CN1292255C - White spot syndrome virus on-spot detection test paper, its preparation and method of application - Google Patents
White spot syndrome virus on-spot detection test paper, its preparation and method of application Download PDFInfo
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- CN1292255C CN1292255C CN 200510042127 CN200510042127A CN1292255C CN 1292255 C CN1292255 C CN 1292255C CN 200510042127 CN200510042127 CN 200510042127 CN 200510042127 A CN200510042127 A CN 200510042127A CN 1292255 C CN1292255 C CN 1292255C
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Abstract
The present invention relates to a test paper for detecting white spot syndrome viruses on site, which comprises a sticker carrier plate, a gold labeling mono-antibody E resisting white spot syndrome viruses secreted by hybridoma cells whose preservation number is CCTCC-C200421, a mono-antibody F resisting white spot syndrome viruses secreted by hybridoma cells whose preservation number is CCTCC-C200423, and sheep anti-mouse IgG, wherein the gold labeling mono-antibody E is labeled by colloidal gold, both ends of the carrier plate are provided with a water absorption layer at the sample adding end, and a water absorption layer at the hand-hold end, the middle part of the carrier plate is provided with a detecting layer of a nitrocellulose membrane, a glass fiber layer block carried with the gold labeling mono-antibody E is arranged at the juncture of the detecting layer, and the water absorption layer at the sample adding end, one end of the layer block is partially arranged below the water absorption layer at the sample adding end, and the other end is partially arranged above the detecting layer; detecting lines and quality control lines are arranged on the detecting layer continued by the layer block, and the detection lines and the quality control lines are respectively coated with the mono-antibody F and the sheep anti-mouse IgG. The test paper has the characteristics of simple preparation method, good stability and repeatability, convenient use, high speed, on-site detection, accuracy and sensitivity.
Description
Technical field
The present invention relates to culture the improvement of disease cause of disease detection technique, specifically is that (it belongs to immunology and virology interleaving techniques field to a kind of Leucodermia virus for white spot syndromevirus, WSSV) on-the-spot detection test paper and preparation thereof, using method.
Background technology
The Leucodermia virus disease is to endanger one of serious disease in the prawn culturing, causes very high prawn mortality ratio, has caused tremendous loss to shrimp culture industry.In view of hickie disease does not still have effective methods of treatment, viral fast and accurately separation detection technology has become one of focus of various countries' scholar's research.At present, the diagnosis of Leucodermia virus disease mainly is to rely in the laboratory Leucodermia virus detected to make a definite diagnosis, and showing the laboratory detection method has: the PCR method; Dna probe in situ hybridization method; The transmission electron microscope observing method; HE dyeing observation under the light microscopic, enzyme-linked immuno-sorbent assay and spot immune trace detection method 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 fast, the on-the-spot purpose that detects.The PCR method is highly sensitive, can be used for the detection of asymptomatic sick shrimp, 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, on-the-spot, the non-false positive that is applicable to the aquiculture disease cause of disease is imperative, in the hope of reaching early the purpose of finding, early preventing.
Summary of the invention
White spot syndrome virus on-spot detection test paper of the present invention and preparation thereof, using method are the present situations at the sick serious harm shrimp culture industry of Leucodermia virus, press for a kind of easy, quick, on-the-spot, accurate test method and hydrobiont physilogical characteristics such as low and design invention, to reach quick, easy, on-the-spot, testing goal accurately.
The objective of the invention is to realize that by following technical scheme developed a kind of white spot syndrome virus on-spot detection test paper, it comprises: the adhesive sticker carrier board of appropriate size; Preserving number is: the monoclonal antibody E anti-Leucodermia virus that the CCTCC-C200421 hybridoma is secreted, colloid gold label (is called for short: gold mark monoclonal antibody E); Monoclonal antibody F) and goat anti-mouse igg (abbreviation: sheep anti-mouse igg) preserving number is: the monoclonal antibody F of the anti-Leucodermia virus that the CCTCC-C200423 hybridoma is secreted (is called for short:.At the both ends of this carrier board, be respectively equipped with application of sample end water accepting layer and handheld terminal water accepting layer; Be provided with the detection layers of nitrocellulose membrane at this carrier board middle part, at this detection layers and application of sample end water accepting layer intersection, be provided with the glass layer piece that is loaded with gold mark monoclonal antibody E, this layer piece one end parts is arranged under the application of sample end water accepting layer, and its other end branch is arranged on this detection layers; With the detection layers of this layer piece continuity on be provided with detection line and nature controlling line, this detection line and nature controlling line place are coated with monoclonal antibody F and sheep anti-mouse igg respectively.
