Vero 细胞 HCP(宿主残留蛋白)ELISA试剂盒说明书分析
HCP残余检测
专属性试剂盒开发根源：宿主细胞的分泌型蛋白和部分死亡细胞释放的结构蛋白成分复杂，不同的工艺，加上相关基因产物需要经过独特的翻译后修饰，这些都大大增加了HCP的品种数量和生化复杂性指导原则：中检院曾指出过“对于不同的目的蛋白质，根据分子量大小和等电点的不同，会采取不同的分离纯化工艺。
不同的分离纯化工艺会导致细胞蛋白残留量和组成上的较大差异。
只有根据不同的生产工艺，制备工艺特异的细胞蛋白标准品和特异的抗血清，建立相应的检测方法，才能更真实反应供试品的’细胞蛋白残留量。
”覆盖率验证：Bio-Rad Laboratories公司发布的HCP残留检测验证流程使用优化的2D分离条件与转印方法可有效分离检测CHO、酵母、大肠杆菌等细胞系的全蛋白谱。
将2D Gel上的全蛋白转移至膜上，经ELISA试剂盒（商品化通用型或自制工艺特异型）内多克隆抗体孵育后进行Western Blot化学发光检测，以确认ELISA试剂盒中的多克隆抗体能够与哪些宿主细胞蛋白结合。
通过Western Blot检测结果（多抗能检测到的HCP数量）与2D膜上检测结果（总HCP数量）的比较，即得出用于评价ELISA试剂盒特异性及适用性的Match Rate。
2D-WB法的局限性▪灵敏度低，只适用于上游样品的分析；▪样品需要经过变性处理，一些表位可能会被破坏而不能被抗体识别；▪银染的胶与WB膜只能通过人工匹配操作，存在人为误差；▪银染与WB的灵敏度本身存在差异，导致可信度不够高；方法，抗体的制备：一种是将空细胞按照已定的生产工艺进行发酵和纯化，然后将获得的东西免疫动物制备抗体。
还有一种就是将工艺获得原液去除抗体后再免疫动物。
标准品的制备：使用空载质粒转化到宿主细胞，按已定的生产工艺进行发酵纯化，纯化不需要走完纯化流程，可以走纯化流程的前几步即可。
Cygnus除了传统的2D-WB的验证服务以外，还独家推出的更高特异性验证方法，Cygnus还独家研发更高特异性检测的方法：AAE（Antibody Affinity Extraction）。
Capto Core400说明书
Capto Core400说明书
Capto Core 400：去除宿主细胞蛋白(hostcell protein，HCP)和DNA，并且比较纯化结果及工艺参数。
方法:用Capto Core 400与Sepharose 6FF纯化Vero细胞培养的Ⅰ、Ⅱ、Ⅲ型sPV浓缩液。
分别检测纯化后sPV的D抗原含量、HCP残留量和DNA残留量，计算D抗原回收率、HCP和DNA去除率，并用t检验分析差异。
结果:Capto Core 400与Sepharose 6FF纯化Ⅰ、Ⅱ、Ⅲ型sPV的D抗原回收率差异均无统计学意义( t值分别为1.09、1.08、1.02，P值均>0.05)，但前者的Vero细胞HCP( t值分别为3.15、3.23、3.54)和DNA 去除率均高于后者( t值分别为3.41、3.25、3.62)，且差异有统计学意义( P值均<0.05)。
Capto Core 400与Sepharose 6FF的介质使用体积比值为0.05，上样后纯化时间比值为0.04，工作缓冲液使用量比值为0.25，工艺线性流速比值为10，每升介质上样量比值为20，最大耐压力比值为2。
结论:对比Capto Core 400可以更有效地去除Vero细胞HCP与DNA，各个工艺参数得到了优化，提高了sPV层析纯化的工作效率，并节约了时间和成本。
贝克曼库尔特 Vero 细胞宿主细胞蛋白免疫酶联免疫吸附试验盒用于测量 Vero 细胞宿主细胞蛋白说
Vero Cell Host Cell Proteins Immunoenzymetric Assay for the Measurement of Vero Cell Host Cell ProteinsCatalog # F500Intended UseThis kit is intended for use in determining the presence of host cell protein impurities in products manufactured by expression in Vero cells. The kit is for Research and Manufacturing Use Only and is not intended for diagnostic use in humans or animals.Summary and ExplanationExpression of viral vaccines and other therapeutic proteins in Vero cells is a cost effective method for production of commercial quantities of a drug substance. The manufacturing and purification process of these products leaves the potential for impurities by host cell proteins (HCPs) from Vero cells. Such impurities can reduce the efficacy of the therapeutic agent and result in adverse toxic or immunological reactions and thus it is desirable to reduce HCP impurities to the lowest levels practical.Immunological methods using antibodies to HCPs such as Western Blot and ELISA are conventionally accepted. While Western blot is a useful method aiding in the identity of HCPs, it suffers from a number of limitations. Western blot is a complex and technique dependent procedure requiring subjective interpretation of results. Furthermore, it is essentially a qualitative method and does not lend itself to obtaining quantitative answers. The sensitivity of Western blot is severely limited by the volume of sample that can be tested and by interference from the presence of high concentrations of the intended product. While Western Blot may be able to detect HCPs in samples from upstream in the purification process, it often lacks adequate sensitivity and specificity to detect HCPs in purified downstream and final product. The microtiter plate immunoenzymetric assay (ELISA) method employed in this kit overcomes the limitations of Western blots providing on the order of 100 fold better sensitivity. This simple to use, objective, and semi-quantitative ELISA is a powerful method to aid in optimal purification process development, process control, routine quality control, and product release testing. This kit is “generic” in the sense that it is intended to react with essentially all of the HCPs that could pollute the product independent of the purification process. The antibodies have been generated against and affinity purified using mild lysate of Vero cells. The resulting antibodies have then been characterized against