CN1700005A - Avian influenza immunogold diagnosis test strip and test card - Google Patents
Avian influenza immunogold diagnosis test strip and test card Download PDFInfo
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- CN1700005A CN1700005A CN 200510057023 CN200510057023A CN1700005A CN 1700005 A CN1700005 A CN 1700005A CN 200510057023 CN200510057023 CN 200510057023 CN 200510057023 A CN200510057023 A CN 200510057023A CN 1700005 A CN1700005 A CN 1700005A
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Abstract
The invention relates to a new technology for measuring AIV called colloid gold immune color printing technology, which uses this technology to make test paper and test card for diagnosing AIV rapidly. The invention develops AIV monoclonal antibody: Antibody 1, sheep resistant AIV polyclonal antibody: Antibody 2 and rabbit resistant Balb/C small mouse immune protein antibody: Antibody 3. Ab1 labels colloid gold as gold antibody, Ab2 as capture antibody and Ab3 as quality controlled antibody. It uses antibody, pyroxylin film (NC film), covering pad, sample pad, backing, and plastic card and so on to assembly process test paper and test card. The test paper and test card can test AIV in the ill animal's blood and stool. It uses 10 minutes with the sensibility of 10ng/ml.
Description
One technical field
Bird flu colloidal gold immunochromatographitest test strip and test card belong to the diagnostic reagent of a kind of new animal, mainly are to be used for diagnosing the animal that is subjected to avian flu virus infection, and whether secondly also can detect the people infects avian influenza virus.This test strips is utilized immunology antigen-antibody energy specific bond principle, colloid gold label antibody (AIV monoclonal antibody) back combines with antigen (AIV), this antigen-antibody bond again with the AIV antibodies, the sandwich antigen-antibody bond that forms color reaction occurs on the NC film, can visual inspection.So this technology belongs to the immunology diagnosis technical field,, its technology learns field and physianthropy field but being mainly used in animal and veterinary.Product is mainly used in animal quarantine and diagnostic field.
Two background technologies
The main at present technical method that adopts of avian flu virus detection has: the isolation identification of HA, HI, ELISA, agar gel diffusion test, immunofluorescence technique, RT-PCR, fluorescence RT-PCR and virus etc.Technology is ELISA and fluorescence RT-PCR more fast and accurately, but these 2 kinds of technology all need special instrument and equipment, and detection time all more than 2 hours, it is more expensive to detect value.All these technical methods all can not be produced in basic unit, quarantine station and on-the-spot use the such as clinical, and special laboratory must be arranged; Though the immune colloidal gold chromatography technology is a kind of than proven technique, and is variant to the application of different antigen-antibody systems, comprise that raw material sources, reagent preparation, production technology etc. are all different, need carry out new design and test.So domestic test strips and the test card that does not still have bird flu immune colloidal gold chromatography quick diagnosis comprises bibliographical information and patent of invention at present.
This invention is according to colloidal gold immunochromatographimethod technical characterstic and avian influenza virus antigen antibody forming system characteristics, design new starting material, reagent preparation and technological process etc., invent a kind of new avian influenza virus Fast Detection Technique, have simple, fast, characteristics such as sensitivity and specificity be good.Go out the result about 10 minutes, can be applied to the quick diagnosis and the quarantine of basic unit and clinical bird flu.
Three summary of the invention
This invention is a kind of technical method of new fast detecting avian influenza virus, have simple, fast, characteristics such as sensitivity and specificity be good.And value is cheap, is applicable to basic unit or clinical detection avian influenza virus.
This reagent strip and test card invention relate generally to the content of the following aspects:
1 avian influenza virus monoclonal antibody (AIV McAb), utilize hybridoma technology to pass through Fusion of Cells, the avian influenza virus monoclonal antibody of the uniqueness that colony screening is set up, this antibody at the avian influenza virus reaction, are the McAb of avian influenza virus common epitope only.This antibody has determined the specificity of reagent strip.With this antibody labeling collaurum, make golden labeling antibody, can with the AIV specific bond in the sample to be checked, so be sprayed on the pad behind this antibody labeling collaurum.
