CN104360068A - Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper - Google Patents
Semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV), and preparation method of test paper Download PDFInfo
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Abstract
The invention discloses a semi-quantitative rapid detection test paper for white spot syndrome virus (WSSV). The semi-quantitative rapid detection test paper comprises a carrier plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad and the water absorption pad are respectively arranged at the two ends of the carrier plate; the nitrocellulose membrane is arranged in the middle of the carrier plate; the gold-labeled pad loaded with a gold-labeled monoclonal antibody A is arranged at the position where the sample pad intersects with the nitrocellulose membrane; the edge of one end of the gold-labeled pad is overlapped under the sample pad, and the edge of the other end of the gold-labeled pad is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with three detection lines (T1, T2 and T3) which are sequentially arranged, and a quality control line; the three detection lines are respectively enveloped with a monoclonal antibody B, and the monoclonal antibodies B have different concentrations; the concentrations of the monoclonal antibodies B on the T1, T2 and T3 are sequentially reduced from the side of the gold-labeled pad; the quality control line close to the water absorption pad is enveloped with goat anti-mouse IgG. The semi-quantitative rapid detection test paper is rapid in detection; after the test paper is used, the result visible to the naked eyes can be obtained after about 10 minutes, and a pathogen can be detected in a way of relative quantification; the test paper is simple and convenient to operate and low in detection cost, thus being suitable for field test of WSSV in the aquaculture process of prawns, crabs, etc.
Description
Technical field
The present invention relates to a kind of prawn white spot syndrome virus (White Spot Syndrome Virus, WSSV) detection method, more relate to a kind of prawn white spot syndrome virus sxemiquantitative quick detection test paper and preparation method thereof, it belongs to immunology, virology and diagnostic reagent interleaving techniques field.
Background technology
Prawn white spot syndrome virus disease is the explosive disease caused by prawn WSSV, and after prawn infects WSSV, in 3 ~ 12 days, mortality ratio can reach 100%.This disease has frequently occurred since breaking out first from 1993, constrains shrimp culture industry healthy and sustainable development, and effectively controlling outbreak of disease becomes the most outstanding and urgent problem.Owing to lacking effective remedy measures, therefore early stage, quick, quantitative detection WSSV the breaking out and early-warning and predicting this disease of prevention and control that WSSV infects, reduce economic loss significant.
WSSV detection method conventional at present has PCR (PCR), real-time quantitative PCR, indirect immunofluorescence, enzyme linked immunoassay etc., these technology all need instrumentation and professional, be not suitable for the real-time detection of plant of basic unit, therefore, a kind of method that is easy, quick, on-the-spot, that quantitatively detect prawn WSSV is developed imperative.
Colloidal gold immune chromatography test have easily carry, simple and efficient to handle, result naked eyes as seen, do not need professional and equipment, the advantage such as economical and practical, it is a kind of very promising instant or onthe technology of site test, development in recent years is rapid, is widely applied in biomedical sector particularly medical test.In Diagnosing Diseases of Aquatic Animals, although prepared list (many) clonal antibody of multiple cultivated animals cause of disease, field quick detection reagent strip is also well developed, only has several bacillary and viral cause of disease quick detection test paper at present, comprise WSSV quick detection test paper, as: Chinese patent " detects test strips of white spot syndrome virus (WSSV) and preparation method thereof " (CN1932520A), by preparing the VP19 of WSSV, the polyclonal antibody of VP28 expressing protein, WSSV Test paper has been prepared as capture antibody (golden labeling antibody) and detection antibody using it.But traditional immune chromatography test paper can only carry out qualitative detection to cause of disease, can not quantitative test, cannot meet in cultivation site fast quantification cause of disease thus the demand of Real-Time Evaluation disease progression process; In addition, capture antibody and detection antibody use same antibody, can cause the epitope that the two competition is identical, affect the detection sensitivity of test paper, and polyclonal antibody easily cross reaction occur, produce false positive.
Summary of the invention
For the problems referred to above, the object of this invention is to provide a kind of prawn WSSV sxemiquantitative quick detection test paper of easy, quick, highly sensitive, high specificity, its testing result naked eyes are visible, do not need special instrument equipment, the sxemiquantitative being applicable to WSSV in the shell-fish such as the shrimps of cultivation site, crab class detects fast.
Another object of the present invention is to provide the preparation method of above-mentioned prawn WSSV sxemiquantitative quick detection test paper.
