CN101470116B - Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit - Google Patents
Colloidal gold immunity percolation sensitization method for detecting avian influenza virus and its reagent kit Download PDFInfo
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Abstract
The invention provides a colloidal gold immunity infiltration sensitivity raising method for checking avian influenza virus and a reagent kit therefore. The colloidal gold immuno infiltration sensitivity raising method comprises: (1) preparing an immunity infiltration test strip; (2) fixing influenza virus multi-clonal antibody on the immunity infiltration test strip; (3) preparing avian influenza virus gold marking probes; 4) preparing sensitivity raising agent; (5) detecting avian influenza virus. The invention utilizes the oxidation reduction reaction between chloric acid and ac=scorbic acid, to generate gold atoms which can be absorbed by colloidal gold, to position the deposition part of the colloidal gold to develop and enhance color, thereby improving the detection sensitivity on object antigen by 8 to 50 times. Based on colloidal gold spot immunity infiltration test method (double-antibody sandwich), the invention adopts nanometer gold particle sensitivity raising technique, to improve the detection sensivity on avian influenza virus, screen sample of high flux, and adapt avian influenza virus detection in basic layer or in-situ field.
Description
Technical field
The present invention relates to a kind of Detecting method, relate in particular to a kind of colloidal gold immunity percolation sensitization method and kit thereof that detects avian influenza virus.
Background technology
Bird flu (Avian Influenza, AI) is a kind of acute highly pathogenic infectious disease from respiratory disease to multiple symptoms such as serious septicemia of the bird that caused by A type influenza virus.Except bird, A type influenza virus also infects the animal of other kinds, such as horse, pig etc., finds first that highly pathogenic bird flu H5N1 can infect the mankind in 1997.From bird flu in 1878 since Italy finds, cause huge economic loss to poultry husbandry.Harm is maximum in history, and the bird flu (H5N1) that economic loss is the most serious breaks out in nineteen eighty-three U.S. Binzhou Prefecture, and U.S. government has spent more than 6,000 ten thousand dollars for this reason altogether, and indirect economic loss is estimated to reach 3.48 hundred million dollars.China's Hongkong is the bird flu of outburst in the recent period, and loss approximately reaches 8,000 ten thousand Hongkong dollars according to estimates.1997 and 2003 are in Hong Kong H5N1 bird flu disturbed one 8 people, and 4 people are dead, begin this virus the end of the year in 2003 and have caused the up to a hundred people to infect in Asian countries, and tens people are dead.Since in February, 2006, bird flu epidemic situation grows in intensity, and the states such as Malaysia, Russia, China, Bosnia-Herzegovena, Romania, France, India, Egypt, Germany, Austria, Iran, Greece, Slovenia, Italy have reported bird flu epidemic situation in succession.Therefore the detection of, high sensitivity easy, quick to avian influenza virus, high specific becomes the task of top priority of the diffusion that controls bird flu epidemic situation.
Existing avian flu virus detection method mainly is divided into antibody test and antigen detection method.Antibody detection method comprises: hemagglutination-inhibition test (Hemagglutination inhibition, HI), agar immunodiffusion (Agarose gel immunodiffusion, AGID), enzyme linked immunosorbent assay (Enzyme-linkedImmunosorbent Assay, ELISA), complement fixation test (CFT) (Complement Fixation Assays, CF), neuraminidase inhibition test (Neuraminidase inhibitor test, NIT) and virus neutralization tests (Virusneutralization Test, VNT) etc.Because antibody detection method can not be distinguished manual injection's vaccine and virus infections thereby animal quarantine is lacked the practical application meaning.Therefore, avian flu virus detection is mainly antigen detection method at present, method is ELISA and fluorescence RT-PCR relatively fast, but there is following defective in these two kinds of methods in widespread adoption: the instrument and equipment that (1) needs are expensive, and testing cost is higher and be difficult for promoting in grass-roots unit; (2) all these technical methods all can not produce in basic unit, quarantine station and on-the-spot use the such as clinical, special laboratory must be arranged; (3) operation is more loaded down with trivial details, and all more than a few hours, directly affected the prevention and control speed of epidemic situation detection time.