This antibody of the anti-Balb/C mouse immuning ball protein of 2 rabbits antibody is to use the immunoglobulin (Ig) of Balb/C mouse as antigen, immunizing rabbit, and the antibody globulin in the extraction immunize rabbit serum is made.Be used on the nature controlling line on the NC film, can combine with golden labeling antibody, with the validity of this testing reagent bar.
This antibody of 3 goat-anti AIV antibody is that the immunoglobulin (Ig) in the extraction immune sheep serum is made with behind the purifying AIV antigen immune goat.This antibody is used in catching on the line on the reagent strip NC film, can combine with golden labeling antibody-AIV antigen antibody complex, forms sandwich antigen antibody complex.With the AIV in this test sample, be one of this invention important content.
The colloid gold label of 4AIV monoclonal antibody and purifying AIV labeling of monoclonal antibody collaurum adopt the special formulation mark, and the antibody that is marked with collaurum adopts proprietary reagent that golden labeling antibody is suspended and preserves through centrifugal purification.Golden labeling antibody after the suspension can keep more than 1 year in room temperature or in reagent strip.
The special processing sample pad of 5 sample pad and pad behind 37 ℃ of dry 2h, is cut into special width after proprietary damping fluid soaks, be used for the visible component of filtered sample, and can not make the sex change of AIV antigen.Be placed on reagent strip (card) foremost, in the lower end of reagent card sample well.The pad processing mode is identical with sample pad, but use different damping fluids to soak, dry back metal spraying labeling antibody, place under the sample pad, and with the special processing sample pad of NC film 5 sample pad and pad after proprietary damping fluid soaks, behind 37 ℃ of dry 2h, be cut into special width, the visible component that is used for filtered sample, and can not make the sex change of AIV antigen.Be placed on reagent strip (card) foremost, in the lower end of reagent card sample well.The pad processing mode is identical with sample pad, but uses different damping fluids to soak, and dry back metal spraying labeling antibody places under the sample pad, and links to each other with the NC film.Pad is the place that golden labeling antibody combines with AIV in the sample, moves to the NC film by the chromatography effect then.
The design test cartridge of 6 test cassettes is by two chimeric forming of plastic plate up and down.Following piece has the groove of a reagent strip, places reagent strip, and upper plate has a well and a reading window.Well is the position that adds sample, and the below is over against sample pad, and reading window is the position of NC film, reads the window side and is printed on T and C letter, represents the position of NC film detection line and the position of nature controlling line respectively.
The technological process of production of 7 these reagent strips mainly is made up of following step:
7.1 the preparation of AIV gold labeling antibody prepares colloid gold particle with gold chloride.Grain size 40nm.Then AIV monoclonal antibody and colloid gold particle are mixed into row labels in proportion, the intact back of mark is sealed with bovine serum albumin(BSA) (BSA).The golden labeling antibody that mark is good carries out purifying with supercentrifugal process, dispels free antibodies and albumen and other small-molecule substance.
7.2 the processing of pad and sample pad is put into different damping fluids with pad and sample pad and is soaked, drying, and the good golden labeling antibody of pad spray mark, dry then.Pad is cut into the width of 50mm, and sample pad is cut into the 40mm width.
7.3 the preparation of reagent batten tears the self-adhesive paper on the backing off, sticks NC film, pad (being sprayed with golden labeling antibody), sample pad, suction packing paper respectively.
7.4 the spray sample of NC film is sprayed capture antibody (goat-anti AIV antibody) and Quality Control antibody (the anti-Balb/C globulin antibody of rabbit) respectively with the NC film on the reagent batten for preparing.Live width 0.8mm.Back 37 ℃ of dryings have been sprayed.
7.5 the reagent batten that the assembling of reagent batten slitting and test card is sprayed with antibody with the NC film is cut into the reagent strip of 50 * 10mm, puts into the aluminium foil bag of drying agent immediately, seals.Or the reagent strip that agent plate is cut into 30 * 10mm put into test cassette, put into aluminium foil bag immediately and seal.
7.6 the check of reagent strip by every batch 1% pick test, comprises susceptibility, specificity and the storage life accelerated test etc. of outward appearance, reagent strip, the reliability of checking reagent strip.
Four specific embodiments
Following example just proves the science and the operability of this invention and proves susceptibility and the specificity that this diagnose test paper has, and can not include all the elements of invention, and concrete use can exceed this scope.
