CN101726597A - Test paper for detecting avian influenza virus and preparation method thereof - Google Patents
Test paper for detecting avian influenza virus and preparation method thereof Download PDFInfo
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- CN101726597A CN101726597A CN200810224933A CN200810224933A CN101726597A CN 101726597 A CN101726597 A CN 101726597A CN 200810224933 A CN200810224933 A CN 200810224933A CN 200810224933 A CN200810224933 A CN 200810224933A CN 101726597 A CN101726597 A CN 101726597A
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Abstract
The invention discloses a piece of test paper for detecting avian influenza virus (AIV). The test paper comprises a soleplate 1, a nitrocellulose membrane 3, a combined pad 2, a combined pad 2'', a nitrocellulose membrane 3 and two pieces of hydrophilic filter paper 4, wherein the nitrocellulose membrane 3 contains a gold marked AIV monoclonal antibody, AIV blood serum and goat anti-mouse IgG; the combined pad 2 and the combined pad 2'' are respectively glass fiber membranes containing degreased casein, the gold marked AIV monoclonal antibody, the AIV blood serum and the goat anti-mouse IgG; and the nitrocellulose membrane 3 contains a gold marked AIV monoclonal antibody, AIV blood serum and goat anti-mouse IgG. The invention further limits the relationships of lengths and positions of the parts. The invention further discloses a sample processing solution for processing excrement and secretion of animals infected by the AIV and a preparation method of the test paper. The test paper has the advantages of good sensitivity and strong specificity.
Description
Technical field
The present invention relates to a kind of test strips that detects avian influenza virus and this test strips the preparation method, the invention still further relates to a kind of excreta of avian influenza virus animal and sample preparation solution of secretion of infecting, belong to the immunodiagnosis field.
Background technology
Bird flu is the deadly infectious disease of a kind of poultry and birds, but under the situation of chance also infected person and causing a disease, even dead.Bird flu in 2004 has swept across since the Asia, involves European and American countries, causes the large quantities of death of poultry, and infects the mankind, causes nearly people more than 200 death, and popular repercussions are not calmed down so far.It is this time popular that to involve scope wide, more than the number of the infected be history.Moreover, the variation of its virus may cause being very popular of people's parainfluenza.Bird flu has caused World Focusing to the mankind's threat.How effectively to prevent and control the popular of this disease, become various countries scientist's important subject.A lot of countries all begin to carry out the research of this respect, are included in aspects such as vaccine, diagnostic techniques and medicine.
The immunochromatography colloidal gold technique is a new diagnostic techniques that grew up in recent years, and it combines the principle of immune response and chromatography, is a kind of immune chromatography method of uniqueness.This technology is a solid phase with the fibre strip chromatographic material, by capillary action sample solution is moved on chromatography strip, antigen in the sample combines with corresponding antibody on the fibrous material high special and high affine immune response takes place, and be detained or be enriched in surveyed area, the colour generation phenomenon appears by reaction substrate.This is a kind of composite immune chromatographic technique with fastest developing speed at present.Detections such as the short sexual gland (HCG) of clinical widely used human chorionic, hepatitis B surface antibody (HbsAg), cocaine now all are based on this method, its advantage is quick, easy, accurate, have high degree of specificity and hypersensitivity, just become the research focus of ecsomatics and diagnostics since the appearance.The principle of immunogold silver staining (IGSS) is that the gold grain on the product measured of golden immunological technique can become silver ion reduction silver-colored particle, forms the darker black layer of color and luster on the surface of gold grain, thereby has strengthened the susceptibility of golden immunological technique.Concrete grammar is on the basis of immuno-gold staining, under the situation that p-dihydroxy-benzene exists, by the reduction reaction in the developer solution that contains silver ion, make and around the gold particle at antigen-antibody reaction position, form a lot of beds of precipitation, light microscopic just can see that the positive reaction position is black-and-blue clearly down, thereby show difficult goldc grains of being located, demonstrate the position of antigen in the tissue by light microscopic.This method not only improves sensitivity, and golden labelled antibody can dilute back application more than 10 times simultaneously, can avoid using the organic pigment with carcinogenic danger in addition.The major advantage of IGSS method is: (1) susceptibility height.Compare with other immune colloidal gold technique, the IGSS method is considered to the most responsive method, is particularly suitable for only containing the sample detection of micro-antigen.(2) accurate positioning.The silver-colored particle deposition of IGSS method is at the antigen-antibody reaction position, and general nothing spreads, and it is comparatively accurate to locate.(3) easy, the safety of method, cost low, economical, simultaneously also can long preservation.(4) replace silver nitrate and actol with silver acetate, not only kept the characteristics of susceptibility, the specificity of original method, low background, good contrast, and whole developing process can carry out under ordinary light.