Because colloid gold particle has very strong absorption merit effect to numerous protein, can with the non-covalent combinations of many kinds of substance such as SPA, IgG, toxin, glycoprotein, enzyme, microbiotic and hormone, thereby make it become immunoreactive good label, and so that immobilon-p immunoassay (Solid Phase Membrane-basedImmunoassay) is easier.Immobilon-p commonly used is cellulose nitrate (nitrocellulose, NC) film.Spot immune determination method (Dot-Immunoboding Assay, DIBA) is the technology that improvement gets up on the immunoblot assay basis.Because its susceptibility, specificity are suitable with enzymoimmunoassay (ELISA), and simple to operate, quick and develop into diverse ways.Comprising spot immune diafiltration determination method (DotImmuno Filtration Assay, DIFA) and spot immune measurement in chromatography method (Dot ImmunoChromatographic Assay, DICA).Spot immune diafiltration determination method take collaurum as label is called colloidal gold immunity percolation determination method (Dot-Immunogold FiltrationAssay, DIGFA).Yet above-mentioned several method detects avian influenza virus and is difficult to satisfy highly sensitive requirement.People mainly utilized silver-colored staining technique to improve the detection sensitivity of DIGFA in the past.Yet the shortcoming of this technology is blurred background, and effect of enhanced sensitivity is relatively not strong, thereby has limited its range of application.
Summary of the invention
The purpose of this invention is to provide a kind of colloidal gold immunity percolation sensitization method and kit thereof that detects avian influenza virus.The present invention is on the basis of colloidal gold dot immunity percolation determination method (double-antibody sandwich), adopt nanogold particle enhanced sensitivity technology, can significantly improve the sensitivity that detects avian influenza virus, has sensitivity, fast and the characteristics such as specificity is good, and cheap, can carry out the high flux examination of sample, be applicable to basic unit or Site Detection avian influenza virus, thereby overcome the not high problem of present colloidal gold immunity percolation determination method (DIGFA) detection sensitivity.
The present invention detects the colloidal gold immunity percolation sensitization assay method of avian influenza virus, and step is as follows:
1. prepare the immunity percolation test strips
The immunity percolation test strips is one of A or B;
Preparation immunity percolation test strips A: being provided with pitch of holes along long direction for 40-110mm, wide rectangle tygon (PE) plastic base length of a film for 3-6mm is that to be the diameter that delegation distributes be the circular sample holes of 0.5-3mm for 8-15 of 2-4mm; Cellulose nitrate (NC) diaphragm identical with tygon (PE) plastic substrate size adhered to below it; During application of sample with tygon (PE) plastic substrate of sample holes upper;
Preparation immunity percolation test strips B: the circular cellulose nitrate diaphragm of cutting 8-15 diameter 0.5-3mm adheres to the long 40-110mm of being, widely is the rectangle tygon (PE) of 3-6mm. and plastic substrate, diaphragm spacing are 3-4mm; The plastic substrate of each diaphragm below is provided with two suction holes, the suction hole be with overlapping with the diaphragm center of circle, parallel with plastic base length of a film major axis than the long 0.3mm of diaphragm diameter of a circle, removed the hole that obtains by the part that diaphragm covers with the wide parallel minor axis of plastic substrate than the ellipse of the short 0.2mm of diaphragm diameter of a circle, during application of sample band absorb water the hole plastic polyethylene (PE) substrate below;
2. the immobilization of avian influenza virus polyclonal antibody on the immunity percolation test strips
1) the immunity percolation test strips of preparation immobilization avian influenza virus polyclonal antibody
A. prepare avian influenza virus Anti-TNF-α liquid solution, with 800mL dissolved in distilled water 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g sodium hydrogen phosphate (Na
2HPO
4) and 0.24g potassium dihydrogen phosphate (KH
2PO
4), pH value to 7.4 with hydrochloric acid (HCl) regulator solution, add water to 1L, shake up, make the phosphate buffer (PBS, Phosphate Buffer Saline) of pH7.4, with phosphate buffer PBS dilution avian influenza virus polyclonal antibody, make that the polyclonal antibody weight percent concentration is 0.05-0.3mg/mL in the solution, preferred weight percent concentration is 0.1-0.2mg/mL, makes avian influenza virus Anti-TNF-α liquid solution;