four commercial cell lines used to produce various viral and protein products. This analysis indicated the vast majority of HCPs are conserved among multiple Vero cell lines and product purification processes. If you have a need of a more sensitive method to demonstrate coverage to HCPs in your process Cygnus Technologies recommends a method that is superior to Western blot called Antibody Affinity Extraction (AAE). AAE is has greatly increased sensitivity and specificity to Western blot which makes it a better predictor of how the antibodies will perform in the ELISA. For additional information on AAE please visit our website and read the posted articles under Technical Documents or contact our Technical Services Department.Special procedures were utilized in the generation of these antibodies to ensure that low molecular weight and less immunogenic impurities as well as high molecular weight components would be represented. As such, this kit can be used as a process development tool to monitor the optimal removal of host cell impurities as well as in routine final product release.This highly sensitive ELISA kit has been qualified for testing of final product HCPs using actual in-process and final drug substance samples from 4 vaccine products all with somewhat different growth and purification processes. Each user of this kit is encouraged to perform a similar qualification study to demonstrate it meets their analytical needs. Provided this kit can be satisfactorily qualified for your samples, the application of a more process specific assay may not be necessary, in that such an assay would only provide information redundant to this generic assay. However, if your qualification studies indicate the antibodies in this kit are not sufficiently reactive with your process specific HCPs it may be desirable to also develop a more process specific ELISA. This later generation assay may require the use of a more specific and defined antisera. Alternatively, if the polyclonal antibody used in this kit provides sufficient sensitivity and broad antigen reactivity, it may be possible to substitute the standards used in this kit for ones made from the impurities that typically co-purify through your purification process and thus achieve better accuracy for process specific HCPs. The use of a process specific assay with more defined antigens and antibodies in theory may yield better specificity, however such an assay runs the risk of beingtoo specific in that it may fail to detect new or atypical impurities that might result from some process irregularity or change. For this reason it is recommendedth at a broadly reactive “generic” host cell protein assay be used as part of the final product purity analysis even when a process specific assay is available. If you deem a more process specific assay is necessary, Cygnus Technologies is available to apply its proven technologies to develop such antibodies and assays on custom basis.The Vero cell assay is a two-site immunoenzymetric assay. Samples containing Vero cell HCPs are reacted simultaneously with a horseradish peroxidase (HRP) enzyme labeled anti-Vero cell antibody (goat polyclonal) in microtiter strips coated with an affinity purified capture goat polyclonal anti-Vero cell antibody. The immunological reactions result in the formation of a sandwich complex of solid phase antibody-HCP-enzyme labeled antibody. The microtiter strips are washed to remove any unbound reactants. The substrate, tetramethylbenzidine (TMB) is then reacted. The amount of hydrolyzed substrate is read on a microtiter plate reader and is directly proportional to the concentration of Vero cell HCPs present.Storage & Stability•All reagents should be stored at 2︒C to 8︒C for stability until the expiration date printed on the kit. •After prolonged storage, you may notice a salt precipitate and/or yellowing of the washconcentrate. These changes will not impact assayperformance. To dissolve the precipitate, mix thewash concentrate thoroughly and dilute asdirected in the ‘Preparation on Reagents’ section. •Reconstituted wash solution is stable until the expiration date of the kit. •Microtiter plate reader spectrophotometer with dual wavelength capability at 450 & 650nm. (Ifyour plate reader does not provide dualwavelength analysis you may read at just the450nm wavelength.)