The object of the invention is to be realized by following technical scheme:
A kind of prawn white spot syndrome virus sxemiquantitative quick detection test paper, comprise carrier board, sample pad, gold mark pad, nitrocellulose filter and adsorptive pads, sample pad and adsorptive pads are positioned at the two ends of carrier board, and nitrocellulose filter is positioned at the middle part of carrier board; Sample pad and nitrocellulose filter intersection are provided with and are loaded with the gold mark pad that gold marks monoclonal antibody (monoclonal antibody) A, and gold mark pad one end imbricate is under sample pad, and other end imbricate is on nitrocellulose filter; Nitrocellulose filter is provided with three detection lines T1, T2, T3 and a nature controlling line of being arranged in order, and three described detection lines are coated with the monoclonal antibody B of variable concentrations, and the concentration of the upper monoclonal antibody B of T1, T2, T3 reduces successively from gold mark pad side; Nature controlling line near adsorptive pads is coated with goat anti-mouse igg.
The different epi-positions of described monoclonal antibody A and B anti-WSSV respectively, be respectively by two strain titles: hybridoma cell strain WSSV-K1, WSSV-K2, preserving number is respectively: CCTCC NO:C2014160 and CCTCC NO:C2014161, depositary institution is: China typical culture collection center, address: Wuhan City, Hubei Province Wuhan University, preservation date: the hybridoma on September 2nd, 2014 secretes gained respectively.The preparation method of described monoclonal antibody A and B, comprises the steps: with the WSSV of purifying as antigen immune Balb/C mouse; Merge by the splenocyte of immune mouse and myeloma cell; Through immunology detection screening technique, filter out hybridoma WSSV-K1 and WSSV-K2 secreting anti-WSSV monoclonal antibody, the antibody of its secretion is anti-WSSV monoclonal antibody A and B.
Observe under inverted microscope, growth conditions good hybridoma division vigorous, outward appearance is full, perfectly round, refractivity is strong, cell size is homogeneous, well adherent; Above-mentioned hybridoma has unlimited division and proliferation ability; The above-mentioned hybridoma cellar culture 2-3 days grown fine, its nutrient culture media changes yellow into by pink, a large amount of anti-WSSV monoclonal antibody containing hybridoma secretion in this nutrient culture media.
The preparation method of prawn WSSV sxemiquantitative quick detection test paper, comprises the following steps:
(1) preparation and purification of monoclonal antibody A and monoclonal antibody B
Recover and cultivate hybridoma cell strain WSSV-K1 and WSSV-K2 secreting anti-WSSV monoclonal antibody, respectively lumbar injection BALB/c mouse, gathering ascites, caprylic acid-ammonium is purified and is obtained anti-WSSV monoclonal antibody A and monoclonal antibody B;
(2) preparation of gold mark pad
1. the preparation of collaurum and gold mark monoclonal antibody A:
Adopt micro-wave oven-trisodium citrate reduction method to obtain the collaurum that grain size is 20 ~ 30 nm, regulate collaurum pH value to be 8.2 with 0.1 mol/L solution of potassium carbonate; Anti-WSSV monoclonal antibody A is added in collaurum in suitable ratio, then adds bovine serum albumin(BSA) (BSA), hang with gold mark conserving liquid after centrifugal purification, be gold mark monoclonal antibody A liquid;
2. the preparation of the gold mark pad of gold mark monoclonal antibody A is loaded with:
By the gold mark monoclonal antibody A liquid spray made on glass fibre, freeze drying;
(3) preparation of nitrocellulose filter
Respectively the anti-WSSV monoclonal antibody B of 3 parts of variable concentrations is sprayed at nitrocellulose filter as detection line T1, T2, T3 according to a determining deviation and concentration order, goat anti-mouse igg is sprayed at nitrocellulose filter as nature controlling line, dries, soak in confining liquid;
(4) making of prawn WSSV sxemiquantitative quick detection test paper
Carrier board sticks nitrocellulose filter, adsorptive pads, gold mark pad and sample pad successively, then is cut into the wide test strips of 3mm with cutting cutter, test paper of the present invention.Be sealed in preservation in aluminium foil bag.
Utilize the method that prawn WSSV half-quantitative detection test paper detects WSSV, its step is as follows:
About 10s in measuring samples is immersed in sample pad 1 one end by adsorptive pads 4 one end of hand-held test paper, and immersion depth is about 0.5cm, keeps flat test paper and is about 10min, visual results, according to detection line T1, T2, T3 colour developing situation quantitate virus content.