B. the immobilization of avian influenza virus polyclonal antibody on the immunity percolation test strips
The immobilization on the immunity percolation test strips of avian influenza virus polyclonal antibody adopts point sample immobilization method or soaks one of immobilization method;
Point sample immobilization method: the above-mentioned avian influenza virus Anti-TNF-α of some 1-3 μ L liquid solution on each diaphragm of each sample holes of above-mentioned immunity percolation test strips A or immunity percolation test strips B; Point sample can be 1 time-3 times, and is for seep, air-dry; Being fixed test strips A or B;
Or soak the immobilization method: get the above-mentioned avian influenza virus Anti-TNF-α of 500-1000 μ L liquid solution and place small test tube, 4 ℃ are soaked 5-10 bar immunity percolation test strips A or B 10-15 hour, take out, air-dry; Being fixed test strips A or B;
2) sealing of test strips
With above-mentioned immobilization test strips A or the B that obtains with point sample method or infusion method, the weight percent concentration of putting into the distilled water dilution is the bovine serum albumin solution (BSA) of 1-5%, 37 ℃ of reaction 20-60min; Take out, air-dry, obtain stand-by immobilization test strips; 4 ℃ of preservations, for subsequent use;
3. preparation avian influenza virus gold is marked the monoclonal antibody probe
1) preparation colloidal gold solution
A. the reagent and the volume proportion thereof that prepare colloidal gold solution:
Distilled water 100mL,
The weight percent concentration of fresh preparation be 1% aqueous solution of chloraurate 1mL,
Weight percent concentration is 1% trisodium citrate aqueous solution 1.0-1.5mL;
The wherein wt percent concentration is that the optimum ratio of 1% trisodium citrate aqueous solution is 1.2mL;
B. prepare colloidal gold solution
Required distilled water heating is boiled; The weight percent concentration that adds required fresh preparation is 1% aqueous solution of chloraurate, adds rapidly required weight percent concentration and be 1% trisodium citrate aqueous solution, constantly stirs, and obtains the colloidal gold solution of claret;
2) colloid gold label avian influenza virus monoclonal antibody
A. prepare the avian influenza virus monoclonal antibody
Prepare the hybridoma cell strain that to secrete the bird flu monoclonal antibody with the avian influenza virus immune mouse, obtain one of monoclonal antibody specific of general A type avian influenza virus, H5 type avian influenza virus, H7 type avian influenza virus or H9 type avian influenza virus; Each subtype avian influenza virus monoclonal antibody only with the avian influenza virus antigen generation specific reaction of corresponding hypotype, thereby determined the specificity of test strips; Use above-mentioned colloidal gold labeled monoclonal antibody, make golden labeling antibody, can with sample to be checked in the combination of avian influenza virus specific;
B. prepare colloid gold label avian influenza virus monoclonal antibody
Getting the colloidal gold solution 10mL of above-mentioned preparation, is 1% sal tartari (K with weight percent concentration
2CO
3) aqueous solution adjusting pH to 8.5-9.2 (use front adjusting, clustering phenomena can appear in too early adjusting), preferred pH is 8.7-9.0; With 800mL dissolved in distilled water 0.2g potassium chloride (KCl), 1.44g sodium hydrogen phosphate (Na
2HPO
4) and 0.24g potassium dihydrogen phosphate (KH
2PO
4), the pH value to 7.4 with hydrochloric acid (HCl) regulator solution adds water to 1L, shakes up, and makes phosphate buffer (PB, Phosphate Buffer); That to be diluted to protein content be 0.1-1mg/mL, pH is identical with the pH value of colloidal gold solution after the above-mentioned adjusting with the above-mentioned avian influenza virus monoclonal antibody for preparing with phosphate buffer (PB), get 0.1-0.5mL, lower centrifugal 1 hour of 13000rpm, 4 ℃, obtain supernatant; Under the rapid stirring supernatant dropwise joined in the above-mentioned colloidal gold solution of regulating the pH value, to the mini mum proteins of liquid be 10-20 μ g/mL, preferred minimum protein content is 10-16 μ g/mL; Room temperature is placed 5min; The weight percent concentration that adds after filtering respectively again is 10%BSA solution 1ml, continue to stir 10-15 minute, then centrifugal 1 hour of 13000rpm, 