Vero 细胞 HCP(宿主残留蛋白)ELISA试剂盒说明书

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。

ELISA试剂盒操作方法ELISA（Enzyme-Linked Immunosorbent Assay）是一种常用的免疫学试验技术，用于检测体内或体外的蛋白质、抗体、抗原等物质。

ELISA试剂盒是用于进行ELISA实验的一种常见装置。

下面将为您介绍ELISA试剂盒的操作方法。

1.准备工作a.预热-将试剂盒中的所有试剂预热到适当的温度，通常为室温至37°C之间。

b.样品处理-准备好待测样品，可以是血清、细胞上清液、组织提取物等。

如果样品中含有大量的蛋白质或杂质，可以进行样品处理，如稀释、蛋白质去除等。

c.标准品准备-试剂盒通常包含一系列浓度已知的标准品，用于制作标准曲线。

按照说明书的要求，用缓冲液或适当的稀释液将标准品稀释为不同浓度的工作液。

2.涂膜板的处理a.涂膜板选择-根据实验需求选择合适的涂膜板，通常有96孔、384孔等。

b.板钉定位-使用板钉或其他适当的方法将涂膜板固定在适当的工作台上。

c.预涂底物溶液加入-将试剂盒提供的预涂底物溶液加入涂膜板的孔中，注意保持各孔液面平整。

3.样品与试剂添加a.标准品和样品加入-按照实验设计的需要，将标准品和待测样品分别加入涂膜板的孔中。

b.阳性对照和阴性对照加入-根据试剂盒的要求，加入阳性对照和阴性对照。

c.洗涤液加入-在每次试剂或样品加入后，使用专用的洗涤液将孔洗涤，以清除无关物质。

4.反应与显色a.结合抗体加入-按照试剂盒说明书要求，加入适当的结合抗体，用于与待测物质结合形成复合物。

b.二抗加入-加入带有底物标记的二抗，与结合抗体结合形成复合物。

c.显色底物加入-加入含有适当底物的显色底物试剂，使底物与酶结合并发出信号。

5.反应停止与测量a.反应停止液加入-在适当的时间点，根据试剂盒的说明，加入反应停止液，停止底物的酶反应。

b.光密封膜或保护板加盖-加盖光密封膜或保护板，防止溶液蒸发或污染。

6.读取和数据处理a.读取吸光度-使用ELISA读板仪读取各孔的吸光度值。

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。

PRRSV N蛋白抗体检测间接ELISA方法的建立1 材料：1.1 蛋白、血清、酶标二抗原核表达PRRSV N蛋白；标准阳性血清和阴性血清，待检血清；自制HRP标记兔抗猪IgG；1.2 主要试剂和溶液包被液（50 mmol/L pH9.6的碳酸盐缓冲液）Na2CO3 1.59 gNaHCO3 2.93 g准确称取后，溶于950 mL的蒸馏水中，调pH值为9.6，定容到1 L；PBS-T洗涤液1000 mL 10 mmol/L pH7.4的PBS中加入0.5 mL Tween-20；封闭液和血清稀释液含10%小牛血清的PBS-T缓冲液，10%马血清的PBS-T缓冲液，含5%BSA的PBS-T缓冲液，含10%BSA的PBS-T缓冲液，含5%脱脂奶的PBS-T缓冲液；底物溶液100 mmol/L的柠檬酸溶液（21 g柠檬酸C6H6O7•H2O溶于去离子水，定容至1 L）24.3 mL，200 mmol/L Na2HPO4•12H2O（71.6 g Na2HPO4•12H2O溶于去离子水，定容至1 L）25.7 mL混匀，加入50 mg的四甲基联苯胺（TMB），临用前加入50 μL的30% H2O2；终止液（2 mol/L H2SO4）每200 mL终止液，由蒸馏水177.8 mL和浓硫酸22.2 mL 混和而成。

1.3 主要仪器设备电热恒温培养箱；4℃冰箱；酶标仪。

2 步骤：2.1抗原包被浓度和二抗稀释度的确定将原核表达的N蛋白分别作2.0 μg/mL，1.0 μg/mL，0.5 μg/mL，0.25 μg/mL，0.1μg/mL，0.05 μg/mL连续稀释6个稀释度，每个稀释度重复两孔，100 μL/孔，4℃包被酶标反应板过夜。

将自制HRP标记的兔抗猪二抗分别做1:50、1:100、1:200和1:400一系列倍比稀释，南京金益柏生物技术有限公司Tel:025-********Fax*************每个稀释度重复两孔，100 μL/孔，组成方阵确定重组蛋白的最佳包被浓度和二抗稀释度。

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®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。

百泰派克生物科技
SDS-PAGE检测宿主蛋白HCP残留
HCP（host cell protein ）宿主细胞蛋白或称宿主蛋白残留，是生物制药过程中来源于宿主生物体的药物产品中的低水平的蛋白质杂质。

