CN102109526A - Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof - Google Patents
Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof Download PDFInfo
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Abstract
The invention relates to test paper for quickly detecting grass carp reovirus (GCRV). The test paper comprises a GCRV-resistant and colloidal gold-labeled monoclonal antibody C which is secreted by a hybridoma cell with the collection number of CCTCC-C201103, a GCRV-resistant monoclonal antibody D and goat anti-mouse immunoglobulin G (IgG) which are secreted by a hybridoma cell with the collection number of CCTCC-C201104, and an adhesive sticker carrier plate, wherein the middle of the carrier plate is provided with a detection layer prepared from a nitrocellulose membrane attached and coated with the monoclonal antibody D serving as a detection line and the goat anti-mouse IgG serving as a quality control line; water absorption paper is arranged at the two extending ends of the detection layer to form a loading end water absorption layer and a handheld end water absorption layer; a glass fiber membrane loaded with the gold-labeled monoclonal antibody C is arranged at the junction of the far end of the detection line of the detection layer and the loading end water absorption layer; and a part of one end of the membrane is arranged below the loading end water absorption layer, and a part of the other end of the membrane is arranged above the detection layer. The invention has the characteristics that: the test paper has high stability and is simple, quick, accurate and sensitive.
Description
Technical field
The present invention relates to culture the improvement of disease cause of disease detection technique, specifically is that (Grass carp reovirus, GCRV) quick detection test paper and preparation thereof, using method belong to immunology and virology interleaving techniques field to a kind of GCRV.
Background technology
The GCRV disease is to endanger one of serious disease during grass carp is cultured, and causes very high grass carp mortality ratio, can reach the mortality ratio of 70-80%, has caused tremendous loss for the grass carp aquaculture.The disease that this virus causes is called hemorrhagic disease of grass carp again, normal and the bacterial hemorrhage very difficult discriminating of its bleeding, still do not have effective methods of treatment in view of hemorrhagic disease of grass carp again, thus easy, detection method especially shows important fast, find in the hope of reaching early, the purpose of prevention early.Viral fast and accurately separation detection technology has become one of focus of scholar's research.
At present, the diagnosis of viral hemorrhagic disease of grass carp mainly is to rely in the laboratory GCRV detected to make a definite diagnosis, and showing the laboratory detection method has: staphylococcal protein a coagglutination test (Yang Guang intelligence 1991); Enzyme-linked immuno-sorbent assay (river forest cultivation 1993); Fluorescent antibody technics detection method (river forest cultivation 1993); Spot immune trace detection method (Shao Jian loyalty 1996); Immunoadsorption electron microscopic observation method (Yuan Aihua 1990); Reverse transcriptase polymerase chain reaction method (Wang Tie brightness 1997) etc.These methods, the operation formula is comparatively complicated, needs certain Laboratory Instruments equipment, and consuming time longer, and whole process need is several and even tens hours, does not reach the purpose of fast detecting.The reverse transcriptase polymerase chain reaction method is highly sensitive, can be used for the detection of asymptomatic sick fish, but its high sensitivity easily causes false positive results; Enzyme-linked immuno-sorbent assay and spot immune trace detection method are detection methods commonly used at present, but the endogenous enzymes of tested tissue can produce interference to its testing result, causes false-positive appearance.Therefore a kind of development of detection method of quick, accurate, easy, quick, the non-false positive that is applicable to the aquiculture disease cause of disease is imperative, finds in the hope of reaching early, the purpose of prevention early.
Summary of the invention
The present invention is directed to the present situation of the sick serious harm grass carp of GCRV aquaculture, at the hydrobiont physilogical characteristics, design invention provides a kind of GCRV quick detection test paper that can reach quick, easy, accurate testing goal and preparation thereof, using method.
Technical scheme of the present invention is achieved as follows
The GCRV quick detection test paper, it comprises: preserving number is: the monoclonal antibody C of the secreted anti-GCRV of CCTCC-C201103 hybridoma, colloid gold label (is called for short: gold mark monoclonal antibody C); Monoclonal antibody D) and goat anti-mouse igg preserving number is: the monoclonal antibody D of the anti-GCRV that the CCTCC-C201104 hybridoma is secreted (is called for short:; The adhesive sticker carrier board of appropriate size; Detection line) and goat anti-mouse igg (but also title: the detection layers that nitrocellulose membrane nature controlling line) is made its special character is that the pars intermedia at this carrier board is provided with and is coated with monoclonal antibody D by stickup and (claims not only:; With two ends that this layer continues mutually thieving paper is set and constitutes application of sample end water accepting layer and handheld terminal water accepting layer respectively; At the far-end and the application of sample end water accepting layer intersection of the detection line of detection layers, be provided with the glass fibre membrane that is loaded with gold mark monoclonal antibody C, this film one end parts is arranged under the application of sample end water accepting layer, and its other end branch is arranged on the detection layers.