Described gold mark monoclonal antibody E, its preparation method is as follows:
(1) preparation of collaurum: the preparation technology with known collaurum mixes chlorauric acid solution with sodium citrate, and colloidal gold solution is made in heating;
(2) preparation of gold mark monoclonal antibody E: monoclonal antibody E is joined in above-mentioned (1) colloidal gold solution in step, slowly mix even, add bovine serum albumin(BSA), the precipitation after centrifugal hang with the phosphate buffer that contains bovine serum albumin(BSA), Sodium azide, makes the golden monoclonal antibody E liquid of marking;
(3) be loaded with the preparation of the glass layer piece of gold mark monoclonal antibody E: with the gold mark monoclonal antibody E liquid that above-mentioned (2) step makes, be sprayed on the glass fibre membrane begin to ooze out to liquid till, freeze drying, 4 ℃ of preservations are standby.
A kind of using method of white spot syndrome virus on-spot detection test paper: at first, use viral extraction element, from the detected shrimp gill, extract Leucodermia virus apace, the preparation test sample; Then, the handheld terminal of hand-held this test paper, the application of sample end water accepting layer of this test paper is inserted or drips this test sample thereon, the inherence was positioned at the detection line of detection layers lower end and showed red at the nature controlling line place that is being positioned at the detection layers upper end in 5 minutes, promptly two red lines, the expression Leucodermia virus is positive and the detection test paper is effective.
The process of described preparation test sample is: use is by test tube and smash the viral extraction element that rod is formed to pieces, will add liquid in the test tube earlier, then; get the tested shrimp cheek and be added in the test tube; with smashing rod to pieces it is fully smashed to pieces, virus is released in this test tube liquid, promptly make test sample.
Liquid in the described adding test tube, it is a pond water, distilled water, tap water, the phosphate buffer of pH 6.0-8.0, any one in the Tris-sodium chloride damping fluid of pH 6.0-8.0.
The invention has the advantages that: the present invention is with the actual features of prior art, on-the-spot fast detection test paper of a kind of Leucodermia virus and preparation thereof have been developed, using method, employing is the simple virus of extracting from target organ, directly the test sample liquid that contains virus is detected, its principle is that white spot syndrome virus on-spot detection test paper adopts the sandwich immunoassay chromatographic theory, promptly mark monoclonal antibody E with the antigen-reactive in the test sample liquid by gold, formation is by gold mark monoclonal antibody E-Leucodermia virus compound, when this compound chromatography is coated on monoclonal antibody F 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 E+ Leucodermia virus antigen+monoclonal antibody F, finally be that colloid gold particle that monoclonal antibody E goes up mark is fixed and accumulated at this and macroscopic red line occurs, unreacted gold mark monoclonal antibody E 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 E 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 Leucodermia virus positive; Detection line does not show redness, and expression Leucodermia virus feminine gender does not promptly contain Leucodermia virus in the test sample or Leucodermia virus 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 E or sheep anti-mouse igg inactivation, testing result is invalid.Characteristics of the present invention are: (1) white spot syndrome virus on-spot detection test paper of the present invention, have characteristics such as easy, quick, on-the-spot, accurate, sensitivity, and need not specialized facilities and operative technique, be suitable for common raiser's pool side and detect.(2) in the white spot syndrome virus on-spot detection test paper using method of the present invention, by tubule with smash the quick viral extraction element formed of rod to pieces, can directly get pond water or tap water etc. and be used to extract viral medium liquid, need not to add special lysate; Only need the shrimp gill smashed to pieces and get final product, extract method such as grind, centrifugal with tradition virus and compare, this method method is convenient, simple, fast, reaches the on-the-spot purpose of extracting the virus detection.(3) white spot syndrome virus on-spot detection test paper of the present invention, the preparation method is simple, stable, good reproducibility.Prove that through practical application white spot syndrome virus on-spot detection test paper of the present invention has easy, quick, on-the-spot, accurate, sensitive characteristics.
Description of drawings
Embodiments of the invention further describe as follows in conjunction with the accompanying drawings:
Fig. 1 white spot syndrome virus on-spot detection test paper synoptic diagram;
The testing result synoptic diagram of Fig. 2 white spot syndrome virus on-spot detection test paper.
