What are the main advantages of using your mouse monoclonal antibody production services compared to other host species?

Utilizing mouse monoclonal antibody production offers several advantages. Mice have a well-characterized immune system, which translates to a high success rate for generating antibodies against a wide range of antigens. Their relatively short life cycle and high fecundity allow for the rapid generation of large numbers of hybridomas, which is essential for comprehensive screening. Additionally, a vast array of immunological reagents and tools are available for mouse studies, facilitating efficient antibody development and characterization. While other species have specific advantages (e.g., higher affinity from rabbit systems, or reduced immunogenicity from humanized antibodies), mouse hybridomas remain a cost-effective and efficient method for many antibody discovery projects, providing a robust platform for generating high-quality antibodies.

How do you select the optimal mouse strain for hybridoma generation?

Mouse strain impacts hybridoma generation success. Different strains have varying immune responses. We consider several factors. For most antigens, we use BALB/c mice. For others, strains like C57BL/6 or SJL may be better. For poorly immunogenic antigens, we may use genetically modified mice with enhanced antigen-presenting cell function. We also consider client requirements and prior experience with mouse strains. We maintain various strains and have expertise in optimizing the immune response.

What's your mouse immunization protocol, and how do you handle poorly immunogenic antigens?

We employ an optimized immunization protocol to maximize the mouse immune response. This involves multiple immunizations with the target antigen and a suitable adjuvant. We tailor the adjuvant (e.g., Freund's, CFA, IFA, Alum, MPLA, or CpG) and immunization schedule (route, frequency) to the antigen's properties. For poorly immunogenic antigens, we use strategies like conjugating the antigen to a carrier protein (e.g., KLH, BSA) or prime-boost immunization. In some cases, we switch to a mouse strain that responds better.

How do you ensure stable, high-quality antibody production from mouse hybridomas over multiple passages?

Ensuring hybridoma stability is crucial. Post-fusion, we use HAT-selective media to eliminate unfused cells. We then perform limiting dilution to isolate stable single hybridoma clones. Throughout culture, we monitor cell viability, growth, and antibody production using flow cytometry and ELISA. A common problem is chromosome loss, leading to decreased or lost antibody production. We address this with regular subcloning (every 2-3 weeks) to maintain high-producing cells. We also offer cryopreservation using controlled-rate freezing and serum-free media. Regular re-cloning is recommended for long-term projects.

What methods do you use to screen hybridoma supernatants, and how do you handle low initial clone numbers?

We use various screening methods to identify desired hybridoma clones. Initial screening typically involves ELISA. We also use flow cytometry for cell binding, especially for membrane targets. For high-throughput screening, we use automated liquid handling. With few initial positive clones, we re-evaluate the immunization protocol (antigen dose, adjuvant, schedule). We may also try alternative assays like SPR or BLI. Furthermore, we can perform cell sorting to enrich antigen-specific B cells before fusion.

How do you validate the specificity of generated mouse monoclonal antibodies?

Ensuring high specificity is critical. We employ a rigorous validation process. Initially, we perform ELISA with related antigens/proteins to assess cross-reactivity. We also use Western blotting and immunohistochemistry (IHC) to confirm binding to the target protein in cells/tissues. For membrane targets, we use flow cytometry with cells expressing related proteins. Furthermore, we use SPR or BLI to measure binding kinetics. If cross-reactivity is a concern, we perform affinity maturation or antibody engineering.

Mouse Hybridoma based Antibody Discovery Service

Introduction Antigen Preparation Immunizations Hybridoma Development Antibody Synthesis Published Data Features FAQ

Creative Biolabs offers services for the construction of mouse monoclonal antibodies. Notably, our exclusive approach to mouse immunization enables the production of mouse monoclonal antibodies within a timeframe of 70 days.

