CN107533059A - 微生物抗原的回收方法 - Google Patents
微生物抗原的回收方法 Download PDFInfo
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- CN107533059A CN107533059A CN201680024144.8A CN201680024144A CN107533059A CN 107533059 A CN107533059 A CN 107533059A CN 201680024144 A CN201680024144 A CN 201680024144A CN 107533059 A CN107533059 A CN 107533059A
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- microorganism
- antigen
- filter membrane
- sample
- recovery
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/56938—Staphylococcus
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/02—Separating microorganisms from the culture medium; Concentration of biomass
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Abstract
本发明提供在不使用特别的仪器的情况下容易地回收微生物所具有的抗原的方法。一种回收微生物的抗原的方法,该方法包括:使包含微生物的标本通过具有不让微生物通过的孔径的滤膜,将所述标本中的微生物捕获于滤膜上,使可破坏微生物的膜的微生物破坏试剂通过捕获了微生物的滤膜而在滤膜上破坏所捕获的微生物,在滤液中回收抗原。
Description
技术领域
本发明涉及回收微生物所含的抗原的方法。
背景技术
败血症等传染病的情况下,早期给予合适的抗菌药对患者的治愈和预后有很大的影响。现有技术中,对于败血症等传染病的检查如下进行。
将2~10mL采集的血液接种于约30mL~100mL细菌培养用液体培养基,在30~37℃进行培养。将培养基的浊度变化、由细菌导致的培养基中的气体生成、pH值的变化等作为指标,进行1~7天的观察,直至确认细菌的增殖。确认菌的生长后,采集培养液,实施革兰氏染色和传代培养(继代培养)。通过革兰氏染色确认大致的菌种分类以及所观察的菌是单一菌种还是多个菌种。通过传代培养增殖的菌的菌落均一(homogeneous)的情况下,假定血液中存在的菌为单一种类,供于鉴定检查、敏感性检查。另一方面,在革兰氏染色中观察到多个菌种、或形状明显不同的菌落增殖的情况下,分别挑取各菌落,反复培养至仅生长单一形状的菌落。像这样从标本的采集至鉴定需要花费数日。被怀疑为败血症的患者的情况下,早期的诊断和合适的抗菌药治疗对预后产生很大的影响。于是,作为以更短的时间准确地鉴定菌的种类的方法,使用如免疫层析法等免疫学方法(参照专利文献1)。使用免疫学方法鉴定细菌的情况下,使特异性识别目标细菌的蛋白质的免疫球蛋白结合。通过预先在结合的免疫球蛋白上标记着色胶乳粒子或荧光物质,可识别细菌的存在。因此,可在不进行纯培养的情况下,由血液培养直接检测菌。但是,血液、培养液、痰、鼻涕、粪便等用于细菌检查的标本中包含各种各样的蛋白质。因此,为了以良好的灵敏度找出目标蛋白质,需要去除夹杂物,通过离心操作或在固体培养基上进行培养来分离菌,从而进行夹杂物的去除。此外,根据目标细菌的蛋白质存在的位置和蛋白质的形状等,有时需要热处理或利用溶液进行的细胞膜的破坏等操作。
