Cygnus CHO HCP ELISA Kit (F015)试剂盒说明书

Cygnus CHO HCP ELISA Kit (F015)试剂盒说明书

Cygnus试剂盒Cygnus technologies热销产品
Cygnus热销产品:cygnus f975、cygnus f410检测试剂盒、cygnus f550
品牌商名称:cygnus
公司官网:

Product Information

Product Type:ELISA Kit
Storage:All kit reagents 2°C to 8°C
Target Expression System:CHO
Species Group:Mammalian
Species:Chinese hamster
Assay format:96 Well Plate
Time to result:~3 hrs. 50 min. or ~5 hrs. 50 min
LOD:400 pg/mL
LLOQ:~4 ng/mL , or ~1 ng/mL
Recommended Diluent:I028

Description

The antibodies in this kit were generated against and affinity-purified from a cell lysate of CHO cells grown in serum supplemented media.

All three Cygnus CHO HCP kits recognize a vast majority of the same HCPs, but because the potential exists that the quantitative value reported for a given sample type may differ between the kits, we cannot predict which kit will be most suitable for your applications. Formal qualification must be performed on each different product to determine which of the three kits is best. For labs looking at CHO HCP impurities for the first time, use our CHO HCP ELISA, 3G (F550). If this method proves inadequate, then suggest evaluate CHO HCP kits CM015 and F015. Please use Sample Diluent, Item No. I028, with these kits; available separately.

描述
该试剂盒中的抗体是针对在补充有血清的培养基中生长的 CHO 细胞的细胞裂解物产生并亲和纯化的。

所有三个 Cygnus CHO HCP 试剂盒都可以识别绝大多数相同的 HCP,但由于可能存在针对给定样品类型报告的定量值可能因试剂盒而异,我们无法预测哪种试剂盒最适合您的应用。 必须对每种不同的产品进行正式鉴定,以确定三个套件中的哪一个是最好的。 对于首次检测 CHO HCP 杂质的实验室,请使用我们的 CHO HCP ELISA, 3G (F550)。 如果这种方法被证明不合适,那么建议评估 CHO HCP 试剂盒 CM015 和 F015。 请在这些套件中使用样品稀释剂,货号 I028; 可单独购买。

Kit Components 试剂盒成分:
Anti-CHO:Alkaline Phosphatase Conjugate
CHO HCP Standards Set, A-F
TBS Wash Concentrate, 20X
PNPP Substrate
Anti-CHO Coated Microtiter Strips

Product FAQs  产品问与答:

  • Can the ELISA Protocol in the Product Insert be Modified?

    The assay protocol recommended in the kits’ product insert have been developed with consideration given to typical limits of detection required in bioprocess impurity analysis. The assays are very robust with respect to protocol options and thus it is possible to modify the protocol to achieve performance parameters more optimal for your analytical needs. Sample volume, incubation times, and use of various sequential (forward or reverse sequential) schemes can lead to significant changes in sensitivity, reduction of non-specific sample matrix affects, or greater upper analytical range. If you change the protocol, it will be necessary to qualify that those changes achieve acceptable accuracy, specificity, and precision. Please refer to the section below on “How to Qualify HCP & Impurity Assays.” For advice on how best to modify an assay to meet your objectives you are encouraged to contact our Technical Services Department.

    产品插页中的 ELISA 方案可以修改吗?
    试剂盒产品插页中推荐的分析方案是在考虑到生物过程杂质分析所需的典型检测限的情况下开发的。 这些分析在方案选项方面非常稳健,因此可以修改方案以实现更适合您的分析需求的性能参数。 样品体积、孵育时间和各种顺序(正向或反向顺序)方案的使用会导致灵敏度显着变化、非特异性样品基质影响的减少或更高的分析范围。 如果您更改协议,则有必要确定这些更改达到可接受的准确性、特异性和精密度。 请参阅下面的“如何鉴定 HCP 和杂质检测”部分。 如需有关如何最好地修改检测以达到您的目标的建议,我们鼓励您联系我们的技术服务部门。

    Calibration of HCP assays

    The absolute quantitation of HCP assays is exceedingly difficult for several reasons. First of all, there is no recognized reference preparation of HCP against which we can calibrate our assays. While ELISA is inherently a quantitative method when applied to a single analyte, ELISAs that attempt to measure simultaneously all of the hundreds of potential HCP impurities in the same well using a single reporter/detector system are at best semi-quantitative assays. Many arbitrary choices and assumptions are used when preparing anti-HCP antibodies and later the choice of material to use when preparing “standards”.