One-Stop Murine Hybridoma Services

We provide comprehensive one-stop murine hybridoma services, customizing protocols at every stage of monoclonal antibody production to meet your exact specifications. This begins with meticulous antigen preparation, offering options from peptide synthesis to protein expression in various platforms like E. coli, yeast, insect, or mammalian cells, ensuring the optimal antigen for your needs. We then employ tailored animal immunization protocols, carefully selecting adjuvants, inoculation routes, dosages, and timing to maximize the immune response. Following this, we utilize refined PEG-mediated fusion methodologies to create hybridomas from splenocytes and myeloma cells. The subsequent hybridoma screening process is also highly customized. We also offer ascites generation or in vitro antibody production. This one-stop approach ensures exceptional specificity and affinity, reaching the picomolar range, and allows for the production of a wide array of functional antibodies. These antibodies can also detect subtle alterations, including post-translational modifications. These tailored antibodies find applications in various techniques, including ELISA, FACS, ICC, IHC, WB, and flow cytometry.

Fig. 1 The workflow of murine hybridoma. (Creative Biolabs Original)

Antigen Preparation

We proficiently express and refine your recombinant proteins utilizing suitable expression systems in E. coli, yeast, insects, or mammalian cells, aligning with the specific objectives of the ensuing antibodies.
Our exclusive technique for chimeric protein expression enables the generation of a soluble form of recombinant protein immunogens within bacterial cells, preserving the original protein's specificity and immunogenicity. This method enhances the likelihood of obtaining monoclonal antibodies yielding positive results in IHC assays, as well as antibodies capable of recognizing the native protein, suitable for employment in ELISA, IP, IF, and Western blotting.
We can devise membrane mimic protein antigens (MMPAs) that maintain structural characteristics akin to multi-span extracellular domains.
Alternatively, we execute peptide synthesis and conjugation methodologies.

Immunizations

We employ tailored protocols for animal immunizations, encompassing appropriate adjuvants, inoculation routes, dosages, and timing.
Our exclusive adjuvant induces a heightened and accelerated immune response in just 21 days.
Our expedited mouse immunization protocol enables the provision of monoclonal antibodies within 70 days.

Development of Hybridoma

Utilizing refined PEG-mediated fusion methodologies, we merge splenocytes with compatible myeloma cells.
Hybridoma clone screening entails bespoke protocols tailored to the intended applications of the requested antibodies, encompassing techniques such as immunoprecipitation, immunoblotting, various ELISA formats, and notably, IHC.
Utilizing a precise methodology to pinpoint monoclonal antibodies suitable for immunohistochemistry (IHC), we introduce our IHC-positive hybridoma screening services.
Our groundbreaking Omni-Hybridoma™ platform enables the efficient selection of numerous hybridoma clones post-fusion, mitigating the risk of fast-growing clones outcompeting potentially valuable slow-growing counterparts.
We deliver positive hybridoma clones either in culture or cryopreserved, along with the corresponding monoclonal antibodies derived from them.

Ascites Generation or In Vitro Antibody Synthesis

Introduction of hybridoma cells into suitable animal hosts to induce ascites production. Typically, we employ immunocompetent mice for ascites generation. Nevertheless, immuno-compromised mice are accessible for non-murine hybridoma cell lines or murine cell lines exhibiting distinct genetic backgrounds. Refined methodologies are employed to initiate tumor formation and accumulate ascitic fluid. The utilization of nude or severe combined immunodeficient (SCID) mice in ascites production serves to eliminate potential contamination from endogenous mouse IgG.
Cultivation of cells in protein-free culture medium. Monoclonal antibody manufacturing is facilitated using stationary flasks or bioreactors ranging from 5-50 liters in capacity. Our antibody fermentation capabilities exceed 300 liters.

Published Data

Schematic diagram of mouse immunization strategy. Fig.1 Mouse immunization strategy and hPD-1-specifc IgG titration curves.¹

Phakham, Tanapati, et al. generated high-potency chimeric anti-PD-1 mAbs using an efficient hybridoma strategy¹. The study integrated various technical procedures into a streamlined workflow, ultimately yielding five potent chimeric anti-PD-1 prototypes. They successfully created mouse hybridoma and mouse/human chimeric clones that produced high-affinity anti-human PD-1 antibodies with robust in vitro PD-1/PD-L1 binding blockade and T cell activation activities.

