文献
消毒供应频道
2019-07-01


BCA定量蛋白测试法的特性和验证方法


Characterization and Validation of the BCA Protein Quantization (Pro1Micro)



The efficacy of automated cleaning procedures must be validated and monitored on acontinuous base. Tissue and blood proteins are common compounds on contaminated instruments. After cleaning, clinical instruments must be completely free of any residue.Methods for the detection of protein are ubiquitous and often used in food or pharmaceutical industries. Recently protein residues have caught much attention since residual protein on surgical instruments surfaces is important because of the continuing risks of transmission of prions (the causative agent of transmissible spongiform encephalopathies such as variant Creutzfeldt-Jakob disease (vCJD))1,2. It is necessary to use protein detection methods to check for the efficient removal of protein from surgical instruments after processing. Protein levels are used as an indication of the amount of prion protein contamination. The Terragene® Chemdye® Pro1Micro is an easy way to monitor the cleanliness of re-usable medical devices in the context of medical and dental practices.

对机械清洗程序的清洗效果进行持续清洗效果验证和监测是必要的。人体组织和血液等蛋白残留污染物是医疗器械上的常见污染物,在清洗后,医疗器械应没有任何残留物。用于检测蛋白质的方法普遍存且常用于食品或制药工业中。最近,蛋白残留污染物的风险开始引起了多方关注,其原因为,手术器械表面上的残留蛋白质可导致朊毒体(传染性海绵状脑病的病原体,如变异克雅氏病(vCJD))的持续传染风险,蛋白残留污染量被作为指标,指示污染的朊毒体数量,因此,使用蛋白残留测试法监测处理后的手术器械是否能有效地去除蛋白质是必要的。Terragene®Chemdye®Pro1Micro(安易测蛋白残留测试棒)适合在包括口腔科在内的医疗机构内监测复用医疗器械的清洗质量,其操作简单易用。


Guidelines for Protein Measurement

蛋白残留测试法相关标准


The U.K. Department of Health published the Health Technical Memorandum 01-01 in March 2016. The established guidelines state that there should be no more than 5μg of protein in-situ on the side of any instrument. Prions are hydrophobic proteins and are easier to remove if they have not dried on the surface of an instrument. The attachment of hydrophobic proteins to surfaces becomes less reversible if they are allowed to dry fully. To enable efficient prion removal the hospital staff should ensure that instruments are transported to the sterile service department (SSD) immediately after the close of the procedure, for cleaning and reprocessing as soon as practically possible.

英国卫生部于2016年3月发布了健康技术备忘录HTM 01-01标准,其规定任何一件医疗器械一整面的残留蛋白数值应不超过5μg。朊毒体是疏水性蛋白质,如朊毒体在医疗器械表面上不处于干燥状态,会更容易被去除。如疏水性蛋白质处于干燥状态,则疏水性蛋白质对器械表面附着性更强。为了有效去除朊毒体,医院操作人员应确保在手术结束后,立即将器械运送到无菌服务部门(CSSD),以便尽快进行清洗和处理。


The HTM 01-01 guidance for decontamination management of surgical instruments establishes that the environment around soiled instruments must be kept at or near saturation humidity3. This prevents full attachment of the hydrophobic proteins such that they are more efficiently removed by cleaning, making the cleaning process more effective and reducing the risks to the patients and staff handling the devices. The guidance also indicates that the upper limit of acceptable protein contamination after processing of a surgical instrument is 5 μg of Bovine Serum Albumin (BSA) equivalent per instrument side.

