CN101470114A - Sensitization detection method of colloidal gold immunity chromatography and use thereof - Google Patents
Sensitization detection method of colloidal gold immunity chromatography and use thereof Download PDFInfo
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Abstract
A sensitivity raising detection method of colloid gold immunity chromatography method comprises: when using general colloid gold immunity chromatography to check object sample, adding an object sample, adding signal amplifying agents of 10 to 80ul between the gold label antibody layer and the detection line; processing the detection line obtained by general colloid gold immunity chromatography method; reacting under room temperature for 1 to 10min; if the object sample contains antigen, the detection carrier is formed with a detection line and a control line as clear black or grey black color, to represent positive property, or if the object sample not contains antigens, the control line is black or grey black color, and the detection line has not color, to represent negative property. The method can significantly improve the sensitivity of colloid gold immunity chromatography reagent kit on the detection of biological macromolecules, having fast color development, lower background interference compared with general silver stain method, wide application prospect and high development value.
Description
Technical field
The present invention relates to a kind of macromolecular substances detection method, particularly relate to the photosensitivity-enhancing method of biomacromolecule material gold-marking immunity chromatography detection and utilize it to make the method for kit.
Background technology
At present, can carry out easy, quick, high selectivity and highly sensitive detection to the biomacromolecule material, all the time be the direction that people make great efforts, fields such as this quick diagnosis in disease, health quarantine, the animal and plant quarantine, food safety detection, environmental monitoring are all significant.Wherein, immunoassay (Immunoassay) based on antigen and antibody specific reaction has high selectivity and convenience because of it, and its principle simply, does not need large-scale experiment equipment or bigger investment can produce uses, running sample testing cost to make it become the first-selected means of many predetermined substance detections far below characteristics such as instrument analytical method simultaneously.
Gold chloride (HAuCl
4) under effects such as reductive agent such as trisodium citrate, white phosphorus, ascorbic acid, tannic acid, polymerization becomes the gold grain of specific size, because electrostatic interaction forms stable colloidal state, so the title collaurum.Because the collaurum surface is electronegative, can with macromolecular positive charge such as protein because of the Electrostatic Absorption strong bonded, do not influence the biological nature of protein simultaneously.Faulk in 1971 and Taytor introduce immunoassay with collaurum, formed immuno-gold labeling technology (Immunogoldlabeling techique), being about to collaurum and being applied to the antigen-antibody mark as tracer, mainly is that carrier is realized immune detection with the nitrocellulose filter.The immuno-gold labeling technology mainly is divided into two kinds of patterns: and (1) immunity percolation analysis (Flowthrough-immunofiltration assay, IFA): immune response is by vertically penetrating (the flow through type) that the nitrocellulose filter that is fixed with part carries out; (2) sidestream immune chromatographic analysis (Lateral flow-immunochromatographicassay, ICA): analysis principle is identical with IFA, just reaction liquid flow be not directly to penetrate mobile, but pass through the cross flow (lateral flow type) that the chromatography effect takes place capillarity, begin to use in nineteen ninety.
Though above-mentioned two kinds of methods all are to be the immune-gold labeled technology of carrier with the nitrocellulose filter, it detects principle bigger difference.Mainly show: after ICA added test solution, the test solution chromatography spread and has realized the separation of sample matrix under capillarity, existed sample substrate to disturb in the detection line hardly; And the washing of IFA by vertically penetrating, detecting spot easily spreads, and is difficult to recognition results.So widely used commercialization test strips (or kit) product adopts the immunochromatography principle mostly.
Yet the maximum deficiency of immuno-gold labeling detection method is that detection sensitivity is low, has limited practical ranges.Past people mainly utilizes silver-colored staining technique to overcome the low problem of detection sensitivity.Promptly amplify reagent as signal and handle detection line on the sample pad with silver nitrate and specific reductant.Yet this method signal amplification effect is not obvious, and is main because the detection signal background is fuzzy, background color is high, is difficult for judging testing result by naked eyes.Therefore at present almost be difficult to see colloidal gold immunochromatographimethod product based on silver-colored staining technique.