    How to obtain the HCP material for immunogen is the first choice that must be made.

    • Do we use a null cell line or a mock transfected cell line or one with the actual product plasmid and what differences in HCP might we see from each? 
    • At what point in the purification process do we obtain the HCPs for the immunogen or standards?

    In almost every case the array and relative concentrations of HCPs in final product are different from the HCP in the ELISA standards.

    • How does one assign a total HCP concentration to an indeterminant mixture of many proteins with different molecular weights?
    • What effect will the different affinities of antibodies to various HCPs as well as the different concentrations of those antibodies have on the ability of the assay to be quantitative?
    • How do we know that we have antibody to all the HCPs present in a given sample type? 

    All of these questions and arbitrary choices mean HCP assays will at best be semi-quantitative methods capable of making only relative estimates of HCP concentrations from one sample to the next. We estimate the potential error in reporting an HCP level in a sample that has a different array of HCPs from the standards to be as high as 4-fold even if you have a very good broadly reactive antibody. The commonly expressed theoretical concern is that HCP assays might under-estimate results due to incomplete coverage of all HCPs. In reality, the quantitative errors in HCP assays due to the arbitrary choices and fundamental limitations of the method as discussed above can result in either over-estimation or under-estimation and tend to do so to about the same degree. In our experience, a well generated and affinity purified antibody will react to more than 70% of individual HCPs as demonstrated by traditional 2D Western blot correlated to silver stain. What is more important than the number of individual proteins with antibody reactivity, is the total mass of those proteins that are detected. In our experience and as you might expect from theoretical considerations, the proteins in highest concentration have the best chance of generating an antibody. As such, an antibody with about 70% reactivity to an individual HCP will react with those proteins that account for more than 95% of the total mass of HCP. Given this level of uncertainty in the absolute HCP concentration, the most important criteria by which we can judge any HCP assay is on other objective parameters by which any analytical method should be qualified or validated. Those criteria include specificity and accuracy as demonstrated by sample dilution linearity and spike recovery experiments, precision, robustness, and sensitivity. Provided the assay has sensitivity to detect at least a relative portion of the HCP in your final drug substance and the assay also meets the other objective analytical parameter specifications, such an assay is a valuable analytical method capable of demonstrating process control and reporting relative levels of HCP contamination from lot to lot as a release test.

    Due to the impracticality of obtaining real final product HCPs that co-purify with product down to the final step, most of our HCP assays will utilize a source of HCPs from very upstream in the purification process. Those HCPs typically do not come from the actual product cell line but rather a null cell or mock transfected cell line. Once we have decided on the source material for the kit standards we first perform some partial purifications to remove non-HCP materials such as growth media additives, buffer salts and extraction reagents. Our initial approach to calibrate this processed material is to simply perform a BCA total protein assay using bovine serum albumin (BSA) as an arbitrary standard. When standards made with the HCP material calibrated by BCA give reasonable “stoichiometric agreement” with the amounts of antibody used in the ELISA then we feel the calibration by BCA is good enough. What is meant by “stoichiometric agreement” is that we know how much antibody is used in the ELISA. It is the quantity of antibody that actually dictates the analytical range and dose response curve of the ELISA. If we assume an average molecular weight for the total HCPs, then we can reasonably estimate HCP concentrations across the valid analytical range of the assay. When the BCA assay concentrations do not reasonably approximate the ELISA stoichiometry, we process the HCP material further. Such processing involves various purification steps to remove components registering in the BCA assay but that are in fact not HCP or at least not immunoreactive HCPs. This processing might also involve affinity purification against the anti-HCP antibody. The purification stops as soon as the BCA concentration gives a realistic stoichiometric agreement in the ELISA.

