CN110944753A - (1→3)-β-D-葡聚糖作为活性霉菌的量度 - Google Patents
(1→3)-β-D-葡聚糖作为活性霉菌的量度 Download PDFInfo
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Abstract
描述了电动装置和方法,目的是从介电流体介质收集可测定试剂。可通过在高压电极处产生等离子体和随后以电压梯度输送带电粒子来诱导电动流。活跃生长的霉菌将碳水化合物细胞壁成分(1→3)‑β‑D‑葡聚糖释放到空气中。本发明认识到空气传播部分是影响呼吸系统健康的部分,并选择性检验可溶于水性介质的游离形式。优选通过所述电动推进方法收集要分析的样品,但可应用任何空气采样方法,例如过滤、冲击器或撞击。
Description
发明背景。
发明领域
本发明涉及在介电介质中可测定试剂的收集和采样。这包括但不限于对于可通过霉菌或霉菌孢子细胞壁成分的生物特异性测定来确定存在或不存在的药剂的空气采样。该领域包括对生物试剂的空气采样,引导到用于测定装置的收集工具,和在其上沉积。试剂特异性测定可包括基于蛋白水解途径的免疫测定或生色测定。测定可包括但不限于比色、荧光、浊度、电化学或伏安法的检测手段。
现有技术描述
(1→3)-β-d-葡聚糖,象过敏原一样(1-3)存在于孢子中,但通过萌发和生长真菌而以不易用显微镜识别为孢子的颗粒形式释放到空气中。因此,虽然(1→3)-β-d-葡聚糖本身不是过敏原,但两者很可能在空气中缔合。在花粉中也发现了(1→3)-β-d-葡聚糖(4)。
结构:
(1→3)-β-D-葡聚糖是大多数真菌、某些细菌、大多数高等植物和许多低等植物的非过敏原水不溶性结构细胞壁成分。葡聚糖最多可占真菌细胞壁干重的60%,其中大部分为(1→3)-β-D-葡聚糖(Klis, 1994)。它们由具有可变分子量和支化度的葡萄糖聚合物组成,即三股螺旋、单股螺旋或无规卷曲结构(Williams, 1997)。在真菌细胞壁中,(1→3)-β-D-葡聚糖与蛋白质、脂质和碳水化合物(如甘露聚糖和壳多糖)相连,并且它们包含(1→3)-b-葡聚糖侧支,该侧支可与相邻的(1→3)-β-D-葡聚糖聚合物连接。
与症状的关系:
空气中的水平与真菌量和症状的严重程度相关。由于从真菌释放的过敏原的贡献,这不是因果关系(5)。B-葡聚糖与50%患有预先存在呼吸系统病症的儿童的呼吸峰值流量变异性(PFV)相关(6)。
Douwes综述了与B-葡聚糖和呼吸系统健康有关的文献(7)。他们得出结论,综述的研究表明在(1→3)-β-D-葡聚糖暴露、气道炎症和症状之间存在某种关联。无法确定与暴露相关的可能潜在炎症机制。包括了使用实验性暴露的种群研究以及动物和人类研究。
测定:
在文献中已使用了三种测定:抑制免疫测定(8)、夹心单克隆免疫测定(9)和鲎变形细胞测定。后者由Associates of Cape Cod商业化。Brooks等人(10)对这3种方法作了实验室间比较。用不同方法得到的结果通常显著相关,因此在相对意义上是可比的,但在实验室和测定之间结果的直接比较是不合适的。
提取:
大多数β-(1,3)-葡聚糖在环境温度为水不溶性,并且两种提取方法为碱提取(通常涡旋,然后在0.3N NaOH中摇动1hr)和热水提取(通常在PBS中的0.05%Tween 20,颠倒旋转,高压釜60min涡旋)。碱提取已用于基于鲎变形细胞的方法,热提取则用于基于EIA的方法。所用的提取方法综述于(10)。对来自滤器的孢子进行测定(11)包括用0.15N NaOH处理。接着用NaOH当量浓度两倍的等体积tris-Cl中和。并非所有文章都提到中和步骤。在花粉出版物(4)中未提及提取。
Madsen等人描述了用NaOH的提取的优化(12),并综述了一些使用或不使用或未提及提取的工作。在这一或其它工作中没有进行不用提取的对照。经常声明需要提取来溶解(1→3)-β-D-葡聚糖。
尺寸范围:
Foto等人对来自受损住宅的大量样品进行了研究(13)。他们声明,(1→3)-β-D-葡聚糖与碎片、菌丝以及孢子有关。没有给出实际的直接数据。Lee等人(14)对(1→3)-β-D-葡聚糖作了室内与室外的比较。他们还提到碎片,但未提供数据。Saleres等人(15)发现了在空气传播的部分中比完整孢子更小的尺寸范围,他们说,这意味更深的肺渗透。Madsen等人(16)在PM1部分中发现了很大部分的(1→3)-β-D-葡聚糖,其中不含孢子。尽管尺寸小,但他们仍说该物质用0.3M NaOH“制成水溶性”。