Summary of the invention
Technical matters solved by the invention provides a kind of sensitization detection method of colloidal gold immunity chromatography, the method is on the basis of conventional colloidal gold immunochromatographimethod assay method, the detection line that the nanogold particle that adopts the reaction of gold chloride and reductive agent to generate obtains conventional colloidal gold immunity chromatography is handled signal is strengthened, can significantly strengthen the detection signal of conventional colloidal gold immunity chromatography and background is clean, background values is low, can improve 8～200 times of detection sensitivities, thereby overcome the low problem of traditional colloidal gold immunochromatographimethod detection method sensitivity.
Another object of the present invention provides preparation method of a kind of colloidal gold immunochromatographimethod high-sensitivity detection kit and products thereof and uses, and this kit is to utilize the sensitization detection method of above-mentioned colloidal gold immunity chromatography to prepare.
A kind of signal that is used for colloidal gold immunity chromatography amplifies reagent, it is the mix reagent that enhanced sensitivity reagent A and enhanced sensitivity reagent B press the volume ratio mixing of 1:1～1:5, the described enhanced sensitivity reagent A concentration that is weight percentage is 0.1%～10% chlorauric acid solution, and enhanced sensitivity reagent B is a reductant solution.It is 0.1～0.5% ascorbic acid solution that wherein said reductant solution is preferably oxammonium hydrochloride solution that volumetric molar concentration is 0.1～10mM or weight percent concentration, also can be other reductive agent, as trisodium citrate, white phosphorus, tannic acid etc.The weight percent concentration of wherein said chlorauric acid solution is preferably 1～4%.The volumetric molar concentration of wherein said oxammonium hydrochloride solution is preferably 2～6mM, or the weight percent concentration of ascorbic acid solution is preferably 0.15～0.3%.
A kind of sensitization detection method of colloidal gold immunity chromatography, when conventional colloidal gold immunity chromatography detects sample to be tested, after adding sample to be tested, between golden labeling antibody layer and detection line, add described signal again and amplify reagent 10～80 μ L, the detection line that conventional colloidal gold immunity chromatography is obtained is handled signal is strengthened, after reacting 1～10 minute under the room temperature, observations.
When method of the present invention is specifically used, wherein said testing sample is animal secretions, serum, organize extracts such as internal organs, testing sample is handled with pre-treating methods such as the centrifugal dilutions of wash-out, get 40～80 μ L and handle drop on the well of conventional colloidal gold immunochromatographimethod kit, each sample repeats 2～3 times, after reacting 1～10 minute under the room temperature, 100 μ L phosphate washing lotion PBST are added in the well residual substance on the washing chromatography strip; Chlorauric acid solution that will be pre-mixed and reductant solution are got 10～80 μ L rapidly and are dripped in well or add on the enhanced sensitivity reagent wells again; Wherein said expansion reagent is to contain the phosphate buffer PB of 0.1mM that weight percent concentration is the surfactant Tween 20 of 1～2% NPE NP40 and 0.1～1%.
The method according to this invention can prepare the special test paper bar and/kit, the preparation method can be following step:
Front end at the upper surface of a plastic base plate is pasted with one deck application of sample end absorbent paper layer, be overlapped with golden labeling antibody layer and detection layers rear end after the described application of sample end absorbent paper layer continuously up to plastic base plate, the rear end of described detection layers upper surface is pasted with the suction side absorbent paper layer that one deck can absorb 200 μ L liquid at least, described golden labeling antibody layer is the cellulose nitrate rete that is coated with antibody, described detection layers is the cellulose nitrate rete, be coated with detection line and control line successively in its position between described golden labeling antibody layer and suction side absorbent paper layer, after preparing, it is pressed 4 mm wides, 8 centimeter length specification slittings are assembled in the plastic casing again.Can many be arranged in the big box, also can one be arranged in the etui, and then supporting required reagent.The upper surface of described box is provided with three perforates: well, add enhanced sensitivity reagent wells and viewport, described well is over against application of sample end absorbent paper layer, the described enhanced sensitivity reagent wells that adds is over against the position between golden labeling antibody layer and the detection line, and described viewport is over against the suction side absorbent paper layer.