Described gold mark monoclonal antibody C preparation:
Be that preparation technology with known collaurum prepares colloidal gold solution, the grain size of collaurum is 10-20nm, monoclonal antibody C is joined in the above-mentioned colloidal gold solution, slowly mix even, add bovine serum albumin(BSA), precipitation after centrifugal is hanged with the phosphate buffer that contains 1-10% bovine serum albumin(BSA) and 0.3-3% Tween-20, adds Sodium azide, makes gold mark monoclonal antibody C.
The described preparation that is loaded with the glass fibre membrane of gold mark monoclonal antibody C:
Gold is marked monoclonal antibody C liquid, be sprayed on the glass fibre membrane by every square centimeter of 700-1000 μ l, freeze drying, 4 ℃ of preservations are standby.
The using method of GCRV quick detection test paper: the application of sample end water accepting layer of this test paper is inserted or drip detected sample thereon, and inherent detection line and the nature controlling line place that is positioned on the detection layers showed redness in 5-10 minute, promptly two red lines, and the expression grass carp exhales intestines
The process of described test sample preparation is: get tested grass carp kidney and be added in the centrifuge tube, by mass/volume 1: (5-10) (g/ml) to add 0.01mol/L pH be the phosphate buffer of 6-8, with smashing rod to pieces the grass carp kidney is smashed to pieces, virus is released in this centrifuge tube liquid, promptly makes test sample.
The detection principle of GCRV quick detection test paper of the present invention:
The application of sample end water accepting layer of GCRV quick detection test paper is inserted or drips the test sample that is contained virion by phosphate buffer from the grass carp kidney extraction of smashing to pieces thereon, according to the sandwich immunoassay chromatographic theory, promptly mark monoclonal antibody C with the antigen-reactive in the test sample liquid by gold, formation is by gold mark monoclonal antibody C-GCRV compound, when this compound chromatography is coated on monoclonal antibody D place on the detection line in advance to nitrocellulose filter, antibody herein can be discerned antigen in this compound, the result of reaction has just formed the sandwich structure of golden mark monoclonal antibody C+ GCRV antigen+monoclonal antibody D, finally be that colloid gold particle that monoclonal antibody C goes up mark is fixed and accumulated at this and macroscopic red line occurs, unreacted gold mark monoclonal antibody C then still continues chromatography and moves ahead, when the point of arrival is coated on nature controlling line sheep anti-mouse igg place in advance, the antibody type is that the gold mark monoclonal antibody C of IgG is lived by its combination, thereby also occur herein being fixed and being accumulated and show macroscopic red line by colloid gold particle, the result is: detection line shows the red expression GCRV positive; Detection line does not show redness, and expression GCRV feminine gender does not promptly contain GCRV in the test sample or GCRV content is extremely low.Nature controlling line shows red, and the expression test paper is effective; The nature controlling line place does not show redness, illustrates that test paper lost efficacy, and promptly or golden labeling antibody C or sheep anti-mouse igg inactivation, testing result is invalid.
The present invention has the following advantages:
1, GCRV quick detection test paper of the present invention, from the breed of reality need, need not specialized facilities and operative technique during use, being fit to common raiser's pool side detects, use simply, have characteristics such as detection is quick, easy, accurate, sensitivity, and have advantages of higher stability;
2, in the GCRV quick detection test paper using method of the present invention, need not to add special lysate; Only need the grass carp kidney smashed to pieces and get final product, extract method such as grind, centrifugal with tradition virus and compare, the method is convenient and simple, but the detection of rapid extraction virus.
Description of drawings
Fig. 1 GCRV quick detection test paper synoptic diagram;
The testing result synoptic diagram of Fig. 2 GCRV quick detection test paper.
Hybridoma cell strain GCRV-C, its deposit number is: CCTCC-C201103; Depositary institution's full name and abbreviation: Chinese typical culture collection center (CCTCC); Depositary institution address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center; Preservation date: on January 17th, 2011.
