CN101893632A - Test paper for detecting fish lymphocystis disease virus and preparation method thereof - Google Patents
Test paper for detecting fish lymphocystis disease virus and preparation method thereof Download PDFInfo
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- CN101893632A CN101893632A CN2010102390850A CN201010239085A CN101893632A CN 101893632 A CN101893632 A CN 101893632A CN 2010102390850 A CN2010102390850 A CN 2010102390850A CN 201010239085 A CN201010239085 A CN 201010239085A CN 101893632 A CN101893632 A CN 101893632A
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Abstract
The invention discloses test paper for detecting fish lymphocystis disease virus, which comprises a vector plate, a sample pad, a combined pad, a nitrocellulose film (NC film) detection layer and a water absorbing pad, wherein the sample pad and the water absorbing pad are positioned at two ends of the vector plate; and the NC film detection layer is positioned in the middle of the vector plate. The test paper is characterized in that: the junction of the sample pad and the NC film detection layer is provided with the combined pad carrying a colloidal gold labeled monoclonal antibody A, the edge at one end of the layer is overlapped under the sample pad, and the edge at the other end is overlapped on the NC film detection layer; and the NC film detection layer is provided with a detection line and a quality control line, the detection line close to the sample pad is coated with a monoclonal antibody B, and the quality control line close to the water absorbing pad is coated with goat anti-mouse IgG. The test paper has the advantages of simple, convenient and quick operation, no need of professional personnel and instruments, sensitive and accurate results, capacity of being transported and stored at normal temperature, and the like.
Description
Technical field
The present invention relates to a kind of test paper for detecting fish lymphocystis disease virus and preparation method thereof, it belongs to immunology and virology interleaving techniques field.
Background technology
Lymphocystis disease virus (Lymphocystis disease virus, LCDV), be subordinate to Iridoviridae (Iridoviridae), Lymphocystivirus (Lymphocystivirus) can cause fresh water, brackish water and the seawater fish generation lymphocyst disease of 34 sections more than 140 kinds all over the world.Fish lymphocyst disease was broken out first on a large scale in China Shandong lefteye flounder plant in 1997, subsequently in Hebei, Guangdong, zhejiang and other places all took place, its infection rate can be up to 80%, mortality ratio can reach 30%, brings enormous economic loss for the aquatic products aquaculture.The epidermis of ill fish, fin, the gill and afterbody etc. locate to occur tumour thing not of uniform size, in case the lymphocyst cell rupture, virion discharges, and can cause systemic infection, also easily form open ulcer surface, cause the secondary infection of other pathogen such as parasite, bacterium.The same with other virosis, the effective medicine of the sick shortage of fish lymphocyst.Therefore, be badly in need of the Fast Detection Technique of LCDV in the breeding production, early find the purpose of prevention early in the hope of reaching.
The method that detects fish LCDV at present both at home and abroad roughly comprises histopathology and transmission electron microscope observing, cell culture method, immunofluorescence technique, spot immune engram technology, PCR method and nucleic acid hybridization etc.The operation of these methods is complicated and consuming time longer, and the technician is had relatively high expectations, and needs certain experimental instrument and equipment, inapplicablely detects in real time in plant of basic unit, and it is imperative therefore to develop a kind of easy quick on-the-spot method that detects fish LCDV.
Summary of the invention
At the problems referred to above, the purpose of this invention is to provide a kind of test paper for detecting fish lymphocystis disease virus.
Test paper for detecting fish lymphocystis disease virus provided by the invention, comprise: carrier board, sample pad, pad, nitrocellulose filter detection layers and adsorptive pads, described sample pad and adsorptive pads are positioned at the two ends of carrier board, the nitrocellulose filter detection layers is positioned at the middle part of carrier board, it is characterized in that sample pad and nitrocellulose filter detection layers intersection are provided with the pad that is loaded with gold mark monoclonal antibody A, pad one end margin overlaps under the sample pad, and other end imbricate is on the nitrocellulose filter detection layers; The nitrocellulose filter detection layers is provided with detection line and nature controlling line, and detection line is coated with monoclonal antibody B near sample pad, and nature controlling line is coated with goat anti-mouse igg near adsorptive pads.