4 ℃, careful abandoning supernatant (was answered colourless liquid, illustrate that then centrifugal speed and time are inadequate if redness occurs), be precipitated thing; With 10ml concentration be 0.01mol/L, pH 8.2 and wherein the weight percent concentration of BSA be 1% tris solution (TBS, Tris Buffered Saline) with above-mentioned sediment dissolving, centrifugal lower 10 minutes 1000rpm/min, 4 ℃ again, remove aggregation, stay supernatant, discard the precipitation of bottom, supernatant 13000rpm, 4 ℃ is centrifugal 1 hour; Containing weight percent concentration with 5ml, to be respectively 0.02% Sodium azide, 1% sucrose, 1% BSA and pH be 8.2 tris solution (TBS) Eddy diffusion bottom loose deposits, obtains bird flu gold mark monoclonal antibody probe; Place 4 ℃ of Refrigerator stores for subsequent use;
4. prepare enhanced sensitivity reagent
Enhanced sensitivity reagent is reagent A and reagent B;
Reagent preparation A: the preparation weight percent concentration is the aqueous solution of chloraurate of 1%-10%; The preferred weight percent concentration of gold chloride is 1-2% in the aqueous solution of chloraurate;
Reagent preparation B: the preparation weight percent concentration is greater than the aqueous ascorbic acid of 0.1-0.5%; The preferred weight percent concentration of ascorbic acid is 0.15% in the aqueous ascorbic acid;
5. detection avian influenza virus
1) drips respectively on the diaphragm of the sample holes of the stand-by immobilization test strips A of above-mentioned preparation or B and be diluted to PBS that the antigen weight percent concentration is the general A type of 0.05-0.1mg/mL in the solution, H5, one of H7 or H9 type fowl stream viral antigen are as the positive control that needs to detect, with the sample to be tested that is dissolved in PBS buffer solution after the pre-treatment of the centrifugal dilution conventional method of wash-out, the negative control without the allantoic fluid of virus infections that obtains by the chicken embryo for the positive control antigen that will detect, each drips 1-3 μ L, each sample drips 1-2 part (namely respectively with 2 parts of identical antigens, testing sample or negative electrode control cells liquid splash into respectively or 2 sample holes or 2 diaphragms on, can do simultaneously Duplicate Samples), for seep, at room temperature react more than 1 minute; Obtain the test strips with positive control antigen, sample to be tested and negative control;
2) get the corresponding gold mark monoclonal antibody probe solution of positive control antigen that detects with the above-mentioned needs of having selected, get 1-3 μ L for every part, drip in each of above-mentioned test strips A with positive control antigen, sample to be tested and negative control and added on each diaphragm of the sample holes of sample or B; Room temperature reaction 0.5～2 minute, the preferred time is 1-2 minute; Add respectively and contain the phosphate washing lotion PBST that concentration of volume percent is 0.02% polysorbas20, wash 2-5 time, each three, every 2-10 μ L obtains test strips to be detected;
3) respectively to the reagent A that dripped first the above-mentioned enhanced sensitivity reagent of 1-3 μ L on above-mentioned test strips A to be detected had added each diaphragm of each sample holes of gold mark monoclonal antibody probe or test strips B, and then the reagent B of dropping 3-8 μ L enhanced sensitivity reagent, after air-dry, repeat again to state operation 1 to 2 time; 1-2 minute observations after last the dropping, if visual inspection has the grey black spot, then think the positive result of avian influenza virus corresponding to this position, with negative control colour developing result without obvious difference, then think the negative result of avian influenza virus corresponding to this position.
If contain one or several of avian flu virus hemagglutinin hypotype (H type) and neuraminidase hypotype (N-type) in human or animal's to be measured secretion, serum, urine or the food leaching liquor, when the NC film contacts with samples to be tested such as secretion, serum, urine or food leaching liquors, when if spot corresponding to avian influenza virus monoclonal antibody is positive, can determine specifically that then avian influenza virus belongs to certain or some plants virus subtype.Adopt enhanced sensitivity reagent to carry out one or many and process, compare with original Sandwich dot immuno-gold assay, the bird flu detection sensitivity can significantly improve 8～50 times.
