CN101187665A - Enterovirus immunofluorescence chromatographic assay test paper and its preparation method - Google Patents

Enterovirus immunofluorescence chromatographic assay test paper and its preparation method Download PDF

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CN101187665A
CN101187665A CN 200710304419 CN200710304419A CN101187665A CN 101187665 A CN101187665 A CN 101187665A CN 200710304419 CN200710304419 CN 200710304419 CN 200710304419 A CN200710304419 A CN 200710304419A CN 101187665 A CN101187665 A CN 101187665A
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antibody
labeled
monoclonal antibodies
reaction
enterovirus
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CN101187665B (en
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荣 周
奇 杨
海 王
王长兵
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北京博晖创新光电技术股份有限公司
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Abstract

The invention relates to a detecting testing paper for enterovirus fluorescence immune chromatographies, which comprises a base plate, a sampling absorbing mat, a marking antibody mat, a reaction film, and a water absorbing mat. The sampling absorbing mat, the marking antibody mat, the reaction film, and the water absorbing mat are connected closely in turn and are adhered on the base plate. The invention is characterized in that one or a plurality of enterovirus fluorescence marking monoclonal antibodies are wrapped by the marking antibody mat, one or a plurality of testing belts and a quality control belt are arranged on the reaction film, an enterovirus monoclonal antibody is fixed on each testing belt, and a sheep anti mouse IgC antibody is arranged on the quality control belt. Enteroviruses which are tested include one or a plurality of Norwalk virus, enteric adenovirus, human astrovirus, and rotavirus. The purpose of qualitative or quantitative tests can be realized through a specific reaction of fluorescein or fluorescence microspheres marking antibody and applying special instrument tests.

Description

一种肠道病毒的免疫荧光层析检测试纸及其制备方法技术领域 TECHNICAL FIELD paper and immunofluorescent detection of one enteroviruses chromatography

本发明涉及一种免疫荧光层析检测试纸,具体地说是一种用于检测肠道常见病毒的免疫荧光层析检测试纸条,可检测的肠道病毒包括诺瓦克病毒、肠道腺病毒、星状病毒和轮状病毒,该试纸可用于流行病监测、临床检验等相关领域。 The present invention relates to a chromatography test strip immunofluorescence, in particular is a common intestinal virus detection immunofluorescence chromatography test strip for the detection of enteroviruses may include Norwalk virus, enteric adenovirus , astrovirus and rotavirus, the paper can be used in the relevant field epidemiological surveillance, clinical testing and so on.

背景技术 Background technique

免疫层析方法是以胶体金、荧光素、酶等作为标记物,利用特异性抗原抗体反应和标记物的特性,对抗原或抗体物质进行定位、 Immunochromatographic method is based on colloidal gold, fluorescein, an enzyme as a label, using the specific properties of antigen-antibody reaction and markers, antigen or antibody material is positioned,

定性乃至定量研究的标记技术。 Qualitative as well as quantitative markers studied. 自Faulk与Taylor在1971年创立胶体金标记技术以来,Geoghegan一l应用免疫金技术检测B淋巴细胞表面抗原,建立了光镜水平的免疫金技术(IGS)。 Since the creation of Faulk and Taylor labeling technique 1971, a l Geoghegan application immunogold B lymphocyte surface antigen detection technique, to establish the level of light microscopy of immunogold technique (IGS). Danscher在此基础上改进并发展了用银显影液增强光镜金颗粒可见性的免疫金染色法(IGSS)。 Danscher improve and enhance the development of light microscopy with gold particles of silver visibility developer immunogold staining (IGSS) on this basis. Holgate也对此法进行了改进。 Holgate also been improved this method. 近年来,胶体金技术亦被引入免疫化学检验领域,称为金标记免疫吸附试验(gold labeled immunosorbent assay, GLISA)。 In recent years, technology has also been introduced into the colloidal gold immunochemical test field, called gold-labeled immunosorbent assay (gold labeled immunosorbent assay, GLISA). 由于胶体金作为标记物时,其试纸条的操作简便,不需要借助较复杂的监测仪器等优越性,因而在免疫组织化学检测中受到了广泛的重视和应用,但一般情况下其检测灵敏度较低。 Due to colloidal gold as a marker, a test strip which is easy to operate, without resorting to more complex monitoring equipment and other advantages, and thus has been widely applied in attention and immunohistochemistry assay, but in general the detection sensitivity low. 以酶类(生物素酶、碱性磷酸酶等)作为标记物时, 涉及加样、温浴、洗涤、显色、终止等多个步骤,耗时较长,对操作人员和环境的要求较高;以荧光素作为标记物时,借助荧光检测仪可以一定程度上提高检测的灵敏度。 When in enzymes (biotin, alkaline phosphatase, etc.) as a marker, to a loading, a plurality of steps in a warm bath, washing, color development and termination, time-consuming, require a higher operating personnel and the environment ; when fluorescein as a label, fluorescence detector means detecting sensitivity can be improved to some extent.

腹泻是人类尤其是儿童、老人及免疫缺陷者的一种常见病和多发病,其中病毒性腹泻占70% ~80%,是导致儿童死亡的重要原因。 Diarrhea is a human, especially children, the elderly and immunocompromised a common and frequently-occurring disease, viral diarrhea which accounts for 70% to 80%, an important cause of child mortality.

其中轮状病毒(Rotavirus, RV)、星状病毒(Astrovirus, AstV)、诺瓦克病毒(Norwalk virus, NV)及肠道腺病毒(Enteric Adenovirus, EAdV)等四种肠道病毒最为常见,3岁前几乎所有儿童都经历过上述腹泻病毒的感染。 Where rotavirus (Rotavirus, RV), astrovirus (Astrovirus, AstV), Norwalk virus (Norwalk virus, NV) and enteric adenovirus (Enteric Adenovirus, EAdV) and other four most common intestinal virus, 3 before the age of almost all children have experienced the above diarrhea virus infection.

轮状病毒引起的腹泻是病毒性腹泻的主要因素,40 % -60 %的腹泻由轮状病毒引起,全球每年约有1.4亿人发生轮状病毒腹泻,其发病率和严重程度居秋冬季腹泻的首位,并导致大约80万儿童死亡, 严重的轮状病毒感染主要见于6~24个月婴幼儿。 Rotavirus diarrhea caused by viral diarrhea is a major factor of 40% -60% of diarrhea caused by rotavirus, each year about 140 million people worldwide rotavirus diarrhea occurs, the incidence and severity of diarrhea in autumn and winter residence the first, and led to about 80 million children died of severe rotavirus infection mainly seen in 6 to 24 month old infants. 肠道腺病毒也是病毒性腹泻的重要病原,主要症状为腹泻,水样便或稀便,腹泻次数较多,病程7-14天,排毒时间10-14天,可伴有发热,呕吐和呼吸道症状,病情相对较轻,少数也能导致严重脱水致死。 Enteric adenovirus viral diarrhea is also an important pathogen, the main symptoms are diarrhea, watery or loose stools, diarrhea more often, duration of 7-14 days, 10-14 days detoxification time, can be accompanied by fever, vomiting, and respiratory tract symptoms are relatively mild, a few can lead to severe dehydration and death. 星状病毒与轮状病毒相似,以2岁以下儿童多发,可以散发,也可以暴发流行。 Astrovirus and rotavirus is similar to many children 2 years of age, can be dissipated, can outbreak. 诺瓦克病毒分布广泛,可导致成人和儿童的急性腹泻的病毒, 是除轮状病毒外引起腹泻的最主要的病毒病原,日本的流行病学调查显示诺瓦克病毒感染率已超过轮状病毒。 Norwalk virus is widely distributed, can cause acute diarrhea in adults and children of the virus, which is caused by rotavirus diarrhea in addition to the most important viral pathogens, Japanese epidemiological survey Norwalk virus infection rate has exceeded the wheel virus. 在美国,成人非细菌性胃肠炎的爆发中有42%由该类病毒所引起,我国还缺乏流行病学调查。 In the United States, adult non-bacterial outbreaks of gastroenteritis in 42% caused by a type of virus, China still lacks an epidemiological investigation.

目前临床上仍无针对这四种肠道病毒的特效治疗药物,虽然有轮状病毒疫苗研制的报道,但其效果还需要观察,所以预防、控制病毒传染显得尤为重要。 The present study is still no specific treatment for these four enteroviruses, although there are reports of rotavirus vaccine, but its effect remains to be seen, so prevention and control of viral infection is very important. 细菌、寄生虫及食物中毒等原因也能引起腹泻,临床症状相仿,但与病毒性腹泻的治疗方案不一样。 Bacteria, parasites and food poisoning can cause diarrhea and other reasons, clinical symptoms similar, but with viral diarrhea treatment programs are not the same. 有报道发现双重病毒或多种病毒感染的腹泻,其症状更严重,病情进展快, 导致死亡的风险更高。 It has been reported double virus diarrhea or more viral infections, more severe symptoms, rapid progression, leading to a higher risk of death.

由于肠道传染病的临床表现和常用的辅助检查缺乏明显的特异性,因此存在着在疾病早期病原体诊断不明确的危险因素,从而存 Because the clinical manifestations and commonly used laboratory examinations of intestinal infectious diseases apparent lack specificity, so there are pathogens in the early diagnosis of the disease is not established risk factors, in order to save

在着疾病早期滥用抗生素、生病期延长而增加患者痛苦和经济负担的客观因素。 Abuse of antibiotics in the early stages of disease, illness extended period increased suffering and economic burden of patients with objective factors. 因此,对此类疾病的深入研究,研制快速准确的诊断技术,对预防疾病的发生和减少死亡率,指导临床对症治疗和避免抗生素滥用有重要意义。 Therefore, further research on these diseases, the development of rapid and accurate diagnostic techniques, to prevent diseases and reduce mortality, clinical symptomatic treatment and avoid abuse of antibiotics is important.

应用高灵敏度的、快速和特异的免疫荧光层析方法进行肠道传染病的病原学诊断,有利于在疾病症状出现的早期就确定引起肠道传染病的病原体。 Application of high sensitivity, fast and specific chromatographic methods immunofluorescence etiological diagnosis of intestinal infectious diseases, facilitate early in the disease symptoms on the determination of intestinal infectious diseases caused by pathogens. 有利于在疾病早期针对性进行有效的抗生素治疗, 缩短疾病的病程,减轻患者的痛苦和经济负担。 Conducive to effective antibiotic therapy targeted at an early stage of disease, shorten the course of the disease, alleviate suffering and economic burden of patients. 对肠道病毒及时而准确的病原学诊断,有利于对肠道病毒的流行病学进行研究,指导在疾病流行的初期进行预防,从而能极大程度的降低肠道传染病的流行范围和程度,降低肠道传染病对社会的危害。 Timely and accurate etiological diagnosis of intestinal virus, enterovirus in favor of epidemiological study guide in the early epidemic prevention, which can greatly reduce the prevalence of the scope and extent of intestinal infectious diseases reduce intestinal infectious diseases harm to society.

目前对于肠道病毒的检测多釆用免疫学检测,传统的免疫检测有乳胶凝集试验(LA)、免疫标记技术[包括酶联免疫测定(EIA)、放射 At present, for the detection of enterovirus preclude the use of multiple immunological detection, the traditional latex agglutination immunoassay (LA), Immunocolloidal [including enzyme immunoassay (EIA), Radiology

免疫测定(RIA)、免疫金染色法(immunogold staining, IGS)等技术], 随着新标记物的应用以及固相载体技术和抗体制备技术的发展,免疫定量技术如时间分辨荧光免疫测定(time-resolved fluoro-immunoassay, TR-IFMA或TrFIA)和定量乳胶凝集试验(quantitative latex agglutination technique, QLAT)等免疫学新技术相继用于病原体的检测,简化了检测步骤,也提高了免疫学方法检测的特异性和灵敏度。 Immunoassay (RIA), immunogold staining (immunogold staining, IGS) technology], with the development and application of technology and the preparation of solid phase support new technologies antibody markers, Immunoassay techniques such as time-resolved fluorescence immunoassay (time -resolved fluoro-immunoassay, TR-IFMA or TrFIA for) and quantitative latex agglutination test (quantitative latex agglutination technique, QLAT) and other immunological new technologies for successive detection of pathogens, simplifies the detection step, also improves the immunological detection method specificity and sensitivity. 可以用于肠道病毒多检的免疫荧光层析检测试 Immunofluorescence chromatography may be used to detect enteroviruses plurality of test sample

纸条法通过不同病原体的特异性单克隆抗体,来对肠道病毒进行病原学诊断,在疾病发生发展的早期就可以进行诊断,从而具有显而易见的临床诊断意义。 Slip method by monoclonal antibodies specific for different pathogens, to etiological diagnosis of intestinal virus, it can be diagnosed at an early stage of disease development, which has obvious clinical diagnosis.

发明内容 SUMMARY

本发明的目的在于提供一种常见肠道病毒的免疫荧光层析快速检测试纸,其具有快速、有效而简便的特点,涉及的病原体为轮状病毒(RV)、星状病毒(AstV)、诺瓦克病毒(NV)及肠道腺病毒 Object of the present invention is to provide a common intestinal virus immunofluorescence chromatographic rapid tests, which is fast, effective and simple features, is directed to the pathogen Rotavirus (the RV), Astrovirus (ASTV), Connaught Wacker virus (NV) and enteric adenovirus

(EAdV)中的一种或多种。 One or more (eadv) in.