At present, this technology is diagnosed in physianthropy field widespread use, but is scarcely out of swaddling-clothes in the research in animal medicine field.Bird flu is the infectious disease that a kind of serious people and animal suffer from altogether, and bird flu in 2004 is popular the Asia, causes aquaculture serious economy loss and people's death.This threat of the scholarly forecast of The World Health Organization (WHO) also will continue, quick diagnosis is prevention and control this sick effective ways.The mainly still agar gel diffusion test of currently used method, HA/HI test ELISA and RT-PCR test.These methods or susceptibility are poor, or complex operation, detect the cost height.The fresh approach of research is a fluorescent RT-PCR technology at present, though accurately special, need expensive equipment and testing cost, need special laboratory, be difficult to popularize.
Application number is method and the dedicated kit thereof that 200410088716.8 patented claims disclose a kind of H5N1 of detection subtype avian influenza virus before the present invention; Application number is that 200510042631.0 patented claim discloses avian influenza virus antigen detection method and golden label fast diagnosis reagent box and preparation method; Application number is that 200510042527.1 patented claim discloses the method that composite quantitative polyase chain reaction detects bird flu and Avian pneumo-encephalitis virus; Application number is that 200510057023.7 patented claim discloses bird flu immune colloid gold diagnosis test paper and test card; Application number is that 03126894.3 patented claim discloses the reagent that colloidal gold chromatography detects sars coronavirus antigen; Application number is that 03126895.1 patented claim discloses the reagent that colloidal gold chromatography detects sars coronavirus antibody; Application number is that 03142652.2 patented claim discloses that immunologic paper is analysed bar and with the method for pathogenic bacteria and toxin in its fast detecting food; Application number is that 02131321.0 patented claim discloses a kind of immunochromatography single stage method and detects the method for beta-adrenin agonist, medicine and the preparation of test paper; Application number is colloidal gold strip and production and the using method that the patented claim of 02139704.X discloses sxemiquantitative fast detecting clenobuterol hydrochloride.
The present invention aim to provide a kind of simple, fast, the method for avian influenza virus in responsive, the special diagnosis ight soil, though the report and relevant patent that is applied to bird flu research aspect arranged at present, but, do not see the report that can detect avian influenza virus in the ight soil simultaneously how detecting animal blood serum, tissue, secretion etc.The present invention sets up the method for avian influenza virus in a kind of immune colloidal gold chromatography method quick diagnosis ight soil, uses in can and detecting in the open-air quick diagnosis of wild animal bird flu.
Summary of the invention
The present invention's one purpose provides a kind of test strips that detects avian influenza virus.
Another object of the present invention provides a kind of ight soil of avian flu virus infection animal and solution of secretion handled.
A further object of the present invention provides a kind of method that detects the avian influenza virus test strips for preparing.
One aspect of the present invention provides a kind of test strips that detects avian influenza virus, and this test strips comprises with the lower part:
Be positioned at the nitrocellulose filter 3 on the backing plate 1, its less than backing plate (1) and with the length difference of backing plate 1 be the 13/30-1/2 of backing plate length, and overlap with backing plate 1, described nitrocellulose filter is consistent in the longitudinal direction with backing plate, and described nitrocellulose filter contains AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark;
Be close to the pad 2 between backing plate 1 and the nitrocellulose filter 3, the length that pad 2 is attached to part between nitrocellulose filter 3 and the backing plate 1 is the 1/5-1/4 of backing plate length, and described pad 2 is the glass fibre membrane that contains AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of degreasing casein, golden mark, preferably, described pad 2 is a pad;
Be close to the pad 2 of nitrocellulose filter 3 upper surfaces "; pad 2 " overlap with nitrocellulose filter 3, the part that the orthogonal projection of this lap and above-mentioned pad 2 are attached between nitrocellulose filter 3 and the backing plate 1 is overlapped at least, described pad 2 " with the length of nitrocellulose filter lap be the 1/30-1/20 of backing plate length, described pad 2 " be the glass fibre membrane of the AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg that contain degreasing casein, golden mark;
Two absorbent filters 4, a slice is close to pad 2 " upper surface; and this absorbent filter, pad 2 ", the orthogonal projection of nitrocellulose filter 3, pad 2 and backing plate 1 has common lap, another sheet is close to nitrocellulose filter upper surface and pad 2 " at a distance of farthest position; this absorbent filter and another absorbent filter, pad 2 " or pad 2 between nearest distance be backing plate length 9/20-7/15, and be overlapped backing plate and nitrocellulose filter in this nearest distance.