因此,需要离心机或微量恒温仪(heat block)等特别的仪器,或者需要像分离培养那样花费时间的操作。另外,离心操作的情况下,弃去上清液时,有时会误吸沉淀的菌,菌数减少,也可能会对正确的判定产生影响。
现有技术文献
专利文献
专利文献1:国际公开第2007/069673号。
发明内容
发明所要解决的技术问题
本发明提供在不使用特别的仪器的情况下容易地回收微生物所具有的抗原的方法。
解决技术问题所采用的技术方案
本发明人对容易地检测病原菌等微生物的方法进行了认真探讨。对于微生物而言,可通过测定该微生物所具有的抗原来检测,本发明人对简单地回收抗原的方法进行了探讨。
其结果发现,藉由通过滤膜而将血液或培养液等标本所含的微生物捕获于膜上,接着对于捕获于膜上的细菌流通包含表面活性剂等的溶液进行处理,可迅速破坏微生物而容易地回收抗原,从而完成了本发明。
即,本发明如下;
[1] 一种回收微生物的抗原的方法,该方法包括:使包含微生物的标本通过具有不让微生物通过的孔径的滤膜,将所述标本中的微生物捕获于滤膜上,使可破坏微生物的膜的微生物破坏试剂通过捕获了微生物的滤膜,从而在滤膜上破坏所捕获的微生物,在滤液中回收抗原;
[2] 根据[1]的回收微生物的抗原的方法,其特征在于,微生物为细菌;
[3] 根据[1]的回收微生物的抗原的方法,其特征在于,微生物破坏试剂为表面活性剂、碱性溶液、或者表面活性剂与碱性溶液的混合物;
[4] 根据[3]的回收微生物的抗原的方法,其特征在于,碱性溶液的pH值为11以上;
[5] 根据[3]的回收微生物的抗原的方法,其中,碱性溶液为氢氧化钠溶液;
[6] 一种微生物的检测方法,该方法包括:通过免疫学测定法对用[1]~[5]中任一项的方法回收的微生物的抗原进行测定;
[7] 根据[6]的微生物的检测方法,其中,免疫学测定法为免疫层析法(immunochromatography)。
本说明书包含作为本申请的优先权基础的日本专利申请号2015-092218号的公开内容。
发明的效果
通过本发明的方法,藉由使包含细菌等微生物的血液、培养液等标本通过滤膜,在滤膜上捕获微生物,可容易地分离标本中所含的微生物和夹杂物质。进而,藉由使包含表面活性剂或碱性溶液的微生物破坏试剂通过滤膜,破坏捕获于膜上的微生物,使微生物所具有的抗原露出,从而可容易地进行回收。本发明的方法不需要特别的仪器,可使微生物的抗原容易地回收。通过对回收的抗原进行测定,可检测微生物的存在,进行微生物传染病的诊断等。
附图说明
图1是表示可用于通过本发明的方法回收的抗原的测定的检测装置的一例的图。
具体实施方式
以下,对本发明进行详细说明。
1.微生物的抗原的回收
可通过本发明的方法回收抗原的微生物无任何限定,包括细菌、藻类、原生生物、酵母和霉菌等真菌、粘菌等真核生物。其中,优选的是引发人和人以外的动物的传染病的病原体微生物。例如可列举:包括耐甲氧西林金黄色葡萄球菌(MRSA)的金黄色葡萄球菌、包括致病性大肠杆菌的大肠杆菌、沙门氏菌、绿脓杆菌、霍乱弧菌、痢疾杆菌(Bacillusdysenteriae)、炭疽杆菌、结核杆菌、肉毒杆菌、破伤风杆菌、链球菌、弯曲杆菌(Campylobacter)、产气荚膜杆菌(Welch bacillus)、副溶血性弧菌、沙眼衣原体、溶血链球菌(Streptococcus haemolyticus)、百日咳博德特氏菌、幽门螺杆菌、钩端螺旋体、梅毒螺旋体(Treponema pallidum)、疏螺旋体(Borrelia)等细菌、白念珠菌(Candida albicans)、毛癣菌(Trichophyton)、曲霉等。