用于手术器械去污管理的HTM 01-01指南确定污染器械周围的环境湿度必须保持在饱和或接近饱和状态。这防止了疏水性蛋白质的完全附着,使得通过清洁更有效地去除它们,使得清洁过程更有效并且降低了患者和操作该器械人员的风险。该指南还表明,手术器械加工后可接受的蛋白质污染的上限为每件器械一整面小于5μg牛血清白蛋白(BSA)。


The Bicinchoninic Acid Method for Protein Detection

BCA蛋白残留测试法


The bicinchoninic acid (BCA) assay, first described by Smith et al.4 is based on the Biuret Assay, and it depends on the conversion of Cu(II) to Cu(I) under alkaline conditions. The Cu(I) is then detected by reaction with BCA, developing a deep purple color to the solution. Since the production of Cu(I) in this assay is a function of protein concentration and incubation time, the protein content of unknown samples can be determined spectrophotometrically by comparison with known protein standards. A further advantage of the BCA assay is that it is generally more tolerant to the presence of compounds that interfere with the Lowry assay. In particular this assay is not affected by rests of detergent. Depending on the proportion between the reactants two assays are usually described:

• a standard assay (0.1-1.0mg/mL) with minimum or no incubation time and

• a microassay (0.5-20μg/mL) with 1 hour incubation time.

在缩二脲测试法的基础上,Smith等人首先阐述了BCA(二辛可酸)测试法,通过在碱性条件下,蛋白残留物让二价铜离子(Cu2+)转换成一价铜离子(Cu+),一价铜离子会与BCA溶液反应,使溶液呈现深紫色。在测试时,一价铜离子的产生与蛋白质浓度、培养时间呈函数关系,因此通过与已知蛋白质浓度的标准品比较,可以通过分光光度法,测定未知样品的蛋白质含量。BCA测试法的另一个优点是,在使用Lowry测试法时,它对产生干扰的化合物具有更强抗干扰能力。而且,该测试法不受清洗剂残留物的影响。根据反应物之间的比例,通常描述两种测定:

•标准测试法(0.1-1.0mg/mL),培养时间最短或无需培养。

•微量测试法(0.5-20μg/mL),培养时间为1小时。


The standard assay is usually used in the food and pharmaceutical industries while the microassay can be adapted for clinical instruments. The microassay uses concentrated reagents and a protocol that utilizes an extended incubation time at an elevated temperature (55-60°C). The result is a sensitive colorimetric protein assay, able to detect proteins in the μg/mL range as required in recent standard revisions.

标准测试法常用于食品和制药行业,而微量测试法用于临床器械。微量测试法使用浓缩反应溶液,其培养温度(55-60°C),并会延长培养时间,其会得到更灵敏的比色,能够检测最近标准修订中所需的mg/mL范围内的蛋白质。


We had optimized the microassay protocol to give reliable results in the 0.50 y 25.00 μg range (Figures 1 and Figure 2). The assay gives both qualitative and quantitative results. It is compatible with detergents, it is practical since this assay has the advantage that it can be carried out as a one-step process compared to other two steps assays, the reagents are stable indefinitely at room temperature and it exhibits less degree of varying response toward different proteins than other protein assay methods.

我们优化了微量测试法,在0.50 y 25.00μg范围内,该测试法可提供可靠的结果(图1和图2)。该测试法可给出定性和定量结果。它不会受到清洗剂干扰,可与清洗剂兼容,具有可操作性,相比其他需2步反应完成的测试法,该测试法可1步直接反应,与其他蛋白测试法相比,其在室温下稳定性佳,且对不同蛋白质的反应变化差异较小。

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Pro1Micro System: The Test Pen

安易测蛋白残留测试棒


The reaction is time and temperature dependant, i.e. the color develops with time and the speed of color development is slower or faster depending on the temperature. We recommend that results be read within 15 minutes of activation, and then discarded. The reaction takes place in minutes depending on the level of contamination. The test chemistry will turn to purple over a long period of time (4-5 hours) even with the absence of protein. Users may interpret the level of contamination on a surface based on the speed and intensity of the color change. The speed of the reaction and the sensitivity reached is enhanced by incubation. Since the reaction is temperature-dependent, it is important to allow the devices to equilibrate to ambient room temperature (15-25°C) if they have been stored at refrigerated temperatures.