The test strips of the detection avian influenza virus of invention preparation is different from existing test strips, this is because contain the degreasing casein on the pad of test strips of the present invention, so with respect to not containing the caseic test strips of degreasing, test strips of the present invention has the high advantage of detection sensitivity.In addition, the length that pad 2 is attached to part between nitrocellulose filter 3 and the backing plate 1 is backing plate length 1/5-1/4, and this is to lose shape necessary on test strips in order to satisfy testing sample; And pad 2 " with the length of nitrocellulose filter lap be the 1/30-1/20 of backing plate 1 length; this absorbent filter and another absorbent filter, pad 2 " or pad 2 between nearest distance be backing plate length 9/20-7/15, and in this nearest distance overlapped backing plate and nitrocellulose filter, this all is to lose shape necessaryly on test strips in order to satisfy testing sample, and the structure of this test strips is seen Fig. 1.At pad 2, pad 2 " to go up the degreasing casein be to soak in treating fluid by pad, and dry more afterwards pad obtains, and described treating fluid contains degreasing casein and phosphate buffer.And pad 2, pad 2 " and nitrocellulose filter on AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark be that dry afterwards pad and nitrocellulose filter obtain by AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of the golden mark of spraying on these two pads and nitrocellulose filter.
Preferably, the pad 2 that wherein is close between backing plate 1 and the nitrocellulose filter 3 is two pads, space between these two pads on the length direction is the 9/20-7/15 of backing plate length, pad 2 " overlap with nitrocellulose filter; the part that the orthogonal projection of this lap and described pad 2 are attached between nitrocellulose filter 3 and the backing plate 1 is overlapped at least, and the orthogonal projection of another pad 2 partially overlaps at least apart from pad 2 " absorbent filter 4 of highest distance position.Therefore the test strips of this structure more helps the both sides balance with respect to the structure of aforementioned test strips many pads 2 between backing plate 1 and nitrocellulose filter 3.
Preferably, the pad 2 of described test strips and pad 2 " also contain 1) Arabic gum, 2) p-dihydroxy-benzene and 3) silver acetate or silver nitrate.
Preferably, described nitrocellulose filter also contains 1) Arabic gum, 2) p-dihydroxy-benzene and 3) silver acetate or silver nitrate.
The pad of test strips of the present invention and nitrocellulose filter contain Arabic gum, citrate, p-dihydroxy-benzene and silver acetate or silver nitrate.These compositions are not present in existing test strips, and the colour developing that contains the test strips of these compositions is better than existing test strips.When containing avian influenza virus in the determinand, on test strips of the present invention, be black-and-blue, and on existing test strips, take on a red color, the sharpness of existing simultaneously test strips colour developing is also not as test strips of the present invention.
Another aspect of the present invention provides processing to infect the ight soil of avian influenza virus animal and the solution of secretion, and this solution comprises phosphate buffer, polyglycol and the normal butyl alcohol of pH value for 7.0-7.4.The animal of the infection avian influenza virus among the present invention is not only poultry, also can be to infect this viral domestic animal or people.
In the prior art, be not specifically designed to processing and infect the ight soil of avian influenza virus animal and the solution of secretion, the present invention then provides this sample preparation liquid, and the purpose of using this treating fluid is to be released in the treating fluid that is provided for the ight soil that will infect the bird flu animal and the virus in the secretion.In addition, the virus of release antiviral antibody easier and on the test strips combines, and makes that the colour developing of test strips is more sensitive.
Preferably, to comprise Macrogol 2000 0, the normal butyl alcohol of 8%-12% mass concentration, the pH value of 0.5%-1.5% mass concentration be 7.2 phosphate buffer to described solution.
More preferably, to comprise the normal butyl alcohol of Macrogol 2000 0,10% mass concentration of 1% mass concentration and 0.05mol/L pH value be 7.2 phosphate buffer to described solution.