本发明的另一个目的在于提供制备上述检测试纸的方法。 Another object of the present invention to provide a process for preparing such a test strip. 本发明的免疫荧光层析快速检测试纸包括底板、附着在底板上依次紧密相连的样品吸收垫、标记抗体垫、反应膜和吸水垫。 Immunofluorescence chromatographic test strip of the present invention includes a base plate fast adhered sample on the base plate closely sequentially absorbent pad, the labeled antibody pad, absorbent pad and the reaction film. 其中所述标记抗体垫包埋有一种或多种肠道病毒的荧光标记单克隆抗 Wherein said labeled antibody pad embedded with one or more fluorescent labeled enterovirus monoclonal anti

体;所述反应膜上具有一条或多条检测带和一条质控带,每条检测 Thereof; said reaction membrane having one or more test line and a control line, each detection

带上固定有一种肠道病毒的单克隆抗体,检测带的条数以及固定的肠道病毒单克隆抗体与标记抗体垫包埋的标记抗体相适应,质控带固定有能与荧光标记单克隆抗体特异结合的抗抗体。 A monoclonal antibody is fixed tape enterovirus, the number of test strips and a fixed enterovirus antibody labeled with monoclonal antibodies labeled antibody pad adapted embedded, the control line can be secured with a fluorescently labeled monoclonal an anti-antibody specific binding.

其中,所述的肠道病毒单克隆抗体为轮状病毒单克隆抗体、肠道腺病单克隆抗体、星状病毒单克隆抗体或诺瓦克病毒单克隆抗体中的一种或多种。 Wherein said enterovirus monoclonal antibody monoclonal antibody rotavirus, adenovirus intestinal monoclonal antibodies, or monoclonal antibodies Astrovirus Norwalk virus monoclonal antibody of one or more. 其中,制备轮状病毒单克隆抗体的免疫原优选为 Wherein the immunogen is preferably prepared as monoclonal antibodies rotavirus

轮状病毒VP6蛋白;制备肠道腺病毒单克隆抗体的免疫原优选为腺病毒40/41型六邻体蛋白(hexon);制备星状病毒单克隆抗体的免疫原优选为I型星状病毒CP蛋白;制备诺瓦克病毒单克隆抗体的免疫原优选为诺瓦克病毒VP1蛋白。 Rotavirus VP6 protein; immunogen prepared enteric adenovirus monoclonal antibody is preferably 40/41 type adenovirus hexon protein (Hexon); astrovirus immunogen preparation of monoclonal antibodies Astrovirus preferably type I CP protein; Norwalk virus immunogen preparation of monoclonal antibodies is preferably Norwalk virus VP1 protein. 上述的免疫原是釆用大肠杆菌原核表达系统表达,利用分子生物学技术扩增出所需的核酸序列,将核酸连接到表达质粒载体上,转染到大肠杆菌中诱导表达,再通过镍亲和柱纯化出目的蛋白。 Preclude the use of the above-described immunogen prokaryotic expression system in E. coli, using molecular biology techniques amplified nucleic acid sequences required to connect to a nucleic acid expression vector, transfected into E. coli and induced expression, and then by nickel affinity column and purified protein. 釆用弗氏免疫佐剂与纯化目的蛋白乳化物免疫实验动物Balb/c小鼠,每只注射量为50jag蛋白,免疫部位为腹服沟和腹腔,免疫间隔期为14天,免疫4次(第一次选用的佐剂为弗氏完全佐剂,后三次为弗氏不完全佐剂);釆取小鼠血清测试Elisa抗体效价,选取效价大于l: 5000的小鼠进行加强免疫,蛋白免疫量为lOOiag/只,免疫部位为脾脏,3天后可进行融合试验。 Preclude the use of Freund's adjuvant immunization purified protein emulsified in experimental animals immunized Balb / c mice, each injection volume 50jag protein, the site of immunization and peritoneal abdominal serving groove, immunization interval is 14 days, immunization 4 times ( the first choice of the adjuvant is Freund's complete adjuvant, the incomplete Freund's adjuvant for the three); Bian take Elisa test mouse serum antibody titers, titers greater than select l: 5000 mice are boosted, an amount of protein immunogen lOOiag / only, as the site of immunization spleen 3 days after the fusion experiment. 无菌取其脾脏细胞并与SP2/0—Ag14骨髓瘤细胞杂交融合,融合剂为PEG3350。 Sterile whichever spleen cells and fused with myeloma cell hybridoma SP2 / 0-Ag14, the fusion agent is PEG3350. 用间接ELISA方法筛选阳性克隆,通过有限稀释法筛选出特异分泌肠道病毒重组蛋白的单克隆细胞株。 Positive clones were screened by indirect ELISA, screened enterovirus specific secreted recombinant protein monoclonal cell lines by limiting dilution. 以体内诱生法进 In vivo induced method into

行腹水制备,以Protein-A、 protein-G或辛酸-硫酸铵法提纯单克隆抗体。 Ascites preparation line to Protein-A, protein-G or caprylic acid - ammonium sulfate purified monoclonal antibody. 制备的单克隆抗体纯化物保存在-2(TC冰箱中。单克隆抗体细胞株经过扩大培养,加入冻存液保存在液氮中。 Preparation of purified monoclonal antibody was stored at -2 (TC refrigerator. Expand the monoclonal antibody cell line after incubation, was stored frozen in liquid nitrogen.

其中,所述质控带固定的能与荧光标记单克隆抗体特异结合的抗抗体为羊抗鼠IgG抗体。 Wherein the control line can be secured with a fluorescent labeled monoclonal antibodies specific antibody conjugated goat anti-mouse IgG antibody.

其中,所述标记抗体是以荧光素或荧光微球作为标记物,与特异性的肠道病毒单克隆抗体进行交联获得。 Wherein said antibody is a fluorescein labeled or fluorescent beads as markers, the monoclonal antibody obtained with a specific crosslinking enterovirus. 荧光素为Molecular Probes公司的Alexa Fluor系列荧光素(Alexa Fluor@350, Alexa Fluor@405, Alexa Fluor@488, Alexa Fluor@555, Alexa Fluor@568, Alexa Fluor@594, Alexa Fluor@647, Alexa Fluor@660, Alexa Fluor@680, AlexaFluor@750等)或安玛西亚公司的Cy系列荧光素(Cy3, Cy3,5' Cy5等)。 Luciferin Molecular Probes company of Alexa Fluor series fluorescein (Alexa Fluor @ 350, Alexa Fluor @ 405, Alexa Fluor @ 488, Alexa Fluor @ 555, Alexa Fluor @ 568, Alexa Fluor @ 594, Alexa Fluor @ 647, Alexa Fluor @ 660, Alexa Fluor @ 680, AlexaFluor @ 750 etc.) or Amersham's Cy series fluorescein (Cy3, Cy3,5 'Cy5, etc.).

其中,所述反应膜为硝酸纤维素膜。 Wherein the reaction membrane is a nitrocellulose membrane.

本发明还提供了一种制备上述检测试纸的方法,其包括如下步 The present invention further provides a method of preparing the above-described test paper, comprising the steps

骤: Step:

1) 在反应膜的不同位置上按照抗体的种类分别固定肠道病毒单克隆抗体以及抗抗体,形成检测带和质控带; 1) the reaction at different locations on the membrane are fixed enterovirus antibodies and monoclonal antibodies, formed in accordance with the detection zone and the control antibody species;

2) 制备标记抗体,并在样品标记抗体垫中包埋标记抗体; 2) Preparation of labeled antibody, the labeled antibody and embedded in a sample pad labeled antibody;

3) 组装底板、样品吸收垫、标记抗体垫、反应膜和吸水垫; 3) assembling the base plate, the sample absorbent pad, the labeled antibody pad, absorbent pad and the reaction membrane;

4) 切割、包装。 4) cutting and packaging.

在使用时,在层析膜的样品垫上加入样品液(假定含有病毒抗原),必要时添加稀释液,在毛细作用下,样品液向吸水垫一端泳动, 在标记抗体垫处与标记抗体形成免疫复合物,再进一步泳动,不同的免疫复合物分别与相应的检测带处的单克隆抗体结合形成类似双抗体夹心的免疫复合物,未结合的标记抗体则会与质控带抗抗体相结合。 In use, the sample added to the sample pad of the chromatographic membrane solution (assuming containing viral antigen), dilution was added as necessary, by capillary action, the liquid sample to the absorbent pad at one end migrate, the labeled antibody is formed at a pad with a labeled antibody immune complexes, further electrophoresis, the different binding immune complexes are detected with the respective monoclonal antibody is formed at a similar double antibody sandwich immune complex, the labeled antibody will not bind to the control line with anti-antibody combined.

标记抗体若釆用的是同一种荧光素或荧光微球,则应当按照不 Preclude the use of a labeled antibody when the same is fluorescein or fluorescent microspheres, it should not be in accordance with

同的包被抗体将质控带彼此分离,在检测时,若质控带处未出现条带,则说明试纸已过期;若质控带处出现条带,而检测带处未出现条带,则说明样品中不含有病毒;若在某个或某些检测带处出现条带,则说明样品液中存在相应的病毒。 Coating antibody with the control line separated from each other in the detection, if the control line at the band does not appear, then the test strip has expired; if the control line at the occurrence of banding, and the detection zone of the strip is not present, then the sample does not contain a virus; if bands appear in one or some of the test strip, then the corresponding virus in the sample liquid.

所述检测抗体、质控抗体(羊抗鼠IgG多抗)的固定是指按照普通胶体金制作工艺,利用点膜机将检测抗体、质控抗体划线到硝酸纤维素膜(固相载体)上并对固相载体进行封闭,其目的是在检测时作为检测带和质控带。 The detection antibody, quality control antibody (goat anti-mouse IgG polyclonal antibody) fixing means according to the conventional production process of colloidal gold, the use of membrane dryer point detection antibody, antibody chain line quality control to a nitrocellulose membrane (solid support) and the solid support for the closure, which aims at detecting a test line and a control line.

所述的抗体标记是指使单克隆抗体和荧光素或荧光微球通过化学交联法形成复合物。 It refers to the antibody labeled monoclonal antibodies and complexes fluorescein or fluorescent microspheres formed by chemical crosslinking. 荧光素标记单克隆抗体:荧光素的活性酯与抗 Fluorescein labeled monoclonal antibodies: Fluorescein active ester with an anti-

体上的羧基反应形成共价键;荧光微球标记单克隆抗体:荧光微粒 The reaction on the carboxyl groups formed covalent bond; fluorescent microspheres labeled monoclonal antibody: fluorescent particles

是釆用高分子聚苯乙烯材料包裹荧光素染料制备而成的,直径为 Polystyrene is a polymer prepared preclude the wrapping material is formed by fluorescein dye, diameter

200 ~ 300nm,荧光微球表面有功能基团羧基。 200 ~ 300nm, fluorescent beads carboxyl surface functional groups. 在双功能交联剂(EDAC和NHS)作用下,荧光微球与单克隆抗体结合。 In the cross-linker (EDAC and NHS) effect, fluorescent microspheres bound to the monoclonal antibody.

所述的标记抗体结合垫制备是将荧光素标记的抗体或荧光微球标记的抗体用稀释液(0.5。/。BSA+3。/。海藻糖+0.1。/oTween-20, 10mM PH7.5Tris-HCl)进行稀释,然后将抗体标记垫(玻璃纤维膜、聚酯膜)浸泡在稀释液中,经过烘干或者真空冷冻干燥后,再装配到试纸条底衬上。 The binding of the labeled antibody is an antibody prepared pads or fluorescently labeled microspheres labeled with fluorescein dilutions (0.5./.BSA+3./. Trehalose + 0.1. / OTween-20, 10mM PH7.5Tris -HCl) was diluted, then the labeled antibody pad (glass fiber membrane, a polyester film) was immersed in the diluted solution, after drying in vacuo or freeze-dried, and then assembled to the backing strip.

所述的试纸条组装和切割是在包被有捕获抗体和质控抗体的硝酸纤维素膜的一侧贴上样品垫和标记抗体结合垫,另一侧贴上高强度吸水纸,利用切割机进行试纸条的切割。 The test strip and the cutting assembly is the side of the nitrocellulose membrane is in the package with a capture antibody and the control sample pad labeled antibody and labeled antibody conjugate pad, the other side of the high-strength absorbent paper paste, by dicing strip cutting machine.