在重组蛋白药物的表达过程中，宿主细胞系统可以表达许多内源性蛋白质，这些重组生物治疗药物中的宿主细胞蛋白会显著影响药物疗效并引起机体发生免疫反应，存在潜在的风险性，因此必须对这类药物进行严格的质量管控。

宿主蛋白残留检测就是检测重组蛋白药物中是否含有其他宿主杂质蛋白，SDS-PAGE 凝胶电泳根据蛋白质的分子质量对蛋白进行可视化的分离，相对分子质量不同的蛋白质可被电泳分离成不同的蛋白条带，通过观察电泳结束后蛋白条带的情况即可快速判断蛋白样品的纯度。

将蛋白类药物进行SDS-PAGE电泳，电泳结束后若有且仅有一条分子质量与蛋白类药物相同的条带，说明该蛋白类药物不含其他宿主蛋白；反之，则说明该蛋白类药物含有其他宿主蛋白。

SDS-PAGE不仅可以判断重组蛋白类药物的纯度，还可检测HCP纯化的结果。

百泰派克生物科技使用Bio-Rad Mini-PROTEAN® Tetra凝胶系统以及Thermo公司最新推出的Obitrap Fusion Lumos质谱仪，结合nanoLC-MS/MS纳升色谱，提供基于SDS-PAGE和质谱的宿主蛋白残留（HCP）分析服务技术包裹，您只需将您的需求告诉我们并寄送样品，我们负责项目所有后续。

百泰派克生物科技还可提供定制化的分析服务，满足不同的实验需求，欢迎免费咨询。

宿主hcp残留质量标准宿主HCP（Host Cell Protein）残留是指在生物制药过程中，宿主细胞中产生的蛋白质残留物。

这些残留物可能会对最终产品的安全性、稳定性和有效性产生不利影响，因此需要严格控制宿主HCP的水平。

为了确保制药产品质量，监管机构和制药行业制定了一系列质量标准和指南，以指导和规范宿主HCP残留的控制。

以下是一些相关的参考内容：1. ICH Q6B指南：国际药品注册协调会议（ICH）发布的Q6B 指南提供了对生物制品宿主细胞蛋白质残留的评估和控制的指导。

其中包括宿主HCP残留的分析方法、关联性和安全评估等内容。

2. FDA指南：美国食品药品监督管理局（FDA）发布了《药物制品开发中的宿主细胞蛋白质残留》，其中详细描述了宿主HCP残留的控制策略和分析方法，以及对HCP的削减和清除效果的要求。

3. EMA指南：欧洲药物管理局（EMA）发布了《生物类似药物发展的质量问题》，其中包括对宿主HCP残留的控制要求和分析方法的指导。

4. USP方法：美国药典（USP）提供了宿主HCP残留的分析方法，如限制性胶体凝胶电泳法（IIEF）和酶联免疫吸附法（ELISA）。

此外，USP还发布了关于衍生宿主HCP测定和特定宿主HCP测定的指南。

5. ASTM标准：美国材料与试验协会（ASTM）发布了关于生物制药中宿主HCP残留的几个标准，如《细胞培养和发酵物中宿主蛋白质测定的标准指南》和《制备0.1%宿主细胞蛋白质溶液的标准测试方法》等。

在这些指南和标准的基础上，制药企业需要根据自身产品特点和国家监管要求制定适合的宿主HCP残留控制策略。

这包括确定合适的宿主HCP残留限度、选择适用的分析方法和监测方案、优化生物制药过程以减少宿主HCP产生等。

此外，制药企业也应该确保生产设施的清洁和消毒措施得到充分执行，以避免宿主HCP的交叉污染。

对于高风险的制造步骤，如细胞培养和纯化过程，应加强监控和验证，确保宿主HCP残留控制的有效性。

宿主hcp残留质量标准
宿主体高级复合物（HCP）是在生物制品生产过程中，常常伴随着目标
产品（如蛋白质药物）的一种杂质。

HCP残留是指在制备的最终产品中仍然存在的HCP。

针对HCP残留的质量标准可以根据不同的国家和地区、不同的药物监
管机构以及不同的生物制品类别而有所不同。

一般而言，针对HCP残
留的质量标准通常包括以下几个方面：
1. HCP的定量限制：确定最终产品中可接受的HCP残留水平，通常以
微克（μg）或百万分之一（ppm）为单位。