The pre-treating method of described measuring samples is as follows:
Get and suffer from the leukodermal Chinese prawn gill, every 1 g gill adds 5 mL TNE damping fluids (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na
20.372 g, H
2o 1 000 mL, pH are 7.5) 25% sucrose solution prepared, ice bath fully grinds 3 ~ 5 min, is utilized by lapping liquid yarn thin,tough silk to filter, and gets supernatant as measuring samples liquid.
The present invention adopts double-antibody sandwich immunochromatography principle.If the WSSV containing more amount in measuring samples, when sample diffusion is to gold mark pad, gold mark monoclonal antibody A will be combined with WSSV, form a large amount of gold mark monoclonal antibody A-WSSV compounds, due to capillarity, this compound enters nitrocellulose filter along with sample liquid, when sample liquid arrives detection line T1, part gold mark monoclonal antibody A-WSSV compound be coated on monoclonal antibody B herein and be combined, form the double-antibody sandwich structure of gold mark monoclonal antibody A-WSSV-monoclonal antibody B, on monoclonal antibody A, the colloid gold particle of mark is fixed herein and is accumulated and makes detection line T1 manifest macroscopic redness, due to detection line T1 to wrap the monoclonal antibody B concentration of quilt the highest, T1 then combines the gold mark monoclonal antibody A-WSSV compound of most volume.Remaining gold mark monoclonal antibody A-WSSV compound continues to move ahead along film with sample liquid, and when arriving detection line T2 and detection line T3, same reaction occurs, and macroscopic redness also appears in detection line T2 and detection line T3.When final sample liquid proceeds to nature controlling line, the free gold mark monoclonal antibody A(monoclonal antibody A Antibody types do not reacted with WSSV is IgG) be combined with the goat anti-mouse igg be coated on nature controlling line in advance, form gold mark monoclonal antibody A-goat anti-mouse igg compound, also occur at nature controlling line place being fixed by colloid gold particle and accumulating manifesting macroscopic redness.When WSSV concentration in measuring samples reduces, the gold mark monoclonal antibody A-WSSV compound of formation reduces, and the amount that can arrive the gold mark monoclonal antibody A-WSSV compound of detection line T3 also reduces, and detection line T3 does not develop the color gradually; Along with WSSV concentration continues to reduce, detection line T2, T1 do not develop the color successively gradually.When detection line T1 had both been the detectability of test strips by manifesting the concentration that light red is changed to WSSV when not developing the color.The quantification range of WSSV is from the detection WSSV concentration be limited to when making T1, T2, T3 all develop the color.Entirely to develop the color according to T1, T2, T3 tri-detection lines, or the colour developing of T1, T2 two detection lines, or T1 detection line colour developing, can quantitative WSSV content in sample.If do not prescribe a time limit lower than ELISA test strip containing WSSV or WSSV concentration in detection sample, 3 detection lines neither present redness, only have nature controlling line to manifest redness; If nature controlling line and detection line place do not show redness, illustrate that test paper lost efficacy, testing result is invalid.
The present invention has the following advantages: detect fast, and naked eyes direct-view result appears in about 10min, and can detect cause of disease by relative quantification; Easy and simple to handle, do not need professional; Testing cost is low, without the need to special instrument equipment, is applicable to cultivation site and detects; Highly sensitive, high specificity, tool is repeatable; It is convenient to store, good stability, the term of validity 6 months under room temperature, the term of validity 12 months at 4 DEG C.Be applicable to WSSV in the breeding process such as prawn, crab class quick, easy, accurately detect.
Accompanying drawing explanation
Fig. 1. the structural representation of WSSV sxemiquantitative quick detection test paper of the present invention.
1, sample pad; 2, gold mark pad; 3, nitrocellulose filter; 4, adsorptive pads; 5, carrier board; 6, detection line T1; 7, detection line T2; 8, detection line T3; 9, nature controlling line.
Fig. 2. the testing result schematic diagram of WSSV sxemiquantitative quick detection test paper of the present invention.
I: WSSV concentration >=6.26 μ g/ml; II: 3.13 μ g/ml≤WSSV concentration < 6.26 μ g/ml; III: 783ng/ml≤WSSV concentration < 3.13 μ g/ml; IV: WSSV concentration < 783ng/ml; V: test strips lost efficacy.