Another object of the present invention provides a kind of preparation method of test paper for detecting fish lymphocystis disease virus.
The preparation method of test paper for detecting fish lymphocystis disease virus provided by the invention is as follows: preparation recovery and the cultivation preserving number of (1) monoclonal antibody A and monoclonal antibody B are the hybridoma cell strain of CCTCC-C200419 and CCTCC-C200420, the lumbar injection BALB/c mouse, gather ascites, sad-ammonium sulfate method is purified and is obtained monoclonal antibody A and monoclonal antibody B.
(2) the 1. preparation of collaurum of the preparation of pad: adopt micro-wave oven-trisodium citrate reduction method to make the collaurum that grain size is 20～30nm.
2. the preparation of gold mark monoclonal antibody A: monoclonal antibody A is added in the above-mentioned 1. collaurum that the step makes in suitable ratio, add bovine serum albumin(BSA) again, preserve liquid with the gold mark behind the centrifugal purification and hang, be gold mark monoclonal antibody A liquid.
3. be loaded with the preparation of the pad of gold mark monoclonal antibody A: the gold that the above-mentioned 2. step makes is marked monoclonal antibody A liquid spray to glass fibre, freeze drying.
(3) preparation of nitrocellulose filter detection layers monoclonal antibody B and the goat anti-mouse igg that will adjust concentration is sprayed on the nitrocellulose filter detection layers, respectively as detection line and nature controlling line, dries in the rear enclosed liquid and soaks.
(4) being produced on of test paper for detecting fish lymphocystis disease virus sticked nitrocellulose filter detection layers, adsorptive pads, pad and sample pad in turn on the carrier board, is cut into the wide test paper of 3mm with cutting cutter again, test paper of the present invention.
The present invention adopts double-antibody sandwich immunochromatography principle, that is: if contain LCDV in the test sample, gold mark monoclonal antibody A combines with LCDV, form gold mark monoclonal antibody A-LCDV compound, when this compound chromatography during to the detection line of nitrocellulose filter detection layers, be coated on LCDV in the monoclonal antibody B recognition complex herein in advance, form the sandwich structure of gold mark monoclonal antibody A-LCDV-monoclonal antibody B, the colloid gold particle of the last mark of monoclonal antibody A is fixed herein and accumulation manifests macroscopic redness, the free gold mark monoclonal antibody A with the LCDV reaction does not continue to move ahead, when arriving the goat anti-mouse igg place that is coated in advance on the nature controlling line, the antibody type is that the gold mark monoclonal antibody A of IgG combines with it, form gold mark monoclonal antibody A-goat anti-mouse igg compound, also occur at the nature controlling line place fixing and accumulating manifesting macroscopic redness by colloid gold particle, consequently detection line and nature controlling line place all manifest redness, the expression LCDV positive; If do not contain LCDV in the test sample, the detection line place does not manifest redness, only manifests redness at the nature controlling line place, expression LCDV feminine gender; If the nature controlling line place does not manifest redness, illustrate that test paper lost efficacy, testing result is invalid.
The present invention has the following advantages: (1) is detected fast, and the naked eyes visible result can appear in 5～10min; (2) easy and simple to handle, do not need the professional; (3) the detection cost is low, need not professional instrument and equipment, is fit to on-the-spot the detection; (4) highly sensitive, high specificity, tool repeatability; (5) store convenient, good stability, the term of validity is 6 months under the room temperature, 4 ℃ of following terms of validity 12 months.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Fig. 2 is a testing result synoptic diagram of the present invention.
Fig. 3 is a testing result pictorial diagram of the present invention.
Wherein, 1, carrier board; 2, sample pad; 3, pad; 4, nitrocellulose filter detection layers; 5, adsorptive pads; 6, detection line; 7, nature controlling line.
Embodiment
Further specify the present invention below in conjunction with accompanying drawing and by specific embodiment.
