CN102109526B - 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 the improvement of the farming disease harms cause of disease detection technique, specifically is a kind of GCRV (Grass carp reovirus, GCRV) quick detection test paper and preparation thereof, using method, belongs to immunology and virology interleaving techniques field.
Background technology
The GCRV disease is to endanger one of serious disease in the grass carp cultivation, causes very high grass carp mortality ratio, can reach the mortality ratio of 70-80%, has caused huge loss for the grass carp aquaculture.The disease that this virus causes is called again hemorrhagic disease of grass carp, normal and the bacterial hemorrhage very difficult discriminating of its bleeding, there is no effective methods for the treatment of in view of hemorrhagic disease of grass carp again, thus easy, detection method is especially aobvious important fast, find in the hope of reaching early, the purpose of prevention early.Virus is separated detection technique has fast and accurately 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 is detected to make a definite diagnosis, and existing 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); Immunity absorption 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, for the hydrobiont physilogical characteristics, design invention provides a kind of rapid detecting test paper for grass carp reovirus that can reach quick, easy, accurate testing goal and preparation thereof, using method.
Technical scheme of the present invention is achieved as follows
Rapid detecting test paper for grass carp reovirus, 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:; The two ends that continue mutually with this layer arrange thieving paper and consist of respectively application of sample end water accepting layer and handheld terminal water accepting layer; At 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 is divided and is arranged on the detection layers.
Described gold mark monoclonal antibody C preparation:
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 ℃ save backup.
The using method of rapid detecting test paper for grass carp reovirus: 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, namely two red lines represent that GCRV is positive and Test paper is effective.
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, namely makes test sample.
The detection principle of rapid detecting test paper for grass carp reovirus of the present invention:
The application of sample end water accepting layer of rapid detecting test paper for grass carp reovirus 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, namely by gold mark monoclonal antibody C with the antigen-reactive in the test sample liquid, 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 to the nitrocellulose filter in advance, antibody herein can be identified 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 the colloid gold particle of the upper mark of monoclonal antibody C 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, Antibody types is that the gold mark monoclonal antibody C of IgG is combined with by it, thereby also occur herein being fixed and being accumulated and show macroscopic red line by colloid gold particle, the result is: detection line shows that red expression GCRV is positive; Detection line does not show redness, and expression GCRV feminine gender does not namely 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 namely or golden labeling antibody C or sheep anti-mouse igg inactivation, testing result is invalid.
The present invention has the following advantages:
1, rapid detecting test paper for grass carp reovirus of the present invention, the cultivation needs from reality need not specialized facilities and operative technique during use, being fit to common raiser's pool side detects, use simply, have and detect the characteristics such as quick, easy, accurate, sensitive, and have higher stability;
2, in the rapid detecting test paper for grass carp reovirus 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 the 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 rapid detecting test paper for grass carp reovirus synoptic diagram;
The testing result synoptic diagram of Fig. 2 rapid detecting test paper for grass carp reovirus.
Hybridoma cell strain GCRV-C, its deposit number is: CCTCC-C201103; Depositary institution's full name and abbreviation: Chinese Typical Representative 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 Representative 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 by reference to the accompanying drawings:
Referring to Fig. 1,2 rapid detecting test paper for grass carp reovirus 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; Carrier board 7 (the application of sample end is A, and handheld terminal is B) with adhesive sticker.At the pars intermedia with the carrier board 7 of 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 detection layers 5; The two ends that continue mutually with this layer arrange thieving paper and consist of respectively application of sample end water accepting layer 1 and handheld terminal water accepting layer 6; At 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 is divided and is arranged on the detection layers 5.
Embodiment 1.
Described monoclonal antibody C colloidal gold labeling 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, for subsequent use;
(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 again Sodium azide concentration is transferred to 0.01-0.03%, namely 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 ℃ save backup.
Rapid detecting test paper for grass carp reovirus detection layers preparation method of the present invention is: after the anti-GCRV monoclonal antibody D freeze drying that caprylic acid-ammonium 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, with goat anti-mouse igg, be prepared into 1-2mg/ml, 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 respectively monoclonal antibody C and D 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 to get 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 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 coated detection line monoclonal antibody.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 take same concentration, with these 5 kinds of monoclonal antibodies of volume as coated antibody, detect simultaneously same GCRV sample, the result: when monoclonal antibody D is coated antibody, best results (seeing Table 1).Thereby, select monoclonal antibody C to mark monoclonal antibody as gold, and monoclonal antibody D is the monoclonal antibody of coated detection line.
Table 1 different mode detects the gold-marking immunity tomographic results of GCRV
Annotate: ++ expression detection line displaing amaranth is than strong positive; + expression detection line is aobvious red positive;
Embodiment 5.
The using method of rapid detecting test paper for grass carp reovirus of the present invention, be the detecting 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 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, expression infects without GCRV or its virus contains extremely low;
Testing result is invalid: application of sample 10 minutes, and nature controlling line 4 places occur without red line on the detection layers 5, represent that this test paper lost efficacy, and testing result is invalid.(seeing Fig. 2)
Being determined as follows of the minimum virus quantity that rapid detecting test paper for grass carp reovirus of the present invention detects: 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; + expression detection line is aobvious red positive;-expression detection line is colourless negative; TCID
50=10
-6.3/ 0.1ml.
The Stability Determination of rapid detecting test paper for grass carp reovirus detection using method of the present invention is 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 identification of rapid detecting test paper for grass carp reovirus of the present invention: the GCRV seed culture of viruses of getting altogether 2 kinds of different regions, viral haemorrhagic septicaemia virus (Egtved virus) and mammal reovirus (Orthoreovirus), with rapid detecting test paper for grass carp reovirus it is detected respectively, testing result sees Table 3
The specificity identification of table 3 rapid detecting test paper for grass carp reovirus
Annotate :+expression detects positive, and-expression detects negative
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 (available from Chinese Shanghai reagent one factory, analyzing pure); 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 above-described embodiment, and it all is possible adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (4)
1. rapid detecting test paper for grass carp reovirus, it comprises: preserving number is: the secreted anti-GCRV of CCTCC-C201103 hybridoma, the monoclonal antibody C of colloid gold label, referred to as gold mark monoclonal antibody C; Preserving number is: the monoclonal antibody D of the anti-GCRV that the CCTCC-C201104 hybridoma is secreted, and referred to as monoclonal antibody D, and goat anti-mouse igg; The adhesive sticker carrier board of appropriate size;
It is characterized in that pars intermedia at this carrier board is provided with by stickup is coated with monoclonal antibody D, i.e. detection line and goat anti-mouse igg, the i.e. detection layers made of the nitrocellulose membrane of nature controlling line; The two ends that continue mutually with this layer arrange thieving paper, consist of respectively application of sample end water accepting layer and handheld terminal water accepting layer; At 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 is divided and is arranged on the detection layers.
2. described rapid detecting test paper for grass carp reovirus according to claim 1 is characterized in that: the monoclonal antibody C of described colloid gold label, and 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 ℃ save backup.
3. the using method of the described rapid detecting test paper for grass carp reovirus 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, namely two red lines represent that GCRV is positive and Test paper is effective.
4. the using method of described rapid detecting test paper for grass carp reovirus according to 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, namely makes test sample.
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| 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 |
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| CN1664587A (en) * | 2005-03-07 | 2005-09-07 | 中国海洋大学 | White spot syndrome virus on-spot detection test paper, its preparation and method of application |
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