The preparation method of wherein said golden labeling antibody layer comprises the steps:
(1). the 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 prepared fresh is 1～5% aqueous solution of chloraurate 1mL
Weight percent concentration is trisodium citrate aqueous solution 1.0～1.5mL of 1～5%;
B. preparation method
With the above-mentioned distilled water of 100mL microwave heating to 65 ℃, the weight percent concentration that adds required prepared fresh is 1～5% aqueous solution of chloraurate 1ml, microwave heating to 95 ℃, the required weight percent concentration of adding is 1～5% trisodium citrate aqueous solution rapidly, constantly stir, gold chloride is under the effect of sodium citrate reductive agent, its collaurum liquid color by black → indigo plant → purple → wine is red, obtain the colloidal gold solution of 20～40nm particle diameter of claret after the cooling, the collaurum that this moment is synthetic can be preserved 12 months under 4 ℃;
(2). prepare golden labeling antibody layer
Get the colloidal gold solution 10mL of above-mentioned preparation, with weight percent concentration is that 1% wet chemical is regulated pH to 8.5～9.2 (preferred 8.7～9.2), with 800mL dissolved in distilled water 0.2g potassium chloride, 1.44g sodium hydrogen phosphate and 0.24g potassium dihydrogen phosphate, pH value to 7.4 with hydrochloric acid conditioning solution, add water to 1L, shake up, make 0.1mM phosphate buffer PB, is 0.1～1mg/mL with described phosphate buffer PB with antibody dilution to protein content, get 0.1～0.5mL, at 13000rpm, centrifugal 30min under 4 ℃, taking out respective volume the supernatant after centrifugal makes and joins that the mini mum proteins of liquid is 10～20 μ g/mL (preferred 10～16 μ g/mL) behind the colloidal gold solution, stir fast and down antibody is slowly dropwise joined in the above-mentioned colloidal gold solution of regulating pH, room temperature is placed 5min; Weight percent concentration after add filtering respectively again is 10% BSA solution 1mL, continues to stir 10～15 minutes, and respectively at 10000rpm, 4 ℃ centrifugal 30min down, abandoning supernatant obtains sediment then; Then with 10mL, concentration be 0.01mol/L, pH 8.2 and wherein the weight percent concentration of BSA be 1% tris solution (TBS, contain 1%BSA) sediment is dissolved, at 10000rpm/min, 4 ℃ of following centrifugal 30min, abandoning supernatant, containing weight percent concentration with 10mL respectively, to be respectively 0.02% Sodium azide, 1% sucrose, 1% BSA and pH be 8.2 the tris solution bottom loose deposits that suspends again, obtains golden labeling antibody probe solution; Till at last golden labeling antibody probe solution being begun to ooze out to nitrocellulose filter metal spraying to liquid with the metal spraying instrument, at 37 ℃ of following 2 hours golden labeling antibody layers of dry formation.
The preparation method of wherein said detection layers comprises: the control line that is coated with the detection line that forms at the specific polyclone of sample to be tested or monoclonal antibody and forms at the specific IgG antibody of golden labeling antibody on nitrocellulose filter: concrete preparation method gets many anti-or monoclonal antibodies, with launching reagent accent concentration is 1mg/mL, sprays detection line in the nitrocellulose filter stage casing with Membrane jetter; Get IgG antibody 1mg/mL again, in the nitrocellulose filter stage casing, apart from detection line 0.5cm place, the spray control line is provided with spray film amount, 37 ℃ of dryings of spray film 2 hours according to 20 μ L/10cm with Membrane jetter.
The products such as test strips of the method and preparation thereof can be widely used in biomacromolecule detection or the medical diagnosis.
Method of the present invention, adopt enhanced sensitivity reagent enhanced sensitivity, utilize gold chloride and oxammonium hydrochloride generation redox reaction to generate the characteristics that gold atom can be adsorbed by collaurum, locate the reinforcement that develops the color of collaurum deposition site, improved the detection sensitivity of biomacromolecule to be checked.Use method of the present invention, can significantly improve the macromolecular sensitivity of colloidal gold immunochromatographimethod kit detection of biological, and colour developing is rapid, background interference is littler than the silver staining of routine, the prospect that is widely used and exploitation are worth.Adopting enhanced sensitivity reagent of the present invention to carry out one or many handles, compare with original colloidal gold immunochromatographimethod determination method, sensitivity can significantly improve 8-200 times, and easy and simple to handle, fast, do not need special instrument and equipment, can be widely used in clinical diagnosis, fields such as antibody, detection of antigens, health quarantine, environment measuring, the prospect that is widely used is worth with exploitation.