Another aspect of the present invention also provides a kind of method for preparing described test strips, and this method may further comprise the steps:
A. glass fibre membrane fully is soaked in the treating fluid, the glass fibre membrane drying after will handling again obtains described pad, and described treating fluid contains degreasing casein and phosphate buffer;
B. AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark is sprayed at pad that described step a obtains and blank nitrocellulose filter and carries out drying;
C. nitrocellulose filter that absorbent filter, step b are obtained and pad are fixed on the described backing plate according to aforesaid position relation, obtain described test strips.
Preferably, between described step b and step c, also use EDC (available from long-livingization of Shanghai Yan development in science and technology company limited dried pad and the nitrocellulose filter that is coated with AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark, article number 4108, trade name: 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) solution soaks, drying places silver ion treatment liquid immersion and dry afterwards again.
Preferably, the concentration of the AIV monoclonal antibody of described golden mark is 3.75-4.25mg/ml, and the concentration of AIV serum is 4.75-5.25mg/ml, and the concentration of sheep anti-mouse igg is 4.75-5.25mg/ml.
Preferably, it is 3.5 citrate buffer, p-dihydroxy-benzene and silver acetate solution that described silver ion treatment liquid contains Arabic gum, pH value, and the volume of Arabic gum: the pH value is the volume of 3.5 citrate buffer: the volume of p-dihydroxy-benzene: the volume of silver acetate solution is 6: 1: 2: 1.
Preferably, the mass concentration of wherein said Arabic gum is that the mass concentration of 14-16%, p-dihydroxy-benzene is that the concentration of 80%-90%, silver acetate is 9.5-10.5%.
More preferably, the mass concentration of described Arabic gum is 15%, the mass concentration of p-dihydroxy-benzene is 85%, the concentration of silver acetate solution is 10%.
Preferably, the degreasing casein and the 0.025-0.075mol/L phosphate buffer that contain the 0.75-1.25% mass concentration among the described step a in the treating fluid.
More preferably, contain the phosphate buffer that the degreasing casein of 1.0% mass concentration and pH value are 7.2 0.05mol/L among the described step a in the treating fluid.
Preferably, the AIV serum among the present invention is goat-anti AIV serum or mouse-anti AIV serum.
Test strips of the present invention is used for detecting the avian influenza virus of poultry ight soil, when AIV antigen with after golden labeling antibody in the pad combines, by chromatography move to nitrocellulose filter again with goat-anti AIV antibodies, precipitation line appears, this line is called for short detection line or T line, do not continue motion and combine, form the 2nd precipitation line, this line abbreviation nature controlling line or C line with sheep anti-mouse antibody on the NC film in conjunction with intact antigen and golden labeling antibody bond.If a band above test-strips, only occurs, then be expressed as feminine gender; If 2 blue-black colour bands above test-strips, occur, then be expressed as the positive; If colour band above test-strips, do not occur, represent that then test-strips lost efficacy.
Test strips of the present invention has following beneficial effect: 1. sensitivity is good: when carrying out sensitivity test, with AIV antigen by 0,0.25,0.5,1.0,5.0,7.5,10,20,40, the 80ng/ml dilute concentration detects, but blue-black minimum measured quantity appears in detection line, i.e. the sensitivity of reagent strip for this reason.The result shows that when the concentration of AIV antigen was 7.5ng/ml, the check line was black-and-blue.2. specificity test: the ight soil and the allantoic fluid normally that detect the poultry of the newcastle disease virus (the English NDV of abbreviation) that has same concentrations, avian infectious bronchitis virus (the English IBV of abbreviation), infectious bursal disease virus (the English IBDV of abbreviation) with this test strips, the observation test result sees to have or not false positive reaction, cross reaction.The result shows, the test strips of the present invention's development is positive to the AIV reaction, and is negative to the detection of the poultry ight soil that contains NDV, IBV, IBDV and normal allantoic fluid.This illustrates this test strips no cross reaction and false positive reaction.
Description of drawings
Fig. 1 is the synoptic diagram of the structure of test strips of the present invention, 1-backing plate among the figure, 2-pad (the plain film of glass fibre), 3-nitrocellulose filter, 2 "-pad, the 4-absorbent filter.
Fig. 2 is the synoptic diagram of the another kind of structure of test strips of the present invention, 1-backing plate among the figure, 2-pad (the plain film of glass fibre), 3-nitrocellulose filter, 2 "-pad, the 4-absorbent filter.
Fig. 3 is the schematic appearance of test strips of the present invention, among the figure 1 '-dress test strips shell, 2 '-well, 3 '-viewport.