Research unit, company that general A type avian influenza virus antigen required for the present invention and each subtype avian influenza virus antigen, avian influenza virus polyclonal antibody can arrive relevant speciality buy or customization; Required instrument, equipment, medicine all have commercially available.
Description of drawings
Fig. 1: the immunity percolation test strips is the front elevation of A.
Fig. 2: the immunity percolation test strips is the vertical view of A.
Fig. 3: the immunity percolation test strips is the front elevation of B.
Fig. 4: the immunity percolation test strips is the vertical view of B.
Embodiment
The present invention is further described in detail by following examples.
Embodiment 1: the preparation of various reagent
1. aqueous solution of chloraurate: take by weighing the 1.0g gold chloride, be dissolved in the 100mL distilled water, form weight percent concentration and be 1% aqueous solution of chloraurate, shake up.
2. trisodium citrate aqueous solution: take by weighing the 1.0g trisodium citrate, be dissolved in the 100mL distilled water, form weight percent concentration and be 1% trisodium citrate aqueous solution, shake up.
3. phosphate buffer (PBS): with 800mL dissolved in distilled water 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g sodium hydrogen phosphate (Na
2HPO
4) and 0.24g potassium dihydrogen phosphate (KH
2PO
4), the pH value to 7.4 with hydrochloric acid (HCl) regulator solution adds water to 1L, shakes up.
4. phosphate buffer (PB): with 800mL dissolved in distilled water 0.2g potassium chloride (KCl), 1.44g sodium hydrogen phosphate (Na
2HPO
4) and 0.24g potassium dihydrogen phosphate (KH
2PO
4), the pH value to 7.4 with hydrochloric acid (HCl) regulator solution adds water to 1L, shakes up.
5. phosphate washing lotion (PBST): with 800mL dissolved in distilled water 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g sodium hydrogen phosphate (Na
2HPO
4) and 0.24g potassium dihydrogen phosphate (KH
2PO
4), the pH value to 7.4 with hydrochloric acid (HCl) regulator solution adds water to 1L, shakes up, and adds the polysorbas20 of 200 μ L, shakes up.
6. bird flu polyclonal antibody: with the PBS dilution, shake up, make that the polyclonal antibody weight percent concentration is 0.05-0.3mg/mL in the solution.
Embodiment 2: avian influenza virus colloidal gold immunity percolation sensitization kit and using method
1. the assembling of avian influenza virus colloidal gold immunity percolation sensitization method kit
1) the immobilization diafiltration test strips A or the B100 bar that prepare are as stated above preserved in 4 ℃ of refrigerators at ordinary times;
Below in conjunction with accompanying drawing test strips A or B are described further:
Such as Fig. 1, Fig. 2, the plastic substrate 1 of test strips A long for 40mm, wide be 3mm, along long direction be provided with pitch of holes be 8 of 2mm to be the diameters that delegation distributes be the circular sample holes 3 of 0.5mm, cellulose nitrate diaphragm 2 overlapping place it below identical with the plastic substrate size.Or the plastic substrate 1 of test strips A long for 110mm, wide be 6mm, along long direction be provided with pitch of holes be 15 of 4mm to be the diameters that delegation distributes be the circular sample holes 3 of 3mm, adhere to below it with the big or small identical cellulose nitrate diaphragm of plastic substrate 2 is overlapping;
Such as Fig. 3, Fig. 4, test strips B is that the circular cellulose nitrate diaphragm 1 of 8 diameter 0.5mm of cutting adheres to the long 40mm, wide on the plastic substrate 2 of 3mm that is, each diaphragm spacing is 3mm; The plastic substrate of each diaphragm below is provided with two suction holes 3, the suction hole be with overlapping with the diaphragm center of circle, parallel with plastic base length of a film major axis for than the long 0.3mm of diaphragm diameter of a circle be 0.8mm, with the wide parallel minor axis of plastic substrate for being that the ellipse of 0.3mm is removed the hole that obtains by the part that diaphragm covers than the short 0.2mm of diaphragm diameter of a circle, during application of sample band absorb water the hole plastic polyethylene (PE) substrate below; Or test strips B is that 2 of the circular nitrocellulose filters of 15 diameter 