Description of drawings
Fig. 1 is the vertical view of the kit of preparation among the embodiment 7;
Fig. 2 is the A-A cut-open view of Fig. 1;
Fig. 3 is the result that commercially available colloidal gold strip detects avian influenza virus NP expressing protein among the embodiment 2;
Fig. 4 detects the result of avian influenza virus NP expressing protein for utilizing collaurum photosensitivity-enhancing method of the present invention among the embodiment 2;
Fig. 5 is the result that commercially available colloidal gold strip detects avian influenza virus in the chick embryo allantoic liquid among the embodiment 3;
Fig. 6 detects the result of avian influenza virus in the chick embryo allantoic liquid for utilizing collaurum photosensitivity-enhancing method of the present invention among the embodiment 3;
Fig. 7 is the result that commercially available colloidal gold strip detects the infected chicken avian influenza virus among the embodiment 4;
Fig. 8 detects the result of infected chicken avian influenza virus for utilizing collaurum photosensitivity-enhancing method of the present invention among the embodiment 4;
Fig. 9 is the result that commercially available colloidal gold strip detects pregnant woman's urine sample among the embodiment 5;
Figure 10 detects the result of pregnant woman's urine sample for utilizing collaurum photosensitivity-enhancing method of the present invention among the embodiment 5;
Figure 11 is the result that commercially available colloidal gold strip detects the Bacterium enteritidis nutrient solution among the embodiment 6;
Figure 12 detects the result of Bacterium enteritidis nutrient solution for utilizing collaurum photosensitivity-enhancing method of the present invention among the embodiment 6;
Figure 13 is the result that the self-control colloidal gold strip detects Avian pneumo-encephalitis virus poison alive among the embodiment 10;
Wherein the result of test strips when Figure 14 lives poison for utilizing among the embodiment 10 kit of the present invention to detect Avian pneumo-encephalitis virus.
Embodiment
Embodiment 1:The preparation of all ingredients
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 1.0～1.5g trisodium citrate, be dissolved in the 100mL distilled water, form weight percent concentration and be 1～1.5% 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, adds the surfactant Tween20 of 200 μ L again, shakes up.
6. how anti-or monoclonal antibody: make with the PBS dilution that many anti-or monoclonal antibody weight percent concentration are 1mg/mL in the solution.
7. expansion reagent: add 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 the phosphate buffer (PB, Phosphate Buffer) of 0.1mM.Add weight percent concentration and be 1% NPE (NP40) and 0.5% Tween 20 surfactants.
8. enhanced sensitivity reagent:
The enhanced sensitivity reagent A: the preparation weight percent concentration is 0.1%～10% gold chloride, to launch the reagent dilution; The preferred weight percent concentration of gold chloride is 1～4% in the aqueous solution of chloraurate;
Enhanced sensitivity reagent B: the preparation volumetric molar concentration greater than the oxammonium hydrochloride of 0.1～10mM or weight percent concentration greater than 0.1～0.5% aqueous ascorbic acid, to launch the reagent dilution.The preferred volumetric molar concentration of oxammonium hydrochloride is 5mM, and the preferred weight percent concentration of ascorbic acid is 0.15% in the aqueous ascorbic acid.
9.IgG antibody: make with PBS dilution that IgG antibody weight percent concentration is 1mg/mL in the solution.
(10.BSA bovine serum albumin(BSA)) solution: make with distilled water dilution that the BSA weight percent concentration is 10% in the solution.
Embodiment 2:Avian influenza virus NP expressing protein enhanced sensitivity detects contrast test
To manually express avian influenza virus NP albumen with 200 times of developping agent dilutions, after carry out continuous doubling dilution, until being diluted to 200 * 2
8Doubly, each concentration sample is measured with the universal gold mark detection test paper bar of commercially available avian influenza virus (Anigen company), each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive.
Utilize sensitization detection method provided by the invention, enhanced sensitivity reagent A (0.5% gold chloride) is mixed according to the volume ratio of 1:5 with enhanced sensitivity reagent B (oxammonium hydrochloride of 10mM), make signal and amplify reagent, reagent 50 μ L processing is amplified in the number of winning the confidence respectively, and concrete disposal route is to add this signal to amplify reagent between the golden labeling antibody layer of test strips and detection line.After processing was negative the result to negative control, parallel enhanced sensitivity was handled 1 test strip of each concentration sample.Detection line, control line show that clearly grey black is the sample positive.When using test strips to detect, as seen minimum 1600 times of dilute samples react the band (see figure 3), test strip is carried out after enhanced sensitivity handles, and as seen minimum 25600 times of dilute samples react the band (see figure 4), and this gold mark photosensitivity-enhancing method can make avian influenza virus NP Protein Detection bottom line improve 16 times.