Singh等人(17, 18)用NIOSH采样器进行了尺寸分级,并在低到PM1部分的3个尺寸份额(cut)中发现等量的(1→3)-β-D-葡聚糖。
Chen等人比较了内部与外部以及人类活动的影响(19, 20)。他们发现最小的尺寸受人类活动影响最小,而较大尺寸的颗粒中(1→3)-β-D-葡聚糖较多。
替代测定:
Vogelmark等人(21)对孢子的青霉菌、曲霉菌、Stachrybotis培养物进行了测定。他们未提到任何提取方法。他们建议用(1→3)-β-D-葡聚糖测定作为霉菌暴露的替代。
Chew等人(22)使用真空除尘的尘土提取物(22)(21),并且对(1→3)-β-D-葡聚糖与活孢子作了关联。他们说,尘土是空气传播暴露的替代。
物种形成:
Iossifova等人对霉菌用EPA标准多重qPCR与尘土中的霉菌种类进行了比较(23)。在(1→3)-β-D-葡聚糖与从qPCR得出的相对霉变指数(RMI)之间没有相关性。他们用复杂的多元统计分析来显示与主要种类的相关性。枝孢霉属(Cladosporium)和曲霉属(Aspergillus genera)以及黑附球菌(Epicoccum nigrum)、短密青霉菌和Wallemia sebi.贡献者。与链格孢属(Alternaria)没有相关性。这不意味链格孢属不产生(1→3)-β-D-葡聚糖。
参考文献:
1. Green BJ, Schmechel D, Summerbell RC. Aerosolized fungal fragments(气溶胶化的真菌碎片). Adan OCG, Samson RA, editors. Wageningen:Wageningen AcadPubl; 2011.
2. Green BJ, Tovey ER, Sercombe JK, Blachere FM, Beezhold DH, SchmechelD. Airborne fungal fragments and allergenicity(空气传播的真菌碎片和过敏原性).Medical Mycology. 2006 Sep;44:S245-S55.
3. Green BJ, Mitakakis TZ, Tovey ER. Allergen detection from 11 fungalspecies before and after germination(在萌发之前和之后从11个真菌种类的过敏原检测). J Allergy Clin Immunol. [Article]. 2003 Feb;111(2):285-9.
4. Rylander R, Fogelmark B, McWilliam A, Currie A. (1 -> 3)-beta-D-glucanmay contribute to pollen sensitivity((1->3)-β-D-葡聚糖可能对花粉过敏性有贡献). Clin Exp Immunol. 1999 Mar;115(3):383-4.
5. Rylander R. Indoor air-related effects and airborne (1 -> 3)-beta-D-glucan(室内空气相关的效应和空气传播的(1-> 3)-β-D-葡聚糖). Environ HealthPerspect. 1999 Jun;107:501-3.
6. Douwes J, Zuidhof A, Doekes G, van der Zee S, Wouters I, Boezen HM,等人. (1 -> 3)-beta-D-glucan and endotoxin in house dust and peak flowvariability in children(房屋尘土中的(1-> 3)-β-D-葡聚糖和内毒素及儿童的峰值流量变异性). American Journal of Respiratory and Critical Care Medicine.[Article]. 2000 Oct;162(4):1348-54.