Embodiment
The preparation of colloidal gold solution
To the 4ml mass concentration 1% trisodium citrate (Na
3C
6H
5O
72H
2O) add the 0.5ml mass concentration in and be 1% tannic acid and the K of 0.5ml 25mmo/L
2CO
3Solution (K
2CO
3Solution equates with the volume that tannic acid adds), mend to trisodium citrate-tannic acid-K with DDW
2CO
3The mixed solution final volume be 20ml, be heated to 60 ℃.Get 1ml concentration and be 1% HAuCl
4Add in the DDW of 79ml, water-bath is heated to 60 ℃, adds trisodium citrate-tannic acid-K then rapidly
2CO
3Mixed solution, under 60 ℃ of temperature, keep certain hour, treat that solution colour becomes peony, approximately behind the 30-60min, will contain HAuCl again
4Mixed solution be heated to boiling, keep boiling 5min to get final product.Be cooled to room temperature, obtain colloidal gold solution, degerming after filtration in the clean glass bottle of packing into, is preserved standby down at 4 ℃.Solution in the present embodiment all disposes with ultrapure water or distilled water, and uses filtering with microporous membrane, removes the impurity in the water.
The AIV MONOCLONAL ANTIBODIES SPECIFIC FOR
Prepare high serum with H9N2 hypotype AIV as antigen immune inoculation SPF chicken.From this serum, extract immunoglobulin G (IgG) with the saturated ammonium sulfate method, prepare Freund vaccine immunity SPF BALB/c mouse with this IgG, get its splenocyte and murine myeloma cell SP2/0 merges under the polyglycol effect, adopt the screening of indirect ELISA detection method to obtain the hybridoma of 3 strains (2H1D1,2H1G4,3H2D7) secretion monoclonal antibody specific, this three strain of hybridoma is secreted the AIV monoclonal antibody.
The preparation of the high immune serum of AIV
Freund antigen immune goat and mouse will be made, preparation goat-anti AIV hyper-immune serum and mouse-anti AIV hyper-immune serum behind the avian influenza virus antigen purifying.
Sheep anti-mouse igg is available from Beijing Bomai Century Biotechnology Co.Ltd, antibody article No.: A8129, trade name sheep anti-mouse igg.
The AIV MONOCLONAL ANTIBODIES SPECIFIC FOR of colloid gold label
Get the collaurum 20mL of 18nm, use K
2CO
3Solution (pH11,0.05mol/L) transferring its pH is 7.5-8.5; Adding 200ml concentration respectively is the AIV monoclonal anti liquid solution that 3.75mg/mL and 4.25mg/mL embodiment 2 obtain, room temperature reaction 4-5min; Add the PBS 200 μ L that contain 10%BSA, 10% sucrose, centrifugal 30min under 4 ℃ of 30000g centrifugal force abandons supernatant; With 0.05mol/L pH 7.2PBS 20mL dissolution precipitation, centrifugal 30min under 4 ℃ of 30000g centrifugal force abandons supernatant; With the PBS 20mL dissolution precipitation that contains 3% glycerine, 5%PEG, 0.05mol/L pH7.2, Zhi concentration is the AIV monoclonal antibody of 3.75mg/mL and 4.25mg/mL gold mark respectively, and 4 ℃ of storages are standby
The preparation of pad and nitrocellulose filter
At first glass fibre membrane being placed 0.75% degreasing casein and 0.05mol/L pH value is that the mixed solution that 7.2 phosphate buffer is formed carries out immersion treatment, take out after 1 hour and dry 15 minutes, obtain pad, this pad can be used as the pad 2 in the accompanying drawing, also can be used as pad 2 ".
The sheep anti-mouse igg that the AIV monoclonal antibody of the golden mark of the 3.75mg/mL that embodiment 3 is obtained and the working concentration that embodiment 2 prepares AIV serum are adjusted to 4.75mg/ml and 4.75mg/ml is sprayed at described pad and blank nitrocellulose filter, and at 35-40 ℃ drying in oven 20-30min.Pad after will handling again and nitrocellulose filter place EDC solution to soak 15min, behind the air-dry 5min, pad are placed silver ion treatment liquid to soak air-dry 15min 2 hours again.The prescription of described silver ion treatment liquid is: volume ratio is 6: 1: 2: 1 mass concentration is that 15% Arabic gum, pH value are that 3.5 citrate buffer solution, mass concentration are that 85% p-dihydroxy-benzene and mass concentration are to obtain silver ion treatment liquid after 10% silver acetate solution mixes.The pad of present embodiment contains the glass fibre of AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of degreasing casein, golden mark; The AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg that contain golden mark on the nitrocellulose filter of present embodiment.