Features of Murine Hybridoma Services in Creative Biolabs

Exceptional specificity and affinity in the picomolar range;
Capacity to produce extensive arrays of functional antibodies;
Capability to discern subtle alterations including post-translational modifications;
Identification of novel epitopes;
Outstanding outcomes even for challenging antigens;
Availability of assays including ELISA, FACS, ICC, IHC, WB, and flow cytometry;

Creative Biolabs boasts a wealth of expertise garnered from numerous case studies in hybridoma technology services, and we cordially invite you to engage in discussions regarding any challenges or solutions pertinent to your project. Please do not hesitate to reach out to us at your convenience.

FAQ

Q: What are the main advantages of using your mouse monoclonal antibody production services compared to other host species?
A: Utilizing mouse monoclonal antibody production offers several advantages. Mice have a well-characterized immune system, which translates to a high success rate for generating antibodies against a wide range of antigens. Their relatively short life cycle and high fecundity allow for the rapid generation of large numbers of hybridomas, which is essential for comprehensive screening. Additionally, a vast array of immunological reagents and tools are available for mouse studies, facilitating efficient antibody development and characterization. While other species have specific advantages (e.g., higher affinity from rabbit systems, or reduced immunogenicity from humanized antibodies), mouse hybridomas remain a cost-effective and efficient method for many antibody discovery projects, providing a robust platform for generating high-quality antibodies.
Q: How do you select the optimal mouse strain for hybridoma generation?
A: Mouse strain impacts hybridoma generation success. Different strains have varying immune responses. We consider several factors. For most antigens, we use BALB/c mice. For others, strains like C57BL/6 or SJL may be better. For poorly immunogenic antigens, we may use genetically modified mice with enhanced antigen-presenting cell function. We also consider client requirements and prior experience with mouse strains. We maintain various strains and have expertise in optimizing the immune response.
Q: What's your mouse immunization protocol, and how do you handle poorly immunogenic antigens?
A: We employ an optimized immunization protocol to maximize the mouse immune response. This involves multiple immunizations with the target antigen and a suitable adjuvant. We tailor the adjuvant (e.g., Freund's, CFA, IFA, Alum, MPLA, or CpG) and immunization schedule (route, frequency) to the antigen's properties. For poorly immunogenic antigens, we use strategies like conjugating the antigen to a carrier protein (e.g., KLH, BSA) or prime-boost immunization. In some cases, we switch to a mouse strain that responds better.
Q: How do you ensure stable, high-quality antibody production from mouse hybridomas over multiple passages?
A: Ensuring hybridoma stability is crucial. Post-fusion, we use HAT-selective media to eliminate unfused cells. We then perform limiting dilution to isolate stable single hybridoma clones. Throughout culture, we monitor cell viability, growth, and antibody production using flow cytometry and ELISA. A common problem is chromosome loss, leading to decreased or lost antibody production. We address this with regular subcloning (every 2-3 weeks) to maintain high-producing cells. We also offer cryopreservation using controlled-rate freezing and serum-free media. Regular re-cloning is recommended for long-term projects.
Q: What methods do you use to screen hybridoma supernatants, and how do you handle low initial clone numbers?
A: We use various screening methods to identify desired hybridoma clones. Initial screening typically involves ELISA. We also use flow cytometry for cell binding, especially for membrane targets. For high-throughput screening, we use automated liquid handling. With few initial positive clones, we re-evaluate the immunization protocol (antigen dose, adjuvant, schedule). We may also try alternative assays like SPR or BLI. Furthermore, we can perform cell sorting to enrich antigen-specific B cells before fusion.
Q: How do you validate the specificity of generated mouse monoclonal antibodies?
A: Ensuring high specificity is critical. We employ a rigorous validation process. Initially, we perform ELISA with related antigens/proteins to assess cross-reactivity. We also use Western blotting and immunohistochemistry (IHC) to confirm binding to the target protein in cells/tissues. For membrane targets, we use flow cytometry with cells expressing related proteins. Furthermore, we use SPR or BLI to measure binding kinetics. If cross-reactivity is a concern, we perform affinity maturation or antibody engineering.

Reference

Phakham, Tanapati, et al. "Highly efficient hybridoma generation and screening strategy for anti-PD-1 monoclonal antibody development." Scientific Reports 12.1 (2022): 17792. Distributed under Open Access license CC BY 4.0, without modification.

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