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
本发明提供一种检测禽流感病毒的胶体金免疫渗滤增敏方法及其试剂盒,步骤如下:1)制备免疫渗滤试纸条;2)禽流感病毒多克隆抗体在免疫渗滤试纸条上的固定化;3)制备禽流感病毒金标单克隆抗体金标探针;4)制备增敏试剂;5)检测禽流感病毒。本发明利用氯金酸与抗坏血酸发生氧化还原反应生成金原子可被胶体金吸附的特点,来定位胶体金沉积部位进行显色加强,提高了待检抗原的检测灵敏度8~50倍。本发明是在胶体金斑点免疫渗滤测定法(双抗体夹心)的基础上,采用纳米金颗粒增敏技术,能提高检测禽流感病毒的检测灵敏度,能进行样品的高通量筛查,适用于基层或现场检测禽流感病毒。The invention provides a colloidal gold immunofiltration sensitization method for detecting avian influenza virus and a test kit thereof, the steps are as follows: 1) preparing an immunofiltration test strip; 2) avian influenza virus polyclonal antibody on the immunofiltration test strip 3) preparation of a gold-labeled monoclonal antibody gold-labeled probe for avian influenza virus; 4) preparation of a sensitizing reagent; 5) detection of avian influenza virus. The present invention utilizes the characteristic that gold atoms produced by oxidation-reduction reaction between chloroauric acid and ascorbic acid can be adsorbed by colloidal gold to locate colloidal gold deposition sites for color enhancement, and improve the detection sensitivity of the antigen to be detected by 8-50 times. The present invention is based on colloidal gold dot immunofiltration assay (double antibody sandwich), adopts nano gold particle sensitization technology, can improve the detection sensitivity of detecting avian influenza virus, can carry out high-throughput screening of samples, and is suitable for Detection of avian influenza virus at the grass-roots level or on-site.
Description
技术领域 technical field
本发明涉及一种病毒的检测方法,尤其涉及一种检测禽流感病毒的胶体金免疫渗滤增敏方法及其试剂盒。The invention relates to a virus detection method, in particular to a colloidal gold immunofiltration sensitization method for detecting avian influenza virus and a kit thereof.
背景技术 Background technique
禽流感(Avian Influenza,AI)是由A型流感病毒引起的禽类的一种从呼吸病到严重败血症等多种症状的急性高致病性传染病。除了禽类,A型流感病毒还感染其他种属的动物,如马、猪等,1997年首次发现高致病性禽流感H5N1可感染人类。自禽流感于1878年在意大利发现以来,给养鸡业造成巨大的经济损失。历史上危害最大,经济损失最严重的一次禽流感(H5N1)爆发于1983年美国滨州地区,美国政府为此共花费了6000多万美元,间接经济损失估计达3.48亿美元。我国香港地区近期爆发的禽流感,据估计损失约达8000万港币。1997年及2003年在香港H5N1禽流感已干扰18人,4人死亡,2003年年底开始该病毒在亚洲国家已造成上百人感染,几十人死亡。2006年2月以来,禽流感疫情愈演愈烈,马来西亚、俄罗斯、中国、波黑、罗马尼亚、法国、印度、埃及、德国、奥地利、伊朗、希腊、斯洛文尼亚、意大利等国相继报道了禽流感疫情。因此对禽流感病毒简便、快捷、高灵敏度、高特异性的检测成为控制禽流感疫情扩散的当务之急。Avian Influenza (AI) is an acute and highly pathogenic infectious disease of poultry caused by type A influenza virus with various symptoms ranging from respiratory disease to severe sepsis. In addition to birds, type A influenza viruses also infect other species of animals, such as horses and pigs. In 1997, it was first discovered that highly pathogenic avian influenza H5N1 can infect humans. Since bird flu was discovered in Italy in 1878, it has caused huge economic losses to the chicken industry. The outbreak of avian influenza (H5N1), which caused the greatest harm and the most serious economic losses in history, occurred in the Binzhou area of the United States in 1983. The US government spent more than 60 million U.S. dollars for this, and the indirect economic loss was estimated to reach 348 million U.S. dollars. The recent outbreak of bird flu in Hong Kong, my country, is estimated to have cost about 80 million Hong Kong dollars. In 1997 and 2003, the H5N1 bird flu in Hong Kong had disturbed 18 people and killed 4 people. Since the end of 2003, the virus has caused hundreds of people to be infected and dozens of people died in Asian countries. Since February 2006, the outbreak of bird flu has intensified. Malaysia, Russia, China, Bosnia and Herzegovina, Romania, France, India, Egypt, Germany, Austria, Iran, Greece, Slovenia, Italy and other countries have reported bird flu outbreaks one after another. Therefore, the simple, fast, highly sensitive and highly specific detection of avian influenza virus has become an urgent task to control the spread of avian influenza epidemic.
现有禽流感病毒检测方法主要分为抗体检测及抗原检测方法。抗体检测方法包括:血凝抑制试验(Hemagglutination inhibition,HI)、琼脂免疫扩散试验(Agarose gel immunodiffusion,AGID)、酶联免疫吸附试验(Enzyme-linkedImmunosorbent Assay,ELISA)、补体结合试验(Complement Fixation Assays,CF)、神经氨酸酶抑制试验(Neuraminidase inhibitor test,NIT)以及病毒中和试验(Virusneutralization Test,VNT)等。由于抗体检测方法不能区分人工注射疫苗和病毒感染因而对动物检疫缺乏实际应用意义。因此,目前禽流感病毒检测主要为抗原检测方法,相对快速的方法为ELISA和荧光RT-PCR,但这两种方法在大范围应用中存在以下缺陷:(1)需要昂贵的仪器设备,检测成本较高且不易在基层单位推广;(2)所有这些技术方法都不能在基层生产场、检疫站和临床等现场使用,必须有专门的实验室;(3)操作较繁琐,检测时间均在数小时以上,直接影响了疫情的防控速度。Existing avian influenza virus detection methods are mainly divided into antibody detection and antigen detection methods. Antibody detection methods include: hemagglutination inhibition test (Hemagglutination inhibition, HI), agar immunodiffusion test (Agarose gel immunodiffusion, AGID), enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, ELISA), complement fixation assay (Complement Fixation Assays, CF), neuraminidase inhibitor test (NIT) and virus neutralization test (Virusneutralization Test, VNT), etc. Since the antibody detection method cannot distinguish between artificially injected vaccines and virus infections, it has no practical significance for animal quarantine. Therefore, at present, the detection of avian influenza virus is mainly an antigen detection method, and the relatively fast methods are ELISA and fluorescent RT-PCR, but these two methods have the following defects in large-scale applications: (1) expensive instruments and equipment are required, and the detection cost is relatively high. (2) All these technical methods cannot be used in grass-roots production sites, quarantine stations and clinics, and must have special laboratories; (3) The operation is cumbersome, and the detection time is within a few days. Hours or more directly affected the speed of epidemic prevention and control.