这个限制可以基于临床试
验的安全性和有效性数据，以及对HCP相关潜在危险的评估。

2. 分析方法要求：确保制定和使用能够准确和可靠地检测和分析HCP
的方法。

这些方法应具备高灵敏度、高选择性和良好的准确性，以满
足对HCP残留的定量要求。

3. 跟踪和控制要求：制定相应的控制策略，以确保生产过程中对HCP
的有效控制和减少，以最大程度地降低HCP残留水平。

这包括通过改
进生产工艺和采用有效的清除和净化步骤等措施。

4.验证要求：建立验证方法，确保制定的控制策略能够可靠地控制HCP 的残留水平，并能够稳定地满足相关质量标准。

HCP残留的质量标准主要关注对HCP残留水平的限制、分析方法的要求、生产过程的控制和验证要求等方面，以确保药物制品的质量和安全性。

Vero细胞使用说明书产品基本信息产品货号YC-A001细胞名称Vero中文名称非洲绿猴肾细胞细胞形态上皮样,贴壁细胞传代比例1:3~1:6培养体系90%DMEM+10%FBS冻存液体系50%DMEM+40%FBS+10%DMSO特殊备注细胞接收1)冻存细胞：如果是干冰运输的冻存细胞，收到后请立即转入液氮储存或短暂（24H)放至-80℃冰箱保存，或直接进行细胞复苏。

2)活细胞：如果是T25瓶活细胞运输，收到后用75%的酒精对T25瓶外表面进行消毒，之后放在5%CO2、37℃的细胞培养箱静置2h，静置后取出细胞瓶在显微镜下观察细胞贴壁情况和细胞汇合度，分别在100X和40X下各拍2个不同视野的细胞拍照记录。

如果汇合度达到80%以上的传代密度，可以进行传代操作，如果细胞汇合度没有达80%以上不够传代，弃掉瓶内培养基，更换新鲜完全培养基。

(灌满细胞培养基不能正常用来培养细胞。

)注意：收到细胞后，活细胞首先观察细胞瓶是否完好，培养液是否漏液、浑浊等现象。

冻存细胞若发现干冰已挥发完、冻存管瓶盖脱落、破损等异常情况，请务必拍照保留，并于收货24h内与我们联系（电话：400-688-9033；https://）。

细胞复苏1)准备工作：将完全培养液置37℃水浴锅预热30分钟，随后将冻存的细胞从液氮中取出，转移到-80℃冰箱，放置数分钟让残余液氮挥发；2)在超净台内用吸管吸取6-7mL完全培养液至15mL离心管中；3)将细胞从-80℃冰箱取出暂时放置于干冰里，复苏时稍稍甩动，去除残留的干冰和液氮，再迅速用镊子夹住盖子放入37℃水浴中快速晃动（注意：水不能没过盖子），使其在1分钟左右完全融化；4)在超净台内，用酒精棉球擦拭冻存管外壁消毒，稍稍晾干。

用单道移液器将所有融化的细胞悬液转至提前准备好的完全培养液中，盖上盖子，1100rpm室温离心4分钟收集细胞；5)超净台内小心吸弃上清，用单道移液器吸取1mL新鲜完全培养液重悬细胞至单细胞悬液，再转移至装有4mL完全培养液的T25cm2培养瓶(或者6cm的皿)中，写上细胞名称、复苏日期、代次，放置37℃、5%CO2饱和湿度培养箱内培养。

本试剂盒只能用于科学研究，不得用于医学诊断小鼠小鼠（（MouseMouse））肿瘤坏死因子可溶性受体肿瘤坏死因子可溶性受体ⅠⅠ（TNFsR-TNFsR-ⅠⅠ）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）。

往预先包被肿瘤坏死因子可溶性受体Ⅰ（TNFsR-Ⅰ）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肿瘤坏死因子可溶性受体Ⅰ（TNFsR-Ⅰ）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。