Embodiment 5
The preparation of pad and nitrocellulose filter
At first glass fibre membrane being placed 1.25% degreasing casein and 0.075mol/L pH value is that the mixed solution that 7.2 phosphate buffer is formed carries out immersion treatment, take out after 1 hour and dry 15 minutes, obtain pad, this pad can be used as pad 2, also can be used as pad 2 ".
The sheep anti-mouse igg that the AIV monoclonal antibody of the golden mark of the 4.25mg/ml that embodiment 3 is obtained and the working concentration of the AIV serum that embodiment 2 prepares are adjusted to 5.25mg/ml and 5.25mg/ml is sprayed at described pad and blank nitrocellulose filter, and at 35-40 ℃ drying in oven 20-30min.Pad after will handling again and nitrocellulose filter place EDC solution to soak 15min, behind the air-dry 5min, pad are placed silver ion treatment liquid to soak air-dry 15min 2 hours again.The prescription of described silver ion treatment liquid is: volume ratio is 6: 1: 2: 1 mass concentration is that 16% Arabic gum, pH value are that 3.5 citrate buffer solution, mass concentration are that 90% p-dihydroxy-benzene and mass concentration are to obtain silver ion treatment liquid after 10.5% silver acetate solution mixes.The pad of present embodiment contains the glass fibre of AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of degreasing casein, golden mark; The AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg that contain golden mark on the nitrocellulose filter of present embodiment.
Embodiment 6
The preparation of test strips
The fixing pad for preparing of embodiment 4 on a side of backing plate 1 upper surface, this pad is as pad 2, its length is 1/4 of backing plate length, align with an end of backing plate 1, the nitrocellulose filter that embodiment 4 is obtained is fixed on the upper surface of pad 2 and the upper surface of backing plate opposite side again, nitrocellulose filter is 1/2 of a backing plate length, again at the fixing pad that obtains of embodiment 4 of the upper surface of the nitrocellulose filter of a side identical with pad 2, this pad is as pad 2 "; pad 2 " and the orthogonal projection and the part of pad 2 between nitrocellulose filter 3 and backing plate 1 of nitrocellulose filter 3 laps overlap, pad 2 " with the length of nitrocellulose filter lap be 1/30 of backing plate 1 length; at last at pad 2 " upper surface with pad 2 " upper surface of relative nitrocellulose filter opposite side fixes two absorbent filters respectively, obtains being used to detect the test strips of avian influenza virus.With pad 2 " absorbent filter, the pad 2 of a relative end " or pad 2 between nearest distance be 7/15 of backing plate length, the test strips for preparing is seen Fig. 1.Adopt adhesive sticker fixedly nitrocellulose filter, pad and absorbent filter in the present embodiment.This backing plate is the plastic base plate (available from Shanghai gold mark bio tech ltd, trade name: plastics are diagnosed offset plate) of white.
Embodiment 7
The preparation of test strips
Fixing two pads preparing of embodiment 5 respectively in the both sides of backing plate upper surface, this pad is as pad 2, two pads equally long, be 1/4 of backing plate length, so between two pads, have the space, the nitrocellulose filter that embodiment 5 is obtained is fixed on the upper surface of these two pads 2 again, nitrocellulose filter is 17/30 of a backing plate length, the nitrocellulose filter that embodiment 5 is obtained is fixed on the upper surface of these two pads again, again at a side secure bond pad 2 of nitrocellulose filter upper surface "; pad 2 " and the orthogonal projection and the part of pad 2 between nitrocellulose filter 3 and backing plate 1 of nitrocellulose filter 3 laps overlap, pad 2 " with the length of nitrocellulose filter lap be 1/30 of backing plate 1 length; at pad 2 " the fixing a slice absorbent filter of upper surface, with pad 2 " opposite side of relative nitrocellulose filter upper surface fixes another sheet absorbent filter; with pad 2 " absorbent filter of a relative end, pad 2 " or pad 2 between nearest distance be 7/15 of backing plate length; obtain being used to detect the test strips of avian influenza virus, the test strips for preparing is seen Fig. 2.Adopt adhesive sticker fixedly nitrocellulose filter, pad and absorbent filter in the present embodiment.This backing plate is the plastic base plate (available from Shanghai gold mark bio tech ltd, trade name: plastics are diagnosed offset plate) of white.