由于胶体金颗粒对许多蛋白质都有很强的吸附功作用,可与SPA、IgG、毒素、糖蛋白、酶、抗生素和激素等多种物质非共价结合,从而使其成为免疫反应的优良标记物,并使得固相膜免疫测定技术(Solid Phase Membrane-basedImmunoassay)更加简便。常用的固相膜为硝酸纤维素(nitrocellulose,NC)膜。斑点免疫测定法(Dot-Immunoboding Assay,DIBA)是在免疫印迹技术基础上改良发展起来的一项技术。由于其敏感性、特异性与酶联免疫测定法(ELISA)相当,且操作简单、快速而发展为不同的方法。其中包括斑点免疫渗滤测定法(DotImmuno Filtration Assay,DIFA)和斑点免疫层析测定法(Dot ImmunoChromatographic Assay,DICA)。以胶体金为标记物的斑点免疫渗滤测定法称为胶体金免疫渗滤测定法(Dot-Immunogold FiltrationAssay,DIGFA)。然而,上述几种方法检测禽流感病毒难以满足高灵敏度的要求。以往人们主要利用银染色技术来提高DIGFA的检测灵敏度。然而,该技术的缺点是背景模糊,增敏效果相对不强,因而限制了其应用范围。Because colloidal gold particles have a strong adsorption effect on many proteins, they can be non-covalently combined with various substances such as SPA, IgG, toxins, glycoproteins, enzymes, antibiotics and hormones, making them an excellent marker of immune response and make Solid Phase Membrane-based Immunoassay easier. The commonly used solid-phase membrane is nitrocellulose (NC) membrane. Dot-Immunoboding Assay (DIBA) is a technology improved and developed on the basis of western blot technology. Because its sensitivity and specificity are comparable to those of enzyme-linked immunoassay (ELISA), and it is simple and fast to operate, it has developed into a different method. These include dot immunofiltration assay (DotImmuno Filtration Assay, DIFA) and dot immunochromatographic assay (Dot ImmunoChromatographic Assay, DICA). The dot immunofiltration assay using colloidal gold as a marker is called colloidal gold immunofiltration assay (Dot-Immunogold FiltrationAssay, DIGFA). However, it is difficult for the above-mentioned methods to detect avian influenza virus to meet the requirements of high sensitivity. In the past, people mainly used silver staining technique to improve the detection sensitivity of DIGFA. However, the disadvantage of this technique is that the background is blurred and the sensitization effect is relatively weak, which limits its application range.
发明内容 Contents of the invention
本发明的目的是提供一种检测禽流感病毒的胶体金免疫渗滤增敏方法及其试剂盒。本发明是在胶体金斑点免疫渗滤测定法(双抗体夹心)的基础上,采用纳米金颗粒增敏技术,能显著提高检测禽流感病毒的灵敏度,具有敏感、快速和特异性好等特点,并且价格低廉,能进行样品的高通量筛查,适用于基层或现场检测禽流感病毒,从而克服了目前胶体金免疫渗滤测定法(DIGFA)检测灵敏度不高的问题。The purpose of the present invention is to provide a colloidal gold immunofiltration sensitization method for detecting avian influenza virus and a kit thereof. The present invention is based on colloidal gold dot immunofiltration assay (double-antibody sandwich), adopts nano-gold particle sensitization technology, can significantly improve the sensitivity of detecting avian influenza virus, and has the characteristics of sensitivity, rapidity and good specificity. And it is cheap, can carry out high-throughput screening of samples, and is suitable for grass-roots or on-site detection of avian influenza virus, thereby overcoming the problem of low detection sensitivity of the current colloidal gold immunofiltration assay (DIGFA).
本发明检测禽流感病毒的胶体金免疫渗滤增敏测定方法,步骤如下:The present invention detects the colloidal gold immunofiltration sensitization assay method of avian influenza virus, and the steps are as follows:
1.制备免疫渗滤试纸条1. Preparation of immunofiltration test strips
免疫渗滤试纸条为A或B之一;The immunofiltration test strip is one of A or B;
制备免疫渗滤试纸条A:沿长为40-110mm、宽为3-6mm的矩形聚乙烯(PE)塑料基片长的方向设有孔间距为2-4mm的8-15个呈一行分布的直径为0.5-3mm的圆形进样孔;将与聚乙烯(PE)塑料基片大小相同的硝酸纤维素(NC)膜片粘附于其下面;加样时带有进样孔的聚乙烯(PE)塑料基片在上;Preparation of immunofiltration test strip A: along the long direction of a rectangular polyethylene (PE) plastic substrate with a length of 40-110mm and a width of 3-6mm, 8-15 holes with a spacing of 2-4mm are arranged in a row. A circular injection hole with a diameter of 0.5-3mm; a nitrocellulose (NC) membrane of the same size as the polyethylene (PE) plastic substrate is adhered to it; Ethylene (PE) plastic substrate on top;
制备免疫渗滤试纸条B:切制8-15个直径0.5-3mm的圆形硝酸纤维素膜片粘附于长为40-110mm、宽为3-6mm的矩形聚乙烯(PE).塑料基片,膜片间距为3-4mm;每个膜片下方的塑料基片上设有两个吸水孔,吸水孔是将与膜片圆心重叠、与塑料基片长平行的长轴比膜片圆的直径长0.3mm、与塑料基片宽平行的短轴比膜片圆的直径短0.2mm的椭圆未被膜片覆盖的部分去除得到的孔,加样时带吸水孔的塑料聚乙烯(PE)基片在下面;Preparation of immunofiltration test strip B: Cut 8-15 circular nitrocellulose membranes with a diameter of 0.5-3mm and adhere to rectangular polyethylene (PE) with a length of 40-110mm and a width of 3-6mm. Plastic Substrate, the distance between the diaphragms is 3-4mm; there are two water absorption holes on the plastic substrate under each diaphragm, and the water absorption holes are the long axis that overlaps with the center of the diaphragm circle and is parallel to the length of the plastic substrate than the diameter of the diaphragm circle. The diameter is 0.3mm long, and the minor axis parallel to the width of the plastic substrate is 0.2mm shorter than the diameter of the diaphragm circle. The hole obtained by removing the part not covered by the diaphragm, plastic polyethylene (PE) with water-absorbing holes when adding samples ) substrate is below;