Fig. 3. the result figure that WSSV sxemiquantitative quick detection test paper of the present invention detects shrimp samples.
Embodiment
The present invention is further illustrated by specific embodiment below in conjunction with accompanying drawing.
following examples instrument and reagent:
Inverted microscope (purchased from Olympus company); High speed freezing centrifuge (Sigama company); Spray film instrument XYZ3060 (purchased from Biodot company); Semi-automatic pad pasting instrument LM5000 (purchased from Biodot company); Slitting instrument (Bidot company); Trisodium citrate (purchased from Chinese Shanghai reagent one factory, analyzing pure); Gold chloride (purchased from Chinese Shanghai reagent one factory, analyzing pure); Goat anti-mouse igg (purchased from Sigama company); Bovine serum albumin(BSA) (purchased from Sigama company); Tween-20(is purchased from Sigama company); Nitrocellulose filter (purchased from Whatman company).
embodiment 1:the preparation of prawn WSSV sxemiquantitative quick detection test paper
1. the development of anti-WSSV monoclonal antibody A and monoclonal antibody B and ascites preparation and purifying
(1) Fusion of Cells: mix as antigen immune BALB/c mouse using the WSSV of purifying and Freund's complete adjuvant equivalent (V/V), after 2 weeks, WSSV and freund 's incomplete adjuvant equivalent (V/V) mix rear booster immunization once; Then tail vein injection booster immunization was adopted once every 1 week with the WSSV of purifying, third time booster immunization after 3 days, aseptic taking-up mouse spleen carries out Fusion of Cells with the P3-X63-Ag8U1 myeloma cell being in exponential phase with polyglycol solution, be added drop-wise in 96 well culture plates after cell suspension after fusion is mixed, put into CO
2concentration is cultivate in 37 DEG C of incubators of 4.5%.Inverted microscope observation of cell growing state, gets Hybridoma Cell Culture supernatant and detects after two weeks.
(2) screening and clone: after fusion, starts to detect when hybridoma group grows to the hole floorage 1/3 of 96 well culture plates, adopts indirect ELISA screening positive hybridoma cell, specific as follows:
1. be coated in by the WSSV of purifying in (50 μ L/ hole) in 96 hole ELISA Plate, 4 DEG C are spent the night;
2. sucking-off coating buffer, wash with the phosphate buffer (PBST: containing the 0.01 mol/L PBS of 0.05% Tween-20, pH 7.4) containing 0.05% Tween-20, each 5min, washes 3 times;
3. every hole adds the bovine serum albumin(BSA) (PBS preparation) of 200 μ L 3%, 37 DEG C of closed 1h, and same 2. method washs 3 times;
4. Hybridoma Cell Culture supernatant is added to ELISA Plate by every hole 50 μ L, hatch 1h for 37 DEG C; Same 2. method washs 3 times;
5. the sheep anti-Mouse Ig(1:4000 of alkali phosphatase enzyme mark dilutes) add to ELISA Plate by every hole 50 μ L, hatch 1h for 37 DEG C; Same 2. method washs 3 times;
6. every hole adds 200 μ L substrate nitrite ions, and dark place reaction 5-20min, every hole adds the NaOH solution cessation reaction of 50 μ L 2 mol/L, measures OD value by 405nm operation wavelength.
With bag by PBS for negative control, survey wavelength each hole absorbance value when being 405nm, calculating the ratio (P/N) of each experimental port and negative control absorbance value, be the positive when P/N >=2.1.Limiting dilution assay is adopted to carry out clone and frozen to the positive hybridoma cell detected.
(3) preparation of ascites: hybridoma cell strain WSSV-K1, WSSV-K2(preserving number screening the anti-WSSV monoclonal antibody of specific secretion is: CCTCC NO:C2014160 and CCTCC NO:C2014161, preservation date: on September 2nd, 2014), two strain monoclonal antibodies identify the different epi-position of WSSV respectively, are IgG type.First lumbar injection BALB/c mouse sterile liquid paraffin in 8 ~ 12 week age, every injected in mice 0.3 mL.After one week, with RPMI 1640 by the hybridoma of cultivation from Tissue Culture Plate gently purge get off, be collected in 10mL sterile centrifugation tube, centrifugal 3 min of 1 000 rpm, repeated washing 1 time, with appropriate RPMI1640 by cell precipitation resuspended mixing gently, every mouse peritoneal injects 0.3 mL hybridoma liquid.Close observation mouse state, after 7 ~ 10 days, mouse web portion obviously expands, and collects ascites in sterile centrifugation tube.