•Pipettors - 50μL and 100μL•Repeating or multichannel pipettor - 100μL •Microtiter plate rotator (400 - 600 rpm) •Sample Diluent (recommended Cat # I028) •Distilled water• 1 liter wash bottle for diluted wash solution•For Research or Manufacturing use only. •Stop reagent is 0.5M H2SO4. Avoid contact with eyes, skin, and clothing.•This kit should only be used by qualified technicians.•Bring all reagents to room temperature. •Dilute wash concentrate to 1 liter in distilled water, label with kit lot and expiration date, and store at4︒C.Assay Protocol•The assay is very robust such that assay variables like incubation times, sample size, and othersequential incubation schemes can be altered tomanipulate assay performance for moresensitivity, increased upper analytical range, orreduced sample matrix interference. Beforemodifying the protocol from what is recommended,you are advised to contact Technical Services forinput on the best way to achieve your desiredgoals.•The protocol specifies use of an approved orbital microtiter plate shaker for the immunologicalsteps. These can be purchased from mostlaboratory supply companies. If you do not havesuch a device, it is possible to incubate the platewithout shaking however, it will be necessary toextend the immunological incubation step in theplate by about one hour in order to achievecomparable results to shaking protocol. Do notshake during the 30-minute substrateincubation step, as this may result in higherbackgrounds and worse precision.800-F500, Rev. 3, 26DEC2019 Vero Cell HCP ELISA Product Insert 2•Bring all reagents to room temperature. Set-up plate spectrophotometer to read dual wavelengthat 450nm for the test wavelength and ~650nm forthe reference.•Thorough washing is essential to proper performance of this assay. Automated platewashing systems or other vacuum aspirationdevices are not recommended. The manualmethod described in the assay protocol ispreferred for best precision, sensitivity andaccuracy. A more detailed discussion of thisprocedure can be obtained from our TechnicalServices Department or on our web site. Inaddition, a video demonstration of proper platewashing technique is available in the ‘TechnicalHelp’ section of our we b site.•All standards, controls, and samples should be assayed at least in duplicate.•Maintain a repetitive timing sequence from well to well for all assay steps to ensure that all incubationtimes are the same for each well.•Make a work list for each assay to identify the location of each standard, control, and sample. •It is recommended that your laboratory assay appropriate quality control samples in each run toensure that all reagents and procedures arecorrect. You are strongly urged to makecontrols in your typical sample matrix usingHCPs derived from your cell line. Thesecontrols can be aliquoted into single use vialsand stored frozen for long-term stability.•If the substrate has a distinct blue color prior to assay it may have been contaminated. If theabsorbance of 100μL of substrate plus 100μL ofstop against a water blank is greater than 0.1 itmay be necessary to obtain new substrate or thesensitivity of the assay may be compromised. •Strips should be read within 30 minutes after adding stop solution since color will fade over time.Limitations•Before relying exclusively on this assay to detect host cell proteins, each laboratory should qualifythat the kit antibodies and assay procedure yieldacceptable specificity, accuracy, and precision. Asuggested protocol for this qualification can beobtained from our Technical Services Departmentor our web site.