用于通过本发明的方法分析微生物的存在的标本也无限定,例如可列举:咽拭子(throat swab)、鼻拭子、鼻腔吸出物(nasal aspirate)、粪便悬浮液、血浆、血清、尿、唾液、羊水、脊髓液(spinal fluid)、脓、器官提取液(organ extract)、各种组织提取液等生物试样,食品提取液、培养上清液、自来水、污水、湖水、河水、海水、土壤提取液、污泥提取液等。其中,从病原性微生物检测的观点来看,特别好是生物试样,可优选使用咽拭子、鼻拭子、鼻腔吸出物、鼻腔清洗液、肺泡灌洗液、直肠拭子或粪便悬浮液等。标本可来源于人,也可来源于人以外的动物。此外,还包括对上述标本中所含的微生物进行培养而得的培养液。标本可直接使用,但培养液等微生物过多的标本或者标本的粘性高等情况下,使这些标本适当悬浮于生理盐水或缓冲液而使用。将使标本悬浮的液体称为标本悬浮用液,将使标本悬浮于标本悬浮用液而得的液体称为标本悬浮液。标本如果是全血等包含血液的标本,理想的是预先使用表面活性剂等破坏红细胞,使其完全溶血。为了使红细胞溶血,使用表面活性剂、各种溶剂、低渗溶液等来处理标本即可。
本发明的方法中,藉由使上述的标本通过滤膜,从而将微生物捕获于滤膜上。在此,捕获是指不让微生物通过滤膜,残留于通过标本后的滤膜上。这时,使用具有不让微生物通过的大小的孔径的滤膜。
要检测的微生物为细菌的情况下,使用孔径1.5μm以下、较好是1.2μm以下、更好是0.8μm以下、更好是孔径0.45μm以下的滤膜,例如孔径0.1~1.2μm、较好是0.22μm、0.45μm、0.8μm或1.2μm的滤膜即可。此外,要检测尺寸较大的霉菌、酵母的情况下,使用孔径大于0.5μm、例如孔径为0.8μm的滤膜即可。作为滤膜,例如可使用具有上述孔径的由混合纤维素酯(MCE: Mixed cellulose esters)、聚偏氟乙烯(PVDF)、聚四氟乙烯(PTFE)、亲水性PTFE、聚醚砜(PES)、亲水性聚醚砜(hydrophilic polyethersulfone)、亲水性聚丙烯(GHP)、尼龙(NYL)、乙酸纤维素(CA: cellulose acetate)、聚砜(PSF)、丙烯酸类共聚物(acryliccopolymer)、聚酰胺、尼龙66、聚酯、聚碳酸酯、硝化纤维素、硝化纤维素与纤维素酯的混合物等制成的滤膜。此外,对于这些滤膜,作为预滤器,可组合使用硼硅酸盐玻璃纤维(GF)或多层玻璃纤维(GxF)等玻璃纤维制预滤器。例如可列举:尼龙制滤膜与多层玻璃纤维(GxF)制预滤器的组合、聚醚砜(PES)制滤膜与多层玻璃纤维(GxF)制预滤器的组合、乙酸纤维素(CA)制滤膜与硼硅酸盐玻璃纤维(GF)制预滤器的组合、尼龙(NYL)制滤膜与硼硅酸盐玻璃纤维(GF)制预滤器的组合等。
将微生物捕获于滤膜上后,破坏所捕获的微生物,使微生物的抗原露出。微生物的破坏优选是指微生物的细胞膜的破坏。通过微生物的破坏而露出的抗原可容易地回收。在此,微生物的抗原可列举蛋白质、脂质、多糖类等能够制作抗体的物质。这意味着利用抗原抗体反应来检测回收的抗原。具体来说,可列举上述微生物的构成成分、上述微生物所产生的毒素、上述细菌的细菌抗原等。例如,可列举MRSA的PBP2'(青霉素结合蛋白2',Penicillin Binding Protein 2')。