(4) purifying of ascites: get ascites by 1: 2(V/V) add acetate buffer, dropwise add caprylic acid (every 1 mL ascites adds 33 μ L caprylic acids) and stir 30 min, after 4 DEG C of standing 2 h, centrifugal 30 min at 12 000 rpm 4 DEG C, supernatant is through 0.45 μm of membrane filtration, add 0.1 mol/L PBS(KCl 0.2 g of 1/10 volume, NaCl 8.0 g, KH
2pO
40.2 g, Na
2hPO
412H
2o 2.9 g, H
2o 1 000 mL), pH to 7.4 is regulated by 1 mol/L NaOH solution, adding saturated ammonium sulfate solution makes the saturation degree of ammonium sulfate be 45%, stir 30 min, after 4 DEG C of standing 2 h, centrifugal 30 min at 12 000 rpm 4 DEG C, precipitate and dissolve with appropriate 0.01 mol/L PBS, and dialyse 3 ~ 4 times, every minor tick 4h.The protein concentration recording the anti-WSSV monoclonal antibody of two strains A and B with Coomassie Brilliant Blue is respectively 10 mg/mL and 2 mg/mL.
2. the preparation of colloid gold label monoclonal antibody A
(1) preparation of collaurum
Get 0.01% chlorauric acid solution 100 mL, put into micro-wave oven, high-grade fire boiling 2 min, disposablely rapidly add a certain amount of 1% citric acid three sodium solution, put into micro-wave oven, low and middle-grade fire continues heating 3 min, be cooled to room temperature and supply dehydration, make the collaurum that grain size is 20 ~ 30 nm.Be 8.2,4 DEG C of preservations by 0.1 mol/L solution of potassium carbonate adjust ph.
(2) the optimum mark amount of collaurum and monoclonal antibody A
Get 7 test tubes and respectively add 1 mL collaurum, then add the monoclonal antibody A to be marked of 0,5,10,15,20,25,30 μ g respectively, concussion mixing 10 min, room temperature leaves standstill 10 min; In above-mentioned each pipe, add 100 μ L 10% NaCl solution respectively again, concussion mixing 10 min, room temperature leaves standstill 2 h.Measure the OD value of each pipe at 520 nm places, with OD value for ordinate, albumen consumption is that horizontal ordinate makes curve, get the minimum monoclonal antibody amount that albumen consumption located that curve is close with transverse axis is at first stable colloid gold, add the actual amount that 20% is monoclonal antibody needed for stable colloid gold on this basis.
(3) determination of collaurum and monoclonal antibody A optimum mark pH value
Get 7 test tubes and respectively add 1 mL collaurum, with 0.1 mol/L K
2cO
3collaurum pH value is regulated to be respectively 4,5,6,7,8,9,10; Often pipe adds the monoclonal antibody A of 50 μ g, and after 10 min that vibrate, room temperature places 10 min; Add 100 μ L 10% NaCl solution again, after 10 min that vibrate, room temperature places 2 h.Measure the OD value of each pipe at 520 nm places, with OD value for ordinate, pH value is that horizontal ordinate makes curve, and the pH value (X) that recording curve OD value maximum is corresponding is the optimal pH of colloid gold label monoclonal antibody A.
(4) preparation of gold mark monoclonal antibody A
Get collaurum and add monoclonal antibody A in suitable ratio, after slowly stirring 30 min, then to add BSA wherein to final concentration be 1%, continues stirring 20 min.By above-mentioned solution through 3 000 rpm, 4 DEG C of centrifugal 20 min, remove precipitation and get supernatant, again by supernatant through 13 500 rpm, 4 DEG C of centrifugal 30 min, abandoning supernatant must precipitate, precipitate with golden labeling antibody cleansing solution (containing the 0.01 mol/L PBS of 1% BSA, pH 7.4) washing, centrifugal, suck the precipitation that supernatant obtains and be gold mark monoclonal antibody A.Precipitation is suspended in golden labeling antibody conserving liquid (containing 1% sucrose, 0.01 mol/L PBS of 1% BSA, 0.1% Tween-20 and 0.02% Sodium azide, pH 7.4), 4 DEG C of preservations.