7. Douwes J. (1 -> 3)-beta-D-glucans and respiratory health:a review ofthe scientific evidence((1-> 3)-β-D-葡聚糖与呼吸系统健康:科学证明的综述).Indoor Air. 2005 Jun;15(3):160-9.
8. Douwes J, Doekes G, Montijn R, Heederik D, Brunekreef B. Measurementof beta(1->3)-glucans in occupational and home environments with aninhibition enzyme immunoassay(利用抑制酶免疫测定来测定职业和住宅环境中的β(1->3)-葡聚糖). Applied and Environmental Microbiology. 1996 Sep;62(9):3176-82.
9. Sander I, Fleischer C, Borowitzki G, Bruning T, Raulf-Heimsoth M.Development of a two-site enzyme immunoassay based on monoclonal antibodiesto measure airborne exposure to (1 -> 3)-beta-D-glucan(基于单克隆抗体研发两点酶免疫测定以检测对(1-> 3)-β-D-葡聚糖的空气传播暴露). Journal of ImmunologicalMethods. 2008 Aug;337(1):55-62.
10. Brooks CR, Siebers R, Crane J, Noss I, Wouters IM, Sander I,等人.Measurement of beta-(1,3)-glucan in household dust samples using Limulusamebocyte assay and enzyme immunoassays:an inter-laboratory comparison(使用鲎变形细胞测定和酶免疫测定来测定家庭尘土样品中的β-(1,3)-葡聚糖:实验室间比较).Environmental Science-Processes & Impacts. 2013;15(2):405-11.
11. Foto M, Plett J, Berghout J, Miller JD. Modification of the Limulusamebocyte lysate assay for the analysis of glucan in indoor environments(室内环境中葡聚糖分析的鲎变形细胞溶解物测定的改进). Anal Bioanal Chem. 2004 May;379(1):156-62.
12. Madsen AM, Frederiksen MW, Allermann L, Peitersen JH. (1 -> 3)-beta-D-glucan in different background environments and seasons(不同背景环境和季节中的(1-> 3)-β-D-葡聚糖). Aerobiologia. [Article]. 2011 Jun;27(2):173-9.
13. Foto M, Vrijmoed LLP, Miller JD, Ruest K, Lawton M, Dales RE. Acomparison of airborne ergosterol, glucan and Air-O-Cell data in relation tophysical assessments of mold damage and some other parameters(与霉损坏和一些其它参数的物理评估有关的空气传播麦角固醇、葡聚糖和空气-O-细胞数据的比较).Indoor Air. 2005 Aug;15(4):257-66.
14. Lee T, Grinshpun SA, Kim KY, Iossifova Y, Adhikari A, Reponen T.Relationship between indoor and outdoor airborne fungal spores, pollen, and(1 -> 3)-beta-D-glucan in homes without visible mold growth(没有可见霉菌生长的住宅室内和室外空气传播的真菌孢子、花粉和(1-> 3)-β-D-葡聚糖的关系).Aerobiologia. 2006 Sep;22(3):227-36.
15. Salares VR, Hinde CA, Miller JD. Analysis of Settled Dust in Homesand Fungal Glucan in Air Particulate Collected during HEPA Vacuuming(住宅中沉降尘土和HEPA真空除尘中收集的空气颗粒中的真菌葡聚糖的分析). Indoor and BuiltEnvironment. [Article]. 2009 Dec;18(6):485-91.
16. Madsen AM, Schlunssen V, Olsen T, Sigsgaard T, Avci H. AirborneFungal and Bacterial Components in PM1 Dust from Biofuel Plants(来自生物燃料工厂的PM1尘土中的空气传播真菌和细菌成分). Ann Occup Hyg. 2009 Oct;53(7):749-57.