Used instrument of the present invention and reagent: inverted microscope (available from OLYMPUS company); High speed freezing centrifuge (SIGMA company); Spray film instrument (available from BIODOT company); Gold chloride (, analyzing pure) available from Chinese Shanghai reagent one factory; Trisodium citrate (, analyzing pure) available from Chinese Shanghai reagent one factory; RPMI 1640 (available from HYCLONE company); Goat anti-mouse igg (available from SIGMA company); Bovine serum albumin(BSA) (available from SIGMA company); Tween-20 (available from SIGMA company); Nitrocellulose filter (available from WHATMAN company).
The preparation of embodiment 1. test paper for detecting fish lymphocystis disease virus.
(1) preparation of monoclonal antibody A and monoclonal antibody B recovery and cultivation preserving number are the hybridoma cell strain of CCTCC-C200419 and CCTCC-C200420, the lumbar injection whiteruss is in healthy BALB/c mouse in 8～10 ages in week, every 0.5mL, pneumoretroperitoneum injections in 7 days are cultured to the hybridoma of logarithmic phase, every 1 * 10
6Individual cell.Collect ascites after 10～15 days, 3000rpm, 4 ℃ of centrifugal 10min remove precipitation and get supernatant, and supernatant is purified through sad-ammonium sulfate method and is obtained monoclonal antibody A and monoclonal antibody B.
(2) preparation of colloid gold label monoclonal antibody A 1. the preparation of collaurum get 0.01% chlorauric acid solution 100mL, put into micro-wave oven, high-grade fire boiling 2min, a certain amount of 1% citric acid three sodium solution of rapid disposable adding, put into micro-wave oven, low and middle-grade fire continue heating 3min, are cooled to room temperature and supply dehydration, make the collaurum that grain size is 20～30nm.Regulating the pH value with the 0.1mol/L solution of potassium carbonate is 8.2,4 ℃ of preservations.
2. ocular estimate determines that the optimum mark of collaurum and monoclonal antibody A measures 7 test tubes and respectively add the 1mL collaurum, adds the monoclonal antibody A to be marked of 0,5,10,15,20,25,30 μ g more respectively, and concussion mixes 20min, and room temperature leaves standstill 10min.Then add 100 to every pipe? L 10% sodium chloride solution, concussion mixes 10min, and room temperature leaves standstill the above observations of 2h.The test tube that does not add monoclonal antibody or monoclonal antibody quantity not sufficient, the unstable formation of collaurum becomes hepatic coagulation phenomenon by redness, adds the amount arrival of monoclonal antibody or surpasses the quantitative test tube of minimum steady, and it is red constant that the collaurum color keeps.Make collaurum keep red minimum monoclonal antibody amount to be the needed minimum monoclonal antibody amount of mark collaurum, 20% the monoclonal antibody amount of adding on this basis is the actual use amount of monoclonal antibody of mark collaurum.
3. the preparation of gold mark monoclonal antibody A is got collaurum and is added monoclonal antibody A in suitable ratio, slowly stir 30min after, be 1% to wherein adding bovine serum albumin(BSA) (BSA) to final concentration again, continue to stir 20min.With above-mentioned solution through 3000rpm, 4 ℃ of centrifugal 20min, remove precipitation and get supernatant, again with supernatant through 13500rpm, 4 ℃ of centrifugal 30min, abandoning supernatant must precipitate, and precipitation is washed with golden labeling antibody cleansing solution (the 0.01mol/L pH 7.4PBS that contains 1%BSA), centrifugal once more, inhale the precipitation of going supernatant to obtain and be gold mark monoclonal antibody A.Precipitation is suspended in golden labeling antibody preserves in the liquid (containing 1% sucrose, 1%BSA, the 0.01mol/L pH 7.4PBS of 0.1%Tween-20 and 0.02% Sodium azide) 4 ℃ of preservations.
(3) preparation of pad 3 is marked monoclonal antibody A liquid with gold and is evenly sprayed on the glass fibre freeze drying.