3. the preparation of gold mark pad 2
Gold being marked monoclonal antibody A uniform liquid sprays on glass fibre, freeze drying.
4. the preparation of nitrocellulose filter 4
With 0.01 mol/L PBS (pH 7.4), monoclonal antibody B concentration is adjusted to 1 mg/mL, 0.5mg/mL and 0.25mg/mL respectively, be sprayed on nitrocellulose filter as T1 with the monoclonal antibody B that concentration is 1mg/mL by spray film instrument, be that the monoclonal antibody B of 0.5mg/mL is sprayed on nitrocellulose filter as detection line T2 using concentration, be that the monoclonal antibody B of 0.25mg/mL is sprayed at as detection line T3 on nitrocellulose filter using concentration, at a distance of 2mm between detection line T1, T2, T3; The concentration of adjustment goat anti-mouse igg is 500 μ g/mL, is sprayed at as nature controlling line 9 on nitrocellulose filter, and nature controlling line 9 and detection line T3 are at a distance of 8 mm, and nature controlling line 9 is near adsorptive pads 4, and detection line is near sample pad 1.By bag by after good nitrocellulose filter drying, in 37 DEG C of confining liquids (containing the 0.01 mol/L PBS of 2% BSA, pH 7.4), soak 1 h, PBST rinsing, dry.
5. the making of prawn WSSV half-quantitative detection test paper of the present invention
Put on one's gloves, on the side of carrier board 5 is pasted in turn, nitrocellulose filter 3, gold mark pad 2, sample pad 1 and adsorptive pads 4, be assembled into test paper plate.Colored self-adhesive paper is sticked with gold mark pad 2 surface in the sample pad 1 of test paper plate.Wherein the coboundary of nitrocellulose filter 3 overlaps under adsorptive pads 4 lower limb, and nitrocellulose filter 3 lower limb overlaps under gold mark pad 2 coboundary, and the coboundary of sample pad 1 overlaps on gold mark pad 2 lower limb (Fig. 1).
The test paper plate posting self-adhesive paper is put into the test paper that slitting trough internal cutting becomes 3 mm wide, the test paper cut is put into the aluminium foil bag that drying agent is housed and seals, test paper of the present invention.
embodiment 2:the detectability of prawn WSSV half-quantitative detection test paper and quantification range measure
1. the purifying of WSSV
Get and suffer from the leukodermal Chinese prawn gill, every 1 g gill adds 5 mL TNE damping fluids (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na
20.372 g, H
2o 1 000 mL, pH is 7.5) 25% sucrose solution prepared, ice bath fully grinds 3 ~ 5 min, lapping liquid is filled in 50 mL centrifuge tubes, respectively through 3 000 at 4 DEG C, 5 000, centrifugal 20 min of 8 000 rpm, supernatant centrifugal 1.5 h of Hitachi's ultracentrifuge 20 000 rpm, precipitation add appropriate 25% sucrose solution resuspended after, through discontinuous sucrose density gradient centrifugal 2 h at 25 000 rpm 4 DEG C, with the virus band between syringe sucking-off 46% ~ 52% sucrose layer, by 1: 5(V/V) add TNE damping fluid, 20 000 rpm are centrifugal 1.5 h again, precipitate resuspended with appropriate 0.01 mol/L PBS.The virion that electron microscopic observation essence is carried, Coomassie Brilliant Blue measures its concentration, and-80 DEG C save backup.
2. detectability and WSSV quantification range
The WSSV of purifying is diluted for concentration is respectively 8 variable concentrations of 12.5 μ g/ml, 6.26 μ g/ml, 3.13 μ g/ml, 1.57 μ g/ml, 783ng/ml, 391ng/ml, 196ng/ml, 97.8ng/ml, the WSSV test paper strip for semi-quantitative detecting using embodiment 1 to make respectively detects it, and negative control is 0.01 mol/L PBS solution.Result shows, and when WSSV protein concentration is 12.5 μ g/ml and 6.26 μ g/ml, detection line T1, T2, T3 present redness; When WSSV protein concentration is 3.13 μ g/ml, ELISA test strip line T1, T2 present redness, and T3 does not develop the color; When WSSV protein concentration is 1.57 μ g/ml, ELISA test strip line T1 presents redness, and T2, T3 do not develop the color; When WSSV protein concentration is 783ng/ml, ELISA test strip line T1 still manifests redness, is that 391ng/ml, 196ng/ml and 97.8ng/ml detection line T1 does not develop the color at WSSV protein concentration.The detection line of negative control does not develop the color.The nature controlling line of all test strips is all significantly red.Determine thus, when detection line T1, T2, T3 and nature controlling line all present redness, WSSV protein concentration >=6.26 μ g/ml; When detection line T1, T2 and nature controlling line manifest redness, 6.26 μ g/ml > WSSV protein concentration >=3.13 μ g/ml; When only having detection line T1 and nature controlling line to manifest redness, 3.13 μ g/ml > WSSV protein concentration >=783ng/ml; When only having nature controlling line to manifest redness, show WSSV protein concentration < 783ng/ml or do not contain WSSV.The detection of this test strips is limited to 783ng/ml, and quantitatively detecting WSSV concentration range is 783ng/ml ~ 6.26 μ g/ml.