2.禽流感病毒多克隆抗体在免疫渗滤试纸条上的固定化2. Immobilization of polyclonal antibody against avian influenza virus on immunofiltration test strips
1)制备固定化禽流感病毒多克隆抗体的免疫渗滤试纸条1) Preparation of immunofiltration test strips for immobilized avian influenza virus polyclonal antibody
a.制备禽流感病毒多克隆抗体溶液,用800mL蒸馏水溶解8g氯化钠(NaCl)、0.2g氯化钾(KCl)、1.44g磷酸氢二钠(Na2HPO4)和0.24g磷酸二氢钾(KH2PO4),用盐酸(HCl)调节溶液的pH值至7.4,加水至1L,摇匀,制成pH7.4的磷酸盐缓冲液(PBS,Phosphate Buffer Saline),用磷酸盐缓冲液PBS稀释禽流感病毒多克隆抗体,使溶液中多克隆抗体重量百分比浓度为0.05-0.3mg/mL,优选重量百分比浓度为0.1-0.2mg/mL,制得禽流感病毒多克隆抗体溶液;a. To prepare polyclonal antibody solution to avian influenza virus, dissolve 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g of dihydrogen phosphate in 800mL of distilled water Potassium (KH 2 PO 4 ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1L, shake well to make phosphate buffer saline (PBS, Phosphate Buffer Saline) with pH 7.4, use phosphate buffer Dilute the polyclonal antibody to avian influenza virus with liquid PBS, so that the polyclonal antibody weight percent concentration in the solution is 0.05-0.3mg/mL, preferably the weight percent concentration is 0.1-0.2mg/mL, to prepare the avian influenza virus polyclonal antibody solution;
b.禽流感病毒多克隆抗体在免疫渗滤试纸条上的固定化b. Immobilization of polyclonal antibody against avian influenza virus on immunofiltration test strips
禽流感病毒多克隆抗体在免疫渗滤试纸条上固定化采用点样固定化法或浸泡固定化法之一;The immobilization of the polyclonal antibody of the avian influenza virus on the immunofiltration test strip adopts one of spotting immobilization method or soaking immobilization method;
点样固定化法:向上述免疫渗滤试纸条A的每个进样孔或免疫渗滤试纸条B的每个膜片上点1-3μL上述禽流感病毒多克隆抗体溶液;点样可为1次-3次,待渗,风干;得到固定化试纸条A或B;Spotting immobilization method: point 1-3 μL of the above-mentioned avian influenza virus polyclonal antibody solution to each injection hole of the above-mentioned immunofiltration test paper A or each diaphragm of the immunofiltration test paper B; It can be 1-3 times, wait for infiltration and air-dry; get immobilized test strip A or B;
或浸泡固定化法:取500-1000μL上述禽流感病毒多克隆抗体溶液置于小试管中,4℃浸泡5-10条免疫渗滤试纸条A或B 10-15小时,取出,风干;得到固定化试纸条A或B;Or immersion immobilization method: take 500-1000μL of the above-mentioned avian influenza virus polyclonal antibody solution in a small test tube, soak 5-10 immunofiltration test strips A or B at 4°C for 10-15 hours, take it out, and air dry; Immobilized test strip A or B;
2)试纸条的封闭2) Sealing of test strips
将上述用点样法或浸泡法得到的固定化试纸条A或B,放入用双蒸水稀释的重量百分比浓度为1-5%的牛血清白蛋白溶液(BSA)中,37℃反应20-60min;取出,风干,得到待用固定化试纸条;4℃保存,备用;Put the above-mentioned immobilized test strip A or B obtained by spotting method or soaking method into bovine serum albumin solution (BSA) with a concentration of 1-5% by weight diluted with double distilled water, and react at 37°C 20-60min; take it out, air-dry to get ready-to-use immobilized test strips; store at 4°C for later use;
3.制备禽流感病毒金标单克隆抗体探针3. Preparation of avian influenza virus gold-labeled monoclonal antibody probe
1)制备胶体金溶液1) Preparation of colloidal gold solution
a.制备胶体金溶液的试剂及其体积配比:a. Reagents and their volume ratios for preparing colloidal gold solution:
双蒸水 100mL、Double distilled water 100mL,
新鲜制备的重量百分比浓度为1%的氯金酸水溶液 1mL、Freshly prepared 1% chloroauric acid aqueous solution with a weight percentage concentration of 1mL,
重量百分比浓度为1%的柠檬酸三钠水溶液 1.0-1.5mL;1.0-1.5mL aqueous solution of trisodium citrate with a concentration of 1% by weight;
其中重量百分比浓度为1%的柠檬酸三钠水溶液的优选配比为 1.2mL;Wherein the preferred proportioning of the trisodium citrate aqueous solution of 1% by weight percentage concentration is 1.2mL;
b.制备胶体金溶液b. Preparation of colloidal gold solution
将所需的双蒸水加热煮沸;加入所需的新鲜制备的重量百分比浓度为1%的氯金酸水溶液,迅速加入所需的重量百分比浓度为1%的柠檬酸三钠水溶液,不断搅拌,得到酒红色的胶体金溶液;The required double-distilled water is heated and boiled; adding the required freshly prepared aqueous auric acid chloride solution with a concentration of 1% by weight, quickly adding the required concentration of trisodium citrate with a concentration of 1% by weight, constantly stirring, Obtain wine red colloidal gold solution;
2)胶体金标记禽流感病毒单克隆抗体2) Colloidal gold-labeled avian influenza virus monoclonal antibody
a.制备禽流感病毒单克隆抗体a. Preparation of avian influenza virus monoclonal antibody
用禽流感病毒免疫小鼠制备获得能够分泌禽流感单克隆抗体的杂交瘤细胞株,获得通用A型禽流感病毒、H5型禽流感病毒、H7型禽流感病毒或H9型禽流感病毒的特异性单克隆抗体之一;各亚型禽流感病毒单克隆抗体只与相应亚型的禽流感病毒抗原发生特异性反应,从而决定了试纸条的特异性;用上述胶体金标记抗体,制成金标抗体,可与待检样品中的禽流感病毒特异性的结合;Immunize mice with avian influenza virus to prepare a hybridoma cell line capable of secreting avian influenza monoclonal antibody, and obtain the specificity of universal A-type avian influenza virus, H5-type avian influenza virus, H7-type avian influenza virus or H9-type avian influenza virus One of the monoclonal antibodies; the monoclonal antibodies of each subtype of avian influenza virus only react specifically with the corresponding subtype of avian influenza virus antigen, thus determining the specificity of the test strip; the above-mentioned colloidal gold is used to label the antibody to make a gold The labeled antibody can specifically bind to the avian influenza virus in the sample to be tested;