ELISA检测试剂盒使用指南ELISA（酶联免疫吸附试验）是一种常用的免疫学实验方法，用于检测病原体、抗体或其他分子的存在和浓度。

它具有高灵敏度、高特异性、易操作和较低成本的优势，被广泛应用于生物医学研究、临床诊断和免疫学领域。

本篇文章将介绍ELISA检测试剂盒的使用指南，包括实验准备、试剂的使用步骤和结果解读。

一、实验准备1.阅读检测项目的说明书：在使用试剂盒前，仔细阅读说明书，了解试剂的使用方法、灵敏度和特异性等关键信息。

2.样本准备：根据实验要求，准备样本。

如果需要检测血清中的抗体水平，可以采集血液样本，离心分离血清；如果需要检测细胞培养上清中的分子，将上清收集，并使其清晰，避免细胞或杂质的污染。

3.样本预处理：根据实验需求，对样本进行必要的预处理。

例如，可以通过加热、稀释、酶解等方式来处理样本，以改变样本组分和浓度。

4.准备品质控制样品：制备阳性和阴性控制样品，用于评估试剂盒和实验操作的稳定性和准确性。

5.实验器材准备：准备所需实验器材，如酶标板、移液器、微孔板洗涤仪等。

确保器材的干净和完整，并按照说明书要求对其进行预处理。

二、试剂使用步骤1.试剂准备：根据说明书要求，将试剂从冰箱中取出，并在室温下放置一段时间使其恢复到室温。

确保试剂瓶盖紧闭，并避免暴露在直接阳光下。

2.实验操作：按照说明书的要求，将试剂加入到酶标板中，并根据实验设计进行标准曲线的设置。

标准曲线用于测量未知样品的数量，并计算出浓度。

3.孵育：根据试剂盒的要求，将酶标板放入孵育箱中进行孵育。

孵育温度和时间应根据实验要求进行调整。

4.洗涤：使用洗涤缓冲液对酶标板上的不特异性结合物进行洗涤。

洗涤过程应准确控制洗涤孔板次数和洗涤液的体积。

5.补液：在洗涤完成后，加入辣根过氧化物酶标况稀释液，促进酶标物与特异性结合物的反应。

6.孵育：根据试剂盒的要求，将酶标板放入孵育箱中进行二次孵育。

7.反应停止：根据试剂盒的要求，加入相应的停止液，停止酶反应。

宿主蛋白残留(HCP)分析服务许多生物药物（抗体、疫苗、重组蛋白等）的制备还是通过生物体系合成的。

尽管采用多种纯化方式，生物药物中还是可能会有微量的宿主蛋白残留（HCP）。

酵母，尤其是毕赤酵母和酿酒酵母，是生物技术工业中常用的宿主细胞之一，它们在表达我们需要的蛋白质的时候也会表达其自身的蛋白，即酵母宿主蛋白，或酵母宿主残留蛋白。

生物制品中残留的宿主细胞蛋白（HCP）作为外源蛋白可能会在不同程度上引发机体的免疫应答，最终导致过敏反应或其他不良反应，因此必须建立合适的检测HCP的方法来监控生物制品的质量。

ELISA是目前应用最广泛的HCP检测方法。

该技术相对简单、精度良好，方便设定控制范围和建立技术规范。

《中华人民共和国药典》2010年版三部中的HCP检测均是要求采用酶联免疫法测定。

在该检测方法中，用存在于生物制品中的HCP作为免疫原生产出的抗体质量至关重要。

无论是商品化ELISA试剂盒，还是自制的多克隆抗体，如果特异性和适用性不够高，会带来漏检HCP的风险，从而对生物制品质量安全带来风险。

ELISA在生物制品开发过程中的应用。

酵母宿主蛋白残留分析——ELISA法的优点1）高灵敏度：ELISA法能够检测到极低浓度的目标蛋白，这对于检测微量的宿主蛋白残留至关重要。

2）高特异性：通过使用针对酵母宿主蛋白的专一性抗体，ELISA可以区分目标蛋白和其他非特异性蛋白，从而确保准确性。

3）量化能力：ELISA不仅能定性检测宿主蛋白残留，还可以定量地评估蛋白的浓度。

4）适应性强：ELISA法可以针对多种类型的酵母宿主蛋白进行调整，适用于多种生产过程和药物制剂。

5）高通量：ELISA法使用微孔板进行操作，可以同时处理多个样本，大大提高了分析效率。

6）成本相对较低：与其他高级的蛋白质分析方法相比，ELISA在材料和设备成本上相对较低。

百泰派克生物科技（BTP）依托高精度紫外-可见分光光度计，根据酶联免疫吸附原理，开发了宿主蛋白残留ELISA分析平台，并通过CNAS/ISO9001双重质量认证体系，可高效、精准的对宿主蛋白残留进行定性、定量分析，广泛用于多种生物制品的质量和安全检测，欢迎免费咨询！此外，针对精准检测生物药物可能残留的宿主蛋白，百泰派克生物科技还提供基于2D DIGE的HCP抗体有效性检测服务。

中国药典 HCP 残留标准概述中国药典（Chinese Pharmacopoeia，简称CP）是我国药品质量标准的法定性文件，也是中国药品监督管理的重要依据。

其中，HCP（Host Cell Protein）残留标准是指在生物制品的生产过程中，宿主细胞产生的蛋白质残留物的限定标准。

本文将深入探讨中国药典中关于HCP残留标准的内容。

HCP残留标准的重要性HCP是指在生物制品（如重组蛋白、抗体等）的生产过程中，宿主细胞产生的蛋白质。

这些HCP残留物可能对制品的质量和安全性产生负面影响，因此需要进行监测和控制。

HCP残留标准的制定和实施，可以保证生物制品的质量和安全性，提高药品的疗效和可靠性。

HCP残留标准在中国药典中的制定1. CP中的相关章节中国药典中关于HCP残留标准的内容主要包括在《生物制品》、《蛋白质制品》等章节中进行规定和说明。

2. 标准制定的过程制定HCP残留标准的过程主要包括以下几个步骤： - 收集国内外相关研究和标准的文献资料； - 组织专家进行讨论和评审，确定标准的技术要求和限定参数； - 进行实验验证和临床试验，获取相关数据和结果； - 最终确定标准，并经过相关部门的审批和发布。