embodiment 3:the detection method of prawn WSSV half-quantitative detection test paper
(1) preparation of sample is detected
Get and suffer from the leukodermal Chinese prawn gill, every 1 g gill adds 5 mL TNE damping fluids (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na
20.372 g, H
2o 1 000 mL, pH are 7.5) 25% sucrose solution prepared, ice bath fully grinds 3 ~ 5 min, is utilized by lapping liquid yarn thin,tough silk to filter, and gets supernatant as detection sample liquid.
(2) application of prawn WSSV half-quantitative detection test paper
Sample pad 1 one end is immersed and is detected about 10s in sample by adsorptive pads 4 one end of hand-held test paper of the present invention, and immersion depth is about 0.5cm, keeps flat test paper and is about 10min, visual inspection testing result, according to detection line T1, T2, T3 colour developing situation quantitate virus content.
(3) result judges
I: detection line T1, T2, T3 and nature controlling line all manifest redness, show WSSV concentration >=6.26 μ g/ml.
II: detection line T1, T2 and nature controlling line manifest redness, show 3.13 μ g/ml≤WSSV concentration < 6.26 μ g/ml.
III: detection line T1 and nature controlling line manifest redness, show 783ng/ml≤WSSV concentration < 3.13 μ g/ml.
IV: only have nature controlling line to manifest redness, show that WSSV concentration is less than 783ng/ml or does not contain WSSV.
V: detection line and nature controlling line do not present redness, show that operation has problem or test paper to lose efficacy.(Fig. 2)
embodiment 4:the specificity of prawn WSSV half-quantitative detection test paper, repeatability and stability test
(1) specificity
Select 4 groups of samples, the 1st group of sample: concentration is the prawn WSSV refining liquid of 15 μ g/ml.2nd group of sample: concentration is the prawn WSSV refining liquid of 5 μ g/ml.3rd group of sample: concentration is the prawn WSSV refining liquid of 1 μ g/ml.4th group of sample: the tissue homogenate supernatant of normal prawn.With more than detection paper 4 groups of samples, testing result shows: the 1st group of sample T1, T2, T3 and nature controlling line all manifest redness (I), 2nd group of sample T1, T2 and nature controlling line manifest redness (II), 3rd group of sample T1 and nature controlling line manifest redness (III), and the 4th group of sample only has nature controlling line to manifest redness (IV) (Fig. 3).
(2) repeatability
With more than the detection paper of different batches 4 groups of samples, each batch of test paper duplicate detection 5 times, result no significant difference.
(3) stability
At test paper being put into room temperature and 4 DEG C, every 15 days with more than detection paper 4 kinds of samples once.Result: when preserving under room temperature, the term of validity is 6 months, when preserving at 4 DEG C, the term of validity is 12 months.
Those of ordinary skill in the art can understand, and it is all possible in protection scope of the present invention, modifying for above-described embodiment, adding and replacing, and it does not all exceed protection scope of the present invention.
Claims (5)
1. the monoclonal antibody detected for prawn white spot syndrome virus, it is characterized in that described monoclonal antibody comprises monoclonal antibody A and the monoclonal antibody B of the different epi-positions of anti-WSSV, be called by two strain names respectively: hybridoma cell strain WSSV-K1, WSSV-K2, preserving number is respectively: CCTCC NO:C2014160 and CCTCC NO:C2014161, depositary institution is: China typical culture collection center, address: Wuhan City, Hubei Province Wuhan University, preservation date: the hybridoma secretion gained on September 2nd, 2014.