    HCP 检测的校准
    由于几个原因,HCP 测定的绝对定量非常困难。首先,没有公认的 HCP 参考制剂可以用来校准我们的检测。虽然 ELISA 在应用于单个分析物时本质上是一种定量方法,但尝试使用单个报告器/检测器系统同时测量同一孔中的所有数百种潜在 HCP 杂质的 ELISA 充其量是半定量分析。在制备抗 HCP 抗体时使用了许多任意选择和假设,然后在制备“标准”时选择使用的材料。

    如何获得免疫原的HCP材料是必须要做的第一选择。

    我们是使用无效细胞系还是模拟转染细胞系,还是使用带有实际产品质粒的细胞系?我们可以从中看到 HCP 的哪些差异?
    我们在纯化过程中的哪个阶段获得免疫原或标准品的 HCP?
    在几乎所有情况下,最终产品中 HCP 的阵列和相对浓度都不同于 ELISA 标准中的 HCP。

    如何将总 HCP 浓度分配给许多具有不同分子量的蛋白质的不确定混合物?
    抗体对各种 HCP 的不同亲和力以及这些抗体的不同浓度对测定的定量能力有什么影响?
    我们如何知道我们对给定样本类型中存在的所有 HCP 都有抗体?
    所有这些问题和任意选择意味着 HCP 测定充其量只能是半定量方法,只能对从一个样品到下一个样品的 HCP 浓度进行相对估计。我们估计,即使您拥有非常好的广泛反应性抗体,在 HCP 阵列与标准不同的样本中报告 HCP 水平的潜在错误也会高达 4 倍。普遍表达的理论担忧是,由于对所有 HCP 的覆盖不完整,HCP 检测可能会低估结果。实际上,由于上​​述方法的任意选择和基本限制导致的 HCP 测定中的定量误差可能导致高估或低估,并且倾向于以大致相同的程度这样做。根据我们的经验,与银染色相关的传统 2D 蛋白质印迹证明,良好生成和亲和纯化的抗体将与 70% 以上的单个 HCP 发生反应。比具有抗体反应性的单个蛋白质的数量更重要的是检测到的这些蛋白质的总质量。根据我们的经验以及您可能从理论上的考虑,最高浓度的蛋白质最有可能产生抗体。因此,对单个 HCP 具有约 70% 反应性的抗体将与那些占 HCP 总质量 95% 以上的蛋白质发生反应。鉴于 HCP 绝对浓度的这种不确定性水平,我们判断任何 HCP 测定的最重要标准是其他客观参数,任何分析方法都应通过这些客观参数进行鉴定或验证。这些标准包括由样品稀释线性和加标回收实验证明的特异性和准确性、精密度、稳健性和灵敏度。如果该测定对检测最终原料药中至少一部分 HCP 具有敏感性,并且该测定还符合其他客观分析参数规范,则该测定是一种有价值的分析方法,能够证明过程控制并报告 HCP 的相对水平。批次间 HCP 污染作为放行测试。

    由于获得与产品共同纯化直至最后一步的真正最终产品 HCP 是不切实际的,我们的大多数 HCP 测定将利用纯化过程中非常上游的 HCP 来源。这些 HCP 通常不是来自实际的产品细胞系,而是来自空细胞或模拟转染细胞系。一旦我们确定了试剂盒标准的来源材料,我们首先会进行一些部分纯化以去除非 HCP 材料,例如生长培养基添加剂、缓冲盐和提取试剂。我们校准这种加工材料的初始方法是使用牛血清白蛋白 (BSA) 作为任意标准简单地执行 BCA 总蛋白测定。当使用 BCA 校准的 HCP 材料制成的标准与 ELISA 中使用的抗体量给出合理的“化学计量一致性”时,我们认为 BCA 的校准已经足够好。 “化学计量一致性”的意思是我们知道在 ELISA 中使用了多少抗体。抗体的数量实际上决定了 ELISA 的分析范围和剂量反应曲线。如果我们假设总 HCP 的平均分子量,那么我们可以合理地估计有效分析范围内的 HCP 浓度的化验。 当 BCA 测定浓度不能合理地接近 ELISA 化学计量时,我们会进一步处理 HCP 材料。 这种处理涉及各种纯化步骤,以去除在 BCA 测定中登记但实际上不是 HCP 或至少不是免疫反应性 HCP 的成分。 该处理还可能涉及针对抗 HCP 抗体的亲和纯化。 一旦 BCA 浓度在 ELISA 中给出真实的化学计量一致,纯化就会停止。

    What is the Storage and Stability of ELISA Kits?