HCP残留标准的技术要求1. 检测方法CP对HCP残留的检测方法进行了规定，主要包括酶联免疫吸附试验（ELISA）、质谱分析等。

2. 残留限定CP对不同类型的生物制品，如重组蛋白、抗体等，制定了不同的HCP残留限定标准。

这些标准通常以微克/剂量单位或以特定的HCP蛋白质浓度进行限定。

3. 安全性评估对于HCP残留标准的制定，除了考虑技术可行性和合理性外，还需要进行安全性评估。

这包括对HCP残留物的潜在毒性和过敏原性进行评估和风险分析。

HCP残留标准的应用与挑战1. 应用领域HCP残留标准主要应用于生物制品的生产和质量控制过程中，用于监测和控制HCP 残留物的含量。

这些生物制品广泛应用于生物医药、生物技术等领域。

宿主vero细胞DNA残留量操作规程试剂（1）DNA标记和检测试剂盒（2）TaKaRa universal Genomic DNA Extraction Kit ver3.0（DV811A）(3 ) TaKaRa DNA Fragment Purification Kit(807A)（4）DNA杂交膜: 尼龙膜（5）2%蛋白酶K溶液：称取蛋白酶K0.20g，溶于灭菌水10ml中，分装后储存于-20℃备用。

(6) 3%牛血清白蛋白溶液：称取牛血清白蛋白0.30g,溶于灭菌水10ml中。

(7) 1mol/l三羟甲基氨基甲烷（Tris）溶液（Ph8.0）:用盐酸调pH至8.0。

(8) 5.0mol/l氯化钠溶液（9）0.5mol/l乙二胺四乙酸二钠溶液（pH8.0）用10mol/l氢氧化钠溶液调节至8.0。

（10）20%十二烷基硫酸钠(SDS)溶液：用盐酸调pH至7.2。

（11）蛋白酶缓冲液（pH8.0）量取1mol/l Tris溶液1.0ml（pH8.0），5mol/l氯化钠溶液2.0ml，0.5mol/l乙二胺四乙酸二钠溶液（pH8.0）2.0ml，20%SDS溶液2.5ml，加灭菌水至10ml。

（12）TE缓冲液（Ph8.0）量取1mol/l Tris溶液10ml，0.5mol/l乙二胺四乙酸二钠溶液（pH8.0）2ml，加灭菌水至1000ml。

（13）1%鱼精DNA溶液：精密称取鱼精DNA0.10g,置10ml量瓶中，用TE缓冲液溶解并稀释至刻度，摇匀，用7号针头反复抽打以剪切DNA成为小分子，分装后贮藏于-20℃备用。

