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ClonaCell™-HY杂交瘤试剂盒

完整的杂交瘤生成试剂盒

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产品号 #（选择产品）

产品号 #03800_C

完整的杂交瘤生成试剂盒

产品优势

节省时间：将杂交瘤的选择和克隆结合为一步
节省资源：单细胞来源的杂交瘤在半固体培养基中形成可见的离散菌落，易于筛选和转移到液体培养基中进行扩增
克隆效率：单个细胞悬浮并固定在半固体培养基中，防止因过度生长而丢失稀有的高产克隆

产品组分包括

ClonaCell™-HY Medium A, 500 mL（目录#03801）

ClonaCell™-HY Medium B, 500 mL（目录#03802）

ClonaCell™-HY Medium C, 100 mL（目录#03803）

ClonaCell™-HY Medium D, 90ml（目录#03804）

ClonaCell™-HY Medium E, 500 mL（目录#03805）

ClonaCell™-HY PEG, 1.5 mL（目录#03806）

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总览

产品说明书及文档

应用领域

概述

使用ClonaCell™-HY杂交瘤试剂盒中的培养基和试剂，完成杂交瘤发育和单克隆抗体生产的所有步骤：
• ClonaCell™-HY Medium用于骨髓瘤和杂交瘤培养
• ClonaCell™-HY Medium用于杂交瘤融合
• ClonaCell™-HY Medium用于杂交瘤融合恢复
• ClonaCell™-HY Medium用于杂交瘤的选择和克隆
• ClonaCell™-HY Medium用于杂交瘤生长
• ClonaCell™衔接挂钩支持杂交瘤融合

ClonaCell™-HY方法使用基于甲基纤维素的半固体选择性培养基，将杂交瘤的选择和克隆结合在一起。单个亲本杂交瘤克隆及其后代在半固体培养基中生长形成不同的菌落时保持在一起。这可以防止由于快速生长的细胞过度生长而导致稀有无性系的损失，这种情况在液体培养基中选择时可能发生。通过人工或机器人的方法，可以很容易地从半固体培养基中挑选杂交瘤菌落，并分散到液体培养基中进行筛选和扩增。

该试剂盒已被证实用于小鼠和大鼠水螅的发育和单克隆抗体的生产，据报道，该试剂盒适用于杂交瘤的生产、克隆和扩增，杂交瘤使用的淋巴细胞来自多种宿主动物，包括人、小鼠、大鼠和仓鼠。为了您的方便，套件组件也可以单独购买。

ClonaCell™-HY方法采用甲基纤维素基半固体选择培养基，将杂交瘤筛选与克隆整合为单一步骤。独立的亲代杂交瘤克隆及其子代细胞在半固体基质中定位生长并保持聚集，最终形成可区分的集落。该特性可避免液体培养基筛选时快速增殖的细胞对稀有克隆的竞争性抑制，同时便于单克隆集落的分离。通过人工或自动化方法可轻松从半固体培养基中挑取克隆，再分散至液体培养基进行筛查与扩增。

该培养基经验证适用于小鼠和大鼠杂交瘤开发，据报告也可用于生产、克隆和扩增人、小鼠、大鼠和仓鼠等多种宿主动物的淋巴细胞产生的杂交瘤。试剂盒组分也可单独购买，方便您的应用。

Subtype
Semi-Solid Media, Specialized Media

Cell Type
Hybridomas

Species
Mouse, Other, Rat

Application
Cell Culture, Hybridoma Generation

Brand
ClonaCell

Area of Interest
Antibody Development, Cell Line Development, Drug Discovery and Toxicity Testing, Hybridoma Generation

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Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type

Product Name

Catalog #

Lot #

Language

Document Type

Product Information Sheet 1

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Document Type

Product Information Sheet 2

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Document Type

Product Information Sheet 3

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Document Type

Product Information Sheet 4

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Document Type

Technical Manual

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Document Type

Safety Data Sheet

Product Name

ClonaCell™-HY Hybridoma Kit

Catalog #

03800

Lot #

All

Language

English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area

Workflow Stages

Workflow Stages for Hybridoma Production

Pre-Enrichment and Fusion

Selection and Cloning

Expansion and Characterization

Resources and Publications

Why is there HT (hypoxanthine, thymidine) in Medium E?

Hybridomas are selected using HAT (hypoxanthine, aminopterin, thymidine). Aminopterin blocks the de novo pathway for synthesizing nucleotide precursors for DNA synthesis. The inhibition of the de novo pathway can persist even after the cells are removed from selection. Hypoxanthine and thymidine (HT) provide the necessary nucleotide precursors for hybridoma cells to synthesize DNA using the salvage pathway. Once the cells are growing well in Medium E, they can be gradually switched to Medium A or another medium without HT.

Is the serum in ClonaCell™-HY media heat inactivated?

Yes, all serum used in ClonaCell™-HY media is heat inactivated.

Is there any IgG in clonacell™-HY media?

While we don't add IgG to the ClonaCell™-HY media, we do add serum, which contains an undefined amount of IgG. We selectively use serum lots with low IgG levels in the production of ClonaCell™-HY media, however, levels vary from lot to lot. IgG levels in a specific lot of ClonaCell™-HY medium are available in the lot-specific Certificate of Analysis.

Are there antibiotics in ClonaCell™-HY media?

These products contain gentamycin rather than penicillin/streptomycin/amphotericin B, because gentamycin is more stable and is a broad spectrum antibiotic that is non-toxic to most mammalian cells in culture.

What is the optimal number of colonies per plate?