所述的免疫层析反应包括双抗体夹心法和间接法两种免疫学方法。 The immunochromatographic reaction include double antibody sandwich method and two kinds of indirect immunological method. 双抗体夹心法是检测带的反应方法,待测抗原在标记抗体垫处与荧光素标记的抗体形成免疫复合物,该复合物在硝酸纤维素膜上进行泳动并与膜上的检测带抗体进行结合,形成固化的免疫复合物; 间接法是质控带的反应方法,荧光素标记的抗体在膜上泳动并与质控带上的抗抗体进行结合,形成固化的免疫复合物。 Double antibody sandwich method is a method of detecting the reaction zone, the antigen to form an immune complex of the antibody with a labeled antibody pad fluorescein-labeled, the composite was subjected to electrophoresis on a nitrocellulose membrane, and the membrane with the detection antibody They are combined to form a cured immunological complex; the indirect method is a method of quality control of the reaction zone, fluorescein labeled antibody binding to the membrane and migrating band and quality control antibodies, immune complex formation cured.

结果检测是利用专用的荧光检测仪对质控带和检测带进行检 Results fluorescence detection is the use of a dedicated control line on the detector and the detection zone for detecting

测,检测带的荧光强度和待测样品中特异抗原的含量成正比。 Measuring, with a fluorescence intensity proportional to the concentration of the test sample and specific antigen.

与现有的肠道病毒检测方法相比较,本发明具有以下优势: Compared with the conventional method for detecting enteroviruses, the present invention has the following advantages:

1) 将荧光素或荧光微球与传统的胶体金免疫层析技术相结合, 通过荧光强度的变化来反应样品中的抗原含量,可在定性的同时实现病原体的定量检测。 1) The fluorescent beads or fluorescein and GICA conventional technology, the antigen content of the sample to the reaction by the change in fluorescence intensity, the quantitative detection of pathogens can be achieved at the same time qualitatively.

2) 结果客观和灵敏度高。 2) Results objective and high sensitivity. 和传统的胶体金试纸条检测方法来比较,荧光素强度的釆集靠荧光检测仪来完成,结果客观而灵敏,灵 And colloidal gold strip conventional detection methods to compare the intensity of fluorescein fluorescence detector by Bian set to complete, and sensitive results objective, spirit

敏度可达到传统胶体金试纸条的数io倍以上。 Sensitivity can be achieved in a conventional colloidal gold strip number io times.

3) 能够同时进行多种常见肠道病毒的检测。 3) can simultaneously detect many common intestinal virus.

附图说明 BRIEF DESCRIPTION

图1是肠道病毒4联检的免疫荧光层析快速检测试纸条的结构示意图; FIG 1 is a schematic diagram of the Unit 4 enterovirus immunofluorescence test strip of flash chromatography;

图2是诺瓦克病毒、肠道腺病毒、星状病毒、轮状病毒均为阳性的结果示意图; FIG 2 is a schematic view of a norovirus, enteric adenovirus, astrovirus, were rotavirus positive results;

图3是肠道腺病毒、轮状病毒为阳性的结果示意图; FIG 3 is enteric adenovirus, rotavirus is a schematic view of a positive result;

图4是单个病毒检测试纸的结构示意图。 FIG 4 is a schematic diagram of a single virus in the test strip.

图中,l为底板、2为反应膜、3为吸水纸、4为标记抗体垫、5 为样品吸收垫、T1〜T4为检测带、C为质控带。 Drawing, l is the base plate, 2 is a reaction film, absorbent paper 3, mat 4 to the labeled antibody, the sample absorbent pad 5, T1~T4 to detect a band, C is the control line.

具体实施方式 Detailed ways

下面结合具体的实施例来进一步阐述本发明。 Specific embodiments of the present invention is further illustrated below in conjunction. 应当理解,这些实施例仅用于说明本发明,而不能限制本发明的保护范围。 It should be understood that these embodiments are merely to illustrate the invention, but not intended to limit the scope of the present invention. 实施例l肠道病毒四项联检试纸的制备 Preparation Example l enterovirus four strips embodiment Unit

参照附图1,本实施例的肠道病毒多检试纸包括底板l、粘附于底板上的包埋有质控带(C带)和检测带(Tl带、T2带、T3带、 T4带)的硝酸纤维素膜2、覆盖于硝酸纤维素膜一侧的高强度吸水 Enterovirus embodiment according to the present embodiment with reference to drawings of the subject multi-strip comprises a base plate 1 L, adhering to the bottom plate embedded with the control line (C-band) and the detection zone (Tl band, T2 band, T3 band, T4 with ) 2 nitrocellulose membrane, nitrocellulose membrane covering one side of a high strength absorbent

纸3、覆盖于硝酸纤维素膜的另一侧的标记抗体垫4、样品吸收垫5。 Paper 3, the other side of the labeled antibody to the nitrocellulose membrane covering mat 4, the sample absorption pad 5.

所述的硝酸纤维素膜的C带区域包埋有羊抗鼠IgG抗体,Tl带包埋有诺瓦克病毒的单克隆抗体,T2带包埋有肠道腺病毒的单克隆抗体,T3带包埋有星状病毒的单克隆抗体,T4带包埋有轮状病毒的单克隆抗体。 The C-band area embedded nitrocellulose membrane sheep anti-mouse IgG antibody, Tl embedded with a monoclonal antibody with norovirus, T2 tape embedded with a monoclonal antibody of enteric adenovirus, T3 with monoclonal antibodies star embedded with viruses, T4 monoclonal antibody embedded with rotavirus. 并且,制备轮状病毒单克隆抗体的免疫原为轮状病毒VP6蛋白;制备肠道腺病毒单克隆抗体的免疫原为腺病毒40/41型六邻体蛋白;制备星状病毒单克隆抗体的免疫原为I型星状病毒抗原;制备诺瓦克病毒单克隆抗体的免疫原为诺瓦克病毒VP1蛋白。 And, rotavirus immune monoclonal antibodies prepared rotavirus VP6 protein original; immunize enteric adenovirus monoclonal antibodies original type adenovirus hexon protein 40/41; Preparation of monoclonal antibodies Astrovirus type I star immunogenic viral antigen; immunize Norwalk virus monoclonal antibodies original norovirus VP1 protein. l.单克隆抗体的制备 Preparation of l monoclonal antibodies

1.1、轮状病毒VP6蛋白单克隆抗体的制备如下: 1.1, rotavirus VP6 protein monoclonal antibody prepared as follows:

目的片段来源于临床分离株,按照GENEBANK上序列号为AF260931的VP6基因序列,按照其序列和pQE-30原核表达载体设计引物如下: Fragment derived from the clinical isolates, according to the sequence number GENEBANK VP6 gene sequence AF260931, in accordance with the sequence and pQE-30 prokaryotic expression vector The primers were designed as follows:

上游引物为5'-TTGGCATGCATGGAGGTTCTGTACTCATTGTC 下游引物为5'-GGGGTCGACACTTAATCAACATGCTTCTAATGG An upstream primer 5'-TTGGCATGCATGGAGGTTCTGTACTCATTGTC downstream primer 5'-GGGGTCGACACTTAATCAACATGCTTCTAATGG

进行聚合酶链式反应(PCR),按照所釆用的DNA聚合酶设置反应体系,其中各引物的添加量均为0.6pmol/iaL,反应程序设置如下: Polymerase chain reaction (the PCR), is provided in accordance with the reaction system preclude the use of DNA polymerase, wherein the addition amount of each primer were 0.6pmol / iaL, reaction procedure as follows:

第一步:95°C 2min;第二步:95°C 30sec, 57°C 30sec, 72°C lmin, 36个循环;第三步:72°C 5min。 The first step: 95 ° 2min C; Step: 95 ° C 30sec, 57 ° C 30sec, 72 ° C lmin, 36 cycles; The third step: 72 ° C 5min.

对PCR产物进行测序,测序结果如序列表SEQIDNO: 1所示, 与AF260931的同源性为100%。 The PCR products were sequenced, the sequencing results of the sequence list SEQIDNO: 1, the homology is 100% AF260931.

将提取的pQE-30质粒和纯化的PCR产物进行Sail和Sphl限制性内切酶的双酶切,酶切条件设置为37'C 3hr;将酶切产物用1% 琼脂糖凝胶电泳并回收;用T4 DNA连接酶将载体和目的基因进行连接。 The pQE-30 plasmid was extracted and purified PCR product was double digested endonucleases Sphl and Sail restriction enzyme conditions to 37'C 3hr; The product was digested with 1% agarose gel electrophoresis and recovered ; using T4 DNA ligase and the gene vector is connected. 连接产物转化大肠杆菌M15感受态细胞。 The ligation product was transformed into E. coli competent cell M15. 挑单菌落培养增菌后,提取质粒进行双酶切鉴定。 Single colonies were picked after enrichment cultivation, plasmid was extracted for double digestion.

将测序鉴定正确的带有VP6蛋白基因的重组质粒(命名为 The correct recombinant plasmid was identified by sequencing with the VP6 protein gene (designated

pQE-30/VP6 )及空质粒pQE-30的转化菌,使用LB液体培养基(加入50 ia g/mL的Amp )250rpm 37。 pQE-30 / VP6) and transformants were empty plasmid pQE-30 using LB broth (added 50 ia g / mL of Amp) 250rpm 37. C培养;至菌浓度为OD=0.5-0.6时, 加入终浓度为lmmol/L的IPTG进行诱导表达6hr。 C culture; 6hr expression was induced to a bacterial concentration of OD = 0.5-0.6, the final concentration of lmmol / L of IPTG. 离心沉淀菌体后进行蛋白质SDS-PAGE电泳观察。 SDS-PAGE electrophoresis was observed a protein precipitate after centrifugation cells. 釆用镍结合柱的方法获得纯化的融合蛋白,通过蛋白质SDS-PAGE电泳进行纯化蛋白的纯度验证, 要求纯度大于99%。 Bian binding method using a nickel column to obtain purified fusion protein, the purified protein was performed to verify the purity of proteins by SDS-PAGE electrophoresis, it requires greater than 99%.

采用弗氏免疫佐剂与纯化蛋白乳化物免疫实验动物Balb/c小鼠,每只注射量为50lig蛋白,免疫部位为腹股沟和腹腔,免疫间隔期为14天,免疫4次(第一次选用的佐剂为弗氏完全佐剂,后三次为弗氏不完全佐剂);釆取小鼠血清测试Elisa抗体效价,选取效价大于l: 5000的小鼠进行加强免疫,蛋白免疫量为100ng/只,免疫部位为脾脏,3天后可进行融合试验。 Freund's adjuvant immunization using experimental animals immunized with purified protein emulsion Balb / c mice, each injection volume 50lig protein, abdominal and inguinal sites of immunization, immunization interval is 14 days, immunization 4 times (first selection the adjuvant is Freund's complete adjuvant, the incomplete Freund's adjuvant for the three); Bian Elisa test the serum antibody titers, titers in mice greater than select l: 5000 mice are boosted protein immunizing amount of 100ng / only, as the site of immunization spleen 3 days after the fusion experiment. 无菌取其脾脏细胞并与SP2 /0^Agl4骨髓瘤细胞杂交融合,融合剂为PEG3350。 Sterile whichever spleen cells with SP2 / 0 Agl4 myeloma cell hybridization ^ fusion, the fusion agent is PEG3350. 用间接ELISA 方法筛选阳性克隆,通过有限稀释法筛选出特异分泌抗轮状病毒VP6蛋白的单克隆细胞株。 Positive clones were screened by indirect ELISA, screened against rotavirus VP6 protein secreting specific monoclonal cell lines by limiting dilution. 以体内诱生法进行腹水制备,以Protein-A、 protein-G或辛酸-硫酸铵法提纯单克隆抗体。 Prepared ascites induced in vivo method to Protein-A, protein-G or caprylic acid - ammonium sulfate purified monoclonal antibody. 制备的单克隆抗体纯化物保存在-20。 The purified monoclonal antibody was prepared at -20. C冰箱中。 C refrigerator. 单克隆抗体细胞株经过扩大培养,加入冻存液保存在液氮中。 The monoclonal antibody cell lines through expansion of incubation, frozen liquid stored in liquid nitrogen.

1.2、腺病毒40/41型六邻体蛋白的单克隆抗体制备如下: 1.2, 40/41 monoclonal antibody type adenovirus hexon protein prepared as follows:

目的片段来源于临床分离株,按照GENEBANK上序列号为D13781的hexon基因序列,选取其中的保守区域和pQE-30原核表达载体设计引物如下: Fragment derived from the clinical isolates, according to the GENEBANK hexon Serial No. D13781 of gene sequences, and select one of the conserved regions of pQE-30 prokaryotic expression vector The primers were designed as follows:

上游引物为5'-GGGGCATGCGTGTACAAGCCAGATGTAG 下游引物为5'-AAAGTCGACGCAGGTCGTTACCCAGACTG An upstream primer 5'-GGGGCATGCGTGTACAAGCCAGATGTAG downstream primer 5'-AAAGTCGACGCAGGTCGTTACCCAGACTG

进行聚合酶链式反应(PCR),按照所釆用的DNA聚合酶设置反应体系,其中各引物的添加量均为0.6pmol/iaL,反应程序设置如 Polymerase chain reaction (the PCR), is provided in accordance with the reaction system preclude the use of DNA polymerase, wherein the addition amount of each primer were 0.6pmol / iaL, the reaction procedure as provided

下: under:

第一步:95°C 2min;第二步:95°C 30sec, 57°C 30sec, 72。 The first step: 95 ° C 2min; Step: 95 ° C 30sec, 57 ° C 30sec, 72. C lmin, 36个循环;第三步:72°C 5min。 C lmin, 36 cycles; The third step: 72 ° C 5min.