（14）DNA稀释液：取1%鱼精DNA溶液50μl，加TE缓冲液至10ml。

（15）washing buffer：0.1M 马来酸；0.15M氯化钠；0.3%Tween 20;PH7.5。

（16）马来酸溶液：0.1M 马来酸；0.15M氯化钠；PH7.5。

（17）检测液：0.1M Tris-Hcl；0.15M氯化钠；PH9.5。

ELISA kitInstruction Manual5-plate formatJuly, 2006For research use only.Not for use in diagnostic or therapeutic procedures.ContentsAbbreviations 2 Introduction 3 Contents of the kit 4 Hazard information 4 Materials and reagents required but not provided 4 Working solutions 4 General procedure 5 Coating antibodies 5 Blocking 5 Test samples and standards 5 Biotinylated detector antibodies 5 SPP conjugate 5 Substrate 5 Cytokine standards 6 Storage kit reagents 6 Directions for washing 7 Trouble shooting 7 References 8AbbreviationsAPC Antigen presenting cellsBSA Bovine serum albuminCD Cluster of differentiationCSB Cytokine stabilization bufferDMSO Dimethyl sulfoxideELISA Enzyme linked immunosorbent assayGM-CSF Granulocyte-Macrophage Colony Stimulating Factor IFN InterferonIL InterleukinMHC Major histocompatibility complexOD Optical densityPB Phosphate bufferPBS Phosphate buffered salinePBST PBS containing 0.05% Tween-20PBST-B PBST containing 0.5% bovine serum albuminSPP Streptavidin-HRP polymerT h T helper subsetTMB TetramethylbenzidineTNF Tumor necrosis factorIntroductionCytokines are a group of regulatory proteins critically involved in many physiological processes such as immune recognition, cell differentiation and cell proliferation. They have been identified in many vertebrate species and are produced by a variety of different cell types. Cytokines are usually produced transiently and locally, acting in a paracrine or autocrine manner. They interact with high affinity cell surface receptors specific for each cytokine or cytokine group and are active at very low concentrations mostly in the picogram range.It is well known now that the type of an antigen-specific immune response largely depends on the selection or preferential activation of defined CD4+T cell subsets (i.e. T h1 and T h2). Activation of these subsets is characterized by the secretion of distinct patterns of cytokines. T h1, but not T h2 cells, primarily secrete IL-2 and IFN-γ while T h2, but not T h1 cells, produceIL-4, IL-5, IL-6, IL-10 and IL-13. Other cytokines, such as TNF-α and GM-CSF are produced by both T h subsets. In addition, the production of IL-12 and IL-10, produced by antigen presenting cells (APC) such as macrophages and dendritic cells, critically contributes to the preferential expansion of T h1- or T h2-type of cells. For instance, early production of IL-12 is considered essential for the development of T h1 cells. On the other hand, the absence or low concentrations of IL-12 and IFN-γ in the early phase of an immune response and concomitant production of IL-4 by cells of the mastcell/basophil lineage or T cells themselves is known to favor the development of T h2 cells. In addition to their regulatory effects on T h subset differentiation, the cytokines released by the two types of T h cells also produce distinct effector functions. For instance, IL-4 and IFN-γhave differential or antagonistic activities on immunoglobulin isotype selection or MHC class II expression. Therefore, the properties of an immune response can be best studied by determining the amounts of cytokines produced by the responding T cells and APC.Contents of the kitItemsQuantity(5-plate format)StorageconditionsCoating antibodies 1 vial 4ºC (39ºF)Cytokine standard 5 vials 4ºC (39ºF)Biotinylated detector antibodies 1 vial 4ºC (39ºF)SPP conjugate (Streptavidin-HRP polymer) 1 vial ≤ -20ºC (-4°F)TMB substrate tablets 5 4ºC (39ºF)Substrate buffer capsules 5 Rt*BSA stock solution (10%) 2 vials (24 ml) 4ºC (39ºF)Cytokine stabilization buffer (CSB)** 1 vial (5 ml) 4ºC (39ºF)Tween-20 1 vial (5 ml) Rt*ELISA plates8 Rt*Adhesive cover slips 10 Rt** Room temperature** For serum and plasma samples only; see under “Test samples and standards”Materials and reagents required but not provided•PB stock: dissolve 96.0 g Na2HPO4.2H2O plus 17.5 g KH2PO4in 1.0 L distilled water and adjust pH to 7.4•Sterile distilled water•H2SO4•Dimethyl sulfoxide (DMSO)•Pipetting devices for the accurate delivery of volume required for the assay performance •Plate washer: automated or manual (squirt bottle, manifold dispenser, etc)•Reading device for microtiter-plate set to 370, 450 and/or 655 nmWorking solutions•PBS: add 10 ml PB stock and 8.8 g NaCl to 1 L distilled water. Adjust pH to 7.4.Alternatively, use commercially available liquid PBS from Invitrogen or other suppliers.Do not use commercially available PBS tablets for the preparation of the coating solution (the filler in the tablets interferes with the coating process).•PBST: 0.5 ml Tween-20 dissolved in 1 L PBS.•PBST-B: 2 ml BSA stock solution (10%) added to 38 ml PBST.•Blocking buffer: 2 ml BSA stock solution (10%) added to 18 ml PBS (for 1 ELISA plate). •Substrate buffer: the contents of one capsule is dissolved in 100 ml distilled water (takes approximately 5 minutes). For optimal performance, the buffer solution should be used within60 minutes.•Stopping solution: 2 M H2SO4TMB (tetramethylbenzidine) and sodium perborate (in substrate buffer)General procedureCoating antibodies•Reconstitute the lyophilized antibodies by injecting 250 µl of sterile distilled water into the vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes at room temperature. Avoid vigorous shaking. To coat 96 wells of an ELISA plate 50 µl is pipetted out of the vial (or use a frozen aliquot of 50 µl; see "Storage kit reagents") and added to 5 ml PBS. Mix gently.•Add 50 µl of diluted antibody solution to each well of the ELISA plate and fill up to 100 µl with PBS.•Seal the plate to prevent evaporation.Incubate overnight at 4ºC or alternatively 1 to 2 hours at 37ºC.Blocking•Remove the coating antibody solution and wash the wells at least six times with PBST. •Add 200 µl of blocking buffer.•Seal the plate and incubate at 37ºC for 1 hour.Test samples and standards•Remove the blocking buffer but do not wash.•Add 1/20 volume of CSB to serum or plasma samples but not to other samples such as cell culture supernatants; CSB inhibits the degradation of cytokines in pure serum or plasma. •Dilute standards and test samples in an appropriate diluent (see “Cytokine standards”). •Add 100 µl to each well.•Seal the plate and incubate at 37ºC for 2 hours or overnight at 4ºC.Biotinylated detector antibodies•Remove test samples/standards and wash the wells at least six times with PBST. •Reconstitute the lyophilized antibodies by injecting 0.5 ml of sterile distilled water into the vial. Mix the solution gently for approximately 15 seconds and allow it to stand for 2 minutes at room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial (or use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B.Mix gently.•Add 100 µl of diluted antibody solution to each well.•Seal the plate and incubate at 37ºC for 1 hour.SPP conjugate•Remove detector antibody solution and wash the wells at least six times with PBST. •Reconstitute the contents of the vial by injecting 0.5 ml of sterile distilled water into the vial.Mix the solution gently for approximately 15 seconds and allow it to stand for 1 minute at room temperature. Avoid vigorous shaking. Hundred microliter is pipetted out of the vial (or use a frozen aliquot of 100 µl; see "Storage kit reagents") and added to 10 ml PBST-B. Mix gently.