学 | Immune colloidal gold test strip for rapidly detecting mucosal disease virus of sika deer |
| CN103267853A (en) * | 2013-06-04 | 2013-08-28 | 大连海洋大学 | Edwardsiella ictaluri colloidal gold immunochromatographic strip and preparation method thereof |
| CN103675264A (en) * | 2013-12-05 | 2014-03-26 | 美艾利尔（上海）诊断产品有限公司 | Colloidal gold film for detecting antibodies on sensitization nitrocellulose membrane and preparation method and application thereof |
| CN103675264B (en) * | 2013-12-05 | 2015-08-19 | 美艾利尔（上海）诊断产品有限公司 | A kind of colloidal gold film for detecting antibody on sensitization nitrocellulose filter and its preparation method and application |
| CN105606812A (en) * | 2015-12-07 | 2016-05-25 | 福建省淡水水产研究所 | Colloidal gold immunochromatographic test strip for quinolone drugs |
| CN106719274A (en) * | 2016-12-23 | 2017-05-31 | 福建省淡水水产研究所 | A kind of pond on a hill drainage arrangement and its application method |
| CN107607718A (en) * | 2017-09-13 | 2018-01-19 | 中国海洋大学 | A kind of lefteye flounder Edwardsiella tarda disease quick diagnosis test paper and preparation method thereof |
| CN110806477A (en) * | 2018-08-06 | 2020-02-18 | 国家纳米科学中心 | Pathogenic bacterium detection test strip, sensor and application thereof |
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Application publication date: 20111221 |