对PCR产物进行测序,测序结果如序列表SEQIDNO: 2所示, 与D13781的同源性为100%。 The PCR products were sequenced, the sequencing results of the sequence list SEQIDNO: 2, the homology is 100% D13781.

将提取的pQE-30质粒和纯化的PCR产物进行Sail和Sphl限制性内切酶的双酶切,酶切条件设置为37°C 3hr;将酶切产物用1% 琼脂糖凝胶电泳并回收;用T4 DNA连接酶将载体和目的基因进行连接。 The pQE-30 plasmid was extracted and purified PCR product was double digested endonucleases Sphl and Sail restriction enzyme conditions to 37 ° C 3hr; The product was digested with 1% agarose gel electrophoresis and recovered ; using T4 DNA ligase and the gene vector is connected. 连接产物转化大肠杆菌M15感受态细胞。 The ligation product was transformed into E. coli competent cell M15. 挑单菌落培养增菌后,提取质粒进行双酶切鉴定。 Single colonies were picked after enrichment cultivation, plasmid was extracted for double digestion.

将测序鉴定正确的带有hexon蛋白基因的重组质粒(命名为pQE-30/hexon)及空质粒pQE-30的转化菌,使用LB液体培养基(加入50 ja g/mL的Amp )250rpm 37。 The recombinant plasmid with correct hexon protein gene sequencing (designated pQE-30 / hexon) and transformants were empty plasmid pQE-30 using LB broth (added 50 ja g / mL of Amp) 250rpm 37. C培养;至菌浓度为OD=0.5-0.6时, 加入终浓度为lmmol/L的IPTG进行诱导表达6hr。 C culture; 6hr expression was induced to a bacterial concentration of OD = 0.5-0.6, the final concentration of lmmol / L of IPTG. 离心沉淀菌体后进行蛋白质SDS-PAGE电泳观察。 SDS-PAGE electrophoresis was observed a protein precipitate after centrifugation cells. 釆用镍结合柱的方法获得纯化的融合蛋白,通过蛋白质SDS-PAGE电泳进行纯化蛋白的纯度验证, 要求纯度大于99%。 Bian binding method using a nickel column to obtain purified fusion protein, the purified protein was performed to verify the purity of proteins by SDS-PAGE electrophoresis, it requires greater than 99%.

后续的单克隆抗体制备步骤同轮状病毒VP6蛋白的单克隆抗体制备。 Monoclonal antibody preparation step subsequent preparation of monoclonal antibodies with rotavirus VP6 protein.

1.3、星状病毒CP蛋白单克隆抗体制备如下: 1.3, astrovirus CP monoclonal antibodies can be prepared as follows:

目的片段来源于临床分离株,按照GENEBANK上序列号为AB009985的CP基因序列,选取其中的保守区域和pQE-30原核表达载体设计引物如下: Fragment derived from the clinical isolates, according to the GENEBANK Serial No. AB009985 gene sequence of the CP, and select one of the conserved regions of pQE-30 prokaryotic expression vector The primers were designed as follows:

上游引物为5'-GGGGCATGCAACGGACGCAACAAATATCAATCT; 下游引物为5'-AAAGTCGACGCGCACCAGTGCGAGC; An upstream primer 5'-GGGGCATGCAACGGACGCAACAAATATCAATCT; downstream primer was 5'-AAAGTCGACGCGCACCAGTGCGAGC;

进行聚合酶链式反应(PCR),按照所釆用的DNA聚合酶设置反应体系,其中各引物的添加量均为0.6pmol/ML,反应程序设置如 Polymerase chain reaction (the PCR), is provided in accordance with the reaction system preclude the use of DNA polymerase, wherein the addition amount of each primer were 0.6pmol / ML, the reaction procedure as provided

下: under:

第一步:95°C 2min;第二步:95°C 30sec, 57°C 30sec, 72°C lmin, 36个循环;第三步:72°C 5min。 The first step: 95 ° 2min C; Step: 95 ° C 30sec, 57 ° C 30sec, 72 ° C lmin, 36 cycles; The third step: 72 ° C 5min.

对PCR产物进行测序,测序结果如序列表SEQIDNO: 3所示, 与AB009985的同源性为100%。 The PCR products were sequenced, the sequencing results of the Sequence Table SEQIDNO: 3, the homology with AB009985 was 100%.

将提取的pQE-30质粒和纯化的PCR产物进行Sail和Sphl限制性内切酶的双酶切,酶切条件设置为37匸3hr;将酶切产物用1% 琼脂糖凝胶电泳并回收;用T4 DNA连接酶将载体和目的基因进行连接。 The pQE-30 plasmid was extracted and purified PCR products were digested Sail and Sphl restriction endonuclease, digestion conditions are set Xi 3hr 37; the digestion products by 1% agarose gel electrophoresis and recovered; using T4 DNA ligase and the gene vector is connected. 连接产物转化大肠杆菌M15感受态细胞。 The ligation product was transformed into E. coli competent cell M15. 挑单菌落培养增菌后,提取质粒进行双酶切鉴定。 Single colonies were picked after enrichment cultivation, plasmid was extracted for double digestion.

将测序鉴定正确的带有CP蛋白基因的重组质粒(命名为pQE-30/CP)及空质粒pQE-30的转化菌,使用LB液体培养基(加入50 ug/mL的Amp )250rpm 37。 The correct recombinant plasmid with the CP gene sequencing (designated pQE-30 / CP), and transformants were empty plasmid pQE-30 using a liquid LB medium (addition of 50 ug / mL of Amp) 250rpm 37. C培养;至菌浓度为OD=0.5-0.6时, 加入终浓度为lmmol/L的IPTG进行诱导表达6hr。 C culture; 6hr expression was induced to a bacterial concentration of OD = 0.5-0.6, the final concentration of lmmol / L of IPTG. 离心沉淀菌体后进行蛋白质SDS-PAGE电泳观察。 SDS-PAGE electrophoresis was observed a protein precipitate after centrifugation cells. 釆用镍结合柱的方法获得纯化的融合蛋白,通过蛋白质SDS-PAGE电泳进行纯化蛋白的纯度验证, 要求纯度大于99%。 Bian binding method using a nickel column to obtain purified fusion protein, the purified protein was performed to verify the purity of proteins by SDS-PAGE electrophoresis, it requires greater than 99%.

后续的步骤同轮状病毒VP6蛋白的单克隆抗体制备。 The subsequent steps monoclonal antibodies with rotavirus VP6 protein. 1.4、诺瓦克病毒VP1蛋白单克隆抗体的制备如下: 1.4, Norwalk virus VP1 protein preparation of monoclonal antibodies as follows:

目的片段来源于临床分离株,按照GENEBANK上序列号为EF535854的VP1蛋白基因序列,按照其序列和pET-28a原核表达载体设计引物如下: Fragment derived from the clinical isolates, according to the GENEBANK Serial No. EF535854 VP1 protein gene sequence, as follows according to their sequence and pET-28a prokaryotic expression vector The primers were designed:

上游引物为5,- CGCGGATCCTCGAATGACGCCGCTCCATC 下游引物为5,- TCCGAGCTCTCTTCTACGCCCATTGCCAG Upstream primer is 5, - CGCGGATCCTCGAATGACGCCGCTCCATC downstream primer is 5, - TCCGAGCTCTCTTCTACGCCCATTGCCAG

进行聚合酶链式反应(PCR),按照所釆用的DNA聚合酶设置反应体系,其中各引物的添加量均为0.6pmol/iaL,反应程序设置如下:第一步:95°C 2min;第二步:95°C 45sec, 56°C 45sec, 72°C 80 sec, 36个循环;第三步:72°C 8min。 Polymerase chain reaction (the PCR), is provided in accordance with the reaction system preclude the use of DNA polymerase, wherein the addition amount of each primer were 0.6pmol / iaL, reaction procedure as follows: The first step: 95 ° 2min C; of step two: 95 ° C 45sec, 56 ° C 45sec, 72 ° C 80 sec, 36 cycles; The third step: 72 ° C 8min.

对PCR产物进行测序,测序结果如序列表SEQIDNO: 4所示, 与EF535854的同源性为100%。 The PCR products were sequenced, the sequencing results of the sequence list SEQIDNO: 4, the homology is 100% EF535854.

将提取的pET-28a质粒和纯化的PCR产物进行Sail和BamHI 限制性内切酶的双酶切,酶切条件设置为37°C 3hr;将酶切产物用1%琼脂糖凝胶电泳并回收;用T4DNA连接酶将载体和目的基因进行连接。 The extracted plasmid pET-28a and the purified PCR product was double digested endonucleases Sail and BamHI restriction enzyme digestion conditions to 37 ° C 3hr; The product was digested with 1% agarose gel electrophoresis and recovered ; T4DNA ligase with the vector and the gene of the connection. 连接产物转化大肠杆菌BL21感受态细胞。 The ligation product was transformed into E. coli BL21 competent cells. 挑单菌落培养增菌后,提取质粒进行双酶切鉴定。 Single colonies were picked after enrichment cultivation, plasmid was extracted for double digestion.

将测序鉴定正确的带有VP1蛋白基因的重组质粒(命名为pET-28a/VPl )及空质粒pET-28a的转化菌,使用LB液体培养基(加入50 ji g/mL的Amp )250rpm 37"C培养;至菌浓度为OD=0.5-0.6时, 加入终浓度为lmmol/L的IPTG进行诱导表达6hr。离心沉淀菌体后进行蛋白质SDS-PAGE电泳观察。釆用镍结合柱的方法获得纯化的融合蛋白,通过蛋白质SDS-PAGE电泳进行纯化蛋白的纯度验证, 要求纯度大于99%。 The correct recombinant plasmid was identified by sequencing with VP1 protein gene (designated pET-28a / VPl) and the empty vector pET-28a transformed bacteria using LB broth (added 50 ji g / mL of Amp) 250rpm 37 " C culture; 6hr expression was induced to a bacterial concentration of OD = 0.5-0.6, the final concentration of lmmol / L of IPTG SDS-PAGE electrophoresis was observed after centrifugation protein precipitation method of binding cells preclude the column to obtain a purified nickel. fusion protein, to verify the purity of the purified protein by SDS-PAGE and proteins, required greater than 99%.

后续的步骤同轮状病毒VP6蛋白的单克隆抗体制备。 The subsequent steps monoclonal antibodies with rotavirus VP6 protein.

待检病原体为常见的肠道病毒的诺瓦克病毒,肠道腺病毒,星状病毒和轮状病毒四种。 To be tested for the pathogens common intestinal virus, norovirus, enteric adenovirus, astrovirus and rotavirus four. 2、试纸条的制备 2, the test strip was prepared

2.1、标记抗体垫上包埋有荧光素或荧光微球标记的四种肠道病毒的单克隆抗体。 2.1, labeled antibody pad embedded with a monoclonal antibody or fluorescein labeled fluorescent beads four enteroviruses.

荧光素标记肠道病毒单克隆抗体:将纯化的肠道病毒单克隆抗体用0.1M碳酸氢钠溶液4'C透析过夜,抗体浓度控制在5-20mg/ml; 荧光素(表面有活化的基团:例如四氟苯酯、磺酸氯酴酯、琥铂酰亚胺酯等)用二甲基甲酰胺(DMF)或二甲基亚砜(DMSO)溶解,浓度为10mg/ml;将溶解后的荧光素加入透析后的单克隆抗体,在室 Enterovirus, fluorescein labeled monoclonal antibody: The purified monoclonal antibodies enterovirus 4'C with 0.1M sodium bicarbonate solution was dialyzed overnight, the antibody concentration at 5-20mg / ml; fluorescein (surface activated group group: e.g. tetrafluorophenyl ester, sulfonate ester chloride yeast, succinic ester platinum, etc.) with dimethylformamide (DMF) or dimethylsulfoxide (DMSO) was dissolved at a concentration of 10mg / ml; dissolved after fluorescein monoclonal antibody was added after the dialysis, the chamber

温条件下磁力搅拌l小时,荧光素与单克隆抗体的质量比为1: 10~ Magnetic stirring at a temperature condition of l hour, and the mass ratio of fluorescein monoclonal antibody was 1: 10 ~

20;反应后用1.5M羟胺氯化物终止反应,抗体荧光素标记过程完成; 用SephadexG25凝胶柱将抗体荧光素标记物进行纯化,得到纯度高的单克隆抗体荧光素标记物。 20; After the reaction the reaction was quenched with 1.5M hydroxylamine chloride, fluorescein labeled antibody to complete the process; SephadexG25 gel column with fluorescein-labeled antibody was purified monoclonal antibodies to obtain highly pure fluorescein label. 配置稀释缓冲液:0.5。 Configuring dilution buffer: 0.5. /。 /. BSA+3。 BSA + 3. /。 /. 海藻糖+0.1。 Trehalose +0.1. /。 /. Tween-20, 10mM pH 7.5 Tris-HCl。 Tween-20, 10mM pH 7.5 Tris-HCl. 将四种抗体荧光素标记物进行稀释(抗体浓度保持在O.Ol昭/ml〜0.1昭/ml),再将抗体标记垫(玻璃纤维膜或聚酯膜)浸泡其中,时间控制在2〜5分钟,取出后釆用37'C气套式烘箱烘干或真空冷冻干燥,在45%湿度下保存备用。 The four kinds of fluorescently labeled antibody was diluted (antibody concentration is maintained at O.Ol Publication Sho /ml~0.1 / ml), and then labeled antibody pad (glass fiber or polyester film) wherein the soaking time control in 2 ~ 5 minutes, remove preclude the use of nested 37'C air drying or vacuum freeze-drying oven, were stored at 45% humidity.