所捕获的微生物的破坏通过用包含表面活性剂或作为碱性溶液的微生物破坏试剂处理微生物来进行。微生物破坏试剂可破坏微生物的膜。使用的表面活性剂无限定,可优选使用能够使微生物的膜增溶的表面活性剂。作为这样的表面活性剂,可使用Triton X-100(Triton为注册商标)(聚氧乙烯(10)辛基苯基醚)、Triton X-114(聚氧乙烯(8)辛基苯基醚)、Triton X-405(聚氧乙烯(40)异辛基苯基醚)、NP-40(Nonidet P-40)(聚氧乙烯(9)辛基苯基醚)等聚氧乙烯对叔辛基苯基醚(Triton类表面活性剂);Tween 20(Tween为注册商标)、Tween 40、Tween 60、Tween 80、Tween 65、Tween 85等聚氧乙烯失水山梨糖醇脂肪酸酯(Tween类表面活性剂);Briji 35(Briji为注册商标)(聚氧乙烯(23)月桂基醚)等聚氧乙烯烷基醚(Briji类表面活性剂);十二烷基-β-D-麦芽糖;辛基-β-D-葡萄糖苷等非离子型表面活性剂或十二烷基硫酸钠(SDS)等阴离子型表面活性剂或苯扎氯铵、苄索氯铵、二癸基二甲基铵盐、十二烷基三甲基氯化铵等阳离子型表面活性剂或CHAPS(3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐,3-(3-cholamidepropyl)dimethylammonio-1-propanesulphonate)、氯化烷基多氨基乙基甘氨酸等两性表面活性剂。优选使用醚型表面活性剂(聚氧乙烯烷基醚、聚氧乙烯烷基烯丙基醚、聚氧乙烯聚氧丙烯二醇等)、酯型表面活性剂(高级醇脂肪酸酯等)、酯醚型表面活性剂(聚氧乙烯失水山梨糖醇脂肪酸酯等)非离子型表面活性剂、阴离子型表面活性剂、两性表面活性剂,特别优选使用非离子型表面活性剂。表面活性剂以0.2~5%(w/w)、较好是0.5~2%(w/w)的浓度使用即可。
碱性溶液不仅破坏微生物的膜,还使膜中的蛋白质A变性,由此可抑制对抗体的非特异性反应。作为碱性溶液,使用pH值11以上、较好是12以上、更好是13以上的溶液即可,可优选使用0.1M~1.0M的氢氧化钠溶液、次氯酸钠等。
表面活性剂和碱性溶液可混合使用,例如可使用包含1~2%的Triton X-100等表面活性剂的0.1~0.5M的氢氧化钠。
采用微生物破坏试剂的处理中,使表面活性剂或碱性溶液通过捕获了微生物的滤膜即可。即,使微生物破坏试剂流过,让微生物破坏试剂与被捕获的微生物接触即可。然后,可在液体残留于滤膜内的状态下,静置1~20分钟,较好是2~10分钟。通过静置,能够可靠地破坏微生物。例如,作为滤膜使用直径20~40mm的滤膜的情况下,使1~数mL标本或标本悬浮液通过滤膜,将标本或标本悬浮液中的微生物捕获于滤膜上。接着,使数mL~数百mL微生物破坏试剂通过滤膜,破坏被捕获于滤膜上的微生物的同时,回收包含微生物的抗原的液体。微生物破坏试剂为碱性溶液的情况下,为了进行中和或者降低表面活性剂浓度,添加数mL~数百mL中性附近的缓冲液,较好是所用的微生物破坏试剂的10分之1~3分之2的容量。将向回收的滤液中添加的溶液称为滤液调整液,使用符合测定回收的滤液中的抗原的方法的条件的试剂即可。作为滤液调整液,使用Tris盐酸缓冲液等中性至酸性的缓冲液即可。回收的滤液中包含微生物的抗原。可通过各种方法测定回收的滤液中的抗原。滤液调整液通过微生物破坏试剂流过的滤膜即可。藉由使滤液调整液通过滤膜,可中和微生物破坏试剂,或者降低表面活性剂浓度,同时回收微生物的抗原。
将用于使标本悬浮的标本悬浮溶液、微生物破坏试剂、滤液调整液合称为微生物抗原提取试剂盒。
2.回收的抗原的测定