Embodiment
Referring to Fig. 1,2 white spot syndrome virus on-spot detection test papers of the present invention, it comprises: the adhesive sticker carrier board 7 of appropriate size; Preserving number is: the monoclonal antibody E anti-Leucodermia virus that the CCTCC-C200421 hybridoma is secreted, colloid gold label (is called for short: gold mark monoclonal antibody E); Monoclonal antibody F) and goat anti-mouse igg (abbreviation: sheep anti-mouse igg) preserving number is: the monoclonal antibody F of the anti-Leucodermia virus that the CCTCC-C200423 hybridoma is secreted (is called for short:.At the both ends of this carrier board 7, be respectively equipped with application of sample end 4 water accepting layers and handheld terminal 1 water accepting layer; Be provided with the detection layers 2 of nitrocellulose membrane at these carrier board 7 middle parts, in this detection layers 2 and application of sample end 4 water accepting layer intersections, be provided with the glass layer piece 3 that is loaded with gold mark monoclonal antibody E, this layer piece 3 one end parts are arranged under application of sample end 4 water accepting layers, and its other end branch is arranged on this detection layers 2; With the detection layers 2 of this layer piece 3 continuity on be provided with detection line 6 and nature controlling line 5, this detection line 6 and nature controlling line 5 places are coated with monoclonal antibody F and sheep anti-mouse igg respectively.
Embodiment 1.
Described monoclonal antibody E colloid gold label method is:
(1) preparation of collaurum: the preparation of 18-20nm colloid gold particle, 0.01% chlorauric acid solution 100ml is mixed with 0.1% sodium citrate 2.5ml, add thermic and make colloidal gold solution for 100 ℃, 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 E:
1 μ l antibody is added in the 1ml collaurum, be mixed, add 10% sodium chloride, 100 μ l, observe change color, if become blue, the antibody deficiency then is described, constantly increase the antibody amount again, be as the criterion until the collaurum color is constant, on this basis, this antibody amount is increased 50-100%, and be the suitableeest monoclonal antibody amount of 1ml colloid gold label this moment.Carry out colloid gold label with this suitable monoclonal antibody amount, the collaurum behind the mark does not have precipitation, no non-specific adsorption, reaches experimental standard.By this suitableeest monoclonal antibody amount, when promptly monoclonal antibody E concentration was 0.5-2g/L, the suitableeest monoclonal antibody amount of 1ml colloid gold label was: 15-20 μ l.Monoclonal antibody E is joined in the colloidal gold solution, stir 10min slowly, add 1% bovine serum albumin(BSA), 4 ℃ are spent the night; Then with it under 4 ℃, the centrifugal 110min of 18000g; Get centrifugation, with the 0.01M phosphate buffer (PBS:KCI 0.2g, NaCI 8.0g, the KH that contain 1% bovine serum albumin(BSA), 0.02% Sodium azide
2PO
40.2g, Na
2HPO
412H
2O 2.9g, distilled water 1000ml, pH 7.4) hang, make gold mark monoclonal antibody E.
Embodiment 2.
Be loaded with the preparation of the glass layer piece of gold mark monoclonal antibody E: the gold mark monoclonal antibody E liquid that will make, be sprayed on the glass fibre membrane begin to ooze out to liquid till, freeze drying, 4 ℃ of preservations are standby.
Embodiment 3.
White spot syndrome virus on-spot detection test paper detection layers preparation method of the present invention is: after the anti-Leucodermia virus monoclonal antibody F freeze drying that sad method is purified, be prepared into the anti-Leucodermia virus monoclonal antibody of 4mg/ml F liquid, with Membrane jetter it is sprayed on the nitrocellulose filter, forms detection line 6; Equally,, be prepared into 300 μ g/ml, it be sprayed on the nitrocellulose filter, form nature controlling line 5 with Membrane jetter with goat anti-mouse igg.Two lines are at a distance of 5mm, nature controlling line 5 nearly handheld terminal water accepting layers, detection line 6 nearly application of sample end water accepting layers.Dry under the room temperature, use pH 7.4 again, contain 37 ℃ of sealings of 0.01M PBS 30min of 10% bovine serum albumin(BSA), the PBS rinsing is dried.
Embodiment 4.
Two strain of hybridoma of secreting monoclonal antibody E and F respectively of the present invention, its preparation is as follows with the method for selecting:
(1) be antigen with the Leucodermia virus liquid of purifying, immune Balb/c small white mouse;
(2) with the splenocyte of immune mouse and myeloma cell's fusion, cultivate 520 strain of hybridoma;
(3) adopt indirect immunofluorescence to filter out the positive hybridoma cell strain of anti-Leucodermia virus;
(4) adopt limiting dilution assay that 30 strain positive hybridoma cells are wherein cloned;
(5), filter out the monoclonal antibody of the anti-Leucodermia virus cyst membrane of 20 strains by immuno-electron microscope.