荧光微球标记肠道病毒单克隆抗体:荧光微粒是采用高分子聚苯乙烯材料包裹荧光素染料制备而成的,直径为200~300nm,荧光微球表面有功能基团羧基;将荧光微球用50mMMES(2-(N-morpholino ) ethane sulfonicacid ), pH6.0 )缓冲液透析,浓度调整到10mg/ml , 体积20ml ; 配制9.38%的碳二亚胺 Enterovirus fluorescent microspheres labeled monoclonal antibody: fluorescent particles is prepared using a polystyrene polymer wrapping materials made of fluorescein dye, a diameter of 200 ~ 300nm, fluorescent beads with a surface functional group a carboxyl group; a fluorescent microspheres with 50mMMES (2- (N-morpholino) ethane sulfonicacid), pH6.0) buffer, dialyzed, the concentration was adjusted to 10mg / ml, 20ml volume; formulated carbodiimide 9.38%

(N-(3-Dimethylaminopropyl)画N-ethylcarbodiimide hydrochloride , (N- (3-Dimethylaminopropyl) Videos N-ethylcarbodiimide hydrochloride,

EDC)的MES缓冲液和10%的NHS (N-Hydroxysuccinimide)的MES缓冲液;20ml微粒加入NHS溶液7.2ml, EDAC溶液1.6ml, 用MES缓冲液补充体积到40ml(荧光微球浓度5mg/ml), 37。 EDC) MES buffer and 10% NHS (N-Hydroxysuccinimide) MES buffer; 20ml particles NHS solution was added 7.2ml, EDAC solution 1.6ml, supplemented with MES buffer to a volume of 40ml (concentration of fluorescent beads 5mg / ml ), 37. C不断旋转反应l个小时;取上述活化荧光微粒20mg(4ml),用50mM pH 8.0磷酸缓冲液离心洗涤,并且浓缩成lml (20mg/ml);加入肠道病毒单克隆抗体4mg (50mM pH 8.0磷酸缓冲液透析处理),并补充体积到2ml,使最终反应浓度为:荧光微球10mg/ml、抗体2mg/ml, 37"C旋转反应4个小时;停止反应,将反应后荧光微球12000rpm4°C 离心30分钟,弃去上清,沉淀物用50mg/ml牛血清白蛋白稀释,超声波处理。配置稀释缓冲液:0.5。/。BSA+3。/。海藻糖+0.1。/。Tween-20, 10mMpH7.5Tris-HCl。将四种抗体荧光微球标记物进行稀释(微球浓度保持在0.05mg/ml〜0.2mg/ml),再将抗体标记垫(玻璃纤维膜或 C l constantly rotating hour reaction; take the above activator fluorescent particles 20mg (4ml), was washed with 50mM pH 8.0 phosphate buffer with centrifugation and concentrated to lml (20mg / ml); enterovirus monoclonal antibody was added 4mg (50mM pH 8.0 phosphate buffer dialysis treatment), adding volume to 2ml, the final reaction concentration: fluorescent beads 10mg / ml, the antibody 2mg / ml, 37 "C rotary reactor 4 hours; the reaction was stopped, the reaction fluorescent microspheres 12000rpm4 ° C centrifuged for 30 minutes, the supernatant discarded, the precipitate was diluted with albumin 50mg / ml bovine serum, the ultrasonic processing configurations dilution buffer:. 0.5 ./ BSA + 3. / trehalose +0.1 ./ Tween-... 20, 10mMpH7.5Tris-HCl. the four kinds of fluorescent microspheres labeled antibody was diluted (microsphere concentration is maintained at 0.05mg / ml~0.2mg / ml), and then labeled antibody pad (glass fiber or film

聚酯膜)浸泡其中,时间控制在2〜5分钟,取出后采用37。 Polyester film) wherein the soaking time control in 2 ~ 5 minutes, remove 37 employed. C气套式 Air jacket formula C

烘箱烘干或真空冷冻干燥,在45%湿度下保存备用。 Freeze-drying or vacuum drying oven, were stored at 45% humidity.

2.2、 四种肠道病毒单克隆抗体的包被 2.2 the four intestinal virus monoclonal antibody-coated

四种肠道病毒(轮状病毒、肠道腺病毒、星状病毒、诺瓦克病毒)单克隆抗体用50mMpH7.2磷酸盐缓冲液稀释至lmg/ml。 Four kinds of enterovirus (rotaviruses, enteric adenovirus, astrovirus, Norwalk virus) monoclonal antibodies were diluted to lmg / ml with phosphate buffer 50mMpH7.2. 在硝酸纤维膜上,用喷膜仪按顺序划上稀释的轮状病毒单克隆抗体、肠道腺病毒单克隆抗体、星状病毒单克隆抗体、诺瓦克病毒单克隆抗体,每条线的间距为0.3cm,包被量为1.0〜2.0pl/cm。 In the nitrocellulose membrane, the membrane device by a jet draw order rotavirus diluted monoclonal antibody, enteric adenovirus monoclonal antibodies, monoclonal antibodies Astrovirus, Norwalk virus monoclonal antibodies, each line spacing of 0.3cm, coating amount 1.0~2.0pl / cm.

2.3、 质控带制备 2.3, prepared with quality control

将羊抗鼠IgG多抗用50mM pH7.2磷酸盐缓冲液稀释至2mg/ml,在硝酸纤维素膜检测带下方0.5cm处划上质控带,包被量为1.0〜2.0fil/cm。 The goat anti-mouse IgG polyclonal antibody was diluted with 50mM pH7.2 phosphate buffer to 2mg / ml, draw control line nitrocellulose membrane detected at 0.5cm below the belt, coating amount 1.0~2.0fil / cm.

2.4、 试纸条的装备及切割 2.4, the test strip and cutting equipment

在胶板的中间位置贴上包被抗体(4种肠道病毒单克隆抗体和羊抗鼠IgG多抗)的硝酸纤维素膜;在胶板的左侧贴上玻璃纤维膜, 并在硝酸纤维素膜的交接一端铺上抗体标记垫,抗体标记垫与硝酸纤维素有1〜2mm部位重叠;在胶板的右侧贴上吸水纸,吸水纸与硝酸纤维素有1〜2mm部位重叠。 In the intermediate position of the labeled antibody-coated plastic sheet (4 Enteric virus monoclonal antibody and polyclonal goat anti-mouse IgG) nitrocellulose membrane; paste glass fiber membrane on the left side of the plastic sheet, and the nitrocellulose prime end of the transfer film covered pad labeled antibodies, labeled antibody pad and nitrocellulose have 1~2mm overlapping portion; absorbent paper paste on the right side of the plastic sheet, absorbent paper and nitrocellulose have 1~2mm site overlap. 利用切条机切成3〜5mm宽度的检测试纸条。 Using a cutting machine and cut into test strip 3~5mm width.

2.5、 样品的检测:使用时将试纸条平铺于检测台上,取50pL 的待检样品,滴入到样品垫上,反应5分钟后宜用荧光检测仪进行荧光信号采集,可在C带和T带相应位置采集到荧光图像。 2.5, the test samples: When using the test strip in the detection stage tile taken 50pL sample to be tested is added dropwise to the sample pad, the reaction of 5 Zhonghou Yi of the fluorescent signals collected by fluorescence detector, may be tape C and T a position corresponding to the fluorescence image acquired. 如果C 带出现条带,Tl- T4带出现荧光图像的代表不同的肠道病毒为阳性, 其抗原含量可由荧光检测仪示出;如果C带未出现条带,说明试纸条失效。 If the C-band banding, Tl- T4 representing different enterovirus band fluorescence image appeared positive, antigen content is shown by the fluorescence detector; if the C-band band does not appear, indicating failure of the test strip. ' '

参照附图2, C带、Tl带、T2带、T3带、T4带均出现荧光图像,表明诺瓦克病毒、肠道腺病毒、星状病毒、轮状病毒均为阳性, 2, C-band, Tl band, T2 band, T3 band, T4 occurs with both fluorescence images indicating Norwalk virus, enteric adenovirus, astrovirus, rotavirus were positive DRAWINGS

其抗原含量可由荧光检测仪示出。 Antigen content thereof is shown by fluorescence detector.

参照附图3, C带、T2带、T4带出现荧光图像,表明肠道腺病 3, C-band, T2 band, T4 with reference to the accompanying drawings fluorescence image appears, indicating that the intestinal adenopathy

毒、轮状病毒为阳性,诺瓦克病毒、星状病毒为阴性。 Poison, positive rotavirus, norovirus, astrovirus negative. 肠道腺病毒、 轮状病毒抗原含量可由荧光检测仪示出。 Enteric adenovirus, rotavirus antigen content shown by the fluorescence detector.

实施例2 Example 2

1、肠道病毒四项联检纸条特异性试验 1, the Joint Inspection Enterovirus four specific test strip

选取5份标本(标本1为沙门氏菌感染粪便标本、标本2为单一轮状病毒感染粪便标本、标本3为单一肠道腺病毒感染粪便标本、 标本4位单一星状病毒感染粪便标本、标本5为单一诺瓦克病毒感染粪便标本),用标本稀释液(0.5%Triton-X100+2%NaCl +0.1%Casein, 10mMpH7.2PB) 1: IO稀释,各吸取50(xl加到肠道病毒四项联检纸条的样品垫上,室温下放置15分钟,上荧光检测仪检测结果。 Select 5 specimens (specimens 1 Salmonella infection stool specimens, the specimen 2 is a single rotavirus infection stool specimens, 3 specimens enteric adenovirus infection a single fecal samples, samples 4 single astrovirus, stool specimens, specimens 5 single stool specimens Norwalk virus), with sample diluent (0.5% Triton-X100 + 2% NaCl + 0.1% Casein, 10mMpH7.2PB) 1: IO dilution, each of the suction 50 (xl added four enterovirus sample Unit mat strip, allowed to stand at room temperature for 15 minutes, the fluorescence detector detection result.

表1 肠道病毒四项联检试纸条测试值<table>table see original document page 18</column></row> <table>2、肠道病毒四项联检灵敏度测试 Table 1 Unit enterovirus four strip test value <table> table see original document page 18 </ column> </ row> <table> 2, enterovirus four Unit sensitivity testing

轮状病毒抗原(浓度3pg/ml) IO倍稀释梯度,浓度(ng/ml)分别为300、 30、 3、 0.3、 0,各吸取50nl加到肠道病毒四项联检纸条的样品垫上,室温下放置15分钟,上荧光检测仪检测结果。 Rotavirus antigen (concentration 3pg / ml) IO-fold dilution gradients, concentration (ng / ml) were 300, 30, 3, 0.3, 0, 50nl of each sample was added to absorb enterovirus four Unit mat strip, left at room temperature for 15 minutes, the fluorescence detector detection result. 肠道腺病毒抗原(浓度3昭/ml) IO倍稀释梯度,浓度(ng/ml)分别为300、 30、 3、 0.3、 0,各吸取50pl加到肠道病毒四项联检纸条的样品垫上, 室温下放置15分钟,上荧光检测仪检测结果。 Enteric adenovirus antigen (Sho concentration 3 / ml) IO-fold dilution gradients, concentration (ng / ml) were 300, 30, 3, 0.3, 0, each of the four suction 50pl added enterovirus sample strip Unit pad, left at room temperature for 15 minutes, the fluorescence detector detection result. 星状病毒抗原(浓度 Astrovirus antigen (concentration

3ng/ml) IO倍稀释梯度,浓度(ng/ml)分别为300、 30、 3、 0.3、 0, 各吸取50^il加到肠道病毒四项联检纸条的样品垫上,室温下放置15 分钟,上荧光检测仪检测结果。 3ng / ml) IO-fold dilution gradients, concentration (ng / ml) were 300, 30, 3, 0.3, 0, each sample was added to 50 ^ il suction enterovirus four Unit note pads, placed at room temperature for 15 min, detection result of the fluorescence detector. 诺瓦克病毒抗原(浓度3昭/ml) 10 倍稀释梯度,浓度(ng/ml)分别为300、 30、 3、 0.3、 0,各吸取50^1 加到肠道病毒四项联检纸条的样品垫上,室温下放置15分钟,上荧光检测仪检测结果。 Norwalk virus antigen (Sho concentration 3 / ml) 10-fold dilution gradients, concentration (ng / ml) were 300, 30, 3, 0.3, 0, 50 ^ 1 was added to each of the suction enterovirus four strip Unit the sample pad, allowed to stand at room temperature for 15 minutes, the fluorescence detector detection result. 四种肠道病毒检测灵敏度均在1.0ng/ml以下。 Enteroviruses are four kinds of detection sensitivity in 1.0ng / ml or less.