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23. Iossifova Y, Reponen T, Sucharew H, Succop P, Vesper S. Use of (1-3)-beta-D-glucan concentrations in dust as a surrogate method for estimatingspecific fungal exposures(使用尘土中的(1-3)-β-D-葡聚糖浓度作为估计特定真菌暴露的替代方法). Indoor Air. 2008 Jun;18(3):225-32。
美国专利US5266461,Tanaka,测定(1→3)-β-D-葡聚糖的方法,是我们知道的唯一测定(1→3)-β-D-葡聚糖的专利。他们用抗体来抑制内毒素敏感因子的途径。
在现有技术中,存在很多从空气收集试剂用于生物测定的实例。例如,以下出版物描述了用于测定的过敏原、病原体和毒素收集的各种方法:
1. Yao等人(2009), Aerosol Science 卷40, 页492-502.
2. Noss等人(2008), Applied and Environmental Microbiology, 卷74, 页5621-5627.
3. King等人(2007), Journal of Allergy and Clinical Immunology, 卷120, 页1126-31.
4. Earle等人(2007), Journal of Allergy and Clinical Immunology, 卷119, 页428-433.
5. Peters等人(2007), Journal of Urban Health:Bulletin of the New YorkAcademy of Medicine, 卷84, 页185-197.
6. Yao和Mainelis (2006), Journal of Aerosol Science, 卷37, 页513-527.
7. Platts-Mills等人(2005), Journal of Allergy and Clinical Immunology, 卷116, 页384-389.
8. Sercombe等人(2004), Allergy, 卷60, 页515-520.
9. Custis等人(2003), Clinical and Experimental Allergy, 卷33, 页986-991.
10. Polzius等人(2002), Allergy, 卷57, 页143-145.
11. Tsay等人(2002), Clinical and Experimental Allergy, 卷32, 页1596-1601.
12. Parvaneh等人(2000), Allergy, 卷55, 页1148-1154.
13. McNerney等人(2010), BMC Infectious Diseases, 卷10, 页161-166,以及美国专利7,384,793中的装置。
样品收集的其它已知方法包括在活性炭上捕集挥发性有机化合物(VOC)、解吸和通过质谱分析。参见Phillips等人(2010), Tuberculosis, 卷90, 页145-15和其中的参考文献。由于测定(assay)本质上严格为化学法,并且如本文限定不为生物特异性,因此不认为本发明包括VOC。生物特异性是指其中通过生物特异性(例如核酸特异性、抗体特异性、受体-配体特异性等)来确定结果的测定。尽管可通过VOC分析实现诊断特异性,但这通过限定的有机化合物的基团的存在和量来推断。
先前的现有技术出版物描述了使用泵送和过滤、擦拭、被动沉积、电动输送等的“干式”方法;通常随后进行提取步骤,并将提取物用于测定。
在以下专利文献中已描述了用于在液体流中收集的方法:
Yuan和Lin,美国专利申请2008/0047429A1中,
Saski等人,2002年颁布的美国专利6,484,594中。