Hybridoma cell strain GCRV-D, its deposit number is: CCTCC-C201104; Depositary institution's full name and abbreviation: Chinese typical culture collection center (CCTCC); Depositary institution address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center; Preservation date: on January 17th, 2011.
Embodiment
Embodiments of the invention further describe as follows in conjunction with the accompanying drawings:
Referring to Fig. 1,2 GCRV quick detection test paper of the present invention, it comprises: preserving number is: the monoclonal antibody C of the secreted anti-GCRV of CCTCC-C201103 hybridoma, colloid gold label (is called for short: gold mark monoclonal antibody C); Monoclonal antibody D) and goat anti-mouse igg preserving number is: the monoclonal antibody D of the anti-GCRV that the CCTCC-C201104 hybridoma is secreted (is called for short:; Be coated with the nitrocellulose membrane of monoclonal antibody D and goat anti-mouse igg; Be loaded with the glass fibre membrane 2 of gold mark monoclonal antibody C; The carrier board 7 (the application of sample end is A, and handheld terminal is B) that has adhesive sticker.At the pars intermedia of the carrier board 7 that has adhesive sticker, paste be coated with monoclonal antibody D (claim not only: detection line 3) and goat anti-mouse igg (but also title: nitrocellulose membrane nature controlling line 4) is a detection layers 5; With two ends that this layer continues mutually thieving paper is set and constitutes application of sample end water accepting layer 1 and handheld terminal water accepting layer 6 respectively; At the far-end and application of sample end water accepting layer 1 intersection of the detection line 3 of detection layers 5, be provided with the glass fibre membrane 2 that is loaded with gold mark monoclonal antibody C, this film one end parts is arranged under the application of sample end water accepting layer 1, and its other end branch is arranged on the detection layers 5.
Described monoclonal antibody C colloid gold label method is:
(1) preparation of collaurum: 10-20nm colloid gold particle preparation, 0.01% chlorauric acid solution 1000ml is mixed with 0.1% sodium citrate 25-44ml, be heated to 100 degrees centigrade, make colloidal gold solution, the pH value of this solution: transfer to 8.0-8.4 with 0.2% sal tartari, standby;
(2) preparation of gold mark monoclonal antibody C:
When monoclonal antibody C concentration was 3-5g/L, the suitableeest monoclonal antibody amount of 100ml colloid gold label was 120-150 μ l, in this ratio monoclonal antibody C is joined in the colloidal gold solution, stirred slowly 50-60 minute, added 1% bovine serum albumin(BSA), and 4 ℃ are spent the night; Then with it under 4 ℃, the centrifugal 110-120 of 17000g minute; Get centrifugation, with the 0.01mol/L phosphate buffer (KCL0.2g, NaCL8.0g, the KH that contain 1-10% bovine serum albumin(BSA) and 0.3-3% Tween-20
2PO
40.2g, Na
2HPO
412H
2O 2.9g, distilled water 1000ml, pH 7.4) hang, add Sodium azide again concentration is transferred to 0.01-0.03%, promptly make gold mark monoclonal antibody C.
Be loaded with the preparation of the glass layer piece of gold mark monoclonal antibody C: the gold mark monoclonal antibody C that will make, be sprayed on the glass fibre membrane by every square centimeter of 700-1000 μ l, freeze drying, 4 ℃ of preservations are standby.
GCRV quick detection test paper detection layers preparation method of the present invention is: after the anti-GCRV monoclonal antibody D freeze drying that sad-ammonium sulfate method is purified, be prepared into the anti-GCRV monoclonal antibody of 3-5mg/ml D liquid, with pen machine with its stroke on nitrocellulose filter, form detection line 3; Equally,, be prepared into 1-2mg/ml with goat anti-mouse igg, with pen machine with its stroke on nitrocellulose filter, form nature controlling line 4.Two lines are at a distance of 4-6mm, nature controlling line 4 nearly handheld terminal water accepting layers 6, detection line 3 nearly application of sample end water accepting layers 1,36-37 ℃ of oven dry.
Two strain of hybridoma of secreting monoclonal antibody C and D respectively of the present invention, its preparation is as follows with the method for selecting:
(1) utilizes grass carp kidney cell line amplification in vitro GCRV, utilize the supercentrifugation purified virus.