表2 肠道病毒四项联检灵敏度测试读值 Table 2 Unit enterovirus four reading sensitivity testing

<table>table see original document page 19</column></row> <table>注:荧光读值l为阴阳性判断的临界值。 <Table> table see original document page 19 </ column> </ row> <table> NOTE: threshold fluorescence reading l yin and yang of judging. 实施例3 Example 3

肠道病毒单项检测试纸条制备 Preparation of single test strip enterovirus

参照附图4,本实施例的肠道病毒多检试纸条包括底板l、粘附于底板上的包埋有质控带(C带)和检测带(T带)的硝酸纤维素膜2、覆盖于硝酸纤维素膜一侧的吸水纸3、覆盖于硝酸纤维素膜的另一侧的标记抗体垫4、样品吸收垫5。 Referring to Figure 4, the present embodiment enterovirus subject test strip comprises a plurality of base L, adhering to the bottom plate with a nitrocellulose membrane embedded control line (C-band) and the detection zone (T band) 2 , nitrocellulose membrane covering the absorbent paper side 3, covers the other side of the nitrocellulose membrane labeled antibody pad 4, the sample absorption pad 5.

所述的硝酸纤维素膜的C带区域包埋有羊抗鼠IgG抗体,检测带包埋有轮状病毒单克隆抗体或肠道腺病毒单克隆抗体或星状病毒单克隆抗体或诺瓦克病毒单克隆抗体。 The C-band area embedded nitrocellulose membrane sheep anti-mouse IgG antibody, detection with embedded rotavirus enteric adenovirus monoclonal antibody or monoclonal antibody or a monoclonal antibody or astrovirus Norwalk virus monoclonal antibody.

待检病原体为轮状病毒或肠道腺病毒或星状病毒或诺瓦克病毒。 Pathogens to be tested or intestinal rotavirus virus or adenovirus Norwalk virus or star.

1、标记抗体垫上包埋有荧光素或荧光微球标记的肠道病毒的单克隆抗体。 1, labeled antibody pad embedded with a monoclonal antibody or fluorescein labeled fluorescent beads enterovirus.

荧光素标记肠道病毒单克隆抗体:将纯化的肠道病毒单克隆抗体用0.1M碳酸氢钠溶液4X:透析过夜,抗体浓度控制在5-20mg/ml; 荧光素(表面有活化的基团:例如四氟苯酯、磺酸氯酚酯、琥铂酰亚胺酯等)用二甲基甲酰胺(DMF)或二甲基亚砜(DMSO )溶解,浓度为10mg/ml;将溶解后的荧光素加入透析后的单克隆抗体,在室温条件下磁力搅拌l小时,荧光素与单克隆抗体的质量比为k 10~ 20;反应后用1.5M羟胺氯化物终止反应,抗体荧光素标记过程完成; 用SephadexG25凝胶柱将抗体荧光素标记物进行纯化,得到纯度高的单克隆抗体荧光素标记物。 Enterovirus, fluorescein labeled monoclonal antibody: The purified monoclonal antibodies enterovirus with 0.1M sodium bicarbonate solution 4X: dialyzed overnight, the antibody concentration at 5-20mg / ml; fluorescein (surface activated groups : e.g. tetrafluorophenyl ester, sulfonate ester chlorophenol, succinic ester platinum, etc.) with dimethylformamide (DMF) or dimethylsulfoxide (DMSO) was dissolved at a concentration of 10mg / ml; dissolved after fluorescein monoclonal antibody was added after the dialysis, magnetically stirred at room temperature for l hour, fluorescein monoclonal antibody mass ratio of k 10 ~ 20; after the reaction was quenched with 1.5M hydroxylamine chloride, fluorescein labeled antibody process is complete; SephadexG25 gel column with fluorescein-labeled antibody was purified monoclonal antibodies to obtain highly pure fluorescein label. 配置稀释缓冲液:0.5。 Configuring dilution buffer: 0.5. /。 /. BSA+3。 BSA + 3. /。 /. 海藻糖+0.1。 Trehalose +0.1. /。 /. Tween-20, 10mM pH 7.5 Tris-HCl。 Tween-20, 10mM pH 7.5 Tris-HCl. 将抗体荧光素标记物进行稀释(抗体浓度保持在0.01吗/ml〜0.1iig/ml ),再将抗体标记垫(玻璃纤维膜或聚酯膜)浸泡其中,时间控制在2〜5分钟,取出后釆用37X:气套式烘箱烘干或真空冷冻干燥,在45%湿度下保存备用。 The fluorescein-labeled antibody was diluted (antibody concentration was maintained at 0.01 do /ml~0.1iig/ml), and then labeled antibody pad (glass fiber or polyester film) wherein the soaking time control in 2 ~ 5 minutes, removed after preclude the use of 37X: air drying oven or a vacuum jacket freeze-drying, were stored at 45% humidity.

荧光微球标记肠道病毒单克隆抗体:荧光微粒是釆用高分子聚苯乙烯材料包裹荧光素染料制备而成的,直径为200~300nm,荧光微球表面有功能基团羧基;将荧光微球用50mMMES(2- Enterovirus fluorescent microspheres labeled monoclonal antibodies: preclude the fluorescent particles is a polymer prepared from polystyrene fluorescein dye wrapping material, having a diameter of 200 ~ 300nm, fluorescent beads with a surface functional group a carboxyl group; a fluorescent microspheres balls 50mMMES (2-

(N-morpholino ) ethane sulfonicacid), pH6.0 )缓冲液透析,浓度调整到10mg/ml , 体积20ml ; 配制9.38%的碳二亚胺 (N-morpholino) ethane sulfonicacid), pH6.0) buffer, dialyzed, the concentration was adjusted to 10mg / ml, 20ml volume; formulated carbodiimide 9.38%

(N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride , EDC)的MES缓冲液和10%的NHS (N-Hydroxysuccinimide)的MES缓冲液;20ml微粒加入NHS溶液7.2ml, EDAC溶液1.6ml, 用MES缓冲液补充体积到40ml (荧光微球浓度5mg/ml ), 37。 (N- (3-Dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride, EDC) MES buffer and 10% NHS (N-Hydroxysuccinimide) MES buffer; 20ml particles NHS solution was added 7.2ml, EDAC solution, 1.6ml, in MES to a volume of 40ml buffer added (concentration of fluorescent beads 5mg / ml), 37. C不断旋转反应1个小时;取上述活化荧光微粒20mg(4ml),用50mMpH 8.0磷酸缓冲液离心洗涤,并且浓缩成lml (20mg/ml);加入肠道病毒单克隆抗体4mg (50mMpH8.0磷酸缓冲液透析处理),并补充体积到2ml,使最终反应浓度为:荧光微球10mg/ml、抗体2mg/ml, 37"C旋转反应4个小时;停止反应,将反应后荧光微球12000rpm 4°C 离心30分钟,弃去上清,沉淀物用50mg/ml牛血清白蛋白稀释,超 C constantly rotating one hour the reaction; take the above activator fluorescent particles 20mg (4ml), washed 50mMpH 8.0 phosphate buffer with centrifugation and concentrated to lml (20mg / ml); enterovirus monoclonal antibody was added 4mg (50mMpH8.0 phosphate buffer dialysis treatment), adding volume to 2ml, the final reaction concentration: fluorescent beads 10mg / ml, the antibody 2mg / ml, 37 "C rotary reactor 4 hours; the reaction was stopped, the reaction fluorescent microspheres 12000rpm 4 ° C centrifuged for 30 minutes, the supernatant discarded, the precipitate was diluted with albumin 50mg / ml bovine serum, ultra

声波处理。 Sonication. 配置稀释缓冲液:0.5。 Configuring dilution buffer: 0.5. /。 /. BSA+3。 BSA + 3. /。 /. 海藻糖+0.1。 Trehalose +0.1. /。 /. Tween-20, 10mMpH7.5Tris-HCl。 Tween-20, 10mMpH7.5Tris-HCl. 将抗体荧光微球标记物进行稀释(微球浓度保持在0.05mg/ml〜0.2mg/ml),再将抗体标记垫(玻璃纤维膜或聚酯膜)浸泡其中,时间控制在2〜5分钟,取出后采用37'C气套式烘箱烘干或真空冷冻干燥,在45%湿度下保存备用。 The fluorescent microspheres labeled antibody was diluted (microsphere concentration is maintained at 0.05mg / ml~0.2mg / ml), and then labeled antibody pad (glass fiber or polyester film) wherein the soaking time control in 2 ~ 5 minutes after air jacket removed using 37'C vacuum oven drying or freeze-drying, were stored at 45% humidity.

2、 肠道病毒单克隆抗体的包被 2, enterovirus coated with monoclonal antibodies

肠道病毒(轮状病毒或肠道腺病毒或星状病毒或诺瓦克病毒) 单克隆抗体用50mMpH7.2磷酸盐缓冲液稀释至lmg/ml。 Enterovirus (rotavirus or enteric adenovirus virus or Norovirus or stellate) monoclonal antibodies were diluted to lmg / ml with phosphate buffer 50mMpH7.2. 在硝酸纤维膜上,用喷膜仪划上稀释的轮状病毒单克隆抗体或肠道腺病毒单克隆抗体或星状病毒单克隆抗体或诺瓦克病毒单克隆抗体,包被量为1.0〜2.0|il/cm。 In the nitrocellulose membrane with the diluted spray device designated rotavirus film enteric adenovirus monoclonal antibody or monoclonal antibody or a monoclonal antibody or astrovirus Norwalk virus monoclonal antibody, the amount of coating 1.0~ 2.0 | il / cm.

3、 质控带制备 3, prepared with quality control

将羊抗鼠IgG多抗用50mM pH7.2磷酸盐缓冲液稀释至2mg/ml,在硝酸纤维素膜检测带下方0.5cm处划上质控带,包被量为1.0〜2.0|il/cm。 The goat anti-mouse IgG polyclonal antibody was diluted with 50mM pH7.2 phosphate buffer to 2mg / ml, draw control line nitrocellulose membrane detected at 0.5cm below the belt, the package is in an amount of 1.0~2.0 | il / cm .

4、 试纸条的装备及切割 4, the test strip and cutting equipment

在胶板的中间位置贴上包被抗体(肠道病毒单克隆抗体和羊抗鼠IgG多抗)的硝酸纤维素膜;在胶板的左侧贴上玻璃纤维膜,并在硝酸纤维素膜的交接一端铺上抗体标记垫,抗体标记垫与硝酸纤维素有1〜2mm部位重叠;在胶板的右侧贴上吸水纸,吸水纸与硝酸纤维素有1〜2mm部位重叠。 In the intermediate position of the blanket labeled capture antibody (monoclonal antibody enterovirus and goat anti-mouse IgG polyclonal antibody) nitrocellulose membrane; paste glass fiber membrane on the left side of the plastic sheet, and the nitrocellulose membrane One end of the transfer pad covered with labeled antibodies, labeled antibody to the nitrocellulose pad 1~2mm have overlapping portions; absorbent paper paste on the right side of the plastic sheet, absorbent paper and nitrocellulose have 1~2mm site overlap. 利用切条机切成3〜5mm宽度的检测试纸条。 Using a cutting machine and cut into test strip 3~5mm width.

5、样品的检测:使用时将试纸条平铺于检测台上,取5(HiL的待检样品,滴入到样品垫上,反应15分钟后宜用荧光检测仪进行荧光信号采集,可在C带和T带相应位置釆集到荧光图像。序列表 5, test sample: the tile in the test strip using the inspection station, taking 5 (HiL sample to be tested is added dropwise to the sample pad 15 minutes the reaction Zhong Houyi fluorescence signal acquisition with a fluorescence detector, can be and T C with a corresponding set position to preclude the fluorescence image. sEQUENCE LISTING

〈110〉北京博晖创新光电技术股份有限公司 <110> Beijing Bo Hui innovative Optoelectronic Technology Co., Ltd.