•The standards used in this assay are comprised of Vero cell HCPs solubilized by methods commonlyused in initial harvesting steps for vaccineproducts. 1D Western blot analysis of theantibodies used in this kit demonstrates that theyrecognize the majority of distinct PAGE separatedbands seen using sensitive protein stainingmethods like silver stain or colloidal gold. Becausethe majority of HCPs will show sufficient antigenicconservation among all lines of Vero cells this kitshould be adequately reactive to HCPs from yourcell line. However, there can be no guarantee thatthis assay will detect all proteins or proteinfragments from your process. If you desire a much800-F500, Rev. 3, 26DEC2019 Vero Cell HCP ELISA Product Insert 3more sensitive method than western blot to detectthe reactivity of the antibodies in this kit to yourindividual HCPs Cygnus Technologies is pleasedto perform AAE as a service to provide coverageinformation of the antibodies to the HCPs in yourprocess samples.•Certain sample matrices may interfere in this assay. The standards used in this kit attempt tosimulate typical sample protein and matrices.However, the potential exists that the product itselfor other components in the sample matrix mayresult in either positive or negative interference inthis assay. High or low pH, detergents, urea, highsalt concentrations, and organic solvents are someof the known interference factors. It is advised totest all sample matrices for interference by dilutingthe 200ng/mL standard, 1 part to 4 parts of thematrix containing no or very low HCP impurities.This diluted standard when assayed as anunknown, should give an added HCP value in therange of 30 to 50 ng/mL. Consult CygnusTechnologies Technical Service Department foradvice on how to quantitate the assay inproblematic matrices.•Avoid the assay of samples containing sodium azide (NaN3) which will destroy the HRP activity ofthe conjugate and could result in the under-estimation of HCP levels.1. Complete washing of the plates to remove excess unreacted reagents is essential to good assay reproducibility and sensitivity. We advise against the use of automated or other manually operated vacuum aspiration devices for washing plates as these may result in lower specific absorbances, higher non-specific absorbance, and more variable precision. The manual wash procedure described below generally provides lower backgrounds, higher specific absorbance, and better precision. If duplicate CVs are poor, or if the absorbance of the 0 standard is greater than 0.200, evaluate plate washing procedure for proper performance.2. High Dose Hook Effect or poor dilutional linearity may be observed in samples with very high concentrations of HCP. High Dose Hook Effect is due to insufficient excess of antibody for very high concentrations of HCPs present in samples upstream in the purification process. Samples greater than 1 mg/mL may give absorbances less than the 200 ng/mL standard. It is also possible for samples to have certain HCPs in concentrations exceeding the amount of antibody for that particular HCP. In such cases the absorbance of the undiluted sample may be lower than the highest standard in the kit, however these samples will fail to show acceptable dilutional linearity/ parallelism as evidenced by an apparent increase in dilution corrected HCP concentration with increasing dilution. High Dose Hook and poor dilutional linearity are most likely to be encountered from samples early in the purification process. If a hook effect is possible, samples should also be assayed diluted. If the HCP concentration of the undiluted sample is less than the diluted sample this may be indicative of the hook effect. Such samples should be diluted at least to the minimum required dilutions (MRDs) as established by your qualification studies using your actual final and in-process drug samples. The MRD is the first dilution at which all subsequent dilutions yield the same HCP value within the statistical limits of assay precision. The HCP value to be