2. a prawn white spot syndrome virus sxemiquantitative quick detection test paper, is characterized in that described sxemiquantitative quick detection test paper comprises monoclonal antibody A according to claim 1 and monoclonal antibody B.
3. sxemiquantitative quick detection test paper according to claim 2, it is characterized in that described sxemiquantitative quick detection test paper also comprises carrier board, sample pad, gold mark pad, nitrocellulose filter and adsorptive pads, described sample pad and adsorptive pads are positioned at the two ends of carrier board, and nitrocellulose filter is positioned at the middle part of carrier board; Described sample pad and nitrocellulose filter intersection are provided with and are loaded with the gold mark pad that gold marks monoclonal antibody A, and gold mark pad one end imbricate is under sample pad, and other end imbricate is on nitrocellulose filter; Nitrocellulose filter is provided with three detection lines T1, T2, T3 and a nature controlling line of being arranged in order, and three described detection lines are coated with the monoclonal antibody B of variable concentrations, and the concentration of the upper monoclonal antibody B of T1, T2, T3 reduces successively from gold mark pad side; Nature controlling line near adsorptive pads is coated with goat anti-mouse igg.
4. a preparation method for prawn white spot syndrome virus sxemiquantitative quick detection test paper as claimed in claim 3, is characterized in that it comprises the steps:
(1) preparation and purification of monoclonal antibody A and monoclonal antibody B
Recover and cultivate hybridoma cell strain WSSV-K1 and WSSV-K2 secreting anti-WSSV monoclonal antibody, respectively lumbar injection BALB/c mouse, gathering ascites, caprylic acid-ammonium is purified and is obtained anti-WSSV monoclonal antibody A and monoclonal antibody B;
(2) preparation of gold mark pad
1. the preparation of collaurum and gold mark monoclonal antibody A:
Adopt micro-wave oven-trisodium citrate reduction method to obtain the collaurum that grain size is 20 ~ 30 nm, regulate collaurum pH value to be 8.2 with 0.1 mol/L solution of potassium carbonate; Anti-WSSV monoclonal antibody A is added in collaurum in suitable ratio, then adds bovine serum albumin(BSA) (BSA), hang with gold mark conserving liquid after centrifugal purification, be gold mark monoclonal antibody A liquid;
2. the preparation of the gold mark pad of gold mark monoclonal antibody A is loaded with:
By the gold mark monoclonal antibody A liquid spray made on glass fibre, freeze drying;
(3) preparation of nitrocellulose filter
Respectively the anti-WSSV monoclonal antibody B of 3 parts of variable concentrations is sprayed at nitrocellulose filter as detection line T1, T2, T3 according to concentration successively reduction order, goat anti-mouse igg is sprayed at nitrocellulose filter as nature controlling line, dries, soak in confining liquid;
(4) making of prawn WSSV sxemiquantitative quick detection test paper
Carrier board sticks successively nitrocellulose filter, adsorptive pads, gold mark pad and sample pad, slitting, test paper of the present invention.
5. a detection method for prawn white spot syndrome virus sxemiquantitative quick detection test paper, is characterized in that it comprises the following steps:
(1) preparation of measuring samples
Get and suffer from the leukodermal prawn gill, every 1 g shrimp gill adds 5 mL TNE damping fluids (Tris-HCl 1.566 g, NaCl 5.844 g, EDTA-Na
20.372 g, H
2o 1 000 mL, pH are 7.5) 25% sucrose solution prepared, ice bath fully grinds 3 ~ 5 min, is utilized by lapping liquid yarn thin,tough silk to filter, and gets supernatant as measuring samples liquid;
(2) prawn white spot syndrome virus half-quantitative detection
Immersed sample pad one end of test paper and detect about 10s in sample, immersion depth is about 0.5cm, and after horizontal positioned 10min, visual inspection testing result, according to detection line T1, T2, T3 colour developing situation quantitate virus content; When detection line T1, T2, T3 and nature controlling line all manifest redness, WSSV concentration >=6.26 μ g/ml; Detection line T1, T2 and nature controlling line manifest redness, 3.13 μ g/ml≤WSSV concentration < 6.26 μ g/ml; Detection line T1 and nature controlling line manifest redness, 783ng/ml≤WSSV concentration < 3.13 μ g/ml; Only have nature controlling line to manifest redness, show that WSSV concentration is less than 783ng/ml or does not contain WSSV.
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Application publication date: 20150218 |