<120〉 一种肠道病毒的免疫荧光层析检测试纸及其制备方法 <120> A chromatography immunofluorescence test paper and preparation method enteroviruses

〈130> <130>

〈歸12 <Return 12

〈170> Patentln version 3. 3 <170> Patentln version 3. 3

<210> 1 <210> 1

<211> 1194 <211> 1194

〈212> DNA 〈213>轮状病毒 <212> DNA <213> Rotavirus

〈400> 1 <400> 1

atgg郷ttc tgtactcatt gtcaaaaact cttaaagatg ctagagat肌 aattgttgaa 60 atgg Hongo ttc tgtactcatt gtcaaaaact cttaaagatg ctagagat muscle aattgttgaa 60

ggtacattat attccaatgt tagcgatctc at t caacaat ttaatcaaat 120 ggtacattat attccaatgt tagcgatctc at t caacaat ttaatcaaat 120

atgactttca aactggagga attggtaatt taccagttag aaattggatt 180 atgactttca aactggagga attggtaatt taccagttag aaattggatt 180

tttgattttg gtctattagg tacaacactt ttaaatttgg atgctaatta tgttgaaaat 240 tttgattttg gtctattagg tacaacactt ttaaatttgg atgctaatta tgttgaaaat 240

gcaagaacta cgattgaata tttcattgat tttattgata atgtatgtat ggatg犯atg 300 gcaagaacta cgattgaata tttcattgat tttattgata atgtatgtat ggatg guilty atg 300

gcaag卿gt ctcaaagaaa tggagtagct ccacaatctg 卿cgttgag gaaattatca 360 gcaag Qing gt ctcaaagaaa tggagtagct ccacaatctg State cgttgag gaaattatca 360

ggcattaaat tt肌gaggat aaattttgat aattcatcag 420 ggcattaaat tt muscle gaggat aaattttgat aattcatcag 420

ctacaaaata ga卿cagcg tactggattt gtctttcata aacctaatat atttccatac 480 ctacaaaata ga State cagcg tactggattt gtctttcata aacctaatat atttccatac 480

tcagcttcgt tcactttgaa tagatctcaa ccaatgcatg ataatttgeit gggaactatg 540 tcagcttcgt tcactttgaa tagatctcaa ccaatgcatg ataatttgeit gggaactatg 540

tggcttaatg ctggatcgga gctggttttg attattcatg tgctataaac 600 tggcttaatg ctggatcgga gctggttttg attattcatg tgctataaac 600

gcaccagcaa gtttgeiacat attgtacagc ttagacgtgc actaaccaca 660 gcaccagcaa gtttgeiacat attgtacagc ttagacgtgc actaaccaca 660

gctactataa ctttgttacc tgatgcagaa agatttagtt ttccaagagt tatcaattcg 720 gctactataa ctttgttacc tgatgcagaa agatttagtt ttccaagagt tatcaattcg 720

gctgacggcg c肌ctacatg gttttttaat ccagtcattc taatgtagaei 780 gctgacggcg c muscle ctacatg gttttttaat ccagtcattc taatgtagaei 780

gtag肌tttt tgttgaatgg acaaat t at "t aacacat atc aggctagatt tggtactatt 840 gtag muscle tttt tgttgaatgg acaaat t at "t aacacat atc aggctagatt tggtactatt 840

atcgcaggaa attttgatac aattcgattg tcatttcagt taatgcgtcc 900 atcgcaggaa attttgatac aattcgattg tcatttcagt taatgcgtcc 900

acaccagctg ttaacgcatt atttccgcaa gcgcaacctt ttcaacacca tgca^csgtt 960 acaccagctg ttaacgcatt atttccgcaa gcgcaacctt ttcaacacca tgca ^ csgtt 960

ggactcacat tacgcattga atctgctgtc tgtg犯tcag tgcttgcgg3 tgcg肌tg朋 1020 ggactcacat tacgcattga atctgctgtc tgtg make friends tcag tgcttgcgg3 tgcg muscle tg 1020

actctgttag Cg肌tgtg3C CgC3gtgCgt c犯ga^tatg ctataccagt tggaccggtt 1080 actctgttag Cg muscle tgtg3C CgC3gtgCgt c guilty ga ^ tatg ctataccagt tggaccggtt 1080

tttcca_cca_g gcatg犯ttg attsctaact attcaccatc g卿g朋g3t 1140 tttcca_cca_g gcatg guilty ttg attsctaact attcaccatc g g Qing Peng g3t 1140

aax:ctgca^c gtgtctttac agtagcttcc 3tteig33gca tgttgattM 1194 aax: ctgca ^ c gtgtctttac agtagcttcc 3tteig33gca tgttgattM 1194

〈210〉 2 <211〉 903 <210> 2 <211> 903

<212> 腿<213> 腺病毒 <212> leg <213> Adenovirus

<400> 2 <400> 2

gtgtacaagc cagatgtagc tcagggaacc ataagttcgg cagatctttt aacgcagcag 60 gtgtacaagc cagatgtagc tcagggaacc ataagttcgg cagatctttt aacgcagcag 60

gcagcgccca ctacattggc tttagggata actttatcgg cctgatgtac 120 gcagcgccca ctacattggc tttagggata actttatcgg cctgatgtac 120

tacaactcca caggcaatat gggtg城tg gctgggc卿 cttcacagct aaatgctgta 180 tacaactcca caggcaatat gggtg City tg gctgggc State cttcacagct aaatgctgta 180

gtggacttgc aagacagg犯 cactgagtta tcataccaac ttatgctgga cgcacttggc 240 gtggacttgc aagacagg guilty cactgagtta tcataccaac ttatgctgga cgcacttggc 240

gatcggagca gatatttttc tatgtggaat caggctgttg acagttacga ccccgacgta 300 gatcggagca gatatttttc tatgtggaat caggctgttg acagttacga ccccgacgta 300

aggatcattg agaaccacgg agtggaggac gaactgccaa attactgctt tccgctggga 360 aggatcattg agaaccacgg agtggaggac gaactgccaa attactgctt tccgctggga 360

gggtctgcag ctacagacac gtactctggc ataaaggcca ctgg3ctgca_ 420 gggtctgcag ctacagacac gtactctggc ataaaggcca ctgg3ctgca_ 420

gacgacaMt atgccgacag aggggc聊a attgaatctg ggaacatttt tgccatggaa 480 gacgacaMt atgccgacag aggggc talk a attgaatctg ggaacatttt tgccatggaa 480

atcaMttgg cggccaatct ctggcgcagc ttcttatact ccaatgtagc tttgtacttg 540 atcaMttgg cggccaatct ctggcgcagc ttcttatact ccaatgtagc tttgtacttg 540

cctgactcat acaagattac gccagacaac attacactgc gaacacctat 600 cctgactcat acaagattac gccagacaac attacactgc gaacacctat 600

gcctacatga 3CggtCgggt ggcggttcct agcgccctcg atacctacgt aaacatcggg 660 gcctacatga 3CggtCgggt ggcggttcct agcgccctcg atacctacgt aaacatcggg 660

gcacggtggt ctccagatcc catggacaat gttaacccct tcaatcacca ccgtaacgcc 720 gcacggtggt ctccagatcc catggacaat gttaacccct tcaatcacca ccgtaacgcc 720

ggtctgcgct atcgatccat gctcttgggc aacgggcgtt acgtaccctt ccacattcaa 780 ggtctgcgct atcgatccat gctcttgggc aacgggcgtt acgtaccctt ccacattcaa 780

gtcccccaga agttttttgc cattaaaaat ctcctcctct taccgggttc ctacacctac 840 gtcccccaga agttttttgc cattaaaaat ctcctcctct taccgggttc ctacacctac 840

gagtgg肌ct cgtt犯catg atcctccaga gcagtctggg taacgacctg 900 gagtgg muscle ct cgtt guilty catg atcctccaga gcagtctggg taacgacctg 900

egg 903 egg 903

<210〉 3 <210> 3

<211〉 948 <211> 948

<212〉 DNA <212> DNA

〈213〉星状病毒 <213> astrovirus

<400〉 3 <400> 3

aacggacgca acaaatatca atctaatcaa cgtgtccgta taaacaactc 60 aacggacgca acaaatatca atctaatcaa cgtgtccgta taaacaactc 60

郞ga肌caag gtgtcacagg accaaaacct gcaatttgtc 肌ac3gccac cgcaacactt 120 Lang ga muscle caag gtgtcacagg accaaaacct gcaatttgtc muscle ac3gccac cgcaacactt 120

ggaacaattg gatcaaatac cacaggagc3 郷cgtgcat cctccttaat 180 ggaacaattg gatcaaatac cacaggagc3 Hongo cgtgcat cctccttaat 180

ccagttttgg ttaaggacgc tactgggagt actcaatttg gcccagtgca ggcgctagga 240 ccagttttgg ttaaggacgc tactgggagt actcaatttg gcccagtgca ggcgctagga 240

gcgcagtatt caatgtggaa gcttaaatac ctcaatgtta gattaacatc tetggt鄉t 300 gcgcagtatt caatgtggaa gcttaaatac ctcaatgtta gattaacatc tetggt Township t 300

gcctc3gcag tC犯tggC3C cgtagtgagg atccgacttc cactccttcc 360 gcctc3gcag tC guilty tggC3C cgtagtgagg atccgacttc cactccttcc 360

tctactagtt ggtctgggct tggagcgcgc aaacatctag atgttactgt tggt犯犯at 420 tctactagtt ggtctgggct tggagcgcgc aaacatctag atgttactgt tggt guilty of committing at 420

gcagttttca agtta犯gcc ttctgatctg ggtgggcctc gagatggttg gtggttaaca 480 gcagttttca agtta guilty gcc ttctgatctg ggtgggcctc gagatggttg gtggttaaca 480

ataatgcttc ggtccatcta tacattgggt 540 ataatgcttc ggtccatcta tacattgggt 540

caaactatgt catcttacca gaacacacag tttacaggag gcctatttct ggtgg3gttg 600 caaactatgt catcttacca gaacacacag tttacaggag gcctatttct ggtgg3gttg 600

tcttcagcat ggtgcttcac 3gggt3tgC3 atttagttaa tct3gt犯朋 660 tcttcagcat ggtgcttcac 3gggt3tgC3 atttagttaa tct3gt made 660 friends

tctacagaca agagtgttga tgtcactttt gagggatcag ctgg肌cacc acttattatg 720 tctacagaca agagtgttga tgtcactttt gagggatcag ctgg muscle cacc acttattatg 720

aatgtacctg agcacagtca ttttgcgaga atggctgtgg aacattcctc cctgtccact 780 aatgtacctg agcacagtca ttttgcgaga atggctgtgg aacattcctc cctgtccact 780

tccctttcga gggctggtgg tgagtcatca tctgacactg tttggcaagt cttgaaca_ca_ 840 tccctttcga gggctggtgg tgagtcatca tctgacactg tttggcaagt cttgaaca_ca_ 840

gctgtttcag cagctgagct tgtgactccg ccgccattca 3ttggCtggt ca卿gtggt 900 gctgtttcag cagctgagct tgtgactccg ccgccattca 3ttggCtggt ca Qing gtggt 900

tggtggtttg tcaaacttat tgctgggcgc gctcgcactg gtgcgcgt 948 tggtggtttg tcaaacttat tgctgggcgc gctcgcactg gtgcgcgt 948

〈210〉 4 <210> 4

〈211〉 1623 <211> 1623

〈212〉 DNA <212> DNA

<213〉诺瓦克病毒 <213> Norwalk virus

〈400〉 4 <400> 4

cgacgaatga cgccagccca tctgatgggt ccax:3gccaa cctcgtccca 60 cgacgaatga cgccagccca tctgatgggt ccax: 3gccaa cctcgtccca 60

gaggtcaaca atgaggttat ggctttggag cccgttgttg gtgccgctat tgcggcacct 120 gaggtcaaca atgaggttat ggctttggag cccgttgttg gtgccgctat tgcggcacct 120

gtagcgggcc aacaaaatgt aMtgacccc tggattagga ataattttgt acaagcccct 180 gtagcgggcc aacaaaatgt aMtgacccc tggattagga ataattttgt acaagcccct 180

ggtgg卿gt tcacagtatc ccctagaaac gctccaggtg aaatactatg gagcgcgccc 240 ggtgg Qing gt tcacagtatc ccctagaaac gctccaggtg aaatactatg gagcgcgccc 240

ttaggccctg atctgaatcc atacctttct cacctggcca tggttatgca 300 ttaggccctg atctgaatcc atacctttct cacctggcca tggttatgca 300

ggtggttttg sagtgg郷t aatcctcgcg ggg犯cgcgt tcax:cgccgg 360 ggtggttttg sagtgg Hongo t aatcctcgcg ggg committed cgcgt tcax: cgccgg 360

tttgcagcag tccc3cca^3 tttcccaact gaaggcttga gccccagcca ggtcactatg 420 tttgcagcag tccc3cca ^ 3 tttcccaact gaaggcttga gccccagcca ggtcactatg 420

ttcccccaca taatagtaga tgttaggcaa ttgg犯cctg tgttgatccc cttacctgat 480 ttcccccaca taatagtaga tgttaggcaa ttgg guilty cctg tgttgatccc cttacctgat 480

gttaggaata atttctacca ttacaatcaa tca^atgact ccaccattaa attgatagca 540 gttaggaata atttctacca ttacaatcaa tca ^ atgact ccaccattaa attgatagca 540

atgctgtaca caccacttag ggctaataat gctggggstg atgtcttcac agtctcttgt 600 atgctgtaca caccacttag ggctaataat gctggggstg atgtcttcac agtctcttgt 600

cgagtcctca cgaggccatc ccccgatttt gatttcatat ttctggtgcc acccacagtt 660 cgagtcctca cgaggccatc ccccgatttt gatttcatat ttctggtgcc acccacagtt 660

gagtcaagaa ccaaaccatt caccgtccca attttaactg ttgaggaaat gactaattca 720 gagtcaagaa ccaaaccatt caccgtccca attttaactg ttgaggaaat gactaattca 720

agattcccca ttcctttgga aaaattgttc acgggtccca gcagtgccct tgttgtcc肌 780 agattcccca ttcctttgga aaaattgttc acgggtccca gcagtgccct tgttgtcc muscle 780