(6) select the monoclonal antibody E and the F of the different anti-Leucodermia virus 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: select the monoclonal antibody E maximum with the virus envelope binding site by immuno-electron microscope, be gold mark monoclonal antibody; Select the 4 kind monoclonal antibodies more by immuno-electron microscope with the virus envelope binding site, monoclonal antibody C, monoclonal antibody G, monoclonal antibody A and monoclonal antibody F, respectively with same concentration, be coated antibody with these 4 kinds of monoclonal antibodies of volume, detect same Leucodermia virus sample simultaneously, the result: when monoclonal antibody F is coated antibody, best results (seeing Table 1).Thereby, select monoclonal antibody E to mark monoclonal antibody, and monoclonal antibody F 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 Leucodermia virus
| Sheet is anti- | Monoclonal antibody C, | Monoclonal antibody G, | Monoclonal antibody A | Monoclonal antibody F |
| The gold-marking immunity tomographic results | + | + | + | ++ |
Annotate: ++ expression detection line displaing amaranth is than strong positive; It is red positive that+expression detection line shows;
The using method of white spot syndrome virus on-spot detection test paper of the present invention, i.e. the detection step of Leucodermia virus: at first, use viral extraction element, from the detected shrimp gill, extract Leucodermia virus apace, the preparation test sample; The process of described preparation test sample is: use is by test tube and smash the viral extraction element that rod is formed to pieces, will add liquid in the test tube earlier, then; get the tested shrimp cheek and be added in the test tube; with smashing rod to pieces it is fully smashed to pieces, virus is released in this test tube liquid, promptly make test sample.Concrete operating process is: add pond water, distilled water, tap water, the phosphate buffer of pH 6.0-8.O, in the Tris-sodium chloride damping fluid of pH 6.0-8.0 any one got 1-2 and only become the shrimp gill or whole juvenile prawn to test tube 0.5ml scale place, puts into test tube, fully smash to pieces, make test sample.
Then, application of sample end 4 water accepting layers of test paper are immersed in the test sample of test tube, liquid level surpasses the MAX line, reads the result in 5 minutes.
Positive findings: check layer 2 presents the nature controlling line 5 and the detection line 6 of two redness up and down, and expression has Leucodermia virus to infect;
Negative findings: check layer 2 upper end present a red nature controlling line 5, represent that no Leucodermia virus infects or its virus contains extremely low;
Testing result is invalid: in the application of sample 5 minutes, check layer 2 upper end nature controlling line 5 places do not have red line and occur, and represent that this test paper lost efficacy, and testing result is invalid.(see figure 2)
The mensuration of the minimum virus quantity that white spot syndrome virus on-spot detection test paper of the present invention detects is as follows:
The Leucodermia virus liquid dilution of purifying is measured with white spot syndrome virus on-spot detection test paper, and consequently: the minimum virus quantity of this detection paper is 1 μ g/ml, the results are shown in Table 2.
The measurement result of the minimum virus quantity of table 2
| Coated antibody | Antibody content (mg/ml) | Viral level (μ g/ml) | |||||
| 100 | 50 | 1 | 0.5 | 0.1 | 0.05 | ||
| 4G 9 | 4 | ++ | ++ | + | - | - | - |
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.
Embodiment 7.
White spot syndrome virus on-spot detection test paper of the present invention detects the repeatability of using method and measures as follows with stability:
(1) white spot syndrome virus on-spot detection test paper of the present invention detects and uses repeated experiment:
Select 2 kinds of samples, it is respectively: suffer from the shrimp gill of hickie disease disease, the normal shrimp gill.White spot syndrome virus on-spot detection test paper with the monoclonal antibody E of same batch the anti-Leucodermia virus of colloid gold label preparation detects above selected 2 kinds of samples, and the result shows that the shrimp gill sample of suffering from hickie disease disease is positive, and normal shrimp gill sample is negative.Same sample, the white spot syndrome virus on-spot detection test paper for preparing with the monoclonal antibody E of the anti-Leucodermia virus of colloid gold label of five different batches: the result is the same.
(2) stability experiment: with white spot syndrome virus on-spot detection test paper of the present invention put into respectively 4 ℃ with room temperature under, per 15 days with deposit under the room temperature this more than detection paper 2 kinds of samples once, per 30 days with deposit under 4 ℃ this 2 kinds of samples are once more than detection paper.Result: this test paper term of validity of depositing under 4 ℃ 12 months.This test paper term of validity of depositing under the room temperature 6 months.