(2) be antigen with the GCRV liquid of purifying, immune Balb/c small white mouse;
(3) with the splenocyte of immune mouse and myeloma cell's fusion, cultivate 450 strain of hybridoma;
(4) adopt indirect immunofluorescence to filter out the positive hybridoma cell strain of anti-GCRV;
(5) adopt limiting dilution assay that 20 strain positive hybridoma cells are wherein cloned;
(6) select the monoclonal antibody C and the D of the different anti-GCRV of 2 strains by different mode gold-marking immunity chromatography method, respectively as gold mark monoclonal antibody and the tested survey line monoclonal antibody of bag.Its concrete experimental technique is: 5 kinds of monoclonal antibodies selecting antiviral strong positive by indirect immunofluorescence, monoclonal antibody C, monoclonal antibody E, monoclonal antibody A, monoclonal antibody B and monoclonal antibody D, gold mark monoclonal antibody C with respectively with same concentration, be coated antibody with these 5 kinds of monoclonal antibodies of volume, detect same GCRV sample simultaneously, the result: when monoclonal antibody D is coated antibody, best results (seeing Table 1).Thereby, select monoclonal antibody C to mark monoclonal antibody, and monoclonal antibody D is the monoclonal antibody of the tested survey line of bag as gold.
Table 1 different mode detects the gold-marking immunity tomographic results of GCRV
Annotate: ++ expression detection line displaing amaranth is than strong positive; It is red positive that+expression detection line shows;
The using method of GCRV quick detection test paper of the present invention, be the detection step of GCRV: get the grass carp kidney and put into centrifuge tube and fully smash to pieces, press mass/volume than 1: (5-10) (g/ml) adds the phosphate buffer of 0.01mol/L pH 6.0-8.0, makes test sample.Then, with sample drop application of sample end water accepting layer 1, read the result in 5-10 minute.
Positive findings: present the detection line 3 and the nature controlling line 4 of two redness on the detection layers 5, expression has GCRV to infect;
Negative findings: present a red nature controlling line 4 on the detection layers 5, represent that no GCRV infects or its virus contains extremely low;
Testing result is invalid: application of sample 10 minutes, and nature controlling line 4 places do not have the red line appearance on the detection layers 5, represent that this test paper lost efficacy, and testing result is invalid.(see figure 2)
The mensuration of the minimum virus quantity that GCRV quick detection test paper of the present invention detects is as follows: the results are shown in Table 2.
The measurement result of the minimum virus quantity of table 2
Annotate: ++ expression detection line displaing amaranth is than strong positive; It is red positive that+expression detection line shows;-expression detection line is colourless negative; TCID
50=10
-6.3/ 0.1ml.
GCRV quick detection test paper of the present invention detects the stability of using method and measures as follows: this test paper term of validity of depositing under 4 ℃ 12 months.This test paper term of validity of depositing under the room temperature 8 months.
Embodiment 8.
The specificity of GCRV quick detection test paper of the present invention is identified: the GCRV seed culture of viruses of getting 2 kinds of different regions altogether, viral haemorrhagic septicaemia virus (Egtved virus) and mammal reovirus (Orthoreovirus), with the GCRV quick detection test paper it is detected respectively, testing result sees Table 3
The specificity of table 3 GCRV quick detection test paper is identified
Annotate :+expression detects positive, and-expression detects negative
Used instrument of the present invention and reagent:
Fluorescence inverted phase contrast microscope (available from LEICA company); Pen machine (available from U.S. BIODOT company); Gold chloride (, analyzing pure) available from Chinese Shanghai reagent one factory; 1640 (available from GIBCO companies); Hyclone (available from PAA company); HAT (available from GIBCO company); Goat anti-mouse igg antibody (available from SIGMA company); Bovine serum albumin(BSA) (available from SIGMA company); Nitrocellulose filter (available from MILLIPORE company); Dimethyl sulfoxide (available from SIGMA company).
Those of ordinary skill in the art can understand, and in protection scope of the present invention, makes amendment for the foregoing description, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (4)
1. GCRV quick detection test paper, it comprises: preserving number is: the secreted anti-GCRV of CCTCC-C201103 hybridoma, the monoclonal antibody C of colloid gold label (are called for short: gold mark monoclonal antibody C); Monoclonal antibody D) and goat anti-mouse igg preserving number is: the monoclonal antibody D of the anti-GCRV that the CCTCC-C201104 hybridoma is secreted (is called for short:; The adhesive sticker carrier board of appropriate size;
Detection line) and goat anti-mouse igg (but also title: the detection layers that nitrocellulose membrane nature controlling line) is made it is characterized in that pars intermedia at this carrier board is provided with is coated with monoclonal antibody D by stickup and (claims not only:; The two ends that continue mutually with this layer are provided with thieving paper, constitute application of sample end water accepting layer and handheld terminal water accepting layer respectively; At the far-end and the application of sample end water accepting layer intersection of the detection line of detection layers, be provided with the glass fibre membrane that is loaded with gold mark monoclonal antibody C, this film one end parts is arranged under the application of sample end water accepting layer, and its other end branch is arranged on the detection layers.