回收的抗原的测定可通过任意的方法进行,但较好是使用对要测定的抗原具特异性的抗体,通过利用抗原抗体反应的免疫学测定法进行测定。作为免疫学测定法,可列举免疫染色法(包括荧光抗体法、酶抗体法、重金属标记抗体法、放射性同位素标记抗体法),将采用电泳的分离与采用荧光、酶、放射性同位素等的检测方法组合的方法(包括蛋白质印迹法、荧光双向电泳法(fluorescence two-dimensional electrophoresis)),酶联免疫吸附测定法(ELISA),斑点杂交法,胶乳凝集法(LA:Latex Agglutination-TurbidimetricImmunoassay,胶乳凝集免疫比浊法),免疫层析法等。较好是通过免疫层析法或ELISA法进行测定。
免疫层析法使用免疫层析检测装置进行。免疫层析用检测装置也称为免疫层析法用试验片。该检测装置为由试验片形成的免疫层析试验片,例如图1所示那样配置而构成。包括:标本供给部位1,其中,在片状的固相支承体上供给标本;以及,标记试剂部位2,其中,在固相支承体上以可展开的方式保持标记了与抗原特异性结合的抗体的标记试剂;捕获试剂部位3,其中,固定化有可特异性结合捕获抗原与标记试剂的复合体的捕获试剂。按照向标本供给部位1供给标本后,标本依次通过标记试剂部位2、捕获试剂部位3的方式构成。
本发明的免疫层析检测装置还可在对照部位包括对照用试剂,也可进一步包括吸收部位。对照用试剂无限定,例如可使用标记试剂中的抗体结合的物质。对照用试剂固定化于捕获试剂部位的下游即可,图1中对应于对照部位4。吸收部位是通过吸收通过了捕获试剂部位的标本而控制标本的流动的具有液体吸收性的部位,设于检测装置的最下游即可,图1中对应于吸收部位6。吸收部位例如将纸制品作为吸收垫使用即可。
本发明的免疫层析检测装置中,标本供给部位可直接使用固相支承体的一端,也可用固相支承体以外的其他构件构成。后一种标本供给部位如下配置:以溶液通过毛细管流可展开移动的方式与固相支承体接触,以使一旦吸收标本或标本与标记试剂的混合物,接着将吸收的标本或混合物供给至固相支承体。作为固相支承体以外的其他构件,例如可列举由硝化纤维素、乙酸纤维素、尼龙、聚醚砜、聚乙烯醇、聚酯、玻璃纤维、聚烯烃、纤维素、聚苯乙烯等天然或合成的聚合物或者它们的混合物形成的构件,但无特别限定。
本发明的免疫层析检测装置中,标记试剂是使与抗原特异性结合的抗体和适当的标记物质结合而成的缀合物(conjugate),作为标记物质,可列举胶体金等金属胶体、硒胶体等非金属胶体、着色树脂粒子等不溶性的物质(不溶性载体)。一般来说,标记试剂含浸于固相支承体以外的其他构件,并进行干燥,将其置于与固相支承体连续地连结的位置。或者,标记试剂也可直接涂布于固相支承体上并进行干燥。标本到达包含标记试剂的标记试剂部位后,标记试剂被溶解于标本中,可在固相支承体上展开。即,标记试剂以可展开的方式保持于标记试剂部位。
本发明的免疫层析检测装置中,捕获试剂是与抗原特异性结合的抗体,捕获试剂部位可特异性结合捕获抗原与标记试剂的复合体,形成标记试剂-抗原-捕获试剂复合体。复合体的存在可作为捕获试剂部位2中标记物质形成的条带的颜色的浓度通过肉眼观察确定、或可利用测定装置进行检测。一般来说,捕获试剂通过直接涂布于固相支承体并干燥的方法制成,但并不仅限于此,也可通过将使其含浸于固相支承体以外的其他构件并干燥而得的构件置于固相支承体上的方法制成。此外,捕获试剂向固相支承体的固定化并不仅限于采用吸附的方法,通过利用氨基、羧基等官能团以化学方式结合的方法等公知的方法进行即可。