reported for such samples is the dilution corrected value at or greater than the established MRD. The diluent used should be compatible with accurate recovery. The preferred diluent is our Cat# I028 available in 100mL, 500mL, or 1 liter bottles. This is the same material used to prepare the kit standards. As the sample is diluted in I028, its matrix begins to approach that of the standards, thus reducing any inaccuracies caused by dilutional artifacts. Other prospective diluents must be tested for non-specific binding and recovery by using them to dilute the 200ng/mL standard, as describ ed in the “Limitations” section below.•Precision on duplicate samples should yield average % coefficients of variation of less than10% for samples in the range of 8-200ng/mL. CVsfor samples less than 8 ng/mL may be greaterthan 10%.•It is recommended that each laboratory assay appropriate quality control samples in each run toensure that all reagents and procedures arecorrect.The standards may be used to construct a standard curve with values reported in ng/m L “total immuno-reactive HCP equivalents”. This data reduction may be performed through computer methods using curve fitting routines such as point-to-point, cubic spline, or 4 parameter logistic fit. Do not use linear regression analysis to interpolate values for samples as this may lead to significant inaccuracies! Data may also be manually reduced by plotting the absorbance values of the standard on the y-axis versus concentration on the x-axis and drawing a smooth point-to-point line. Absorbances of samples are then interpolated from this standard curve.800-F500, Rev. 3, 26DEC2019 Vero Cell HCP ELISA Product Insert 4800-F500, Rev. 3, 26DEC2019 Vero Cell HCP ELISA Product Insert 5Performance CharacteristicsCygnus Technologies has qualified this assay by conventional criteria as indicated below. A copy of this qualification report can be obtained on our web site or by request. This qualification is generic in nature and is intended to supplement but not replace certain user and product specific qualification and qualification that should be performed by each laboratory. At a minimum each laboratory is urged to perform a spike and recovery study in their sample types. In addition, any of your samples types containing process derived HCPs within or above the analytical range of this assay should be evaluated for dilutional linearity to ensure that the assay is accurate and has sufficient antibody excess for your particular HCPs. Each laboratory and technician should also demonstrate competency in the assay by performing a precision study similar to that described below. A more detailed discussion of recommended user qualification protocols can be obtained by contacting our Technical Services Department or at our web site.SensitivityThe lower limit of detection (LOD ) is defined as that concentration corresponding to a signal two standard deviations above the mean of the zero standard. LOD is ~0.7 ng/mL.The lower limit of quantitation (LOQ ) is defined as the lowest concentration, where concentration coefficients of variation (CVs) are less than 20%. The LOQ is less than 2 ng/mL.PrecisionBoth intra (n=20 replicates) and inter-assay (n=10 assays) precision were determined on 3 pools with low (~8ng/mL), medium (~25ng/mL), and high concentrations (~75ng/mL). The % CV is the standard deviation divided by the mean and multiplied by 100.Specificity/Cross-Reactivity1D Western blot and ELISA analysis against 4commercial Vero cell strains indicate that most of the proteins are conserved among all cell lines. Therefore, this assay should be useful for detecting HCPs from other Vero cell lines. Western blot, both 1 & 2dimensional, is highly orthogonal to ELISA and to non-specific protein staining methods such as silver stain or colloidal gold. As such, the lack of identity between silver stain and western blot does not necessarily mean there is not antibody to that protein or that the ELISA will not detect