3mm of cutting place the long 110mm, wide on the plastic substrate 1 of 6mm that is, each diaphragm spacing is 4mm; The plastic substrate of each diaphragm below is provided with two suction holes 3, the suction hole be with overlapping with the diaphragm center of circle, parallel with plastic base length of a film major axis for being 3.3mm than the long 0.3mm of diaphragm diameter of a circle, being that the ellipse of 2.8mm is removed the hole that obtains by the part that diaphragm covers with the wide parallel minor axis of plastic substrate than the short 0.2mm of diaphragm diameter of a circle, during application of sample band absorb water the hole plastic polyethylene (PE) substrate below;
2) phosphate buffer (PBS) 100ml places plastic bottle;
3) the enhanced sensitivity reagent reagent A for preparing as stated above; Get gold chloride 1.0g, be diluted to 100mL with distilled water and be placed in the brown reagent bottle, the in-built 10mL of each kit after the packing can detect 100 groups of test strips simultaneously.The enhanced sensitivity reagent reagent B for preparing as stated above; Get ascorbic acid 1.0g, be diluted to 100-150mL with distilled water and be placed in the brown reagent bottle, each reagent is packed in-built 10mL after the packing; Can detect 100 test strips;
4) phosphate washing lotion (PBST): get 200 μ L polysorbas20s, be mixed with 1000mL, the in-built 100mL of each kit after the packing with the PBS damping fluid of pH7.4,0.01mol/L;
5) positive control antigen liquid is diluted to the solution 1ml that antigen weight percent concentration in the solution is one of general A type, H5, H7 or the H9 type avian influenza virus antigen of 0.05-0.1mg/mL with PBS; Can detect 100 test strips;
6) negative controls is for the allantoic fluid 10mL without virus infections of the positive control antigen that will detect by the acquisition of chicken embryo; Can detect 100 test strips;
7) gold mark probe reagent, getting protein content is the avian influenza virus monoclonal antibody probe liquid 1000 μ L for positive control of 10-20 μ g/mL, is placed in the vial, the rear sealing that is evacuated is preserved in 4 ℃ of refrigerators at ordinary times; Can detect 100 test strips;
8) liquid-transfering gun of 10 of plastic dropping bottles, 1-25 μ L range;
9) length and width is respectively greater than 100 of the filter paper of diafiltration test strips A or B.
2. the using method of kit is as follows:
1) takes out one of above-mentioned test strips A or B, be put in keep flat on the table one above the filter paper;
The positive control antigen liquid that 2) will need respectively to detect, with the sample to be tested liquid that is dissolved in PBS buffer solution after the pre-treatment of the centrifugal dilution conventional method of wash-out and negative controls 1-3 μ L, splash into the sample holes of above-mentioned test strips A or the diaphragm of test strips B, each sample is done respectively 2 parts, and is for seep;
3) add respectively the corresponding gold mark monoclonal antibody probe solution of bird flu antigen that 1 μ L and above-mentioned needs detect on the diaphragm of the sample holes of the above-mentioned paper slip A that has added sample or test strips B, put dried;
4) drip respectively 10 μ L phosphate washing lotions (PBST) to the diaphragm of the sample holes of the above-mentioned test strips A that has added gold mark monoclonal antibody probe reagent or test strips B and wash the NC film, each three, to wash 2-5 time, the room temperature underlying is dried;
5) add respectively the reagent A of 2 μ L enhanced sensitivity reagent on the diaphragm of the above-mentioned sample holes of having used the test strips A that phosphate washing lotion (PBST) washed the NC film or test strips B, add again the reagent B of 5 μ L enhanced sensitivity reagent, developed the color 1-2 minute; Can repeat this operation once or for several times;
6) observe the relevant position after developing the color for the last time 1-2 minute and have or not the grey black spot, have and then think the positive result of avian influenza virus corresponding to this position, do not have or color is compared light avian influenza virus corresponding to this position result that is negative that then thinks with corresponding negative control.