用作捕获试剂的抗体与用作标记试剂的抗体可以相同,但抗原中仅存在一个与该物质结合的部位的情况下,无法形成标记试剂-抗原-捕获试剂复合体。因此,该情况下,捕获试剂和标记试剂必须分别与抗原的不同部位结合。
固相支承体只要试样标本可通过毛细管现象被吸收并且流动即可,可以是任意的支承体。例如,支承体选自硝化纤维素、乙酸纤维素、尼龙、聚醚砜、聚乙烯醇、聚酯、玻璃纤维、聚烯烃、纤维素、聚苯乙烯等天然或合成的聚合物或者它们的混合物。固相支承体较好是具有条带状(strip)的形状。
以下,对利用免疫层析法的自血液培养阳性标本的PBP2'直接检测法进行具体说明。
从血液培养分离的菌株中,4成左右为葡萄球菌。葡萄球菌的情况下,出现大量耐甲氧西林金黄色葡萄球菌(MRSA)等对抗生素具有抗性的株。为了迅速并适当地治疗败血症,需要迅速诊断有无抗性。作为与葡萄球菌的耐药性相关的蛋白质,已知PBP2'(青霉素结合蛋白2',Penicillin Binding Protein 2')。
于是,本发明中,提出通过直接对血液培养中葡萄球菌呈阳性的标本进行试验,不经分离培养而在短时间内检测PBP2'的方法。
从怀疑患败血症的患者采集血液,开始采用BacT/ALERT等血液培养装置的培养。装置的提示铃响起(报警)后,采集1ml确认了菌增殖的标本,加入0.1M氢氧化钠与1.5%Triton X-100的混合液1ml,迅速破坏血细胞。使经处理的标本全部通过孔径小于菌体的滤膜。目标菌体被捕获于滤膜上。作为目标蛋白质的PBP2'存在于细菌的细胞膜上,因此为了以良好的灵敏度进行检测,需要破坏菌体。使0.2M氢氧化钠与1.0% Triton X-100的混合液1ml通过滤器,静置5分钟,从而破坏菌体,使PBP2'露出。使含0.6M Tris盐酸盐的中和液通过滤器,将含PBP2'的滤液滴加至以免疫层析法为原理的测试条带,该测试条带包含固定化有特异性捕获PBP2'的抗体的膜和抗体致敏胶乳粒子(antibody-sensitized latexparticles),从而可在短时间内检测PBP2'。
实施例
以下,列举实施例对本发明进行详细说明。本发明并不仅限于这些实施例。
实施例1 PBP2'测定用免疫层析法用试验片的制作
(1)抗体致敏胶乳粒子的制备和干燥
通过常规方法用胃蛋白酶处理抗PBP2'单克隆抗体而获得F(ab')2。将其致敏并结合于粒径0.4μm的胶乳粒子,喷雾于聚苯乙烯无纺布。接着,在减压装置内减压干燥1小时,制成干燥胶乳抗体垫。使用时以4mm的间隔截断,作为标记试剂部位2使用。
(2)PBP2'测定用免疫层析法用试验片的制作
通过常规方法用胃蛋白酶处理识别部位与用于胶乳致敏的抗PBP2'单克隆抗体不同的第二抗PBP2'单克隆抗体而获得F(ab')2。将其用含0.075%CHAPS的柠檬酸缓冲液(pH6)稀释,涂布于硝化纤维素膜(固相支承体5),充分干燥(捕获试剂部位3)。将抗小鼠IgG作为对照试剂同样地涂布于硝化纤维素膜,充分干燥(对照部位4)。
在疏水性片材7上配置包括捕获试剂部位3和对照部位4的固相支承体5,作为标记试剂部位2、标本供给部位1采用玻璃类纤维,作为吸收部位6采用滤纸,配置于任意的位置,制成PBP2'测定用免疫层析法用试验片。
免疫层析法用试验片的结构示于图1。
实施例2 PBP2'提取试剂的制备
作为标本悬浮用液,制备将0.1M氢氧化钠与1.0% Triton X-100混合而得的水溶液。
作为R1试剂(微生物破坏试剂),制备将0.2M氢氧化钠与1.5% Tx-100混合而得的水溶液。
作为R2试剂(滤液调整液),制备0.6M Tris盐酸盐水溶液(pH6.0±0.5)。
将上述的标本悬浮用液、R1试剂和R2试剂合称为PBP2'提取试剂。