that protein. If you desire a much more sensitive and specific method than western blot to detect the reactivity of the antibodies in this kit to your individual HCPs Cygnus Technologies is pleased to perform AAE as a service to provide coverage information of the antibodies to the HCPs in your process samples. This method has been shown to be much more sensitive and specific than Western blots in detecting antibody reactivity to individual HCPs. The same antibody as is used for both capture and HRP label can be purchased separately as Cat# VC 807-AF.Cross reactivity to non-HCP components has not been extensively investigated with this kit. You should evaluate components in your samples for positive interferences such as cross reactivity and non-specific binding. Negative interference studies are described below.Recovery/ Interference StudiesVarious buffer matrices commonly used in purification and final formulation of drug substances expressed in Vero cells were evaluated by adding known amounts of Vero cell HCP preparation used to make the standards in this kit. Because this assay is designed to minimize matrix interference most of these buffers yielded acceptable recovery defined as between 80-120%. The standards used in this kit contain 8mg/mL of bovine serum albumin intended to simulate non-specific protein affects of most sample proteins or virus products. However very high concentrations of some products may interfere in the accurate measurement of HCPs. In general, extremes in pH (less than 5.0 and greater than 8.5), high salt concentration, high polysaccharide concentrations, and most detergents can cause under-recovery. Each user should qualify that their sample matrices yield accurate recovery. Such an experiment can be performed, by diluting the 200ng/mL standard provided with this kit, into the sample matrix in question as described in the “Limitations” section. CygnusTechnologies offers a more concentrated form of theHCP (Cat # F503H at 100μg/mL) used to prepare thekits standards for your spike recovery and preparation ofanalyte controls.Hook CapacityIncreasing concentrations of HCPs greater than 200ng/mL were assayed as unknowns. The hook capacity,defined as that concentration yielding an absorbancereading less than the 200 ng/mL standard was greaterthan 1000 μg/mL.Cygnus Technologies also offers kits for the extractionand detection of CHO Host Cell DNA. The following kitsare available:•Residual Host Cell DNA extraction:Cat # D100W, DNA Extraction Kit in 96 deep well plateCat # D100T, DNA Extraction Kit in microfuge tubesTo place an order or to obtain additional productinformation contact Cygnus Technologies:Cygnus Technologies, LLC4332 Southport Supply Rd. SESouthport, NC 28461 USATel: 910-454-9442Email for all Order inquiries:*****************************Email for Technical Support:**********************************_____________________________________________800-F500, Rev. 3, 26DEC2019 Vero Cell HCP ELISA Product Insert 6。
Vero 细胞 HCP(宿主残留蛋白)ELISA试剂盒说明书分析
Vero 细胞HCP（宿主细胞蛋白）Vero细胞宿主蛋白酶联免疫检测目录#F500预期用途该试剂盒用于检测用Vero 细胞.生产的制品中是否有宿主蛋白残留。
仅供研究和工业生产，不能用于人和动物的诊断。
总结与说明病毒疫苗或其他治疗用蛋白在Vero细胞中表达使商业用大量生产药物原料产品的经济简便方法。
生产和纯化这些产品的过程中会残留Vero细胞中的一些蛋白质（称为宿主细胞蛋白，HCPs）而造成污染。
这种污染可以减少药物的疗效，引发毒副反应和免疫学反应，因此最好在实际生产制品过程中将HCPs降低到最低水平。
一些利用抗体来除HCPs的免疫学方法，如Western Blot 和ELISA被广泛使用。
虽然Western blot是一种检测HCPs的有效方法，但它受到了一些限制。
因为它过程复杂且技术依赖性强，需要操作者对结果进行分析解释。
而且，它在本质上是一种定性检测，不能定量分析。
Western blot的敏感性易受被检测样品量的影响，也受目的产品浓度的干扰。
因此Western blot可用来检测纯化上游的蛋白质，而对纯化下游或终产品的检测灵敏度与特异性较低。
本试剂盒中用到的ELISA方法克服了Western Blot的缺点，将敏感度提高了100倍。
ELISA操作简单、客观、可获得半定量的结果，是纯化工艺，过程控制，常规质量检测的最佳选择。
这个试剂盒可与纯化过程中残留的可独立污染产品的所有HCPs反应，就这个意义上来说，这个试剂盒是通用的。
用Vero细胞轻度裂解物获得抗体并经亲和纯化，得到的最终抗体可以与用于生产各种病毒疫苗和蛋白质产品的四种商用细胞系反应。
这一分析表明，绝大多数HCP存在于各种Vero细胞系和纯化过程中。
如果你需要一个更为敏感和特异性的方法去检测样品中的HCP量，Cygnus Technologies公司为你推荐一个优于2D Western blot的方法，我们将这个方法称为2D HPLC-ELISA。
HCP宿主细胞蛋白
百泰派克生物科技HCP宿主细胞蛋白宿主细胞蛋白（HCP，Host Cell Protein）是在生物制药（如生产重组蛋白药物、单克隆抗体）工艺过程中所产生的杂质，其超过一定含量则很有可能会引起免疫反应或其他不良反应。