(4) preparation of nitrocellulose filter detection layers 4 is 1mg/mL with the concentration that 0.01mol/L pH 7.4PBS adjusts monoclonal antibody B, with spray film instrument it is sprayed on the nitrocellulose filter detection layers, as detection line 6.Equally, is the concentration of adjusting goat anti-mouse igg 500? g/mL is sprayed at it on nitrocellulose filter detection layers, and at a distance of 5mm, nature controlling line 7 is near adsorptive pads 5 as nature controlling line 7, two lines, and detection line 6 is near sample pad 2.Bag by after the good nitrocellulose filter detection layers drying, is soaked 1h, PBST (the 0.01mol/L pH 7.4PBS that contains 0.05%Tween-20) rinsing, drying in 37 ℃ of confining liquids (the 0.01mol/L pH 7.4PBS that contains 2%BSA).
(5) making of test paper for detecting fish lymphocystis disease virus of the present invention 1. the assembling of test paper for detecting fish lymphocystis disease virus plate put on one's gloves, paste nitrocellulose filter detection layers 4, pad 3, sample pad 2 and adsorptive pads 5 in turn in a side of carrier board 1, be assembled into test paper plate.Colored self-adhesive paper is sticked on adsorptive pads 5 surfaces at test paper plate, and the self-adhesive paper that has MAX line label is sticked on sample pad 2 and pad 3 surfaces.Wherein the coboundary of nitrocellulose filter detection layers 4 overlaps under adsorptive pads 5 lower limbs, and nitrocellulose filter detection layers 4 lower limbs overlap under pad 3 coboundarys, and the coboundary of sample pad 2 overlaps on pad 3 lower limbs.The (see figure 1) 2. making of the test paper for detecting fish lymphocystis disease virus test paper plate that will post self-adhesive paper is put into slitting trough inscribe and is slit into the wide test paper of 3mm, the test paper that cuts is put into the aluminium foil bag that drying agent is housed seals, test paper of the present invention.
Fish tissue to be checked (epidermis, the gill, fin, stomach, intestines etc.) is got in the preparation of using method (1) test sample of embodiment 2. test paper for detecting fish lymphocystis disease virus, mix with the TNE damping fluid by 1: 10 (W/V), adding appropriate amount of quartz sand grinds with mortar, the homogenate of 7000～8000rpm ice bath is got supernatant as test sample.
(2) adsorptive pads 5 one ends of the hand-held test paper of the use of test paper for detecting fish lymphocystis disease virus immerse sample pad 4 one ends in the test sample, and liquid level does not surpass markings " MAX ", keeps flat test paper, observations in 5～10min.
(3) result judges 1. positive findings: nitrocellulose filter detection layers 4 nature controlling lines 6 and detection line 7 all occur red, and expression has LCDV to infect.
2. negative findings: 4 nature controlling lines of nitrocellulose filter detection layers 6 occur red, represents no LCDV infection.
3. null result: nitrocellulose filter detection layers 4 nature controlling lines 6 no red lines occur, and the expression operation has problem or test paper inefficacy (see figure 2).
Mensuration (1) the immunochromatographic method measurement sensitivity test of the embodiment 3. test paper for detecting fish lymphocystis disease virus sensitivity fish LCDV purification liquid of detection paper gradient dilution.Result: can detected minimum virus quantity be 1 μ g/mL.
(2) after immunogold silver staining enhancing sensitivity test immunochromatographyassay assay finishes, test paper to be strengthened (is contained 0.5% sodium chloride with cleansing solution, the 0.05mol/L pH 8.2Tris-hydrochloric acid of 0.5%Tween-20) washing 2～3min, balance 5min in the citrate buffer of 0.05mol/L pH 3.8 again.Put into silver-colored developer solution lucifuge colour developing 10～20min, the distilled water washing.Move into 1min in the stop bath (20% sodium thiosulfate, 15% sodium sulphite), distilled water washing, air dry.The result: immunogold silver staining can be increased to the sensitivity of test paper 0.01 μ g/mL.