虽然从空气有效地收集试剂,但这种液体流系统不可避免地引起样品的高度稀释。除非重新浓缩试剂,否则结果有灵敏性的折衷。
Northrup等人在美国专利7,705,739和7,633,606中描述了一种自主运行的系统,用于空气采样和测定其中的空气传播物质。他们没有说明确切的空气采样方法,也没有详述如何将其转移到测定系统。
存在很多基于过滤或静电沉淀的用于空气净化的可商购系统。关于一般描述,请参见美国环境保护局的文章“Guide to Air Cleaners in the Home”(住宅中的空气清洁器指南),U.S. EPA/OAR/ORIA/Indoor Environments Division(MC-6609J)(MC-6609J)EPA 402-F-08-004,2008年5月。存在很多使用高效颗粒空气(HEPA)滤器或静电沉淀滤器的系统的商业实例。这些系统广泛用于从空气去除颗粒物质或过敏原,包括作为家庭供暖、通风和空调(HVAC)系统的一部分。HEPA滤器的优势是可去除小到微米尺寸范围的颗粒,而静电沉淀法的优势是可带来高体积流量,而压差很小或没有。作为静电沉淀系统技术规范的详细实例,参见Bourgeois的美国专利3,191,362。虽然从空气有效去除试剂,但这种空气净化系统本身不适合收集样品用于分析。
熟知过滤方法用于收集检验所用的空气样品。也可用这样的过滤方法捕获包含(1→3)-β-D-葡聚糖的颗粒用于测定。
基于电动的空气清洁系统已由Sharper Image公司以商品名Ionic Breeze开发并且以前已商业化(但现已停产)。Brown在美国专利2,949,550中阐明了原始的电动原理。Lee在美国专利4,789,801中对此进行了进一步改进,以改善气流并最小化臭氧的产生。可商购系统的进一步改进描述于Taylor和Lee的美国专利6,958,134、Reeves等人的7,056,370、Botvinnik的7,077,890、Lau等人的7,097,695和Taylor等人的7,311,762。在使用电动推进的装置的前面描述中,共同的元件为由导线组成的高压电极。由于导线的横截面积很小,因此,与导线正交地产生非常陡的电压梯度。高电压梯度引起产生由带电粒子组成的等离子体,并且通过高电压梯度将动能传递给带电粒子。产生的净空气流通过在带电粒子和不带电粒子之间交换动能而造成,并且净空气流由并置的平面电极引导,这些平面电极处于零电压或与导线电极相反符号的电压。带电粒子静电沉淀到平面电极上,可定期去除以清洁。这个工作体针对空气净化,而不是样品收集。然而,如Custis等人(2003)首先描述的那样,通过用纸巾擦拭电极,Ionic Breeze装置已适应于用于过敏原分析的样品收集。过敏原从纸巾提取,并经过免疫测定。在Peters等人(2007)和Platts-Mills等人(2005)的工作中,也用Ion Breeze收集过敏原,用于免疫测定分析。之前,Parvaneh等人(2000年)描述了一种电离装置,带有“具有导电表面作为集尘板的金属杯”,由其提取过敏原用于测定。尚不清楚样品如何收集在金属杯的内部上而不会粘附到整个表面。该装置由瑞典斯德哥尔摩的Airpoint AB制造。然而,没有关于Airpoint AB制造或销售此种产品的公开信息,没有足够的信息使本领域的技术人员能够了解该装置的细节,同一作者也没有在有关环境过敏原检测的后续出版物中使用类似的装置。没有提及将样品集中到由电压梯度产生的势阱中。
Yao等人(2009)和Yao和Mainelis(2006)已描述了将生物可测定试剂收集到测定工具或装置上的方法。Yao和Manielis(2006)描述了与平面电极电接触的琼脂凝胶块,Yao等人(2009)描述了插入在平面电极之间的微量滴定板。这两项工作都描述了由泵驱动的空气流,并使要分析的试剂静电沉淀到测定工具上。在这些工作中,电极和琼脂块具有基本相同的面积。