因此，HCP残留量检测是抗体药物产品研发与质控中最常进行的分析项目，HCP残留数值越低，则产品质控越好。
宿主细胞蛋白（HCP）是由宿主细胞产生的与工艺相关的杂质，通常在重组生物制药产品中的含量较低，即使HCP杂质的总含量很低同样也可能对药物的性质造成不良影响。
对于患者而言，HCP是异源蛋白，通常大部分异源蛋白都可能具有免疫原性，因此即使很低浓度的HCP也可能引发不良免疫反应。
宿主细胞的HCP中也包含一些蛋白水解活性酶，可使目的蛋白发生片段化，从而导致总产品降低。
除了会导致目的蛋白降解和片段化以外，HCP还会导致蛋白聚集等种种不良影响。
因此，需对产品中的HCP进行监测和控制，以支持对治疗性蛋白产品中HCP相关风险的评估和控制。
通常，在治疗性蛋白的生产中用得最多的HCP检测方法为酶联免疫吸附试验（ELISA），该方法可以采用多克隆抗体直接量化分析HCP的总体丰度。
然而，一般无法采用此法快速定量分析单个HCP组分，且可能检测不到免疫原性较弱或非免疫原性的HCP。
当前已开发出多种互补的分析用于监测HCP，包括1D/2D-PAGE、基于质谱（MS）的等分析技术。
其中，液相色谱-串联质谱联用法（LC-MS/MS）可同时鉴定和定量分析HCP杂质，是ELISA的主要互补分析方法。
百泰派克生物科技提供生物制药分析服务，其中包括宿主蛋白残留（HCP）分析服务。
百泰派克生物科技有经验丰富的技术人员，可以提供从实验设计、样品检测、数据分析等全套专业服务，可精准检测生物药物可能残留的宿主蛋白。
大肠杆菌宿主残留蛋白(E.coli P)-NEWA
本试剂盒只能用于科学研究，不得用于医学诊断大肠杆菌宿主残留蛋白（E.coli P）ELISA检测试剂盒使用说明书检测原理试剂盒采用双抗体一步夹心法酶联免疫吸附试验（ELISA）。
往预先包被大肠杆菌宿主残留蛋白（E.coli P）抗体的包被微孔中，依次加入标本、标准品、HRP标记的检测抗体，经过温育并彻底洗涤。
用底物TMB显色，TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。
颜色的深浅和样品中的大肠杆菌宿主残留蛋白（E.coli P）呈正相关。
用酶标仪在450nm 波长下测定吸光度（OD 值），计算样品浓度。
样品收集、处理及保存方法1. 血清：使用不含热原和内毒素的试管，操作过程中避免任何细胞刺激，收集血液后，3000转离心10分钟将血清和红细胞迅速小心地分离。
2. 血浆：EDTA、柠檬酸盐或肝素抗凝。
3000转离心30分钟取上清。
3. 细胞上清液：3000转离心10分钟去除颗粒和聚合物。
4. 组织匀浆：将组织加入适量生理盐水捣碎。
3000转离心10分钟取上清。
5. 保存：如果样本收集后不及时检测，请按一次用量分装，冻存于-20℃，避免反复冻融，在室温下解冻并确保样品均匀地充分解冻。
自备物品1.酶标仪（450nm）2.高精度加样器及枪头：0.5-10uL、2-20uL、20-200uL、200-1000uL3.37℃恒温箱操作注意事项1. 试剂盒保存在2-8℃，使用前室温平衡20分钟。
从冰箱取出的浓缩洗涤液会有结晶，这属于正常现象，水浴加热使结晶完全溶解后再使用。
2. 实验中不用的板条应立即放回自封袋中，密封（低温干燥）保存。
3. 浓度为0的S0号标准品即可视为阴性对照或者空白；按照说明书操作时样本已经稀释5倍，最终结果乘以5才是样本实际浓度。
4. 严格按照说明书中标明的时间、加液量及顺序进行温育操作。
5. 所有液体组分使用前充分摇匀。
试剂盒组成名称96孔配置48孔配置备注微孔酶标板12孔×8条12孔×4条无标准品0.3mL*6管0.3mL*6管无样本稀释液6mL 3mL 无检测抗体-HRP 10mL 5mL 无20×洗涤缓冲液25mL 15mL 按说明书进行稀释底物A 6mL 3mL 无底物B 6mL 3mL 无终止液6mL 3mL 无封板膜2张2张无说明书1份1份无自封袋1个1个无注：标准品（S0-S5）浓度依次为：0、5、10、20、40、80 ng/mL试剂的准备20×洗涤缓冲液的稀释：蒸馏水按1：20稀释，即1份的20×洗涤缓冲液加19份的蒸馏水。
Vero细胞疫苗中宿主细胞蛋白(HCP)检测研究简介
发布日期20040709栏目生物制品评价>>生物制品质量控制标题Vero细胞疫苗中宿主细胞蛋白（HCP）检测研究简介作者田博部门正文内容Vero细胞疫苗中宿主细胞蛋白（HCP）检测研究简介生物制品组田博Vero细胞HCP是指疫苗中来源于宿主细胞的蛋白成分，其主要成分包括：宿主细胞结构蛋白和转化蛋白（细胞分泌的促生长蛋白），前者的危险性在于引起机体过敏反应，后者的潜在危险性在于引起细胞转化。
以Vero细胞为基质细胞生产疫苗的过程是将病毒接种于Vero细胞，病毒利用细胞环境复制自身，同时导致宿主细胞结构受损、死亡，释放大量的宿主细胞结构蛋白于疫苗中，这部分蛋白有可能成为疫苗中的过敏原；Vero细胞生长的同时向培养液中释放生长因子等促生长蛋白，可引起细胞增殖和分化，Vero细胞结构蛋白和促生长蛋白构成宿主细胞蛋白（以下简称HCP）。
目前，CHO细胞与Vero细胞被WHO和我国药品监督管理局认可，用于生物制品的生产的传代细胞。
Vero细胞（一种非洲绿猴肾的传代细胞系）是一种理想的疫苗生产基质：遗传背景清楚，核型稳定，无外源因子污染，160代以内没有致瘤性，适合大规模培养，可用生物反应器生产，保证了疫苗大批量细胞的均质性和安全性。
Vero细胞已经用于疫苗的生产：80年代法国率先使用Vero细胞生产脊髓灰质炎疫苗和狂犬病疫苗, 一亿二千万人份人群接种的结果证明Vero细胞作为疫苗的细胞基质是安全和有效的。
1994年维尔博狂犬病疫苗进入我国并投放市场。
目前，我国已正式生产Vero细胞乙型脑炎灭活疫苗。
其它正在开发、研究和生产的疫苗有流感疫苗、出血热疫苗、甲肝疫苗、轮状病毒疫苗、SARS疫苗等等。
WHO推荐使用Vero细胞生产疫苗：WHO于1987和1998年分别组织制定了《使用传代细胞生产生物制品规程》和《使用动物细胞生产生物制品规程》。
在前者中提出：使用传代细胞生产生物制品，必须经过纯化，疫苗中残余DNA不得超过10ng/剂量，去除疫苗中毒性蛋白，但对HCP残留量没有提出明确要求。
SHENTEK
SHENTEK®Vero细胞裂解型HCP残留检测试剂盒（一步酶联免疫吸附法）说明书货号：1301309在实验前请完整阅读本说明书，务必重视注意点和常见问题！版本：A/0仅供研究用湖州申科生物技术股份有限公司⏹产品名称通用名：Vero细胞裂解型HCP残留检测试剂盒（一步酶联免疫吸附法）。
⏹包装规格96测试/盒。
⏹预期用途该试剂盒适用于定量检测细胞裂解工艺的各种生物制品的中间品、半成品和成品中Vero宿主细胞蛋白的残留。
该试剂盒仅供研究使用，不可用于诊断。
⏹检测原理本试剂盒基于固相酶联免疫吸附法（Enzyme-linked Immunosorbent Assay，ELISA），采用双抗体夹心的方式对待测样品中残留Vero HCPs进行定量检测。
该试剂盒内的多克隆抗体是通过裂解Vero细胞所得HCPs作为抗原，免疫绵羊获得血清，进而通过亲合纯化方法得到高质量抗体。
抗体通过目前主流的覆盖率分析法规方法评估其覆盖率水平。
该分析方法通过在预包被抗Vero HCPs多克隆抗体的酶标板中加入校准品或待测样品、HRP标记的抗Vero HCPs多克隆抗体进行共孵育；洗涤后，利用加入的TMB底物进行显色反应，最后使用终止液终止酶催化反应。
利用酶标仪在450nm波长下测读吸光度值，其吸光度与校准品和样品中的HCPs浓度成正相关，通过剂量-反应曲线可计算得出样品中Vero HCPs的浓度。
本试剂盒对实际样品无需进行特殊处理，仅需通过合适的稀释比例进行适用性验证即可直接使用。
本试剂盒检测步骤少，快速，专一性强，性能稳定可靠。
图1检测原理示意图试剂盒组分表1.试剂盒组分组分产品号规格说明Vero HCP 校准品PNB0122瓶冻干粉。
精确量取500μL复溶液，溶解，静置约5分钟，溶液应该澄清透明，无肉眼可见不溶物。
具体含量见标识。
抗Vero HCP 预包被酶标板PNA0138孔×12条已包被适量的绵羊抗Vero HCPs多克隆抗体，铝箔袋密封包装，含干燥剂。
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Vero 细胞HCP（宿主细胞蛋白）Vero细胞宿主蛋白酶联免疫检测目录#F500预期用途该试剂盒用于检测用Vero 细胞.生产的制品中是否有宿主蛋白残留。
仅供研究和工业生产，不能用于人和动物的诊断。
总结与说明病毒疫苗或其他治疗用蛋白在Vero细胞中表达使商业用大量生产药物原料产品的经济简便方法。
生产和纯化这些产品的过程中会残留Vero细胞中的一些蛋白质（称为宿主细胞蛋白，HCPs）而造成污染。
这种污染可以减少药物的疗效，引发毒副反应和免疫学反应，因此最好在实际生产制品过程中将HCPs降低到最低水平。
一些利用抗体来除HCPs的免疫学方法，如Western Blot 和ELISA被广泛使用。
虽然Western blot是一种检测HCPs的有效方法，但它受到了一些限制。
因为它过程复杂且技术依赖性强，需要操作者对结果进行分析解释。
而且，它在本质上是一种定性检测，不能定量分析。
Western blot的敏感性易受被检测样品量的影响，也受目的产品浓度的干扰。