    Cygnus kits and reagents have been formulated to withstand shipment and/or temporary storage under ambient temperature conditions without any significant deterioration. Orders within North America are shipped next day delivery service without cold packs. International orders are shipped with cold packs to protect from extreme heat.  Although stability studies have demonstrated that Cygnus kits can be held several weeks at ambient temperatures without any significant deterioration, we recommend storage at 2-8°C upon receipt of the products.   Our components have been challenged at elevated temperature excursions, addressing concerns over extreme summertime temperatures, delays in shipping, custom clearances and/or refrigeration problems at the customer locations.

    Should you have concerns regarding the performance of kits that have been subject to temperature deviations, we recommend an analysis of your control samples and historical specifications that determine the integrity of the kit. If the curve absorbances, sensitivity, and controls are within range, then the kit is acceptable for use.

    ELISA 试剂盒的储存和稳定性如何?
    Cygnus 试剂盒和试剂经过精心配制,可在环境温度条件下经受运输和/或临时储存,而不会出现任何显着变质。北美地区的订单在没有冷包的情况下第二天发货。国际订单随附冷袋以防止过热。虽然稳定性研究表明 Cygnus 套件可以在环境温度下保存数周而不会出现任何明显的劣化,但我们建议在收到产品后将其储存在 2-8°C。我们的组件在高温偏移中面临挑战,解决了对夏季极端温度、运输延迟、海关清关和/或客户所在地制冷问题的担忧。

    如果您对受温度偏差影响的试剂盒的性能有疑虑,我们建议您分析您的对照样品和确定试剂盒完整性的历史规格。如果曲线吸光度、灵敏度和对照在范围内,则可以使用该试剂盒。

    Which Cygnus CHO HCP kit to use: F015, CM015, F550 or F550-1?

    Cygnus currently offers 3 ELISA kits to measure CHO HCPs. Kit F015, the first kit developed more than 18 years ago, utilized a mild lysate of washed CHO cells for the immunogen and for affinity purification of the antibody, as well as for kit standards. This mild lysis procedure generated an array of HCPs similar to those found in culture media after cells have been grown to densities and viabilities similar to product harvest. Cygnus continues to support this kit as many companies have qualified it for their process. However, for first time users we suggest you first try our F550-1 3rd Generation CHO ELISA kit as it has been qualified for many products and has proven to be a broadly reactive generic kit able to detect downstream HCPs in most processes.

    Kit CM015 was developed 16 years ago with the rationale that since most CHO expressed products are released into the growth media (conditioned media) an antibody and/or assay made to those HCPs found in conditioned media should be more specific than a cell lysate assay. However, the CM015 kit has been demonstrated to be more process specific and thus is not a generic kit that might be applied across most products and processes.

    The F550 3rd Generation kit used an even more reactive antibody than earlier kits and incorporates significant improvements in the assay methodology. The antibody used in that kit has been evaluated for coverage by orthogonal method such as Antibody Affinity Extraction (AAE) with 2D-PAGE/Silver Stain. The F550 kit, when supported by comprehensive qualification of the ELISA itself along with data from orthogonal methods like AAE and Mass Spectrometry, has been successfully used as a lot release test in several processes. Due to the depletion of anti-CHO HCP Antibody used in F550 kit, this kit has been replaced with the resupply F550-1 CHO HCP ELISA, 3G.