McNerney等人(2010)描述一种呼吸分析仪装置,其中个人呼吸或咳嗽到呼吸管中,样品收集在管的内表面上,用柱塞刮擦到光学生物感测器上,进行免疫结合反应,且生物感测器利用渐逝波照明系统通过散射光确定结核分枝杆菌(M. tuberculosis)的存在或不存在。
本文的申请人Inspirotec Inc.已商业化了用于测定生物物质的离子捕获装置。此装置和相关装置描述于Inspirotec的美国专利9,618,431,用于捕获介电流体中可测定试剂的电动装置;9,481,904,用于捕获和生物测定空气传播的可测定病原试剂的电动方法;8,038,944,用于捕获介电流体中可测定试剂的电动装置;9,360,402,利用可移除电极捕获介电流体中可测定试剂的电动装置;和9,216,421,用于采样和分析的集成系统。离子捕获技术可用于捕获包含(1→3)-β-D-葡聚糖的空气传播颗粒。
这些现有技术均未提示用(1→3)-β-D-葡聚糖的可溶部分作为霉菌暴露的量度。
发明概述
(1→3)-β-d-葡聚糖是霉菌细胞壁的结构成分,但也可在酵母、霉菌、花粉、细菌中发现。空气中最大的部分可归因于霉菌。
已使用的测定为竞争性免疫测定、夹心免疫测定和鲎变形细胞溶解物(LAL)测定。竞争性免疫测定不太灵敏,夹心免疫测定与LAL相当,据称不太灵敏而特异性更高。LAL测定取决于来自鲎的特异蛋白酶的激活,该蛋白酶从合成底物产生荧光信号。结果为非常灵敏的测定。特异性似乎一直不是问题,大多数持续的工作都使用可商购的LAL测定。
已有很多提议称(1→3)-β-d-葡聚糖的测定是总霉菌暴露的良好替代。
由呼气峰值流量(PEF)显示房屋尘土中(1→3)-β-d-葡聚糖的水平与呼吸系统症状之间的相关性。
在有活跃霉菌生长的住宅中,发现(1→3)-β-d-葡聚糖的尺寸范围为18至0.18微米。这意味很大一部分在比孢子小的颗粒和碎片中。就像尘螨过敏原一样,较小范围的颗粒更有可能保持空气传播更长的时间,且更深地渗透肺。
作为结构细胞壁成分,(1→3)-β-d-葡聚糖通常为不溶形式。因此,至今已进行的所有检验都使用了极端加热步骤或碱处理,以使其可溶用于检验。空气传播形式的很大一部分在颗粒可溶范围内的这一事实已被忽视。在免疫测定之前,我们常会从我们的装置中去除来自样品的所有不溶和颗粒物质。那部分将包含会深深渗透到肺中的较低颗粒尺寸范围。
该部分的碱提取对结果影响很小(参见实施例)。
因此,(1→3)-β-d-葡聚糖的空气传播可溶部分的测定为呼吸道刺激物的直接测定,呼吸道刺激物的释放也与真菌过敏原向空气中的释放平行。现有技术均未提示直接测定为空气传播物质收集的样品中游离形式的(1→3)-β-d-葡聚糖。该游离形式作为可溶部分的测定是本发明的主题。这种游离形式将代表较小尺寸的颗粒,熟知这些颗粒会深深地渗透到肺泡中,从而引发呼吸问题,例如在肺部深处引起过敏反应,这导致哮喘。因此,该测定值为通常与过敏原行为平行的参数,因此,它是用于评估通常对霉菌过敏原暴露的有用替代。
在本发明的优选实施方案中,通过电动推进装置收集包含游离(1→3)-β-D-葡聚糖的可溶部分,该电动推进装置例如如由发明人以名称Inspirotec或Exhale商业化的。然而,根据需要,也可使用本领域的技术人员熟知的其它装置,例如通过具有限定孔径的滤器过滤空气,所述滤器例如微孔薄膜滤器或纤维滤器,或HEPA滤器或Electret滤器,如由3M公司商业化的,或通过冲击捕获限定尺寸等级的颗粒的滤器。实例为Anderson冲击器和NIOSH旋风冲击器(Lindsley等人, Journal of Environmental Monitoring, 2006, 8, 1136-42)或 Spin Con湿式旋风法或基于Bobcat electret的过滤,如由美国密苏里州Drexel的Alburty Labs商业化的。
通过阅读整个说明书,包括附加权利要求和附图,本发明的其它目的、特征和优点将变得显而易见。
发明概述
图1表示通用的电动流动装置。
图2A-2D表示具有可移除电极的组装的载体。
图3为在那些相同传播样品中(1→3)-β-D-葡聚糖与霉菌过敏原存在或不存在相比的比较的统计分析的图形表示。