We recommend 50-150 colonies per plate. An average fusion will result in approximately 1000 colonies per fusion (approx. 100 colonies per plate). Even if the average number of colonies per plate approaches 300, there should still be enough separation between colonies to pick easily.

Why do I have to put my fused cells into liquid medium overnight? Why can't I just plate directly into Medium D?

We recommend waiting up to 24 hours so that all of the fused cells can go through one cell cycle. This will ensure they have a chance to express HPRT (hypoxanthine guanine phosphoribosyltransferase), the enzyme necessary to survive in the presence of aminopterin (present in Medium D). Additionally, fused cells are very fragile immediately after fusion. Waiting a day before mixing the cells with the methylcellulose will improve their survival. Although it is not recommended, fused cells may be plated on the same day as fusion, but the cells should be allowed to recover for several hours in ClonaCell™-HY Medium C prior to plating.

What myeloma and mouse strains should I use?

Myeloma: There are at least two common myeloma cell lines used to generate hybridomas - SP2/0 and P3X63Ag8.683. Both are available from ATCC. Researchers should ensure that the myeloma line is from a reliable source and is negative for mycoplasma. Mycoplasma contamination of the myeloma line can result in decreased efficiency of hybridoma formation. Mouse: We suggest using BALB/c splenocytes and parental myeloma cells of BALB/c for the following reasons: they are highly immune reactive, well characterized and myeloma cells are available from the same genetic strain. Other mouse strains, however, are also compatible with cloning in ClonaCell™-HY media.

Can I grow human/rat/T cell hybridomas in ClonaCell™-HY?

Although we have not tried to generate human, rat or T cell hybridomas during in-house testing, these experiments are expected to be successful using ClonaCell™-HY. The researcher would need to ensure that the cell lines used in the fusion are sensitive to HAT selection and grow well in methylcellulose-based medium.

There are very few colonies growing in my Medium D. Why?

Low numbers of colonies is generally a result of low fusion efficiency, which can have many causes. The fusion efficiency can be affected by the presence of serum during fusion, the presence of mycoplasma, low viability of cells, overexposure to polyethylene glycol or slow-growing myeloma cells prior to fusion.

Why does the ClonaCell™-HY manual suggest two different methods for fusion (A or B)? Can one expect better results with one method over the other?

Which method chosen is a personal preference and there should not be significant differences in efficiency. Method B is faster and has less steps, but Method B requires you to remove all the PEG before the cells are diluted, so you will risk aspirating cells if not very careful. With Method A, you dilute the PEG with Medium B, so you have less opportunity to lose cells.

Why does the ClonaCell™-HY manual suggest two different methods for fusion (A or B)? Can one expect better results with one method over the other?

A: Which method chosen is a personal preference and there should not be significant differences in efficiency. Method B is faster and has less steps, but Method B requires you to remove all the PEG before the cells are diluted, so you will risk aspirating cells if not very careful. With Method A, you dilute the PEG with Medium B, so you have less opportunity to lose cells.

Once I pick the colonies and grow the cells in plates, will the residual methylcellulose interfere with characterization? For example, will I have problems doing an ELISA?

There will likely be some residual methylcellulose contamination when colonies are picked and transferred to the 96-well plate with the liquid growth medium. The concentration of methylcellulose, however, should be low enough that it should not interfere with most assays.

Structural basis for nonneutralizing antibody competition at antigenic site II of the respiratory syncytial virus fusion protein. Mousa JJ et al. Proceedings of the National Academy of Sciences of the United States of America 2016 OCT

Abstract

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix-loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies.

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An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody with prophylactic and therapeutic activity in vivo Wilson JR et al. Antiviral Research 2016 NOV

Abstract

Zoonotic A(H7N9) avian influenza viruses emerged in China in 2013 and continue to be a threat to human public health, having infected over 800 individuals with a mortality rate approaching 40%. Treatment options for people infected with A(H7N9) include the use of neuraminidase (NA) inhibitors. However, like other influenza viruses, A(H7N9) can become resistant to these drugs. The use of monoclonal antibodies is a rapidly developing strategy for controlling influenza virus infection. Here we generated a murine monoclonal antibody (3c10-3) directed against the NA of A(H7N9) and show that prophylactic systemic administration of 3c10-3 fully protected mice from lethal challenge with wild-type A/Anhui/1/2013 (H7N9). Further, post-infection treatment with a single systemic dose of 3c10-3 at either 24, 48 or 72 h post A(H7N9) challenge resulted in both dose- and time-dependent protection of up to 100% of mice, demonstrating therapeutic potential for 3c10-3. Epitope mapping revealed that 3c10-3 binds near the enzyme active site of NA, and functional characterization showed that 3c10-3 inhibits the enzyme activity of NA and restricts the cell-to-cell spread of the virus in cultured cells. Affinity analysis also revealed that 3c10-3 binds equally well to recombinant NA of wild-type A/Anhui/1/2013 and to a variant NA carrying a R289K mutation known to infer NAI resistance. These results suggest that 3c10-3 has the potential to be used as a therapeutic to treat A(H7N9) infections either as an alternative to, or in combination with, current NA antiviral inhibitors.

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Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice. Sapparapu G et al. Nature 2016 NOV

Abstract

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy(1). To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies (mAbs) from subjects with prior ZIKV infection. A subset of mAbs recognized diverse epitopes on the envelope (E) protein and exhibited potently neutralizing activity. One of the most inhibitory mAbs, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African, Asian, and American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. mAb treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human mAbs can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.

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