实施例3
滤膜的尺寸的探讨
使通过血液琼脂培养基培养的MRSA悬浮于生理盐水,调整至1.0×108个作为标本,
1)标本1:混合制成标本悬浮液1,总量定为2mL;
2)将全部2mL用滤膜(材质:乙酸纤维素,孔径:0.45μm,直径:25mm)过滤;
3)使1000μL的R1试剂通过2)的滤膜,将溶液残留于滤膜内静置5分钟;
4)使400μL的R2试剂通过3)的滤膜,回收滤液;
5)滴加至实施例1中制成的PBP2'试验片的标本供给部位,10分钟后进行判定。判定结果示于表1。“++”表示较强的阳性,“+++”表示强阳性。
[表1]
PVDF: 聚偏氟乙烯
acrylic copolymer: 丙烯酸共聚物。
表1的结果显示使用孔径0.22~1.2μm的滤膜的情况下,均可通过本发明的方法检测出MRSA的PBP2'。使用孔径0.22~0.45μm的滤膜的情况下特别好。
实施例4
滤膜的材质的探讨
使通过血液琼脂培养基培养的MRSA悬浮于生理盐水,调整至1.0×108个作为标本,
1)标本1:混合制成标本悬浮液1,总量定为2mL;
2)将全部2mL用滤膜(材质:乙酸纤维素,孔径:0.45μm,直径:25mm)过滤;
3)使1000μL的R1试剂通过2)的滤膜,将溶液残留于滤膜内静置5分钟;
4)使400μL的R2试剂通过3)的滤膜,回收滤液;
5)滴加至实施例1中制成的PBP2'试验片的标本供给部位,10分钟后进行判定。判定结果示于表2。“++”表示较强的阳性,“+++”表示强阳性。
[表2]
PVDF: 聚偏氟乙烯
MCE: 混合纤维素酯
PES: 聚醚砜
hydrophilic polyethersulfone: 亲水性聚醚砜
GHP: 亲水性聚丙烯
NYL:尼龙
CA:乙酸纤维素
GxF:多层玻璃纤维制预滤器
GF: 硼硅酸盐玻璃纤维制预滤器
PTFE: 聚四氟乙烯。
表2的结果显示使用1~13的任一滤膜的情况下,均可通过本发明的方法检测出MRSA的PBP2'。
实施例5
表面活性剂的探讨
使通过血液琼脂培养基培养的MRSA悬浮于生理盐水,调整至1.0×108个作为标本,
1)标本1:混合制成标本悬浮液1,总量定为2mL;
2)将全部2mL用滤膜(材质:乙酸纤维素,孔径:0.45μm,直径:25mm)过滤;
3)使1000μL的R1试剂通过2)的滤膜,将溶液残留于滤膜内静置5分钟;
4)使400μL的R2试剂通过3)的滤膜,回收滤液;
5)滴加至实施例1中制成的PBP2'试验片的标本供给部位,10分钟后进行判定。判定结果示于表3。“++”表示较强的阳性,“+++”表示强阳性。
[表3]
EMULGEN 106:聚氧乙烯(5)月桂基醚
EMULGEN A500:聚氧乙烯二苯乙烯化苯基醚。
表3的结果显示使用任一种表面活性剂的情况下,均可通过本发明的方法检测出MRSA的PBP2'。
实施例6
与现有技术的比较实验
针对再现性将本发明的滤膜法与作为现有技术的离心法进行了比较。
标本:将MRSA菌株用血液培养基(去纤维蛋白马血1:液体培养基4)进行稀释;
1.离心法
1)标本1:混合制成标本悬浮液3,总量定为2mL;
2)以7000g、5分钟、室温进行离心;
3)弃上清液,加入200μL的R1试剂;
4)添加100μL的R2试剂并混合;
5)滴加至实施例1中制成的PBP2'试验片的标本供给部位,10分钟后进行判定。
2.滤膜法
1)标本1:混合制成标本悬浮液1,总量定为2mL;
2)将全部2mL用滤膜(材质:乙酸纤维素,孔径:0.45μm,直径:25mm)过滤;
3)使1000μL的R1试剂通过2)的滤膜,将溶液残留于滤膜内静置5分钟;