•Add 100 µl to each well.•Seal the plate and incubate at 37ºC for 1 hour.Substrate•Remove SPP conjugate and wash the wells at least six times with PBST.•Dissolve one TMB tablet in 1.0 ml DMSO (vortex at high speed for 5 minutes for complete dissolution)and than add 10 ml substrate buffer.•Mix thoroughly and immediately dispense 100 µl into each well. Leave the plate on the laboratory bench at room temperature (color development between 10 and 30 minutes).The substrate produces a soluble end-product that is blue in color and can be read spectrophotometrically at 370 or 655 nm. The reaction can be stopped by adding 50 µl of2 M H2SO4 (resulting in a yellow solution which can be read at 450 nm).Cytokine standardsFor maximum recovery, the vial with lyophilized cytokine standard should be reconstituted in 0.5 ml distilled water and allowed to stand for 1 minute at room temperature. Thereafter, the reconstituted cytokine standard (stock solution) is placed on melting ice and is immediately diluted as indicated below (preferentially within one hour). Use vials with cytokine standards only once.Please note that temperature of buffers and standard solution(s) should now be kept at 0-4ºC until use in the ELISA.The total amount of cytokine standard is indicated on the label of the vial (ng/vial). After reconstitution in 0.5 ml water, the concentration (ng/ml) will become twice the amount on the label [e.g. amount on label is 4.8 ng/vial; after reconstitution, the concentration becomes9.6 ng/ml = 9600 pg/ml].The standard stock solution is diluted to 320 pg/ml in PBST-B (highest concentration cytokine to be used in the standard range).The linear region of the cytokine standard curve is now obtainable in a series of two-fold dilutions in PBST-B ranging from 320 to 5 pg/ml. Always include a blank control (PBST-B only) in the standard range.Before establishing the standard curve, the OD value of the blank control (OD.bl) is subtracted from the measured OD values of the different standard solutions. The standard curve is now plotted as the standard cytokine concentration versus the corresponding (measured) OD value minus OD.bl. In addition, the actual OD values of the test samples are determined by subtracting OD.bl from the measured OD values.The concentration of the cytokine in the test sample can then be interpolated from the standard curve. It is useful to prepare a series of dilutions of the unknown test sample to assure that the OD will fall in the linear portion of the standard curve.Note 1: The OD value measured for the blank control (OD.bl) must be below 0.2.Note 2: for measuring cytokines in cell culture supernatant, samples should be diluted inPBST-B. However, when measuring cytokines in pure serum or plasma, the diluent for the standard and blank control should preferentially be control serum or plasma originating from the same species.Storage kit reagentsThe vials with lyophilized coating antibodies and biotinylated detector antibodies can be safely stored in a refrigerator for a defined length of time (expiry date indicated on the vial). After reconstitution, the antibodies remain fully active for minimal 6 months at 4ºC (39ºF) when kept sterile. However, it is strongly recommended to divide the reconstituted antibody solutions into small aliquots for single use. These aliquots should be stored at ≤-20ºC. Under these conditions the antibodies are stable for at least one year.Upon arrival, the vial with lyophilized SPP conjugate should be stored at ≤ -20°C. Storage of the vial at room temperature or at 4ºC for several months may lead to lower OD readings in the ELISA. After reconstitution, the SPP solution is stable for 2 months at 4°C but rapidly looses activity when stored at room temperature. It is strongly recommended that after reconstitution, the solution is immediately divided into small aliquots for single use and stored at ≤-20°C. Under these conditions SPP is stable for minimal 12 months.Directions for washing•Incomplete washing will adversely affect the assay. All washing must be performed with wash buffer (PBST).•Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into each well. After aspiration, fill the wells with at least 300 µl wash buffer. Let soak for 10 to 20 seconds, then aspirate the liquid. Repeat as directed under "General procedure". After washing, the plate is inverted and tapped dry on absorbent paper.•Alternatively, the wash buffer may be put into a squirt bottle. If a squirt bottle is used, flood the plate with wash buffer, completely filling all wells. After washing, the plate is inverted and tapped dry on absorbent paper.•If using an automated washing device, the operating instructions should carefully be followed.Trouble shooting•Poor consistency of replicates can be overcome by increasing the stringency of washes particularly after the incubation step with detector antibody.•High values of the blank control (optical density > 0.2) can be overcome by shortening the incubation time with the substrate solution or is caused by improper washing procedures. •Inconsistent replicates may be due to cross-contamination of wells by improper pipetting procedures.•If no signal is observed in the wells with the standards•try a new vial with cytokine standard•check the pH of the substrate solution (between 5.0 and 5.5)•verify whether the antibody, SPP conjugate and standardpreparations were properly diluted•Avoid sodium azide in wash buffers and diluents, as this is an inhibitor of peroxidase activity.•Storage of reconstituted SPP at room temperature for several days can lead to a significant loss of SPP activity and consequently low OD readings.ReferencesBooks:•Practice and theory of enzyme immunoassays 1985In: Laboratory techniques in biochemistry and molecular biology, Vol.15 (eds R.H.Burdon and P.H. van Knippenberg)Science Publishers bv, Amsterdam, The Netherlands•ELISA and other Solid Phase Immunoassays.Theoretical and Practical Aspects 1988(eds D.M.Kemeny and S.J.Challacombe)John Wiley & Sons Ltd, Chichester, UK• A practical guide to ELISA 1991(ed D.M.Kemeny) Pergamon Press, Oxford, UKReview of U-CyTech ELISA references:Human cytokines:•Arend, S.M. et al. 2000 J. Infect. Diseases 181: 1850-1854 •Demirkiran, A. et al. 2006 Liver Transpl. 12: 277-284 •Hoogendoorn, M. et al. 2005 Clin. Cancer Res. 11: 5310-5318 •Tang, Y-M. et al. 2006 World J. Gastroenterol. 11: 4575-4578•de Waal, L. et al. 2004 J. Virol. 78: 1775-1781Monkey cytokines:•Fallon, P.G. et al. 2003 J. Infect. Dis. 187: 939-945•Hartman, G. et al. 2005 Vaccine 23: 3310-3317•Kornfeld, C. et al. 2005 J. Clin. Invest. 115: 1082-1091 •Mascarell, L. et al. 2006 Vaccine 24: 3490-3499•Polakos, N.K. et al. 2001 J. Immunol. 166: 3589-3598•de Swart, R.L. et al. 2002 J. Virol. 76: 11561-11569Mouse cytokines:•Eijkelkamp, N. et al. 2004 J. Neuroimmun. 150: 3-9•Kavelaars, A. et al. 2005 J. Neuroimmun. 161: 162-168•Vroon, A. et al. 2005 J. Immunol. 174: 4400-4406Rat cytokines:•Dieleman, J.M. et al. 2006 Life Sci. 79: 551-558•Pacheco-López, G. et al. 2005 J. Neurosci. 25: 2330-2337•Sajti, E. et al. 2004 Brain Behav. Immun. 18: 505-514•Teunis, M.A.T. et al. 2002 J. Neuroimmun. 13: 30-38。