2. according to the described GCRV quick detection test paper of claim 1, it is characterized in that: the monoclonal antibody C of described colloid gold label, its preparation method is as follows:
(1) preparation of collaurum: with known method, by certain proportioning gold chloride is mixed and heats being prepared into colloidal gold solution with trisodium citrate, the grain size of the collaurum that makes is 10-20nm;
(2) preparation of the monoclonal antibody C of colloid gold label: monoclonal antibody C is joined in above-mentioned (1) colloidal gold solution in step, slowly mix even, add bovine serum albumin(BSA), precipitation after centrifugal is hanged with the phosphate buffer that contains 1-10% bovine serum albumin(BSA) and 0.3-3% Tween-20, add Sodium azide, make the monoclonal antibody C of colloid gold label;
(3) be loaded with the preparation of glass fibre membrane of the monoclonal antibody C of colloid gold label: the monoclonal antibody C of the colloid gold label that above-mentioned (2) step is made, be sprayed on the glass fibre membrane by every square centimeter of 700-1000 μ l, freeze drying, 4 ℃ of preservations are standby.
3. the using method of the described GCRV quick detection test paper of claim 1, it is characterized in that: the application of sample end water accepting layer of this test paper is inserted or drip detected sample thereon, inherent detection line and the nature controlling line place that is positioned on the detection layers showed red in 5-10 minute, promptly two red lines, the expression GCRV is positive and the detection test paper is effective.
4. according to the using method of the described GCRV quick detection test paper of claim 3, it is characterized in that: the process of described test sample preparation is: get tested grass carp kidney and be added in the centrifuge tube, by mass/volume than 1: (5-10) (g/ml) to add 0.01mol/LpH be the phosphate buffer of 6-8, with smashing rod to pieces the grass carp kidney is smashed to pieces, virus is released in this centrifuge tube liquid, promptly makes test sample.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104569391A (en) * | 2015-01-30 | 2015-04-29 | 肇庆大华农生物药品有限公司 | ELISA (enzyme linked immunosorbent assay) detection kit for grass carp bleeding disease virus antibody and preparation method of kit |
| CN105319361A (en) * | 2015-11-05 | 2016-02-10 | 浙江万里学院 | Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof |
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| CN105891469A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Portunus trituberculatus reovirus detection test paper and preparation method thereof |
| CN105891469B (en) * | 2014-11-14 | 2018-08-03 | 宁波大学 | A kind of Portunus trituberculatus Miers reovirus test strip and preparation method thereof |
| CN104569391A (en) * | 2015-01-30 | 2015-04-29 | 肇庆大华农生物药品有限公司 | ELISA (enzyme linked immunosorbent assay) detection kit for grass carp bleeding disease virus antibody and preparation method of kit |
| CN104569391B (en) * | 2015-01-30 | 2016-05-25 | 肇庆大华农生物药品有限公司 | A kind of GCHV antibody ELISA detection kit and preparation method thereof |
| CN105319361A (en) * | 2015-11-05 | 2016-02-10 | 浙江万里学院 | Scylla serrata reovirus colloidal gold immunochromatographic assay reagent strip and preparing method and using method thereof |
| CN105319361B (en) * | 2015-11-05 | 2017-06-23 | 浙江万里学院 | Scylla paramamosain reovirus colloidal gold immunochromatographimethod reagent strip and preparation method thereof, application method |
| CN106198992A (en) * | 2016-06-24 | 2016-12-07 | 中国水产科学研究院黄海水产研究所 | A kind of colloidal gold immunochromatographydetection detection test paper for detecting seven band cabrilla nervous necrosis virus antibody and application thereof |
| CN117538318A (en) * | 2024-01-10 | 2024-02-09 | 山东利尔康医疗科技股份有限公司 | Residual peracetic acid test card and preparation method thereof |
| CN117538318B (en) * | 2024-01-10 | 2024-04-05 | 山东利尔康医疗科技股份有限公司 | Residual peracetic acid test card and preparation method thereof |
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