    Starting from January 1, 2021, the 3rd generation CHO HCP ELISA Kit, Item Number F550-1, replaced CHO HCP ELISA Kit, 3G, Item Number F550. Cygnus manufactured, characterized, and qualified large pools of the new reagents that replaced reagents used in F550 kit. The new antibodies were developed and purified in the same way as the original critical reagents in the F550 Kit. The coverage of the new antibodies to the CHO HCP antigen was determined to be ~86% by Antibody Affinity Extraction with 2D-PAGE Silver Stain and 97% by AAE with MS. The antibody in F550-1 kit are very similar to the antibody in the depleted F550 kit. More importantly, Cygnus Technologies has demonstrated the equivalency of the new antibodies (3G-0016-1-AF) supporting 3G CHO ELISA Kit (F550-1) using a panel of CHO samples from a number of independent biomanufacturing processes. Download Cygnus Case Study comparing reactivity of anti-CHO HCP Antibody from F550 (3G-0016-AF) and F550-1 (3G-0016-1-AF) to CHO HCPs.

    Contact our expert technical staff to determine if the F550-1 kit is satisfactory for your product and process or if a process-specific method is needed.

    使用哪个 Cygnus CHO HCP 试剂盒:F015、CM015、F550 或 F550-1?
    Cygnus 目前提供 3 种 ELISA 试剂盒来测量 CHO HCP。试剂盒 F015 是 18 多年前开发的第一个试剂盒,它利用洗涤过的 CHO 细胞的温和裂解物作为免疫原和抗体的亲和纯化以及试剂盒标准品。这种温和的裂解程序在细胞生长到与产品收获相似的密度和活力后,产生了一系列与培养基中发现的 HCP 相似的 HCP。 Cygnus 继续支持该套件,因为许多公司已经对其流程进行了认证。但是,对于初次使用的用户,我们建议您首先尝试我们的 F550-1 第三代 CHO ELISA 试剂盒,因为它已通过许多产品的认证,并且已被证明是一种广泛反应的通用试剂盒,能够在大多数过程中检测下游 HCP。

    试剂盒 CM015 是 16 年前开发的,其基本原理是,由于大多数 CHO 表达产物被释放到生长培养基(条件培养基)中,因此对条件培养基中发现的 HCP 进行的抗体和/或测定应该比细胞裂解物测定更具特异性。然而,CM015 套件已被证明更具工艺特异性,因此不是可应用于大多数产品和工艺的通用套件。

    F550 第三代试剂盒使用比早期试剂盒更具反应性的抗体,并在检测方法上进行了重大改进。该试剂盒中使用的抗体已通过正交方法评估覆盖率,例如使用 2D-PAGE/Silver Stain 进行抗体亲和提取 (AAE)。 F550 试剂盒在 ELISA 本身的全面鉴定以及来自 AAE 和质谱等正交方法的数据的支持下,已成功用作多个过程中的批放行测试。由于 F550 试剂盒中使用的抗 CHO HCP 抗体耗尽,该试剂盒已替换为补给 F550-1 CHO HCP ELISA, 3G。

    从 2021 年 1 月 1 日起,第三代 CHO HCP ELISA 试剂盒,货号 F550-1,取代了 CHO HCP ELISA 试剂盒,3G,货号 F550。 Cygnus 制造、表征和鉴定了大量新试剂,以取代 F550 试剂盒中使用的试剂。新抗体的开发和纯化方法与 F550 试剂盒中的原始关键试剂相同。使用 2D-PAGE Silver Stain 进行抗体亲和提取确定新抗体对 CHO HCP 抗原的覆盖率约为 86%,而 AAE 和 MS 的覆盖率确定为 97%。 F550-1 试剂盒中的抗体与耗尽的 F550 试剂盒中的抗体非常相似。更重要的是,Cygnus Technologies 使用来自多个独立生物制造过程的一组 CHO 样本证明了支持 3G CHO ELISA 试剂盒 (F550-1) 的新抗体 (3G-0016-1-AF) 的等效性。下载 Cygnus 案例研究,比较来自 F550 (3G-0016-AF) 和 F550-1 (3G-0016-1-AF) 的抗 CHO HCP 抗体与 CHO HCP 的反应性。

    请联系我们的专业技术人员以确定 F550-1 套件是否适合您的产品和工艺,或者是否需要特定工艺的方法。