Embodiment 8
Handle the preparation of animal wastes solution
Contain the Macrogol 2000 0 of 1% mass concentration, the normal butyl alcohol of 10% mass concentration in this solution, the pH value is 7.2 phosphate buffer, contains the NaCl of 8.5g/L, the Na of 2.2g/L in this damping fluid
2HPO
4NaH with 0.3g/L
2PO
4, the solution of described other compositions is all used phosphate buffer.
Embodiment 9
Handle the preparation of animal wastes solution
The pH value that contains the normal butyl alcohol of Macrogol 2000 0,8% mass concentration of 0.5% mass concentration and its surplus in this solution is 7.2 phosphate buffer.
Embodiment 10
Handle the preparation of animal wastes solution
The pH value that contains the normal butyl alcohol of Macrogol 2000 0,12% mass concentration of 1.5% mass concentration and its surplus in this solution is 7.2 phosphate buffer.
Embodiment 11
Test strips of the present invention detects the method that whether contains avian influenza virus in the animal wastes
The animal wastes of field acquisition are at first handled with following solution, this solution contain the normal butyl alcohol of Macrogol 2000 0,10% mass concentration of 1% mass concentration and pH value be 7.2, phosphate buffer.Ratio with 12ml solution dissolving 1g animal wastes is dissolved the animal wastes of gathering, fully stirring is fully dissolved up to animal wastes and is scattered in the solution, the supernatant of getting 05-1ml is added drop-wise in the well on the test strips, from viewport, observe 2 blue-black colour bands, show in the animal wastes of collection and contain avian influenza virus, this proof has been verified zoogenetic infection avian influenza virus.Adopt the sensitivity of the viral antigen test strip of variable concentrations survey, the sensitivity of the detection virus of test strips of the present invention can reach 7.5ng/ml.
Embodiment 12
Test strips of the present invention detects the method that whether contains avian influenza virus in the animal saliva
Gather the animal saliva of PI avian influenza virus and at first handle with following solution, this solution contains that the normal butyl alcohol of Macrogol 2000 0,10% mass concentration of 1% mass concentration and pH value are 7.2, the phosphate buffer of 0.05mol/L.The animal saliva of gathering with the ratio dissolving of 1ml solution dissolving 1g saliva, behind the mixing, the drips of solution of getting 05-1ml is added in the well on the test strips, observes 2 blue-black colour bands from viewport, this has shown detected zoogenetic infection avian influenza virus.Adopt the sensitivity of the viral antigen test strip of variable concentrations survey, the sensitivity of the detection virus of test strips of the present invention can reach 7.5ng/ml.
Claims (12)
1. test strips that detects avian influenza virus, this test strips comprises with the lower part:
Backing plate (1), described backing plate are plastic base plate;
Be positioned at the nitrocellulose filter (3) on the backing plate (1), it is the 13/30-1/2 of backing plate length and overlaps with backing plate (1) less than backing plate (1) and with the length difference of backing plate (1), described nitrocellulose filter is consistent in the longitudinal direction with backing plate, and described nitrocellulose filter contains AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark;
Be close to the pad (2) between backing plate (1) and the nitrocellulose filter (3), the length that pad (2) is attached to part between nitrocellulose filter (3) and the backing plate (1) is backing plate length 1/5-1/4, and described pad is the glass fibre membrane of the AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg that contain degreasing casein, golden mark, preferably, described pad (2) is a pad;
Be close to the pad (2 ") of nitrocellulose filter (3) upper surface; and pad (2 ") overlap with nitrocellulose filter (3), the part that the orthogonal projection of this lap and above-mentioned pad (2) are attached between nitrocellulose filter (3) and the backing plate (1) is overlapped at least, pad (2 ") is the 1/30-1/20 of backing plate (1) length with the length of nitrocellulose filter lap, and described pad (2 ") is the glass fibre membrane of the AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg that contain degreasing casein, golden mark;
Two absorbent filters (4), a slice is close to the upper surface of pad (2 "); and the orthogonal projection of this absorbent filter, pad (2 "), nitrocellulose filter (3), pad (2) and backing plate (1) has common lap, another sheet is close to nitrocellulose filter upper surface and pad (2 ") at a distance of farthest position; nearest distance is the 9/20-7/15 of backing plate length between this absorbent filter and another absorbent filter, pad (2 ") or the pad (2), and is overlapped backing plate and nitrocellulose filter in this nearest distance.