b.制备胶体金标记禽流感病毒单克隆抗体b. Preparation of colloidal gold-labeled avian influenza virus monoclonal antibody
取上述制备的胶体金溶液10mL,用重量百分比浓度为1%的碳酸钾(K2CO3)水溶液调节pH至8.5-9.2(用前调节,过早调节会出现聚集现象),优选pH为8.7-9.0;用800mL蒸馏水溶解0.2g氯化钾(KCl),1.44g磷酸氢二钠(Na2HPO4)和0.24g磷酸二氢钾(KH2PO4),用盐酸(HCl)调节溶液的pH值至7.4,加水至1L,摇匀,制成磷酸盐缓冲液(PB,Phosphate Buffer);用磷酸盐缓冲液(PB)将上述制备获得的禽流感病毒单克隆抗体稀释至蛋白含量为0.1-1mg/mL、pH与上述调节后的胶体金溶液的pH值相同,取0.1-0.5mL,在13000rpm、4℃下离心1小时,得到上清液;快速搅拌下将上清液逐滴加入到上述调节过pH值的胶体金溶液中,至液体的最小蛋白量为10-20μg/mL,优选最小蛋白含量为10-16μg/mL;室温放置5min;再分别加入过滤后的重量百分比浓度为10%BSA溶液1ml,继续搅拌10-15分钟,然后在13000rpm、4℃离心1小时,小心弃去上清液(应无色液体,若出现红色则说明离心速度和时间不够),得到沉淀物;用10ml浓度为0.01mol/L、pH 8.2且其中BSA的重量百分比浓度为1%的三羟甲基氨基甲烷溶液(TBS,Tris Buffered Saline)将上述沉淀物溶解,再在1000rpm/min、4℃离心下10分钟,去除聚集物,留上清液,弃去底部的沉淀,将上清液13000rpm、4℃离心1小时;用5ml含有重量百分比浓度分别为0.02%的叠氮钠、1%的蔗糖、1%的BSA且pH为8.2的三羟甲基氨基甲烷溶液(TBS)重新悬浮底部疏松沉淀,得到禽流感金标单克隆抗体探针;置于4℃冰箱保存备用;Take 10 mL of the colloidal gold solution prepared above, and adjust the pH to 8.5-9.2 with an aqueous solution of potassium carbonate (K 2 CO 3 ) with a concentration of 1% by weight (adjust before use, and aggregation will occur if adjusted too early), preferably pH 8.7 -9.0; Dissolve 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g of potassium dihydrogen phosphate (KH 2 PO 4 ) with 800 mL of distilled water, and adjust the concentration of the solution with hydrochloric acid (HCl). pH value to 7.4, add water to 1L, shake well to make phosphate buffer saline (PB, Phosphate Buffer); use phosphate buffer saline (PB) to dilute the avian influenza virus monoclonal antibody prepared above to a protein content of 0.1 -1mg/mL, pH is the same as the pH value of the above adjusted colloidal gold solution, take 0.1-0.5mL, centrifuge at 13000rpm, 4°C for 1 hour to obtain the supernatant; add the supernatant dropwise under rapid stirring In the above-mentioned colloidal gold solution that has adjusted the pH value, the minimum protein content to the liquid is 10-20 μg/mL, preferably the minimum protein content is 10-16 μg/mL; place it at room temperature for 5 minutes; then add the filtered weight percentage concentration of 1ml of 10% BSA solution, continue to stir for 10-15 minutes, then centrifuge at 13000rpm, 4°C for 1 hour, carefully discard the supernatant (it should be colorless liquid, if it appears red, it means that the centrifugation speed and time are not enough), and the precipitate is obtained With 10ml concentration is 0.01mol/L, pH 8.2 and wherein the weight percentage concentration of BSA is 1% Tris hydroxymethyl aminomethane solution (TBS, Tris Buffered Saline) that above-mentioned precipitate is dissolved, again at 1000rpm/min, 4 Centrifuge at ℃ for 10 minutes, remove the aggregates, leave the supernatant, discard the precipitate at the bottom, and centrifuge the supernatant at 13000rpm and 4℃ for 1 hour; use 5ml of sodium azide and 1% sucrose, 1% BSA and a tris solution (TBS) with a pH of 8.2 to resuspend the loose sediment at the bottom to obtain the avian influenza gold standard monoclonal antibody probe; store it in a 4°C refrigerator for later use;
4.制备增敏试剂4. Preparation of Sensitizing Reagent
增敏试剂为试剂A和试剂B;The sensitizing reagents are reagent A and reagent B;
配制试剂A:配制重量百分比浓度为1%-10%的氯金酸水溶液;氯金酸水溶液中氯金酸的优选重量百分比浓度为1-2%;Preparing reagent A: preparing an aqueous solution of auric acid chloride with a concentration of 1%-10% by weight; the preferred concentration of auric acid by weight in the aqueous solution of auric chloride is 1-2%;
配制试剂B:配制重量百分比浓度大于0.1-0.5%的抗坏血酸水溶液;抗坏血酸水溶液中抗坏血酸的优选重量百分比浓度为0.15%;Prepare reagent B: prepare an aqueous solution of ascorbic acid with a weight percentage concentration greater than 0.1-0.5%; the preferred weight percentage concentration of ascorbic acid in the ascorbic acid aqueous solution is 0.15%;
5.检测禽流感病毒5. Detection of Avian Influenza Virus
1)向上述制备的待用固定化试纸条A的进样孔或B的膜片上分别滴加用PBS稀释成溶液中抗原重量百分比浓度为0.05-0.1mg/mL的通用A型、H5、H7或H9型禽流病毒抗原之一作为需要检测的阳性对照、用洗脱离心稀释常规方法前处理后溶于PBS缓冲溶液的待测样本、针对所要检测的阳性对照抗原通过鸡胚获得的未经病毒感染的尿囊液的阴性对照,各滴加1-3μL,每个样品滴加1-2份(即分别将2份相同的抗原、待测样品或阴极对照细胞液分别滴入或2个进样孔或2个膜片上,可同时做平行样),待渗,在室温下反应1分钟以上;得到带阳性对照抗原、待测样本及阴性对照的试纸条;1) To the injection hole of the immobilized test strip A prepared above or the membrane of B, respectively drop the general-purpose type A, H5 diluted with PBS so that the weight percent concentration of the antigen in the solution is 0.05-0.1mg/mL , one of the H7 or H9 avian virus antigens as the positive control to be detected, the sample to be tested that is dissolved in PBS buffer solution after being pretreated by the conventional method of elution, centrifugation and dilution, and the positive control antigen to be detected is obtained through chicken embryos For the negative control of the allantoic fluid without virus infection, add 1-3 μL each, and add 1-2 parts to each sample (that is, drop 2 parts of the same antigen, the sample to be tested or the negative control cell fluid respectively into or 2 injection holes or 2 membranes, parallel samples can be made at the same time), wait for infiltration, and react at room temperature for more than 1 minute; get test strips with positive control antigen, sample to be tested and negative control;