Used instrument of the present invention and reagent:
Freezing microtome (available from LEICA company); Fluorescent microscope (available from OlYMPUS company); Inverted phase contrast microscope (available from OlYMPUS company); Membrane jetter (available from U.S. BIODOT company); Gold chloride (available from Chinese Shanghai reagent one factory, lot number 85-01-02 analyzes pure); 1640 (available from GIBCO companies); Hyclone (available from HYCLONE 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, a kind of white spot syndrome virus on-spot detection test paper, it comprises: the adhesive sticker carrier board of appropriate size; Preserving number is: the monoclonal antibody E anti-Leucodermia virus that the CCTCC-C200421 hybridoma is secreted, colloid gold label; Preserving number is: the monoclonal antibody F of the anti-Leucodermia virus that the CCTCC-C200423 hybridoma is secreted and goat anti-mouse igg is characterized in that: at the both ends of this carrier board, be respectively equipped with application of sample end water accepting layer and handheld terminal water accepting layer; Be provided with the detection layers of nitrocellulose membrane at this carrier board middle part, at this detection layers and application of sample end water accepting layer intersection, be provided with the glass layer piece of the monoclonal antibody E that is loaded with colloid gold label, this layer piece one end parts is arranged under the application of sample end water accepting layer, and its other end branch is arranged on this detection layers; With the detection layers of this layer piece continuity on be provided with detection line and nature controlling line, this detection line and nature controlling line place are coated with monoclonal antibody F and goat anti-mouse igg respectively.
2, according to the described white spot syndrome virus on-spot detection test paper of claim 1, it is characterized in that: the monoclonal antibody E of described colloid gold label, its preparation method is as follows:
(1) preparation of collaurum: the preparation technology with known collaurum mixes chlorauric acid solution with sodium citrate, and colloidal gold solution is made in heating;
(2) preparation of the monoclonal antibody E of colloid gold label: monoclonal antibody E 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 bovine serum albumin(BSA), Sodium azide, makes the monoclonal antibody E liquid of colloid gold label;
(3) be loaded with the preparation of glass layer piece of the monoclonal antibody E of colloid gold label: the monoclonal antibody E liquid of the colloid gold label that above-mentioned (2) step is made, be sprayed on the glass fibre membrane begin to ooze out to liquid till, freeze drying, 4 ℃ of preservations are standby.
3, a kind of using method by the described white spot syndrome virus on-spot detection test paper of claim 1 is characterized in that: at first, use viral extraction element, extract Leucodermia virus apace from the detected shrimp gill, the preparation test sample; Then, the handheld terminal of hand-held this test paper, the application of sample end water accepting layer of this test paper is inserted or drips this test sample thereon, the inherence was positioned at the detection line of detection layers lower end and showed red at the nature controlling line place that is being positioned at the detection layers upper end in 5 minutes, promptly two red lines, the expression Leucodermia virus is positive and the detection test paper is effective.
4, according to the using method of the described white spot syndrome virus on-spot detection test paper of claim 3, it is characterized in that: the process of described preparation test sample is: use is by test tube and smash the viral extraction element that rod is formed to pieces, earlier distilled water will be added in the test tube, the phosphate buffer of pH 6.0-8.0, in the Tris-sodium chloride damping fluid of pH 6.0-8.0 any one, then; get the tested shrimp cheek and be added in the test tube; with smashing rod to pieces it is fully smashed to pieces; virus is released in this test tube liquid, promptly makes test sample.
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| CN101930005A (en) * | 2010-07-16 | 2010-12-29 | 浙江大学 | Quick detection test paper strip for white spot syndrome viruses (WSSV) and preparation method thereof |
| CN101893632A (en) * | 2010-07-29 | 2010-11-24 | 中国海洋大学 | Test paper for detecting fish lymphocystis disease virus and preparation method thereof |
| CN101968488A (en) * | 2010-10-26 | 2011-02-09 | 吉林大学 | Colloidal gold fast diagnostic test strip of goose paramyxovirus disease |
| CN102109526B (en) * | 2011-02-12 | 2013-04-24 | 鲁东大学 | Test paper for quickly detecting grass carp reovirus (GCRV) and preparation method and using method thereof |
| CN104360068A (en) * | 2014-11-16 | 2015-02-18 | 中国海洋大学 | Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper |
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