图4为对一组用NaOH处理或未处理的空气传播部分进行(1→3)-β-D-葡聚糖测定的比较的统计分析的图形表示。
图5为在全美国76个住宅中(1→3)-β-D-葡聚糖的分布的图形表示。
发明详述
通过采样装置对空气采样,采样装置可如在颁予申请人的先前专利中详述,即美国专利8,038,944、9,216,421、9,360,402、9,481,904和9,618,431。这些专利中每一个的说明书通过引用结合到本文中。特别要注意US9,360,402,其使用带有可移除电极的筒进行生物特异性物质分析。该装置通过将拆下的电极直接浸入缓冲提取介质,并用涡旋混合器在离心管中摇动,来提供提取生物特异性试剂的便利,如下所述。
实施例1. 图1表示一个基本的离子推进装置,它具有壳1,以及在横截面中所见和符号表示为电压轮廓的高压导线2,接地板状电极3。产生的离子流由箭头4表示。产生的净空气流入由箭头5表示,流出由箭头6表示。有利地,电极3为可移除的,并且可安装到载体,载体可从壳1移除,如US 9,360,402中所示。
实施例2. 图2A和2B示出可移除的载体组装体21,它可与图1的壳一起使用,以作为固定电极的替代物。在US 9,360,402中详细描述了载体组装体21。载体组装体21包括一件式塑料载体23、闩锁25和捕获电极27。载体23可从壳1移除。另外,捕获电极27可移除地固定到载体23。闩锁25支撑电极27,见图2C和2D。闩锁25适应于将捕获电极27固定到载体23。为了检验,可将载体23从壳1移除。然后可将闩锁25和电极27从载体23移除,以便于检验。
实施例3. 在各种霉菌阳性和霉菌阴性的住宅环境中运行样品。使用来自IndoorBiotechnologies的MARIA试剂盒和BioRad Inc.提供的MagPix仪器,通过多重免疫测定来确定空气传播的霉菌过敏原的存在。常规运行采样器5天,如实施例4中详述地,从拆下的电极提取包含过敏原和(1→3)-β-D-葡聚糖的物质,上清液按照用于动力学速率测定的制造商方案,通过MARIA和通过Glucatell测定试剂盒(Associates of Cape Cod Inc.,EastFalmouth, MA)检验。
图3显示对霉菌过敏原Alt a 1和Asp f 1)及(1→3)-β-D-葡聚糖通过MARIA™多重免疫测定方法测定的霉菌过敏原存在的比较的统计分析。箱形图显示了90%范围和全范围的值。图3中汇总的结果表明,在空气传播样品中不存在霉菌过敏原和不存在(1→3)-β-D-葡聚糖之间的关系显著。与阴性组(2.42)比较,在霉菌过敏原阳性组(7.19)也观察到更高的平均值。来自图3的数据显示空气传播的霉菌过敏原与存在(1→3)-β-D-葡聚糖之间可能的显著关系。
实施例4. NaOH提取对测定的(1→3)-β-D-葡聚糖的影响.
表1.
上述Inspirotec空气采样装置在每个检验环境中连续运行5天。按照检验,将不锈钢电极条从位于装置中的筒中移除,并转移到15ml离心管。将1毫升具有0.02%Tween 20的PBS加入管中,并经10分钟间歇涡旋。从离心管取出样品,并转移到2mL螺帽管中,然后以15,000g离心30分钟。移出上清液,并放入新的2ml螺帽管中。在另一个2mL螺帽管中,通过加入20uL2.5N NaOH使80µl的这些样品达到0.05N NaOH。将样品在室温下在定轨摇床上摇动2.5hr,通过加入100µL 2M Tris – HCL中和(1M Tris – HCL最终)。(1→3)-β-D-葡聚糖水平用Glucatell测定试剂盒(Associates of Cape Cod Inc., East Falmouth, MA)按照制造商的动力学速率测定标准方案测定。
结果也图形显示于图3中,如用JMP软件包,JMP® Pro 13.0.0(SAS InstituteInc. Cary, NC)统计分析的。
最佳拟合直线的斜率为1.41。这表明,在以这种方式收集和分析样品时,(1→3)-β-D-葡聚糖的41%在不溶部分中。本发明把注意力集中在可溶部分,如通过来自离心且无提取的上清液所测定。