The antigen of the at every turn selected a kind of avian influenza virus of above testing process is done positive contrast antigen, selects the monoclonal antibody of the colloid gold label corresponding with this antigen again, in order to determine the kind of the contained avian influenza virus of sample to be tested.General at first selected general A type avian influenza virus antigen is done positive contrast antigen, selects the monoclonal antibody dilution of corresponding with this antigen colloid gold label again, carries out examination; Tests positive, selected subtype virus antigen is done the definite concrete kind of the further detection of monoclonal antibody that positive control reaches the colloid gold label corresponding with it respectively again.Because every kind of kit can only detect for a kind of virus, carry out the qualitative examination of kind such as the need scene, then need carry the corresponding detection kit of viral antigen of required detection.
Embodiment 3: the preparation method of collaurum
The present invention is take 1% gold chloride, 1% trisodium citrate and distilled water as raw material, adopt trisodium citrate reduction method to prepare colloid gold particle, can be by changing the addition of trisodium citrate, preparation different-diameter and size be the colloid gold particle of homogeneous (15-150nm) comparatively;
Get the 100mL distilled water, heating is boiled, and adds 1% aqueous solution of chloraurate (HAuCl of the fresh preparation of 1mL
4), add rapidly 1.2mL 1% trisodium citrate, constantly stir, it is best that the collaurum that is prepared into claret is used for testing effect, and synthetic collaurum is more stable at this moment, can preserve 12 months under 4 ℃.
Embodiment 4: the determining of colloid gold label optimum pH
Get several 5mL test tubes, add respectively the 1mL colloidal gold solution, with 0.1mol/L HCl and 25mmol/L K
2CO
3The pH value of solution is adjusted to respectively 3,4,5,6,7,8,9,10,11,12,13,14; Get one 96 well culture plates, from low to high above-mentioned colloidal gold solution is respectively got 100 μ L by the pH value and added in the hand-hole triplicate; Every hole adds respectively the good bird flu monoclonal antibody of purifying that 3 μ L weight percent concentration are 1mg/mL, mixes in the hole, places 15min under the room temperature; It is 10%NaCl that every hole adds respectively 20 μ L concentration, mixes in the hole, places 10min under the room temperature; The change color of observing colloid gold, and measure respectively its OD
520nmAnd OD
580nm, be recorded in OD
520nmAnd OD
580nmPH (X) when difference is maximum; Repeat the step of front, further refinement pH value gradient is set as X-0.6 with pH value gradient again; X-0.3; X; X+0.3; X+0.6; X+1, observing colloid gold change color until placed 2 hours under the room temperature, is measured respectively its OD
520nmAnd OD
580nmValue is recorded in OD
520nmWith OD
580nmPH value when difference is maximum, the appropriate pH value during judge mark is 8.5-9.2 accordingly, preferably the pH value is 8.7-9.2.
Embodiment 5: determine the minimum protein stabilized amount of colloid gold label
Definite experiment of minimum protein stabilized amount is with 0.1mol/L K
2CO
3Solution is regulated colloidal gold solution and is carried out mark to pH8.5, and the reagent according to the form below relevant to other of the antibody after the dilution operates item by item.
The minimum albumen metering of table 2 spectrophotometric determination stable colloid gold
Take the OD value as ordinate, the protein consumption is that horizontal ordinate is made a curve, gets curve and transverse axis and is minimum protein stabilization amount near that protein consumption.Add again on this basis 10-20% and be actual protein stabilization amount.The result shows, when protein content is 10-20 μ g/mL, namely sees fit; When protein content is 10-16 μ g/mL, more suitable.
Embodiment 6: avian influenza virus NP expressing protein enhanced sensitivity detection sensitivity and specific test
To manually express avian influenza virus NP albumen with 200 times of PBS dilutions, after carry out continuous doubling dilution, until be diluted to 200 * 2
5Doubly, when using ELISA test strip, namely detecting the lowest limit before the enhanced sensitivity is 800 times of dilutions of sample, and it is 6400 times of dilutions of sample that test strip is carried out detecting the lowest limit after enhanced sensitivity is processed, and this gold mark photosensitivity-enhancing method can make avian influenza virus NP Protein Detection bottom line improve 8 times.Use simultaneously Avian pneumo-encephalitis virus (NDV) to detect, ELISA test strip contains PBS dilution and the normal cell nutrient solution of NDV, and is all negative.Universal detection does not produce interference to this explanation NDV to avian influenza virus.