Embodiment 3:H9N2 subtype avian influenza virus collaurum enhanced sensitivity detects test
With H9N2 subtype avian influenza virus inoculated into chick embryo, the results chick embryo allantoic liquid carries out continuous doubling dilution with developping agent, until being diluted to 2
12Doubly, each dilutability allantoic fluid sample is measured with the universal gold mark detection test paper bar of commercially available avian influenza virus (Anigen company), each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive.
Utilize sensitization detection method provided by the invention, enhanced sensitivity reagent A (1% gold chloride) is mixed according to the volume ratio of 1:2 with enhanced sensitivity reagent B (oxammonium hydrochloride of 10mM), make signal and amplify reagent, reagent 40 μ L processing is amplified in the number of winning the confidence respectively, and concrete disposal route is to add this signal to amplify reagent between the golden labeling antibody layer of test strips and detection line.After processing was negative the result to negative control, parallel enhanced sensitivity was handled 1 test strip of each concentration sample.Detection line, control line show that clearly grey black is the sample positive.When using test strips to detect, promptly detect the lowest limit before the enhanced sensitivity and be hemagglutinin tire (HAU) be 2
-5(see figure 5), it is 2 that test strip is carried out enhanced sensitivity processing back HAU
-12, but also not arriving lowest limit (see figure 6), the comparison test findings shows, but H9N2 subtype avian influenza virus collaurum is detected enhanced sensitivity 2
7Doubly.
Embodiment 4:H9N2 subtype avian influenza virus collaurum enhanced sensitivity detects test in the virus infections sample
With H9N2 subtype avian influenza virus inoculation chicken, throat swab is gathered in the back and the cloaca swab detects.The centrifugal back of collected specimens wash-out is diluted with developping agent, each concentration sample is measured with the universal gold mark detection test paper bar of commercially available avian influenza virus (Anigen company), each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive.
Utilize sensitization detection method provided by the invention, enhanced sensitivity reagent A (5% gold chloride) is mixed according to the volume ratio of 1:1 with enhanced sensitivity reagent B (oxammonium hydrochloride of 5mM), make signal and amplify reagent, reagent 80 μ L processing is amplified in the number of winning the confidence respectively, and concrete disposal route is to add this signal to amplify reagent between the golden labeling antibody layer of test strips and detection line.Detection line, control line show that clearly grey black is the sample positive.When using test strips to detect, be to have only chicken throat swab bands visible (see figure 7) before the enhanced sensitivity No. 10, test strip is carried out 1,2,3,6,8, No. 10 chicken throat swab in enhanced sensitivity processing back all can see obvious band (see figure 8), improve detection sensitivity, can find virus infections as early as possible in actual applications.
Embodiment 5:The collaurum very early pregnancy is detected the enhanced sensitivity test of test paper
Get the pregnant woman's urine that is four months pregnant and carry out doubling dilution with developping agent, detecting test paper (production of Blue-Cross Bioceuticals Inc.) with commercially available collaurum very early pregnancy measures each concentration sample, each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive.
Utilize sensitization detection method provided by the invention, enhanced sensitivity reagent A (0.1% gold chloride) is mixed according to the volume ratio of 1:5 with enhanced sensitivity reagent B (0.1% ascorbic acid), make signal and amplify reagent, reagent 10 μ L processing is amplified in the number of winning the confidence respectively, and concrete disposal route is to add this signal to amplify reagent between the golden labeling antibody layer of test strips and detection line.Detection line, control line show that clearly grey black is the sample positive.When using test strips to detect, be not diluted to 2 during enhanced sensitivity
9The time These positive bands of being visible to the naked eye, be all negative (see figure 9) after surplus.Test strip is carried out being diluted to 2 after enhanced sensitivity is handled
15The time naked eyes can see These positive bands, the enhanced sensitivity multiple is 2
6Times (see figure 10).
Embodiment 6:The salmonella enhanced sensitivity detects contrast test
The Bacterium enteritidis cultured solution of broth is carried out continuous doubling dilution, until being diluted to 2
4Doubly, each dilutability sample is measured with the universal gold mark detection test paper bar of commercially available salmonella (Dalian general auspicious Kanggong department), each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive.