2. test strips according to claim 1, the pad (2) that wherein is close between backing plate (1) and the nitrocellulose filter (3) is two pads, space between these two pads on the length direction is the 9/20-7/15 of backing plate length, pad (2 ") is overlapped with nitrocellulose filter (3); the part that the orthogonal projection of this lap and a described pad (2) are attached between nitrocellulose filter (3) and the backing plate (1) is overlapped at least, and the orthogonal projection of another pad (2) partially overlaps the absorbent filter (4) apart from pad (2 ") highest distance position at least.
3. test strips according to claim 1 and 2, and wherein said pad (2) and pad (2 ") also contain 1) Arabic gum, 2) p-dihydroxy-benzene and 3) silver acetate or silver nitrate.
4. according to each described test strips among the claim 1-3, wherein said nitrocellulose filter also contains 1) Arabic gum, 2) p-dihydroxy-benzene and 3) silver acetate or silver nitrate.
5. handle the ight soil of infection avian influenza virus animal and the solution of secretion for one kind, this solution comprises phosphate buffer, polyglycol and the normal butyl alcohol of pH value for 7.0-7.4.
6. solution according to claim 5, Macrogol 2000 0, the normal butyl alcohol of 8%-12% mass concentration, the pH value that wherein said solution comprises the 0.5-1.5% mass concentration is 7.2 phosphate buffer, preferably, described solution comprises the Macrogol 2000 0 of 1% mass concentration, the normal butyl alcohol and the 0.05mol/L pH value of 10% mass concentration is 7.2 phosphate buffers.
7. method for preparing claim 1 or 2 described test strips, this method may further comprise the steps:
A. glass fibre membrane fully is soaked in the treating fluid, the glass fibre membrane drying after will handling again obtains described pad, and described treating fluid contains degreasing casein and phosphate buffer;
B. AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark is sprayed at pad that step a obtains and blank nitrocellulose filter and carries out drying;
C. nitrocellulose filter that absorbent filter, step b are obtained and pad are fixed on the described backing plate according to claim 1 or 2 described position relations, obtain described test strips.
8. method according to claim 7, wherein between described step b and step c, also dried pad and the nitrocellulose filter that is coated with AIV monoclonal antibody, AIV serum and the sheep anti-mouse igg of golden mark soaked also drying with the immersion of EDC solution, the dry silver ion treatment liquid that places again afterwards.
9. according to claim 7 or 8 described methods, the concentration of the AIV monoclonal antibody of wherein said golden mark is 3.75-4.25mg/ml, and the concentration of AIV serum is 4.75-5.25mg/ml, and the concentration of sheep anti-mouse igg is 4.75-5.25mg/ml.
10. according to Claim 8 or 9 described methods, it is 3.5 citrate buffer, p-dihydroxy-benzene and silver acetate solution that wherein said silver ion treatment liquid contains Arabic gum, pH value, and the volume of Arabic gum: the pH value is the volume of 3.5 citrate buffer: the volume of p-dihydroxy-benzene: the volume of silver acetate solution is 6: 1: 2: 1.
11. according to the method described in the claim 10, the mass concentration of wherein said Arabic gum is that the mass concentration of 14-16%, p-dihydroxy-benzene is that the mass concentration of 80-90%, silver acetate is 9.5-10.5%, preferably, the mass concentration of described Arabic gum is 15%, the mass concentration of p-dihydroxy-benzene is 85%, the concentration of silver acetate solution is 10%.
12. according to each described method among the claim 7-11, the degreasing casein and the 0.025-0.075mol/L phosphate buffer that wherein contain the 0.75-1.25% mass concentration in the described treating fluid of step a, preferably, contain the phosphate buffer that the degreasing casein of 1.0% mass concentration and pH value are 7.2 0.05mol/L in the described treating fluid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810224933A CN101726597A (en) | 2008-10-27 | 2008-10-27 | Test paper for detecting avian influenza virus and preparation method thereof |
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|---|---|---|---|
| CN200810224933A CN101726597A (en) | 2008-10-27 | 2008-10-27 | Test paper for detecting avian influenza virus and preparation method thereof |
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| CN101726597A true CN101726597A (en) | 2010-06-09 |
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Application publication date: 20100609 |