The measurement result of the minimum virus quantity of table 1
Annotate: ++ expression is than strong positive; + expression is positive;-expression detection line is colourless negative.
3 groups of samples of specificity, repeatability and stability test (1) specificity test and Selection of embodiment 4. test paper for detecting fish lymphocystis disease virus, the 1st group of sample: fish LCDV crude extract.The 2nd group of sample: the tissue homogenate supernatant of suffering from the sick fish of lymphocyst.The 3rd group of sample: the tissue homogenate supernatant of normal fish.With 3 groups of samples more than the detection paper and with the PCR method relatively.The testing result unanimity: 1st, 2 groups of samples are positive, and the 3rd group of sample is negative.(see figure 3) (2) replica test above 3 groups of samples of the detection paper of different batches, each batch test paper duplicate detection 5 times, no significant difference as a result.
(3) stability test is put into test paper under room temperature and 4 ℃, and per 15 days with 3 groups of samples more than the detection paper once.The result: the term of validity is 6 months when preserving under the room temperature, and the term of validity is 12 months during 4 ℃ of following preservations.
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 (2)
1. test paper for detecting fish lymphocystis disease virus, comprise: carrier board, sample pad, pad, nitrocellulose filter detection layers and adsorptive pads, described sample pad and adsorptive pads are positioned at the two ends of carrier board, the nitrocellulose filter detection layers is positioned at the middle part of carrier board, it is characterized in that sample pad and nitrocellulose filter detection layers intersection are provided with the pad that is loaded with gold mark monoclonal antibody A, pad one end margin overlaps under the sample pad, and other end imbricate is on the nitrocellulose filter detection layers; The nitrocellulose filter detection layers is provided with detection line and nature controlling line, and detection line is coated with monoclonal antibody B near sample pad, and nature controlling line is coated with goat anti-mouse igg near adsorptive pads.
2. the preparation method of test paper for detecting fish lymphocystis disease virus according to claim 1 is characterized in that it comprises the steps:
(1) preparation of monoclonal antibody A and monoclonal antibody B
Recovery and cultivation preserving number are the hybridoma cell strain of CCTCC-C200419 and CCTCC-C200420, and the lumbar injection BALB/c mouse is gathered ascites, and sad-ammonium sulfate method is purified and obtained monoclonal antibody A and monoclonal antibody B;
(2) preparation of pad
1. the preparation of collaurum: adopt micro-wave oven-trisodium citrate reduction method to make the collaurum that grain size is 20～30nm;
2. the preparation of gold mark monoclonal antibody A: monoclonal antibody A is added in the above-mentioned 1. collaurum that the step makes in suitable ratio, add bovine serum albumin(BSA) again, preserve liquid with the gold mark behind the centrifugal purification and hang, be gold mark monoclonal antibody A liquid;
3. be loaded with the preparation of the pad of gold mark monoclonal antibody A: the gold that the above-mentioned 2. step makes is marked monoclonal antibody A liquid spray to glass fibre, freeze drying;
(3) preparation of nitrocellulose filter detection layers
Monoclonal antibody B and the goat anti-mouse igg of adjusting concentration are sprayed on the nitrocellulose filter detection layers,, dry in the rear enclosed liquid and soak respectively as detection line and nature controlling line;
(4) making of test paper for detecting fish lymphocystis disease virus
On carrier board, stick nitrocellulose filter detection layers, adsorptive pads, pad and sample pad in turn, be cut into the wide test paper of 3mm with cutting cutter again, test paper of the present invention.
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| CN103278636A (en) * | 2013-06-04 | 2013-09-04 | 大连海洋大学 | Colloidal gold immune chromatography test paper strip of shewanella smarisflavi and preparation method thereof |
| CN104792781A (en) * | 2015-04-30 | 2015-07-22 | 张舒捷 | Detection test strip for Barium ion in drinking water and water source and detection method thereof |
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| CN104792781A (en) * | 2015-04-30 | 2015-07-22 | 张舒捷 | Detection test strip for Barium ion in drinking water and water source and detection method thereof |
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Application publication date: 20101124 |