Utilize sensitization detection method provided by the invention, enhanced sensitivity reagent A (1% gold chloride) is mixed according to the volume ratio of 1:3 with enhanced sensitivity reagent B (0.5% ascorbic acid), make signal and amplify reagent, reagent 60 μ L processing is amplified in the number of winning the confidence respectively, and concrete disposal route is to add this signal to amplify reagent between the golden labeling antibody layer of test strips and detection line.Detection line, control line show that clearly grey black is the sample positive.When using test strips to detect, have only stoste sample (ID is labeled as 0) as seen to react band (seeing Figure 11), test strip is carried out after enhanced sensitivity handles, minimum 8 times of dilute samples (ID is labeled as-3) as seen react band (seeing Figure 12), and this gold mark photosensitivity-enhancing method can make Bacterium enteritidis detect 8 times of bottom line raisings.
Embodiment 7:The preparation of colloidal gold immunochromatographimethod high-sensitive detecting kit and application thereof
As depicted in figs. 1 and 2, a kind of colloidal gold immunochromatographimethod high-sensitive detecting kit, comprise box body 1 and place its interior test strips, described box body 1 upper surface is provided with three perforates: well 2, add enhanced sensitivity reagent wells 3 and viewport 4, described test strips comprises plastic base plate 11, front end at the upper surface of plastic base plate 11 is pasted with one deck application of sample end absorbent paper layer 5, be overlapped with golden labeling antibody layer 6 and detection layers 9 rear end after the described application of sample end absorbent paper layer 5 continuously up to plastic base plate 11, the rear end of described detection layers 9 upper surfaces is pasted with the suction side absorbent paper layer 10 that one deck can absorb 200 μ L liquid at least, described golden labeling antibody layer 6 is for being coated with the cellulose nitrate rete of antibody, described detection layers 9 is the cellulose nitrate rete, be coated with detection line 7 and control line 8 successively in its position between described golden labeling antibody layer 6 and suction side absorbent paper layer 10, described well 2 is over against application of sample end absorbent paper layer 5, the described enhanced sensitivity reagent wells 3 that adds is over against the position between golden labeling antibody layer 6 and the detection line 7, and described viewport 4 is over against suction side absorbent paper layer 10.
The preparation method of wherein said golden labeling antibody layer 6 comprises the steps:
(1). the 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 prepared fresh is 1～5% aqueous solution of chloraurate 1mL
Weight percent concentration is trisodium citrate aqueous solution 1.0～1.5mL of 1～5%;
B. preparation method
With the above-mentioned distilled water of 100mL microwave heating to 65 ℃, the weight percent concentration that adds required prepared fresh is 1～5% aqueous solution of chloraurate 1ml, microwave heating to 95 ℃, the required weight percent concentration of adding is 1～5% trisodium citrate aqueous solution rapidly, constantly stir, gold chloride is under the effect of sodium citrate reductive agent, its collaurum liquid color by black → indigo plant → purple → wine is red, obtain the colloidal gold solution of 20～40nm particle diameter of claret after the cooling, the collaurum that this moment is synthetic can be preserved 12 months under 4 ℃;
(2). prepare golden labeling antibody layer 6
Get the colloidal gold solution 10mL of above-mentioned preparation, with weight percent concentration is that 1% wet chemical is regulated pH to 8.5～9.2, with 800mL dissolved in distilled water 0.2g potassium chloride, 1.44g sodium hydrogen phosphate and 0.24g potassium dihydrogen phosphate, pH value to 7.4 with hydrochloric acid conditioning solution, add water to 1L, shake up, make 0.1mM phosphate buffer PB, is 0.1～1mg/mL with described phosphate buffer PB with antibody dilution to protein content, get 0.1～0.5mL, at 13000rpm, centrifugal 30min under 4 ℃, taking out respective volume the supernatant after centrifugal makes and joins that the mini mum proteins of liquid is 10～20 μ g/mL behind the colloidal gold solution, stir fast and down antibody is slowly dropwise joined in the above-mentioned colloidal gold solution of regulating pH, room temperature is placed 5min; Weight percent concentration after add filtering respectively again is 10% BSA solution 1mL, continues to stir 10～15 minutes, and respectively at 10000rpm, 4 ℃ centrifugal 30min down, abandoning supernatant obtains sediment then; Then with 10mL, concentration be 0.01mol/L, pH 8.2 and wherein the weight percent concentration of BSA be 1% tris solution (TBS, contain 1%BSA) sediment is dissolved, at 10000rpm/min, 4 ℃ of following centrifugal 30min, abandoning supernatant, containing weight percent concentration with 10mL respectively, to be respectively 0.02% Sodium azide, 1% sucrose, 1% BSA and pH be 8.2 the tris solution bottom loose deposits that suspends again, obtains golden labeling antibody probe solution; Till at last golden labeling antibody probe solution being begun to ooze out to nitrocellulose filter metal spraying to liquid with the metal spraying instrument, at 37 ℃ of following 2 hours golden labeling antibody layers 6 of dry formation.