Embodiment 7:H9N2 subtype avian influenza virus colloidal gold immunity percolation sensitization detects test
With H9N2 subtype avian influenza virus inoculated into chick embryo, the results chick embryo allantoic liquid carries out continuous doubling dilution with PBS, utilizes sensitization detection method provided by the invention, and the enhanced sensitivity reagent A is mixed according to 1: 1 volume ratio with enhanced sensitivity reagent B, make enhanced sensitivity reagent, get respectively enhanced sensitivity reagent 4 μ L and process.When using ELISA test strip, namely detect the lowest limit before the enhanced sensitivity and be hemagglutinin tire (HAU) be 2
-1, it is 2 that test strip is carried out the rear HAU of enhanced sensitivity processing
-5, the comparison test findings shows, but H9N2 subtype avian influenza virus collaurum is detected enhanced sensitivity more than 16 times.
Claims (1)
1. colloidal gold immunity percolation kit that detects avian influenza virus is characterized in that: assembling comprises
1) immobilization diafiltration test strips A or B are 100; The preparation method of described immobilization diafiltration test strips A or B is as follows:
Preparation immunity percolation test strips A: along long for 40-110mm, wide be that to be the diameter that delegation distributes be the circular sample holes of 0.5-3mm for 8-15 of 2-4mm for the long direction of the rectangle vinyon substrate of 3-6mm is provided with pitch of holes; The cellulose nitrate diaphragm identical with vinyon substrate size adhered to below it; During application of sample with the vinyon substrate of sample holes upper;
Preparation immunity percolation test strips B: the circular cellulose nitrate diaphragm of cutting 8-15 diameter 0.5-3mm adheres to the long 40-110mm of being, widely is the rectangle tygon of 3-6mm. and plastic substrate, diaphragm spacing are 3-4mm; The plastic substrate of each diaphragm below is provided with two suction holes, the suction hole be with overlapping with the diaphragm center of circle, parallel with plastic base length of a film major axis than the long 0.3mm of diaphragm diameter of a circle, removed the hole that obtains by the part that diaphragm covers with the wide parallel minor axis of plastic substrate than the ellipse of the short 0.2mm of diaphragm diameter of a circle, during application of sample band absorb water the hole the plastic polyethylene substrate below;
Then with the immobilization on immunity percolation test strips A or B of avian influenza virus polyclonal antibody, adopt point sample immobilization method or soak one of immobilization method;
With above-mentioned immobilization test strips A or the B that obtains with point sample method or infusion method, the weight percent concentration of putting into the distilled water dilution is the bovine serum albumin solution of 1-5% at last, 37 ℃ of sealing 20-60min; Take out, air-dry, obtain stand-by immobilization test strips; 4 ℃ of preservations, for subsequent use;
2) phosphate buffer PBS100ml places plastic bottle;
3) enhanced sensitivity reagent A reagent 10mL can detect 100 groups of test strips; Enhanced sensitivity reagent B reagent 10mL can detect 100 test strips;
Reagent A: the preparation weight percent concentration is the aqueous solution of chloraurate of 1%-10%;
Reagent B: the preparation weight percent concentration is greater than the aqueous ascorbic acid of 0.1-0.5%;
4) phosphate washing lotion PBST 100mL;
5) positive control antigen liquid is diluted to the solution 1ml that antigen weight percent concentration in the solution is one of general A type, H5, H7 or the H9 type avian influenza virus antigen of 0.05-0.1mg/mL with PBS; Can detect 100 test strips;
6) negative controls is without the allantoic fluid 10mL of avian flu virus infection; Can detect 100 test strips;
7) gold mark probe reagent, getting protein content is the avian influenza virus monoclonal antibody probe liquid 1000 μ L for positive control of 10-20 μ g/mL, is placed in the vial, the rear sealing that is evacuated is preserved in 4 ℃ of refrigerators at ordinary times; Can detect 100 test strips;
8) liquid-transfering gun of 10 of plastic dropping bottles, 1-25 μ L range;
9) length and width is respectively greater than 100 of the filter paper of diafiltration test strips A or B.
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