因此Western blot可用来检测纯化上游的蛋白质，而对纯化下游或终产品的检测灵敏度与特异性较低。
本试剂盒中用到的ELISA方法克服了Western Blot的缺点，将敏感度提高了100倍。
ELISA操作简单、客观、可获得半定量的结果，是纯化工艺，过程控制，常规质量检测的最佳选择。
这个试剂盒可与纯化过程中残留的可独立污染产品的所有HCPs反应，就这个意义上来说，这个试剂盒是通用的。
用Vero细胞轻度裂解物获得抗体并经亲和纯化，得到的最终抗体可以与用于生产各种病毒疫苗和蛋白质产品的四种商用细胞系反应。
这一分析表明，绝大多数HCP存在于各种Vero细胞系和纯化过程中。
如果你需要一个更为敏感和特异性的方法去检测样品中的HCP量，Cygnus Technologies公司为你推荐一个优于2D Western blot的方法，我们将这个方法称为2D HPLC-ELISA。
2D HPLC-ELISA相较于2D Western blot 来说敏感性强，特异性高。
要想更多了解此方法，请联系我们公司服务部门。
使用特殊的方法制备抗体，以减少低分子量和低免疫原性污染物以及高分子量组分。
例如本试剂盒，可以作为一个工艺过程开发的工具来监控去除宿主细胞污染物的最佳条件，以及确保最终产品发布。
这种敏感性高的酶联免疫吸附试剂盒已被用于检测终产品的HCP，通过对来自不同生长状态和纯化过程的4种疫苗产品的中间产品和最终产品的实际检测来得到结果。
我们鼓励该试剂盒的的每个使用者进行类似的验证研究以证明它符合他们的分析需求。
这个试剂盒能满足你们的样品需求，不用再运用其他的特异性实验，因为那些实验提供的信息对这个通用的试剂盒来说是重复多余的。
但是，如果你的验证研究表明此试剂盒中的抗体与你的样品中的HCP不能充分反应，那就需要同时运用一种特异性的ELISA，且需要特异性强的抗血清。
或者，如果该试剂盒中使用的多克隆抗体能提供足够的敏感性和广泛的抗原反应性，可用它替代本试剂盒中经共纯化的污染物制作的标准品，从而在特异的HCP检测过程中达到更好的精确性。
使用更多的抗原和抗体所做的特异性分析法的应用理论上具有更好的特异性，但因为它过于特异而不能检测出因为操作不规范或过程改变后产生的一些新的或非典型的污染物。
由于这个原因，我们建议用具有广泛反应性的“通用”宿主细胞蛋白检测实验分析最终产品纯度，即使可使用过程特异性分析。
如果你认为有必要进行过程特异性分析检测， Cygnus Technologies公司将运用其已经验证的方法开发这种抗体并按顾客要求进行检测。
检测原理Vero细胞检测是一个双位点酶联免疫检测。
样品中包含的Vero Cell HCPs蛋白与标记有抗-Vero Cell抗体的辣根过氧化酶同时反应，反应在之前涂有一层吸附性抗-Vero Cell蛋白抗体的微量滴定板中进行。
免疫反构成为夹层结构：固相抗体-HCP-酶标记抗体。
反应结束后清洗微量滴定板，去除未结合反应物。
添加TMB作用底物反应。
微量滴定板读数器上测定水解物浓度，水解物浓度与Vero Cell HCPs蛋白浓度成正比。
试剂&材料（试剂盒提供）成分产品#抗-Vero cell:HRP F501羊抗Vero cell亲和纯化抗体，用辣根过氧化酶标记。
排列在加有防腐剂的试剂盒中，1x12mLAnti-Vero cell coated微量滴定板F502*12×8孔装在含有干燥剂的袋子内Vero cell HCP标准液F503含有Vero cell HCP、牛血清白蛋白与防腐剂。
规格有0, 2, 8, 25, 75和200ng/mL . 1 mL/瓶终止液F006 0.5N硫磺酸1x12mLTMB F005 3,3’,5,5’-四甲基联苯胺. 1x12mL清洗物（20倍浓缩）F004 含防腐剂的Tris缓冲液1x50ml*除了#F505，所有成分都可单独购买储存&稳定性*为了保持稳定，所有的试剂应储存在2°C至8°C，直到打印所示的截止日期为止*若作为底物的试剂在450nm吸光度大于0.1，则不要使用*试剂盒所带的复原液在保质期前都是稳定的材料和器材（试剂盒不提供）能在450&650nm对微量滴定板进行读数的分光光度计（如果你微量滴定板不能不提供双波长的分析，你可以读取450nm波长）移液枪50µL 和100µL多头或多道移液器100µL微量滴定板旋转器 150 - 200 rpm样品稀释液推荐用Cat # 1028蒸馏水1L洗涤瓶，用来稀释洗液警告*仅供研究和工业使用*终止液是0.5N硫磺酸，禁止眼睛、皮肤或衣服与之接触。
试剂盒的其他试剂均无害*该试剂盒只能由有资格的技术人员操作试剂预处理*将所有试剂放到室温下*用蒸馏水将浓缩的洗液稀释到1L，在试剂盒上贴上标签，写上组别和失效日期，储存在4°C注意事项1.正确洗板，除去过量的未反应试剂对保持实验的重复性和灵敏度是必不可少的。
我们强烈建议不要使用自动化或其他手动操作的真空吸尘装置清洗板，因为这样会降低特异性的吸光度，增加非特异的吸光度，使精确度改变。
下面介绍的人工清洗程序在一般情况下只造成较低的背景干扰，且特异性的吸光度高，精确度更好。
2.样品中HCP浓度高时可观察到高剂量钩状效应或低水平的线性稀释。
高剂量钩效应是由于在纯化上游阶段样品的HCP浓度过高，相对来说抗体较低而引起的。
浓度大于1mg/mL 的样品，其吸光度低于200 ng/mL的标准液。
当抗体浓度超过HCP浓度时，也会出现钩效应。
在这种情况下，未稀释的样品的吸光度可能低于试剂盒最高标准液的吸光度。
然而，这些样本不能显示随着稀释度增加，HCP 浓度也增加的线性关系。
钩状效应最常出现在纯化上游阶段。
如果可能出现钩状效应，我们需要对已稀释的样品进行检测。
如果未稀释样品中HCP的浓度低于已稀释样品中HCP的浓度，则可能引起钩状效应。
这样的样品至少要稀释到用实验中间品和终成品验证了的需要稀释的最低限度（MRDS）。
在保证统计学精确度的情况下，一系列能检测出相同HCP值的样品稀释度中，MRD是第一个符合要求的稀释度。
据报道，样品的稀释度与HCP值的关联是在已建立的MRD的基础上修正的。
所用稀释剂应该准确回收。
首选的稀释剂是我们规格为100ml、500ml或1l/瓶的 Cat# I028。
本试剂盒的标准液也是由这种稀释液制备的。
当样品用1028稀释时，其稀释模型开始接近标准，从而减少用其他稀释液所造成的任何错误。
其他要用的稀释液必须测试非特异性的结合，并用它们回收已稀释的200ng/mL 的标准液，方法如下文所述。
局限性*在依靠这种检测来检测宿主细胞残留蛋白之前，每一个实验室都应该验证该试剂盒的抗体和检测程序的特异性、准确性和精确度是否符合要求。
可以从我们公司的服务部门或网站获得验证试验的方法。
*在本实验中使用的标准品包括用常规方法在疫苗产品初次收获液中溶解的Vero细胞HCPs。
在该试剂盒中使用的用来分析抗体的1D Western blot可识别经敏感蛋白染色（如银染和胶体金染色）的大多数的PAGE分离的条带。
由于Vero 细胞系的大多数HCPS会表现出足够的抗原量，该试剂盒应该有足够的抗体活性与你的细胞系充分反应。
但是，我们不能保证该试剂盒能检测出你们生产过程中所有的蛋白质或蛋白质片段。
如果你希望用比 western blot 更为灵敏的方法来检测该试剂盒中的抗体与你的HCPs是否充分反应，我们将为你提供经过ELISA 后用2D-HPLC分离HCPs的方法。
*某些样品基质可能会干扰试验。
本试剂盒使用的标准品试图模拟典型的样品与基质矩阵。
然而，潜在的产品本身或样品基质中的其他成分可能对本实验产生一些正向或负向干扰。
高或低的PH值，去垢剂，尿素，高浓度的盐和有机溶剂是已知的干扰因素。
建议对所有样品基质进行测试以排除干扰，用经稀释的200ng/ml标准液，1/4的基质不含或含非常低的HCP。
如果要用标准品检测未知样品，则需添加30到50 ng/mL标准品的HCP值。
至于如何定量样品基质的值，请咨询公司服务部门。
*避免对含有叠氮化钠（NaN3）的样品进行测定，因为它会破坏辣根过氧化酶的连接活性，测得的HCP值偏低。
实验方案* 该测定是非常强大的，可通过改变孵育时间、样本大小及它后续反应等实验中的可变因素可提高敏感性，增加分析范围或减少样本基质干扰。
在修改我公司推荐的方法之前请联系我们，我们将以最佳方式输入数据以确保达到您期望的目的。
*实验指定使用经认证的微量滴定板振动器或旋转器进行免疫学操作。
这些设备可以从大多数实验室购买，不需要在公司买。
或者你也可以直接在我们公司购买经验证的，预校准的微量滴定板振动器。
如果你没有这样的装置，可以直接在板中孵育，不需要震荡，但要将免疫孵育时间延长到一个小时左右来达到使用振荡器所达到的效果。
孵育时的前30min不要振荡，否则会造成背景污染或降低精确度。
*将所有试剂放到室温下。
设置板的分光光度计的测试波长为450nm，650nm处的读数为作为参考。
*彻底清洗是保证本实验性能的必不可少的步骤。
我们强烈建议不要使用自动化或其他真空吸尘装置清洗板。
本实验推荐的手工清洗方法是保证精确性、敏感性和正确性的首选。
关于此方法更详细的信息可以从公司的服务部门或网站获得。
*所有标准品、对照品和样品至少应做一次重复实验。
*实验板孔加入试剂的顺序应一致，以保证所有反应孔的孵育时间一致。
*作一个工作记录，以确定检测时标准品、对照品和样品各自的位置。
*建议您的实验室对每一个过程的对照品进行测试，以确保所有试剂和程序是正确的。