Embodiment 1 with the avian influenza virus standard antigen of deactivation with normal not immune chicken serum by 1: 10-1: 10000 dilutions, detect with the AIV immunity colloidal gold test paper strip that assembles, the positive occurring, can detected greatest dilution be 1: 8000, and the HA test can only detect 1: 100.With chicken serum dilution newcastle disease virus (NDV) weakness vaccine, test strips detects chicken serum and the normal chicken serum that contains NDV, and is all negative simultaneously.Illustrate that AIV immune colloid gold reagent bar has specificity and susceptibility preferably.
Embodiment 2 with the avian influenza virus antigen of deactivation with normal not immune chicken serum by 1: 10-1: 10000 dilutions, detect with the AIV immune colloidal gold test card that assembles, the positive occurring, can detected greatest dilution be 1: 8000, and the HA test can only detect 1: 100.With chicken serum dilution newcastle disease virus (NDV) weakness vaccine, test card detects chicken serum and the normal chicken serum that contains NDV, and is all negative simultaneously.Illustrate that the AIV immune colloidal gold test card has specificity and susceptibility preferably.
Embodiment 3 with AIV inactivation antigen purifying after, measure protein concentration with ultraviolet spectrophotometer, increase progressively for 10 times with normal not immune chicken serum then and be diluted to 100ug/ml, 10ug/ml, 1ug/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, simultaneously NDV is done same dilution.Detect respectively with AIV immune colloid gold reagent bar and test card.Can to detect the concentration of AIV be 10ng/ml for reagent strip and test card as a result, and that NDV and normal serum detect is all negative.Thereby prove the susceptibility and the specificity of this diagnostic reagent bar and test card.
Embodiment 4 gathers the blood of infected chicken, and positive (two red lines all appear in test card and test strips) appears in bird flu diagnosis test paper and test card detection with development.Its sample is detected confirmation through RT-PCR infect AIV.
Claims (7)
- The immune colloidal gold chromatography diagnostic reagent bar and the test card of 1 one kinds of fast detecting avian influenza virus (AIV) is characterized in that forming reagent strip by treated pad (1), sample pad (2), adsorptive pads (3), backing (4), nitrocellulose filter (5), sign bar (6) etc.; Reagent strip 5cm * the 0.5cm that does not contain sign bar paper puts into a special plastic clip (7) and forms test card.Test sample process sample pad and pad act on through chromatography and occur catching line and nature controlling line (5), with the naked eye judged result on the NC film.It is negative to occur a nature controlling line on the NC film, occurs catching line and nature controlling line is positive, lines do not occur and represents that test strips or test card lost efficacy.Go out the result in 15 minutes.
- 2 immune colloidal gold chromatography diagnosis test paper and its feature of test card content (1) pads according to the described a kind of fast detecting AIV of claim 1 are all-glass paper, produce by U.S. Millipore company, after the borate buffer immersion treatment that contains polyvinyl alcohol (PVA) (PVA), dry, spray avian influenza virus (AIV) monoclonal antibody of colloid gold label, dry again, the AIV monoclonal antibody is the epitope at all avian influenza virus, be connected with sample pad on the pad, be connected with the NC film down.
- 3 immune colloidal gold chromatography diagnosis test paper and test khaki content characteristic (2) sample pad according to the described a kind of fast detecting AIV of claim 1 also are all-glass paper, produce by Millipore company, the same pad of processing mode, but pH of buffer is 7.2, and only soak and dried, in reagent strip and test card, play filtered sample, be positioned on the pad, be attached on the backing.
- 4 immune colloidal gold chromatography diagnosis test paper and test khaki content characteristic (3) absorption pads according to the described a kind of fast detecting AIV of claim 1 are a kind of thick filter paper with better water-intake capacity, by the special-purpose thieving paper of Millipore company production and the filter paper for oil of Hangzhou filter paper factory production, be cut into and require directly use of size.Be attached to the end of (4) backing, play water sorption (chromatography effect).
- 5 immune colloidal gold chromatography diagnosis test paper and test khaki content characteristic (4) backings according to the described a kind of fast detecting AIV of claim 1 are hectograph materials that support sample pad, pad, NC film, absorption pad.Backing is with posting self-adhesive paper on the hectograph, self-adhesive paper is cut into the shape that test strips needs, can pasting pad, sample pad, NC film, adsorptive pads and sign paper etc. respectively after tearing.