2)取与上述已选定的需要检测的阳性对照抗原相对应的金标单克隆抗体探针溶液,每份取1-3μL,滴加于上述带阳性对照抗原、待测样本及阴性对照的试纸条A的每个已加过样品的进样孔或B的每个膜片上;室温反应0.5~2分钟,优选时间为1-2分钟;再分别加入含体积百分比浓度为0.02%的吐温20的磷酸盐洗液PBST,洗2-5次,每次三滴,每滴2-10μL,得到待检测试纸条;2) Take the gold-labeled monoclonal antibody probe solution corresponding to the above-mentioned selected positive control antigen that needs to be detected, take 1-3 μL for each portion, and add dropwise to the above-mentioned positive control antigen, sample to be tested and negative control. Each sample injection hole of test strip A or each diaphragm of B; react at room temperature for 0.5-2 minutes, preferably 1-2 minutes; Tween 20 phosphate washing solution PBST, washed 2-5 times, three drops each time, 2-10 μL per drop, to obtain the test strip to be tested;
3)分别向上述待检测试纸条A已加过金标单克隆抗体探针的每个进样孔或试纸条B的每个膜片上先滴加1-3μL上述增敏试剂的试剂A,然后再滴加3-8μL增敏试剂的试剂B,待风干后,再重复述操作1至2次;最后一次滴加后1-2分钟观察结果,肉眼观察若有灰黑色斑点,则认为该位置对应的禽流感病毒为阳性结果,与阴性对照显色结果无明显区别,则认为该位置对应的禽流感病毒为阴性结果。3) Add 1-3 μL of the above-mentioned sensitizing reagent to each injection hole of the above-mentioned test strip A to be tested to which the gold-labeled monoclonal antibody probe has been added or to each diaphragm of the test strip B. A, then add 3-8 μL of reagent B of the sensitizing reagent dropwise, and repeat the
若待测人或动物的分泌物、血清、尿液或者食品浸提液中含有禽流感病毒血凝素亚型(H型)和神经氨酸酶亚型(N型)的一种或数种,当NC膜与分泌物、血清、尿液或者食品浸提液等待测样本接触时,如果禽流感病毒单克隆抗体对应的斑点呈阳性时,则可特异性地确定禽流感病毒属于某种或某些种病毒亚型。采用增敏试剂进行一次或多次处理,与原有斑点免疫金测定法相比,禽流感检测灵敏度可显著提高8~50倍。If the human or animal secretion, serum, urine or food extract contains one or more of the avian influenza virus hemagglutinin subtype (H type) and neuraminidase subtype (N type) When the NC membrane is in contact with secretions, serum, urine or food extracts, if the spots corresponding to the avian influenza virus monoclonal antibody are positive, it can be specifically determined that the avian influenza virus belongs to a certain or Certain virus subtypes. Using the sensitizing reagent for one or more treatments, compared with the original dot immunogold assay, the detection sensitivity of avian influenza can be significantly increased by 8-50 times.
本发明所需的通用A型禽流感病毒抗原和各亚型禽流感病毒抗原、禽流感病毒多克隆抗体可到相关专业的研究单位、公司购买或定制;所需的仪器、设备、药品均有市售。The general A-type bird flu virus antigen required by the present invention and each subtype bird flu virus antigen, bird flu virus polyclonal antibody can go to relevant professional research unit, company to buy or customize; Required instruments, equipment, medicine all have commercially available.
附图说明 Description of drawings
图1:免疫渗滤试纸条为A的正视图。Figure 1: Front view of immunofiltration test strip A.
图2:免疫渗滤试纸条为A的俯视图。Figure 2: Top view of immunofiltration test strip A.
图3:免疫渗滤试纸条为B的正视图。Figure 3: Front view of immunofiltration test strip B.
图4:免疫渗滤试纸条为B的俯视图。Figure 4: Top view of immunofiltration test strip B.
具体实施方式 Detailed ways
本发明通过以下实施例作进一步具体描述。The present invention is further specifically described by the following examples.
实施例1:各种试剂的配制Embodiment 1: the preparation of various reagents
1.氯金酸水溶液:称取1.0g氯金酸,溶解于100mL双蒸水中,形成重量百分比浓度为1%的氯金酸水溶液,摇匀。1. Chlorauric acid aqueous solution: Weigh 1.0 g of chloroauric acid, dissolve it in 100 mL of double-distilled water to form a chloroauric acid aqueous solution with a concentration of 1% by weight, and shake well.
2.柠檬酸三钠水溶液:称取1.0g柠檬酸三钠,溶解于100mL双蒸水中,形成重量百分比浓度为1%的柠檬酸三钠水溶液,摇匀。2. Trisodium citrate aqueous solution: Weigh 1.0 g of trisodium citrate, dissolve it in 100 mL of double distilled water to form a trisodium citrate aqueous solution with a concentration of 1% by weight, and shake well.
3.磷酸盐缓冲液(PBS):用800mL蒸馏水溶解8g氯化钠(NaCl),0.2g氯化钾(KCl),1.44g磷酸氢二钠(Na2HPO4)和0.24g磷酸二氢钾(KH2PO4),用盐酸(HCl)调节溶液的pH值至7.4,加水至1L,摇匀。3. Phosphate buffered saline (PBS): Dissolve 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g of potassium dihydrogen phosphate in 800mL of distilled water (KH 2 PO 4 ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1L, and shake well.
4.磷酸盐缓冲液(PB):用800mL蒸馏水溶解0.2g氯化钾(KCl),1.44g磷酸氢二钠(Na2HPO4)和0.24g磷酸二氢钾(KH2PO4),用盐酸(HCl)调节溶液的pH值至7.4,加水至1L,摇匀。4. Phosphate buffer (PB): Dissolve 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g potassium dihydrogen phosphate (KH 2 PO 4 ) in 800mL distilled water, and use Hydrochloric acid (HCl) to adjust the pH value of the solution to 7.4, add water to 1L, shake well.