ccacaaaatg gcaggtgcac gactgatggc gtgctcttag gcactaccca actgtccccc 840 ccacaaaatg gcaggtgcac gactgatggc gtgctcttag gcactaccca actgtccccc 840

gtcaacatct gcaccttcag 郷Cg3tgtC acccatattt caggtactcg cabctacaga 900 gtcaacatct gcaccttcag Hongo Cg3tgtC acccatattt caggtactcg cabctacaga 900

atg肌tttgg cttctcaaaa ttggaacaat tatgacccaa cccagcccct 960 atg muscle tttgg cttctcaaaa ttggaacaat tatgacccaa cccagcccct 960

ctgggaactc cagatttcgt gggaaagatc C犯ggC3tgC cacaaaggga 1020 ctgggaactc cagatttcgt gggaaagatc C made ggC3tgC cacaaaggga 1020

gacggctcga cccgcggtca caaagctaca gtgagcactg ggagtgttga ctttactcca 1080 gacggctcga cccgcggtca caaagctaca gtgagcactg ggagtgttga ctttactcca 1080

aagctgggca gcgttcagtt cgccactgat acagacaatg attttgaaac tggccaaaac 1140 aagctgggca gcgttcagtt cgccactgat acagacaatg attttgaaac tggccaaaac 1140

acaaggttca ccccagtcgg tgtcatccag gatggtagta gtgcccacag aaatgaaccc 1200 acaaggttca ccccagtcgg tgtcatccag gatggtagta gtgcccacag aaatgaaccc 1200

c肌c肌tggg tactcccaga ttactcaggt agaactgttc ataatgtaca cctagcccct 1260 c c muscle muscle tggg tactcccaga ttactcaggt agaactgttc ataatgtaca cctagcccct 1260

gccgtagccc ccacttttcc cttcctttct tcagatctac tatgcccgga 1320 gccgtagccc ccacttttcc cttcctttct tcagatctac tatgcccgga 1320

tgcagtgggt atcccaatat ggatttggat tgtctactcc cccaggaatg ggtgCagCELC 1380 tgcagtgggt atcccaatat ggatttggat tgtctactcc cccaggaatg ggtgCagCELC 1380

ttctaccaag aggcagcccc a^tcacaatct gatgtggctt tgttgagatt tgtgaatcca 1440 ttctaccaag aggcagcccc a ^ tcacaatct gatgtggctt tgttgagatt tgtgaatcca 1440

gggttctgtt tg恥tgc肌g cttcataaag caggctatgt cacagtggct 1500 gggttctgtt tg shame tgc muscle g cttcataaag caggctatgt cacagtggct 1500

cacactggcc agcatgattt ggttatcccc cccaatggct actttagatt tgattcctgg 1560 cacactggcc agcatgattt ggttatcccc cccaatggct actttagatt tgattcctgg 1560

gtcaaccagt cctacacact tgcccccatg ggaaatggag cggggcgtag acgtgcatta 1620 t肌1623 gtcaaccagt cctacacact tgcccccatg ggaaatggag cggggcgtag acgtgcatta 1620 t muscle 1623

<210〉 5 <210> 5

<211> 32 <211> 32

<212> DNA 〈213>人工序列 <212> DNA <213> Artificial Sequence

<400> 5 <400> 5

ttggcatgca tggaggttct gtactcattg tc 32 ttggcatgca tggaggttct gtactcattg tc 32

<210> 6 <210> 6

〈211〉 33 <211> 33

〈212〉 DNA <213〉人工序列 <212> DNA <213> Artificial Sequence

<400> 6 <400> 6

ggggtcgaca cttaatcaac atgcttctaa tgg 33 ggggtcgaca cttaatcaac atgcttctaa tgg 33

<210〉 7 <210> 7

<211> 28 <211> 28

<212> DNA <213>人工序列 <212> DNA <213> Artificial Sequence

<400> 7 <400> 7

ggggcatgcg tgtacaagcc agatgtag 28 ggggcatgcg tgtacaagcc agatgtag 28

〈210〉 8 <210> 8

〈211〉 29 <211> 29

<212〉 DNA <213〉人工序列 <212> DNA <213> Artificial Sequence

<400> 8 <400> 8

aaagtcgacg caggtcgtta cccagactg 29 aaagtcgacg caggtcgtta cccagactg 29

<210> 9 <210> 9

<211> 33 <211> 33

<212> DNA <213〉人工序列 <212> DNA <213> Artificial Sequence

<400〉 9 <400> 9

ggggcatgca acggacgcaa caaatatcaa tct 33 ggggcatgca acggacgcaa caaatatcaa tct 33

〈210〉 10 <210> 10

〈211〉 25 <211> 25

〈212〉 廳<213>人工序列 <212> Office <213> Artificial Sequence

<400> 10 <400> 10

aaagtcgacg cgcaccagtg cgagc 25 aaagtcgacg cgcaccagtg cgagc 25

<210> 11 <210> 11

〈211> 29 <211> 29

<212> 腿 <212> leg

<213> 人工序列 <213> Artificial Sequence

<400> 11 <400> 11

cgcggatcct cgaatgacgc cgctccatc 29 cgcggatcct cgaatgacgc cgctccatc 29

<210〉 12 <210> 12

<211> 29 <211> 29

<212〉 廳 <212> Hall

<213〉 人工序列 <213> Artificial Sequence

〈400> 12 <400> 12

tccgagctct cttctacgcc cattgccag 29 tccgagctct cttctacgcc cattgccag 29

Claims (9)

1、一种肠道病毒免疫荧光层析快速检测试纸,包括底板、附着在底板上依次紧密相连的样品吸收垫、标记抗体垫、反应膜和吸水垫,其特征在于所述标记抗体垫包埋有一种或多种肠道病毒的荧光标记单克隆抗体;所述反应膜上具有一条或多条检测带和一条质控带,每条检测带上固定有一种肠道病毒的单克隆抗体,检测带的条数以及固定的肠道病毒单克隆抗体与标记抗体垫包埋的标记抗体相适应,质控带固定有能与荧光标记单克隆抗体特异结合的抗抗体。 A flash chromatography on enterovirus immunofluorescence test strip, comprising a base plate attached to the sample on the base plate closely sequentially absorbent pad, the labeled antibody pad, absorbent pad and the reaction membrane, wherein said labeled antibody pad embedded with one or more enteroviruses fluorescently labeled monoclonal antibody; the reaction membrane having one or more test line and a control line, each detection strip is fixed to an infectious virus monoclonal antibody detection and a fixed number of tape enterovirus antibody labeled with monoclonal antibodies labeled antibody pad adapted embedded, the control line can be secured with a fluorescent labeled monoclonal antibodies specific antibody binding.
2、 根据权利要求l所述的检测试纸,其特征在于,所述的肠道病毒单克隆抗体为轮状病毒单克隆抗体、肠道腺病单克隆抗体、星状病毒单克隆抗体或诺瓦克病毒单克隆抗体中的一种或多种。 2. The test strip according to claim l, wherein said enteric virus monoclonal antibody monoclonal antibody rotavirus, adenovirus intestinal monoclonal antibodies, or monoclonal antibodies Astrovirus Nova One or more grams virus monoclonal antibodies.
3、 如权利要求l所述的检测试纸,其特征在于,所述的抗抗体为羊抗鼠IgG抗体。 3. The test strip according to claim l, wherein said antibody is an anti-mouse IgG antibody.
4、 如权利要求l所述的检测试纸,其特征在于,所述反应膜为硝酸纤维素膜。 4. The test strip according to claim l, wherein said film is a nitrocellulose membrane reaction.
5、 如权力要求l所述,其特征在于,所述荧光标记单克隆抗体的标记荧光素为Molecular Probes公司的Alexa Fluor系列荧光素或安玛西亚公司的花青素Cy系列荧光素。 5, as the power requirement l, wherein said fluorescent label a fluorescent labeled monoclonal antibody to prime Molecular Probes company fluorescein or Alexa Fluor series Amersham anthocyanins Cy fluorescein family.
6、 如权利要求2〜5任一项所述的检测试纸,其特征在于,制备轮状病毒单克隆抗体的免疫原为轮状病毒VP6蛋白;制备肠道腺病毒单克隆抗体的免疫原为腺病毒40/41型六邻体蛋白;制备星状病毒单克隆抗体的免疫原为I型星状病毒CP蛋白;制备诺瓦克病毒单克隆抗体的免疫原为诺瓦克病毒VP1蛋白。 6, test strip as claimed in any one of claims 2 ~ 5, characterized in that the monoclonal antibody immunize original rotavirus VP6 rotavirus protein; immunize enteric adenovirus monoclonal antibodies original 40/41 type adenovirus hexon protein; immunize astrovirus original monoclonal antibodies astrovirus CP type I protein; immunize norovirus monoclonal antibodies original norovirus VP1 protein.
7、 一种制备权利要求1〜6任一项所述试纸的方法,其包括如下步骤:l)在反应膜的不同位置上按照抗体的种类分别固定肠道病毒单克隆抗体以及抗抗体,形成检测带和质控带;2) 制备标记抗体;3) 组装底板、样品吸收垫、标记抗体垫、反应膜和吸水垫,并在标记抗体垫中包埋标记抗体。 7, the test paper method according to any one of claims 1~6 A process for preparing comprising the steps of: l) are fixed enterovirus antibodies and monoclonal antibodies according to the type of antibody reaction at different locations on the film, is formed detection zone and the control zone; 2) preparation of labeled antibodies; 3) assembling the base plate, the sample absorbent pad, the labeled antibody pad, absorbent pad and the reaction membrane, and embedded in the tag labeled antibody pad.
8、 如权利要求7所述的方法,其特征在于,标记单克隆抗体的制备方法是:将纯化的肠道病毒单克隆抗体用0.1M碳酸氢钠溶液4 'C透析过夜,抗体浓度控制在5-20mg/ml;荧光素用二甲基甲酰胺或二甲基亚砜溶解,浓度为10mg/ml;将溶解后的荧光素加入透析后的单克隆抗体,在室温条件下磁力搅拌1小时,荧光素与单克隆抗体的质量比为l: 10~20;反应后用1.5M羟胺氯化物终止反应,抗体荧光素标记过程完成;用SephadexG25凝胶柱将抗体荧光素标记物进行纯化,得到纯度高的单克隆抗体荧光素标记物。 8. A method as claimed in claim 7, characterized in that the method for preparing monoclonal antibodies are labeled: enterovirus purified monoclonal antibodies with 0.1M sodium bicarbonate solution and 4 'C was dialyzed overnight in the antibody concentration control 5-20mg / ml; fluorescein using dimethylformamide or dimethyl sulfoxide is dissolved, at a concentration of 10mg / ml; fluorescent pigment is added after dissolving the monoclonal antibodies dialyzed, magnetic stirring at room temperature for one hour mass fluorescein monoclonal antibody ratio of l: 10 ~ 20; quenched with 1.5M hydroxylamine chloride after the reaction, the process is completed fluorescein-labeled antibody; SephadexG25 gel column with fluorescein-labeled antibody was purified to give monoclonal antibodies of high purity fluorescein label.
9、 如权利要求7所述的方法,其特征在于,标记单克隆抗体的制备方法是:将荧光微球用50mMpH6.0MES缓冲液透析,浓度调整到10mg/ml,体积20ml; 配制9.38%的碳二亚胺的MES缓冲液和10%的NHS的MES缓冲液;20ml微粒加入NHS溶液7.2ml,EDAC 溶液1.6ml,用MES缓冲液补充体积到40ml, 37。 9. A method as claimed in claim 7, characterized in that the method for preparing monoclonal antibodies are labeled: fluorescent beads with 50mMpH6.0MES dialysis buffer, the concentration was adjusted to 10mg / ml, 20ml volume; 9.38% of formulation MES buffer carbodiimide and 10% NHS MES buffer; 20ml particles NHS solution was added 7.2ml, EDAC solution 1.6ml, supplemented with MES buffer volume to 40ml, 37. C不断旋转反应1 个小时;取上述活化荧光微粒20mg,用50mM pH 8.0磷酸缓冲液离心洗涤,并且浓缩成lml;加入肠道病毒单克隆抗体4mg,并补充体积到2ml,使最终反应浓度为:荧光微球10mg/ml、抗体2mg/ml, 37'C旋转反应4个小时;停止反应,将反应后荧光微球12000rpm4。 C constantly rotating one hour the reaction; take the above activator fluorescent particles 20mg, washed with phosphate buffer 50mM pH 8.0 by centrifugation, and concentrated to lml of; enterovirus monoclonal antibody was added 4mg, adding to the volume of 2ml, the final concentration of the reaction : fluorescent beads 10mg / ml, the antibody 2mg / ml, 37'C rotation of the reaction four hours; the reaction was stopped, the reaction fluorescent microspheres 12000rpm4. C离心30分钟,弃去上清,沉淀物用50mg/ml牛血清白蛋白稀释,超声波处理。 C centrifuged for 30 minutes, the supernatant discarded, the precipitate was diluted with albumin 50mg / ml bovine serum, ultrasonic treatment. ' '
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