宿主hcp残留质量标准
宿主HCP（Host Cell Protein）是在生物药物生产过程中残留在最终制品中的宿主细胞蛋白。

由于HCP的存在可能影响药物的安全性和有效性，因此制定了一些质量标准来控制宿主HCP的残留。

根据国际药典（Pharmacopoeia）和相关法规，通常将药物中的宿主HCP的含量限制在一定的范围内。

质量标准通常根据以下几个因素进行制定：
1. 宿主细胞类型：不同的宿主细胞在药物生产过程中会产生不同类型和量的HCP。

因此，质量标准可能会针对具体的宿主细胞类型进行调整。

2. 宿主细胞株的纯度：宿主细胞株的纯度也会影响HCP的含量。

因此，质量标准中可能会包含关于宿主细胞株纯度的要求。

3. 临床安全性：HCP的存在可能引发免疫反应和其他不良反应，因此质量标准通常会根据对患者安全性的考虑制定。

4. 生物药物特性：某些生物药物对HCP的容忍度可能有所不同，对于容忍度低的药物，质量标准可能会更为严格。

根据具体情况，质量标准通常以HCP的含量限制为主要参数。

这些含量限制可能会根据批次或者剂型进行调整。

此外，还可以衡量和监测HCP的特定蛋白质成分，以更全面地评估其质量。

总之，宿主HCP残留质量标准是通过限制和监测药物中的宿主细胞蛋白含量来确保药物的安全性和有效性。

这些标准通常是根据宿主细胞类型、宿主细胞株纯度、临床安全性和生物药物特性等因素进行制定的。