5.磷酸盐洗液(PBST):用800mL蒸馏水溶解8g氯化钠(NaCl),0.2g氯化钾(KCl),1.44g磷酸氢二钠(Na2HPO4)和0.24g磷酸二氢钾(KH2PO4),用盐酸(HCl)调节溶液的pH值至7.4,加水至1L,摇匀,加入200μL的吐温20,摇匀。5. Phosphate washing solution (PBST): Dissolve 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na 2 HPO 4 ) and 0.24g potassium dihydrogen phosphate in 800mL distilled water (KH 2 PO 4 ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1 L, shake well, add 200 μL Tween 20, and shake well.
6.禽流感多克隆抗体:用PBS稀释,摇匀,使溶液中多克隆抗体重量百分比浓度为0.05-0.3mg/mL。6. Avian influenza polyclonal antibody: dilute with PBS, shake well, so that the weight percent concentration of polyclonal antibody in the solution is 0.05-0.3 mg/mL.
实施例2:禽流感病毒胶体金免疫渗滤增敏试剂盒及使用方法Embodiment 2: Avian influenza virus colloidal gold immunofiltration sensitization kit and method of use
1.禽流感病毒胶体金免疫渗滤增敏方法试剂盒的装配1. Assembly of avian influenza virus colloidal gold immunofiltration sensitization method kit
1)按上述方法制备的固定化渗滤试纸条A或B100条,平时于4℃冰箱中保存;1) 100 immobilized percolation test strips A or B prepared by the above method, usually stored in a refrigerator at 4°C;
下面结合附图对试纸条A或B作进一步说明:Below in conjunction with accompanying drawing, test strip A or B are further described:
如图1、图2,试纸条A的塑料基片1长为40mm、宽为3mm,沿长的方向设有孔间距为2mm的8个呈一行分布的直径为0.5mm的圆形进样孔3,与塑料基片大小相同的硝酸纤维素膜片2重叠置于其下面。或试纸条A的塑料基片1长为110mm、宽为6mm,沿长的方向设有孔间距为4mm的15个呈一行分布的直径为3mm的圆形进样孔3,与塑料基片大小相同的硝酸纤维素膜片2重叠粘附于其下面;As shown in Figure 1 and Figure 2, the
如图3、图4,试纸条B为切制8个直径0.5mm的圆形硝酸纤维素膜片1粘附于长为40mm、宽为3mm的塑料基片2上,各个膜片间距为3mm;每个膜片下方的塑料基片上设有两个吸水孔3,吸水孔是将与膜片圆心重叠、与塑料基片长平行的长轴为比膜片圆的直径长0.3mm即0.8mm、与塑料基片宽平行的短轴为比膜片圆的直径短0.2mm即0.3mm的椭圆未被膜片覆盖的部分去除得到的孔,加样时带吸水孔的塑料聚乙烯(PE)基片在下面;或试纸条B为切制15个直径3mm的圆形硝酸纤维素膜2片置于长为110mm、宽为6mm的塑料基片1上,各个膜片间距为4mm;每个膜片下方的塑料基片上设有两个吸水孔3,吸水孔是将与膜片圆心重叠、与塑料基片长平行的长轴为比膜片圆的直径长0.3mm即3.3mm、与塑料基片宽平行的短轴比膜片圆的直径短0.2mm即2.8mm的椭圆未被膜片覆盖的部分去除得到的孔,加样时带吸水孔的塑料聚乙烯(PE)基片在下面;As shown in Figure 3 and Figure 4, the test strip B is to cut 8
2)磷酸盐缓冲液(PBS)100ml,置于塑料瓶中;2) 100ml of phosphate buffered saline (PBS), placed in a plastic bottle;
3)按上述方法制备的增敏试剂试剂A;取氯金酸1.0g,用双蒸水稀释到100mL放在棕色试剂瓶中,分装后每个试剂盒内装10mL,可以同时检测100组试纸条。按上述方法制备的增敏试剂试剂B;取抗坏血酸1.0g,用双蒸水稀释到100-150mL放在棕色试剂瓶中,分装后每个试剂包装内装10mL;可检测100条试纸条;3) The sensitizing reagent reagent A prepared by the above method; take 1.0 g of chloroauric acid, dilute it to 100 mL with double distilled water and put it in a brown reagent bottle. note. The sensitizing reagent B prepared by the above method; take 1.0g of ascorbic acid, dilute it to 100-150mL with double distilled water and put it in a brown reagent bottle. After subpackaging, each reagent package contains 10mL; it can detect 100 test strips;
4)磷酸盐洗液(PBST):取200μL吐温20,用pH7.4、0.01mol/L的PBS缓冲液配制成1000mL,分装后每个试剂盒内装100mL;4) Phosphate washing solution (PBST): Take 200 μL Tween 20, prepare 1000 mL with PBS buffer solution with pH 7.4 and 0.01 mol/L, and fill each kit with 100 mL after aliquoting;
5)阳性对照抗原液,用PBS稀释成溶液中抗原重量百分比浓度为0.05-0.1mg/mL的通用A型、H5、H7或H9型禽流感病毒抗原之一的溶液1ml;可检测100条试纸条;5) Positive control antigen solution, diluted with PBS to become 1ml of solution of one of the general-purpose A-type, H5, H7 or H9 type avian influenza virus antigens with an antigen weight percentage concentration of 0.05-0.1mg/mL in the solution; 100 test strips can be detected note;
6)阴性对照液,针对所要检测的阳性对照抗原通过鸡胚获得的未经病毒感染的尿囊液10mL;可检测100条试纸条;6) Negative control solution, 10 mL of non-virus-infected allantoic fluid obtained from chicken embryos for the positive control antigen to be detected; 100 test strips can be detected;
7)金标探针试剂,取蛋白含量为10-20μg/mL的针对阳性对照的禽流感病毒单克隆抗体探针液1000μL,放在小玻璃瓶中,抽成真空后封口,平时于4℃冰箱中保存;可检测100条试纸条;7) Gold standard probe reagent, take 1000 μL of the positive control avian influenza virus monoclonal antibody probe solution with a protein content of 10-20 μg/mL, put it in a small glass bottle, vacuumize it and seal it, and store it at 4 °C at ordinary times Store in the refrigerator; can detect 100 test strips;
8)塑料滴瓶10个、1-25μL量程的移液枪;8) 10 plastic drop bottles, pipette gun with a range of 1-25μL;
9)长、宽分别大于渗滤试纸条A或B的滤纸100张。9) 100 pieces of filter paper whose length and width are respectively larger than percolation test strip A or B.