该反应是与时间和温度呈正相关的,即反应溶液的颜色随时间和温度发生变化,不同温度和时间显色速度会不同。建议在激活后10分钟内读取结果,然后丢弃。根据不同的污染程度,反应会在数分钟内完成。即使没有蛋白质,测试用化学物质也会在一段时间内(4-5小时)变成紫色。操作者可基于颜色变化的速度和程度,来评价污染水平。测试棒通过培养提高了反应速度和达到的灵敏度。由于反应与温度有关,因此如果设备在冷藏温度下保存,则必须使测试棒平衡至室温(15-25℃)。


Pro1Micro System: Swab Sampling Procedure Validation

安易测蛋白残留测试棒的验证


The conditions in which surgical instruments are handled during and after surgery may ,affect the level of tissue protein, prion attachment and the efficacy of subsequent decontamination regimes and sampling methods. In order to evaluate this we investigated the swabbing recovery of BSA and brain homogenates from surgical stainless steel with respect to time and “wet” and “dry” storage conditions.

手术期间和术后处理手术器械的方式不同,可能影响组织蛋白残留水平,朊毒体附着以及随后的去污方案和取样方法的有效性。为了评估这一点,我们研究了外科不锈钢中BSA和脑组织匀浆的擦拭恢复时间和“湿”、“干”储存条件。


As it is pointed out in the HTM 01-01 guidance the protein measurement is per side of instrument rather than per unit area of an instrument. Nevertheless “side per instrument” varies greatly from instrument to instrument so we decided to base our tests following the ISO 15883-1:2009 standard that requires that about 10 cm² of an instrument is swabbed 6 . Surgical stainless steel tokens were used since it has been shown that tokens represent surgical instrument surfaces more closely. Tokens of 10 cm² were used for the inoculation with drops of the BSA and brain homogenate samples. Samples were left to dry at room temperature, we also did some experiments drying the samples at 60°C. We considered the initial time when all the water from the protein solutions was evaporated. Sample collection was done thoroughly over the token area with pre-moistened swabs with firm swab contact and movement on the surface for maximum sample recovery. Afterwards the swabs were immersed in Pro1Micro solution and the absorbance at 562 nm was measured (Figure 2). A titration of the Pro1Micro solution was done with BSA as reference for total protein concentration.

正如在HTM 01-01标准中指出的那样,蛋白残留监测是针对器械的每一侧面,而不是器械的单位面积。然而,“每把器械的侧面”因而异,因此我们决定根据ISO 15883-1:2009标准进行测试,该标准要求擦拭约10 cm²的器械6。使用外科不锈钢模拟物,因为已经表明不锈钢模拟物更紧密地代表外科器械表面。10cm²的标记用于接种BSA滴和脑匀浆样品。将样品在室温下干燥,我们还进行了一些实验,在60℃下干燥样品。我们考虑了蛋白质溶液中所有水分蒸发的初始时间。样品收集在不锈钢模拟物区域上进行,使用预先润湿的拭子,使拭子牢固接触并在表面上移动,以最大限度地回收样品。然后将拭子浸入Pro1Micro溶液中并测量562nm处的吸光度(图2)。用BSA作为总蛋白质浓度的参考进行Pro1Micro溶液的滴定。


Long drying times have adverse effect on different cleaning methods so we tested how the recovery of protein and brain homogenate to stainless steel varied with time. At initial time we obtained nearly 90% of recovery from both samples (Figure 3 and Figure 4). As the time of drying increases the protein recovery by swabbing decreases. This goes along with the HTM 01-01 guidance that points out to keep moistened the surgical instruments preventing them from drying.

干燥时间对不同的清洗方法有不利影响,因此我们测试了不锈钢中蛋白和脑匀浆的回收率如何随时间变化。在初始时,我们从两个样品中获得了近90%的回收率(图3和图4)。随着干燥时间的增加,通过擦拭方法的蛋白回收率降低。这与HTM 01-01指南一致,指出要保持手术器械的湿润,防止污染物干涸。


In general the recovery was less than 50 % and in some samples it was less than 30% (data not

shown).

一般来说,采样率低于50%,而在一些样品中,采样率低于30%(数据未显示)。

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