实施例5. 在全美国住宅中(1→3)-β-D-葡聚糖水平的分布
使用Inspirotec装置,从跨美国的76个住宅收集空气样品。在各个住宅中,使装置运行1至5天时间。在运行期结束后,将不锈钢电极条从位于装置中的筒中移除,并转移到15ml离心管。将1毫升具有0.02%Tween 20的1xPBS加入管中,并经10分钟时间间歇涡旋。从15mL离心管取出样品,并转移到2mL螺帽管中。然后以15,000g离心30分钟。移出上清液,并放在新的2mL螺帽管中。(1→3)-β-D-葡聚糖浓度用Glucatell测定试剂盒(Associates of CapeCod Inc., East Falmouth, MA)按照动力学速率测定的制造商方案测定。
结果被图形显示于图4中,如用JMP软件包JMP® Pro 13.0.0 (SAS InstituteInc. Cary, NC)统计分析的。将低于用于测定的零时间场对照平均值的值指定为场对照值/2,并且将这些值的log10的出现数绘制在箱中,如x轴上所示。
在62%的住宅中检测到(1→3)-β-D-葡聚糖。
从前述明显看到,在从空气传播物质收集的样品的可溶部分中,(1→3)-β-D-葡聚糖的测定值代表空气中游离(1→3)-β-D的部分,这是从活跃生长的霉菌释放的物质,且将最深地渗透到呼吸系统中,并影响呼吸系统健康。该部分也可能与过敏原从霉菌的释放平行,因此它也是空气传播过敏原暴露的替代测定。在先技术忽略了可溶部分,而集中在可从较大复合物提取的物质。对本领域的技术人员显而易见的是,可溶部分可通过本文所述的优选离心方法或通过其它熟知的方法(例如过滤或沉降)制备。
因此,本文描述一种用于分析气溶胶颗粒的方法,其中所述气溶胶颗粒由空气采样装置捕获,从采样介质提取,并分析可溶部分的(1→3)-β-D-葡聚糖。空气采样装置可基于电动推进或基于静电沉淀。(1→3)-β-D-葡聚糖可通过基于鲎变形细胞的测定或通过免疫测定来测定。采样装置可基于过滤、撞击(impingement)或冲击器(impactor)。
本发明也描述一种用于分析气溶胶颗粒的方法,其中所述气溶胶颗粒从电动推进通过电动推进装置的一定体积空气沉积在所述装置的电极上,所述电极可移除地附接到载体支座;将所述电极从所述载体支座移除,并放入提取容器;将预定体积的提取流体加到所述提取容器;将所述电极在所述提取流体中搅拌预定时间;并将全部或部分所述提取流体加到用于分析所述气溶胶颗粒的(1→3)-β-D-葡聚糖的反应混合物。
Claims (11)
1.一种用于分析气溶胶颗粒的方法,其中所述气溶胶颗粒由空气采样装置捕获,从采样介质提取,并分析可溶部分的(1→3)-β-D-葡聚糖。
2.根据权利要求1所述的方法,其中所述空气采样装置基于电动推进。
3.根据权利要求1所述的方法,其中所述采样装置基于静电沉淀。
4.根据权利要求1所述的方法,其中(1→3)-β-D-葡聚糖通过基于鲎变形细胞的测定来测定。
5.根据权利要求1所述的方法,其中(1→3)-β-D-葡聚糖通过免疫测定来测定。
6.根据权利要求1所述的方法,其中采样装置基于过滤。
7.根据权利要求1所述的方法,其中采样装置基于撞击。
8.根据权利要求1所述的方法,其中采样装置基于冲击器。
9.一种用于分析气溶胶颗粒的方法,其中所述气溶胶颗粒从电动推进通过电动推进装置的一定体积空气沉积在所述装置的电极上,所述电极可移除地附接到载体支座;将所述电极从所述载体支座移除,并放入提取容器;将预定体积的提取流体加到所述提取容器;将所述电极在所述提取流体中搅拌预定时间;并将全部或部分所述提取流体加到用于分析所述气溶胶颗粒的(1→3)-β-D-葡聚糖的反应混合物。
10.根据权利要求11所述的方法,其中(1→3)-β-D-葡聚糖通过基于鲎变形细胞的测定来测定。
11.根据权利要求11所述的方法,其中(1→3)-β-D-葡聚糖通过免疫测定来测定。
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