2.试剂盒的使用方法如下:2. The method of using the kit is as follows:
1)取出上述试纸条A或B一条,放于平放在桌面上的一张滤纸上面;1) Take out the above-mentioned test strip A or B, and place it on a piece of filter paper that is placed flat on the table;
2)分别将需要检测的阳性对照抗原液、用洗脱离心稀释常规方法前处理后溶于PBS缓冲溶液的待测样本液及阴性对照液1-3μL,滴入上述试纸条A的进样孔或试纸条B的膜片,每个样品分别做2份,待渗;2) Drop 1-3 μL of the positive control antigen solution to be detected, the sample solution to be tested and dissolved in PBS buffer solution after pretreatment by the conventional method of elution and centrifugal dilution, and the negative control solution, respectively, into the sample injection of the above test strip A Hole or the diaphragm of test strip B, make 2 copies of each sample, and wait for infiltration;
3)向上述已加入样品的纸条A的进样孔或试纸条B的膜片上分别加入1μL与上述需要检测的禽流感抗原相对应的金标单克隆抗体探针溶液,置干;3) Add 1 μL of the gold-labeled monoclonal antibody probe solution corresponding to the above-mentioned avian influenza antigen to be detected to the injection hole of the paper strip A or the membrane of the test strip B that has been added with the sample, and let it dry;
4)向上述已加入金标单克隆抗体探针试剂的试纸条A的进样孔或试纸条B的膜片分别滴加10μL磷酸盐洗液(PBST)洗NC膜,每次三滴,洗2-5次,室温下置干;4) Add 10 μL of phosphate washing solution (PBST) to the injection hole of test strip A or the membrane of test strip B to which the gold-labeled monoclonal antibody probe reagent has been added, respectively, to wash the NC membrane, three drops each time , washed 2-5 times, and dried at room temperature;
5)向上述已用磷酸盐洗液(PBST)洗过NC膜的试纸条A的进样孔或试纸条B的膜片上分别加入2μL增敏试剂的试剂A,再加入5μL增敏试剂的试剂B,显色1-2分钟;可重复该操作一次或数次;5) Add 2 μL of Reagent A of the sensitizing reagent to the injection hole of the test strip A or the membrane of the test strip B of the NC membrane that has been washed with phosphate washing solution (PBST), and then add 5 μL of the sensitizing reagent Reagent B of the reagent, develop color for 1-2 minutes; this operation can be repeated once or several times;
6)最后一次显色1-2分钟后观察相应位置有无灰黑色斑点,有则认为该位置对应的禽流感病毒为阳性结果,没有或颜色与相应阴性对照相比淡则认为该位置对应的禽流感病毒呈阴性结果。6) 1-2 minutes after the last color development, observe whether there are gray-black spots at the corresponding position. If there is, the avian influenza virus corresponding to the position is considered to be a positive result. If there is no or the color is lighter than the corresponding negative control, the corresponding position is considered to be Avian influenza virus was negative.
以上检测过程每次选定一种禽流感病毒的抗原做阳性的对照抗原,再选用与这种抗原相对应的胶体金标记的单抗,用以确定待测样本所含禽流感病毒的种类。一般首先选定通用A型禽流感病毒抗原做阳性的对照抗原,再选用与这种抗原相对应的的胶体金标记的单抗稀释液,进行筛查;检测呈阳性,再分别选定亚型病毒抗原做阳性对照及与其相对应的胶体金标记的单抗进一步检测确定具体的种类。因为每种试剂盒只能针对一种病毒进行检测,如需现场进行种类的定性甄别,则需携带所需检测的病毒抗原相应的检测试剂盒。In the above detection process, an antigen of an avian influenza virus is selected as a positive control antigen each time, and then a colloidal gold-labeled monoclonal antibody corresponding to this antigen is selected to determine the type of avian influenza virus contained in the sample to be tested. Generally, the universal type A bird flu virus antigen is first selected as a positive control antigen, and then the colloidal gold-labeled monoclonal antibody dilution corresponding to this antigen is selected for screening; the test is positive, and then the subtypes are selected respectively The virus antigen was used as a positive control and the corresponding colloidal gold-labeled monoclonal antibody was further detected to determine the specific species. Because each kit can only detect one type of virus, if you need to perform qualitative screening of the species on site, you need to carry a detection kit corresponding to the virus antigen to be detected.
实施例3:胶体金的制备方法Embodiment 3: the preparation method of colloidal gold
本发明以1%氯金酸、1%柠檬酸三钠与双蒸水为原料,采用柠檬酸三钠还原法制备胶体金颗粒,可通过改变柠檬酸三钠的加入量,制备不同直径且大小较为均一的胶体金颗粒(15-150nm);The present invention uses 1% chloroauric acid, 1% trisodium citrate and double distilled water as raw materials, adopts trisodium citrate reduction method to prepare colloidal gold particles, and can prepare colloidal gold particles with different diameters and sizes by changing the amount of trisodium citrate added Relatively uniform colloidal gold particles (15-150nm);
取100mL双蒸水,加热煮沸,加入1mL新鲜制备的1%氯金酸水溶液(HAuCl4),迅速加入1.2mL 1%柠檬酸三钠,不断搅拌,制备成酒红色的胶体金用于测试效果最好,此时合成的胶体金较稳定,在4℃下可保存12个月。Take 100mL of double distilled water, heat to boil, add 1mL of freshly prepared 1% chloroauric acid aqueous solution (HAuCl 4 ), quickly add 1.2mL of 1% trisodium citrate, and keep stirring to prepare wine red colloidal gold for testing the effect Preferably, the colloidal gold synthesized at this time is relatively stable and can be stored for 12 months at 4°C.