The preparation method of wherein said detection layers 9 comprises: the control line 8 that is coated with the detection line 7 that forms at the specific polyclone of sample to be tested or monoclonal antibody and forms at the specific IgG antibody of golden labeling antibody on nitrocellulose filter: concrete preparation method gets many anti-or monoclonal antibodies, with launching reagent accent concentration is 1mg/mL, sprays detection line 7 in the nitrocellulose filter stage casing with Membrane jetter; Get IgG antibody 1mg/mL again, in the nitrocellulose filter stage casing, apart from detection line 0.5cm place, spray control line 8 is provided with spray film amount, 37 ℃ of dryings of spray film 2 hours according to 20 μ L/10cm with Membrane jetter.
When using this kit, get internal organs extracts such as chicken organ or anus secretion lysate, serum, whole blood, liver spleen, be diluted to 40～80 μ L with developping agent reagent, drip on the well 2 of immune chromatography reagent kit, each sample repeats 2～3 times; After reacting 1～10 minute under the room temperature, in advance enhanced sensitivity reagent A and B are mixed according to the volume ratio of 1:1～1:5, getting 10～80 μ L rapidly drips in adding on the enhanced sensitivity reagent wells 3, after reacting 1～10 minute under the room temperature, contain antigen as above-mentioned secretion, serum, whole blood or internal organs and then on the detection carrier, form detection line, control line two-wire for clearly black is then positive, otherwise control line is black, and it is then negative that detection line does not have color.
Embodiment 8:Determining of the colloid gold label optimum pH of the colloidal gold immunochromatographimethod high-sensitive detecting kit among the embodiment 7
Get several 5mL test tubes, add the 1mL colloidal gold solution respectively, with 0.1mol/L HCl, 25mmol/L K
2CO
3With 1-10mol/LKOH the pH value of solution is adjusted to 3,4,5,6,7,8,9,10,11,12,13,14 respectively; 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 the good monoclonal antibody of purifying that 3 μ L weight percent concentration are 1mg/mL respectively, mixes in the hole, places 15min under the room temperature; It is 10%NaCl that every hole adds 20 μ L concentration respectively, mixes in the hole, places 10min under the room temperature; The change color of observing colloid gold, and measure its OD respectively
520nmAnd OD
580nm, be recorded in OD
520nmAnd OD
580nmPH (X) when difference is maximum; Repeat the step of front, further again refinement pH value gradient is set at X-0.6 with pH value gradient; X-0.3; X; X+0.3; X+0.6; X+1, observing colloid gold change color was placed 2 hours under room temperature, measured its OD respectively
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 in view of the above, preferably the pH value is 8.7～9.2.
Embodiment 9:Determining of the minimum protein stabilized amount of colloid gold label of the colloidal gold immunochromatographimethod high-sensitive detecting kit among the embodiment 7
Salt precipitation determination method: utilize colourimetry to determine golden mark optimal pH, use 0.1mol/LK
2CO
3Regulate collaurum pH value to 8.7～9.2, antibody is removed aggregation through the 10000rpm/min high speed centrifugation, and antigen or antibody are diluted to 0.2mg/mL with the PB damping fluid (pH 8.0) of 0.01mol/L, and the antigen after the dilution, antibody and other reagent are pressed table 1 operation.
Table 1 salt precipitation method is measured the minimum albumen metering of stable colloid gold
Add reagent by last table, static 1～2 hour, contain the little pipe of albumen quality and present coagulation phenomenon by red stain indigo plant, add the solution that protein meets or exceeds in the quantitative test tube of minimum steady and then keep red constant.The protein content that redness is remained unchanged is the suitableeest protective number of protein, and increasing by 20% on this basis is the actual amount of the protein of stable colloid gold.The result shows, when protein content is 10～20 μ g/mL, promptly sees fit; When protein content is 10～16 μ g/mL, more suitable.