- 6 immune colloidal gold chromatography diagnosis test paper and test khaki content characteristic (5) NC films according to the described a kind of fast detecting AIV of claim 1 are a kind of materials that are specifically designed to colloidal gold strip, are produced by Millipore company or Waterman company.Form by one deck filter membrane and glued membrane, and the rolling wound packages (2cm * 100m).2 antibody lines of spray are caught line (AIV antibody) for one on the NC film, and another line (nature controlling line) is the anti-Balb/C mouse immuning ball protein of a rabbit antibody.The NC film is attached to the centre of backing, and two ends are connected with adsorptive pads with pad respectively.
- 7 immune colloidal gold chromatography diagnosis test paper and test khaki content characteristic (6) sign bars according to the described a kind of fast detecting AIV of claim 1, it is a self-adhesive paper that has sign, stick on 2 ends of test strips respectively, having the arrow end is to be attached on sample pad and the pad, and end immerses testing sample thus.Lean on the water sorption of the adsorptive pads of the other end to make sample through the NC film.8 according to the immune colloidal gold chromatography diagnosis test paper of the described a kind of fast detecting AIV of claim 1 and test khaki feature (7) plastic clip be one with the special card of plastics, by up and down 2 form, sheet can be entrenched togather up and down.Following sheet mainly contains one and puts the groove of test strips and the latch that combines with last slice.Last slice mainly contain a latch that detects hole (or claiming to read window) and a sample well and combine with following sheet.Detect the next door, hole and be printed on T and C printed words respectively, T represents to catch the position of line, and C represents the position of nature controlling line.Detecting the hole is the window of observations, and sample well is the position that drips sample.This plastic clip plays the protection test strips, and more attractive in appearance, reduces the safety of polluting and protecting the operator.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510057023 CN1700005A (en) | 2005-04-18 | 2005-04-18 | Avian influenza immunogold diagnosis test strip and test card |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510057023 CN1700005A (en) | 2005-04-18 | 2005-04-18 | Avian influenza immunogold diagnosis test strip and test card |
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| CN1700005A true CN1700005A (en) | 2005-11-23 |
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| CN 200510057023 Pending CN1700005A (en) | 2005-04-18 | 2005-04-18 | Avian influenza immunogold diagnosis test strip and test card |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN101701958A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Fluorescent test paper strip capable of simultaneously testing newcastle disease and bird flu virus and application thereof |
| CN101701957A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Production method of fluorescently-labeled test strip for rapidly testing avian influenza virus |
| CN101067626B (en) * | 2007-06-08 | 2012-04-04 | 北京英诺特生物技术有限公司 | Colloidal gold chromatographic band for detecting IgM, IgG antibody by double-capture method and producing method thereof |
| CN103941005A (en) * | 2014-02-17 | 2014-07-23 | 广西壮族自治区兽医研究所 | Colloidal gold test strip used for detecting H7 subtype avian influenza virus |
-
2005
- 2005-04-18 CN CN 200510057023 patent/CN1700005A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101067626B (en) * | 2007-06-08 | 2012-04-04 | 北京英诺特生物技术有限公司 | Colloidal gold chromatographic band for detecting IgM, IgG antibody by double-capture method and producing method thereof |
| CN101650366A (en) * | 2008-08-11 | 2010-02-17 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN101650366B (en) * | 2008-08-11 | 2014-04-02 | 万志静 | Quick test paper for detecting enterovirus and method for preparing same |
| CN101701958A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Fluorescent test paper strip capable of simultaneously testing newcastle disease and bird flu virus and application thereof |
| CN101701957A (en) * | 2009-03-05 | 2010-05-05 | 中国检验检疫科学研究院 | Production method of fluorescently-labeled test strip for rapidly testing avian influenza virus |
| CN103941005A (en) * | 2014-02-17 | 2014-07-23 | 广西壮族自治区兽医研究所 | Colloidal gold test strip used for detecting H7 subtype avian influenza virus |
| CN103941005B (en) * | 2014-02-17 | 2016-01-20 | 广西壮族自治区兽医研究所 | Detect the colloidal gold strip of H7 subtype avian influenza virus |
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