4)使400μL的R2试剂通过3)的滤膜,回收滤液;
5)滴加至实施例1中制成的PBP2'试验片的标本供给部位,10分钟后进行判定。
让A、B、C这3名实施离心法和滤膜法3次,对判定结果的再现性进行了比较。判定结果示于表4。“-”表示阴性,“+”表示阳性,“++”表示较强的阳性,“+++”表示强阳性。
[表4]
。
表4的结果显示本发明的滤膜法的再现性优于现有技术的离心法。
工业上利用的可能性
通过本发明的方法,可检测病原性微生物的传染病和病原性微生物的存在。
符号的说明
1 标本供给部位
2 标记试剂部位
3 捕获试剂(捕获抗体)部位
4 对照部位
5 固相支承体(硝化纤维素膜)
6 吸收部位(吸收垫)
7 顶部层叠材料或外罩。
本说明书中引用的所有刊物、专利和专利申请直接通过引用纳入本说明书中。
Claims (7)
1.回收微生物的抗原的方法,该方法包括:使包含微生物的标本通过具有不让微生物通过的孔径的滤膜,将所述标本中的微生物捕获于滤膜上,使可破坏微生物的膜的微生物破坏试剂通过捕获了微生物的滤膜而在滤膜上破坏所捕获的微生物,在滤液中回收抗原。
2.根据权利要求1所述的回收微生物的抗原的方法,其特征在于,微生物为细菌。
3.根据权利要求1所述的回收微生物的抗原的方法,其特征在于,微生物破坏试剂为表面活性剂、碱性溶液、或者表面活性剂与碱性溶液的混合物。
4.根据权利要求3所述的回收微生物的抗原的方法,其特征在于,碱性溶液的pH值为11以上。
5.根据权利要求3所述的回收微生物的抗原的方法,其中,碱性溶液为氢氧化钠溶液。
6.微生物的检测方法,该方法包括:通过免疫学测定法对用权利要求1~5中任一项所述的方法回收的微生物的抗原进行测定。
7.根据权利要求6所述的微生物的检测方法,其中,免疫学测定法为免疫层析法。
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JP7313830B2 (ja) * | 2019-01-29 | 2023-07-25 | デンカ株式会社 | 検体の展開を制御し得る、糖鎖抗原を抽出し測定するためのイムノクロマト試験キット |
JP7507017B2 (ja) | 2019-07-05 | 2024-06-27 | 旭化成株式会社 | 呼吸器感染症の起因菌を検出する方法 |
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WO2016175269A1 (ja) | 2016-11-03 |
KR102561659B1 (ko) | 2023-07-28 |
JP2016211853A (ja) | 2016-12-15 |
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CN107533059B (zh) | 2020-10-16 |
ES2894842T3 (es) | 2022-02-16 |
US10436783B2 (en) | 2019-10-08 |
KR20230117629A (ko) | 2023-08-08 |
EP3290920A4 (en) | 2018-11-07 |
US20180143195A1 (en) | 2018-05-24 |
KR20170140252A (ko) | 2017-12-20 |
EP3290920A1 (en) | 2018-03-07 |
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