实施例4:胶体金标记最适pH值的确定Embodiment 4: Determination of the optimum pH value of colloidal gold labeling
取若干个5mL试管,分别加入1mL胶体金溶液,用0.1mol/L HCl和25mmol/L K2CO3将溶液的pH值分别调为3、4、5、6、7、8、9、10、11、12、13、14;取一96孔培养板,按pH值从低到高将上述胶体金溶液各取100μL加入孔中,重复三次;每孔分别加入3μL重量百分比浓度为1mg/mL的纯化好的禽流感单克隆抗体,孔内混合均匀,室温下放置15min;每孔分别加入20μL浓度为10%NaCl,孔内混合均匀,室温下放置10min;观察胶体金的颜色变化,并分别测定其OD520nm和OD580nm,记录在OD520nm和OD580nm差值最大时的pH(X);重复前面的步骤,再进一步细化pH值梯度,将pH值梯度设定为X-0.6;X-0.3;X;X+0.3;X+0.6;X+1,观察胶体金颜色变化,直到室温下放置2小时,分别测定其OD520nm和OD580nm值,记录在OD520nm与OD580nm差值最大时的pH值,据此判断标记时的适宜pH值为8.5-9.2,优选pH值为8.7-9.2。Take several 5mL test tubes, add 1mL colloidal gold solution respectively, and use 0.1mol/L HCl and 25mmol/L K2CO3 to adjust the pH value of the solution to 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14; take a 96-well culture plate, add 100 μL of the above colloidal gold solutions into the wells according to the pH value from low to high, and repeat three times; The purified avian influenza monoclonal antibody was mixed evenly in the wells, and placed at room temperature for 15 minutes; 20 μL of 10% NaCl was added to each well, mixed evenly in the wells, and placed at room temperature for 10 minutes; the color change of the colloidal gold was observed, and measured separately Its OD 520nm and OD 580nm , record the pH (X) when the difference between OD 520nm and OD 580nm is the largest; repeat the previous steps, and then further refine the pH value gradient, and set the pH value gradient to X-0.6; X- 0.3; X; X+0.3; X+0.6; X+1, observe the color change of colloidal gold until it is placed at room temperature for 2 hours, measure its OD 520nm and OD 580nm values respectively, and record when the difference between OD 520nm and OD 580nm is the largest According to the pH value, it is judged that the appropriate pH value for labeling is 8.5-9.2, and the preferred pH value is 8.7-9.2.
实施例5:确定胶体金标记最低蛋白稳定量Example 5: Determining the minimum amount of stable protein labeled with colloidal gold
最低蛋白稳定量的确定实验,以0.1mol/L K2CO3溶液调节胶体金溶液至pH8.5进行标记,稀释后的抗体与其他有关试剂按下表逐项操作。For the determination of the minimum protein stability experiment, adjust the colloidal gold solution to pH 8.5 with 0.1mol/L K 2 CO 3 solution for labeling. The diluted antibody and other related reagents are operated one by one as shown in the table below.
表2 分光光度计测定稳定胶体金最小蛋白计量Table 2 Spectrophotometer Determination of Stable Colloidal Gold Minimum Protein Quantity
以OD值为纵坐标,蛋白质用量为横坐标作一曲线,取曲线与横轴接近那一点的蛋白质用量即为最小蛋白质稳定量。在此基础上再加10-20%即为实际蛋白质稳定量。结果显示,当蛋白含量为10-20μg/mL时,即认为合适;当蛋白含量为10-16μg/mL时,更为合适。Take the OD value as the vertical axis and the protein dosage as the horizontal coordinate to draw a curve, and take the protein dosage close to the point of the curve and the horizontal axis as the minimum stable protein amount. Adding 10-20% on this basis is the actual stable protein amount. The results show that when the protein content is 10-20μg/mL, it is considered suitable; when the protein content is 10-16μg/mL, it is more suitable.
实施例6:禽流感病毒NP表达蛋白增敏检测敏感性和特异性试验Embodiment 6: Sensitivity and specificity test of avian influenza virus NP expression protein sensitization detection
将人工表达禽流感病毒NP蛋白用PBS稀释200倍,后进行连续倍比稀释,直至稀释至200×25倍,在使用试纸条检测时,即增敏前检测底限为样品800倍稀释,对检测试纸条进行增敏处理后检测底限为样品6400倍稀释,该金标增敏方法可以使禽流感病毒NP蛋白检测底线提高8倍。同时用新城疫病毒(NDV)进行检测,试纸条检测含NDV的PBS稀释液和正常细胞培养液,都为阴性。这说明NDV对禽流感病毒通用型的检测不产生干扰。Dilute the NP protein of artificially expressed avian influenza virus 200 times with PBS, and then perform serial dilution until it is diluted to 200× 25 times. When using test strips for detection, the detection limit before the sensitivity enhancement is 800 times the dilution , after sensitizing the test strips, the detection limit is 6400 times dilution of the sample. This gold standard sensitization method can increase the detection limit of avian influenza virus NP protein by 8 times. Detect with Newcastle disease virus (NDV) simultaneously, test strip detects the PBS dilution containing NDV and normal cell culture fluid, all is negative. This shows that NDV does not interfere with the detection of the common type of avian influenza virus.
实施例7:H9N2亚型禽流感病毒胶体金免疫渗滤增敏检测试验Embodiment 7: H9N2 subtype avian influenza virus colloidal gold immunofiltration sensitization detection test
用H9N2亚型禽流感病毒接种鸡胚,收获鸡胚尿囊液用PBS进行连续倍比稀释,利用本发明提供的增敏检测方法,将增敏试剂A与增敏试剂B按照1∶1的体积比混合,制成增敏试剂,分别取增敏试剂4μL处理。在使用试纸条检测时,即增敏前检测底限为血凝素效价(HAU)为2-1,对检测试纸条进行增敏处理后HAU为2-5,比对试验结果表明,对H9N2亚型禽流感病毒胶体金检测可增敏16倍以上。Inoculate chicken embryos with H9N2 subtype avian influenza virus, harvest the allantoic fluid of chicken embryos and carry out serial doubling dilution with PBS, utilize the sensitization detection method provided by the present invention, use sensitization reagent A and sensitization reagent B according to the ratio of 1:1 The volume ratio was mixed to make a sensitizing reagent, and 4 μL of the sensitizing reagent was taken for treatment. When using test strips for detection, the detection limit before sensitization is that the hemagglutinin titer (HAU) is 2 -1 , and the HAU is 2 -5 after the test strips are sensitized. The comparison test results show that , the detection of H9N2 subtype avian influenza virus colloidal gold can be more than 16 times more sensitive.
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1076281A (en) * | 1992-03-11 | 1993-09-15 | 夏良元 | The metal collosol silver development process of immune detection |
-
2008
- 2008-06-12 CN CN 200810114767 patent/CN101470116B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1076281A (en) * | 1992-03-11 | 1993-09-15 | 夏良元 | The metal collosol silver development process of immune detection |
Non-Patent Citations (2)
Title |
---|
Zhanfang Ma,et al..Naked-Eye Sensitive Detection of Immunoglubulin G by Enlargement of Au Nanoparticles In Vitro.《Angew. Chem. Int. Ed.》.2002,第12卷第2176-2177页及图1. * |
王泽霖 等.单抗体介导的斑点免疫金渗滤法(DIGFA)快速检测禽流感病毒.《SARS与禽流感国际学术研讨会》.2004,第299-307页. * |
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