Embodiment 10:The detection example of the colloidal gold immunochromatographimethod high-sensitive detecting kit among the embodiment 7: Avian pneumo-encephalitis virus malicious enhanced sensitivity alive detects contrast test
Use the Avian pneumo-encephalitis virus inoculated into chick embryo, the results chick embryo allantoic liquid carries out continuous doubling dilution with developping agent, until being diluted to 2
12Doubly, (coated antibody is the rabbit hyper-immune serum with the Avian pneumo-encephalitis virus gold mark detection test paper bar for preparing voluntarily, labelled antibody is a monoclonal antibody specific) each dilutability allantoic fluid sample is measured, each sample determination 2 times, every batch of mensuration is left and taken the negative contrast of 1 row culture.In detection, detection line, control line show that clearly peony is the sample positive (seeing Figure 13).
Utilize colloidal gold immunochromatographimethod high-sensitive detecting kit provided by the invention, enhanced sensitivity reagent A (4% gold chloride) is mixed according to the volume ratio of 1:3 with enhanced sensitivity reagent B (0.15 ascorbic acid), make signal and amplify reagent, the number of winning the confidence is amplified reagent 20 μ L and is handled to splash into and add the enhanced sensitivity reagent wells on the kit respectively, react observation viewport 4 after 10 minutes.Detection line, control line show that clearly grey black is the sample positive.Test findings shows, uses colloidal gold immunochromatographimethod high-sensitive detecting kit provided by the invention to detect Avian pneumo-encephalitis virus poison alive, but enhanced sensitivity 2
7Doubly above (seeing Figure 14).
Above-described embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (8)
1. a signal that is used for colloidal gold immunity chromatography amplifies reagent, it is characterized in that: it presses the mix reagent of the volume ratio mixing of 1:1～1:5 for enhanced sensitivity reagent A and enhanced sensitivity reagent B, the described enhanced sensitivity reagent A concentration that is weight percentage is 0.1%～10% chlorauric acid solution, and enhanced sensitivity reagent B is a reductant solution.
2. signal according to claim 1 amplifies reagent, it is characterized in that: described reductant solution is that volumetric molar concentration is that the oxammonium hydrochloride solution of 0.1～10mM or weight percent concentration are 0.1～0.5% ascorbic acid solution.
3. signal according to claim 2 amplifies reagent, and it is characterized in that: the weight percent concentration of described chlorauric acid solution is 1～4%.
4. signal according to claim 3 amplifies reagent, and it is characterized in that: the volumetric molar concentration of described oxammonium hydrochloride solution is 2～6mM, or the weight percent concentration of ascorbic acid solution is 0.15%～0.3%.
5. the sensitization detection method of a colloidal gold immunity chromatography, it is characterized in that: when conventional colloidal gold immunity chromatography detects sample to be tested, after adding sample to be tested, between golden labeling antibody layer and detection line, add each described signal of claim 1～4 again and amplify reagent 10～80 μ L, the detection line that conventional colloidal gold immunity chromatography is obtained is handled signal is strengthened, after reacting 1～10 minute under the room temperature, observations.
6. method according to claim 5, it is characterized in that: described testing sample is animal secretions, serum, organize the extract of internal organs, testing sample is handled with conventional pre-treating method, get 40～80 μ L and handle drop on the well of conventional colloidal gold immunochromatographimethod kit, each sample repeats 2～3 times, after reacting 1～10 minute under the room temperature, 100 μ L phosphate washing lotion PBST are added in the well residual substance on the washing chromatography strip; Chlorauric acid solution that will be pre-mixed and reductant solution are got 10～80 μ L rapidly and are dripped in well or add on the enhanced sensitivity reagent wells again; Wherein said expansion reagent is to contain the phosphate buffer PB of 0.1mM that weight percent concentration is the surfactant Tween20 of 1～2% NPE NP40 and 0.1～1%.
7. each described signal of claim 1～4 amplifies the application of reagent in colloidal gold immunity chromatography.
8. each described sensitization detection method application in biomacromolecule detection or medical diagnosis of claim 5～6.
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