The role of B-cell cloning in the generation of antibody critical reagents: apoptosis-related antigen case study

B-cell cloning, a recent improvement over typical hybridoma technology, is becoming an industry standard tool for the reliable and reproducible production of monoclonal antibodies. Read on to find out how we have successfully utilized B-cell cloning to produce antibody critical reagents, for a recombinant protein, and to learn how this service offering can help you by supplying antibody critical reagents specific to your clinical development needs.^1,2

By expanding our existing capabilities, including in the further refinement of our monoclonal antibody generation services, we can provide you with an unlimited supply of reproducible monoclonal antibodies that can be used in advancing the development of your biotherapeutics, from the preclinical phase all the way through to commercialization.

Here we share a case study that demonstrates how we utilize B-cell cloning and how this technique can help you achieve your goals.¹

We present the case study in the following steps, to mirror our B-cell cloning procedure:

1. Immunization of an animal with the antigen of interest
2. Isolation of peripheral blood mononuclear cells (PBMC) or splenocytes
3. Cell sorting to identify those which are producing antigen-specific antibodies
4. Primary screening and culturing of those cells
5. Identification of B-cells producing the antibodies of interest
6. Cloning the heavy and light chain genes of the selected B-cells
7. Insertion into a mammalian expression vector and creation of a cell line or purification of the antibody

Case Study: B-cell cloning of an apoptosis-related antigen

Using B-cell cloning, we generated highly reproducible monoclonal antibodies against a recombinant protein involved in caspase-related apoptosis, from a sample expressed in E.coli.¹

1. Immunization

The recombinant protein was purified and injected into two rabbits. An enzyme-linked immunosorbent assay (ELISA) was then used to detect and measure the antibody levels in obtained blood samples (see Table 1).¹

Table 1. ELISA Titers¹

2. Isolation of splenocytes

This initial ELISA demonstrated that both of the immunized rabbits had produced significant antibody titers. Subsequently, splenocytes were isolated from one of the rabbits.¹

3. Cell sorting

These cells were incubated with a labelled antigen and sorted through a fluorescence-activated cell sorter (FACS) which separated out any splenocytes reacting to the antigen for B-cell enrichment.¹

4. Primary screening and culturing

Once the sorting process was complete, cells were plated into a 384-welled plate and cultured overnight. Aliquots of cultured B-cell supernatants were tested and confirmed by ELISA.¹

5. Identification of B cells

Table 2. ELISA results: Clones with an OD signal greater than 0.3¹

There were 6.9% (132 clones) of the total clones which had an OD signal greater than 0.3 in the antigen specific ELISA (see Table 2).¹ 

6. Cloning the heavy and light chain genes

Based on the corresponding absorbance readings, 12 clones with the strongest antibody responses were selected, and the heavy and light chain genes from each were cloned into a linear expression module (LEM). The LEM was then transfected into human embryonic kidney (HEK) cells and the culture supernatants were tested by ELISA.¹

Table 3. ELISA results: 12 clones with the strongest antibody responses¹

7. Subcloning into mammalian expression vector and antibody purification

The top 3 ELISA-positive clones were subcloned into an expression vector and transiently transfected into HEK cells. The cells were cultured, and the supernatants were tested by ELISA. The culture supernatants were then harvested, and the secreted antibody was purified by Protein A affinity chromatography.¹ 

Table 4. ELISA results: Top 3 ELISA-positive clones¹

Critical Reagent Management

Generation of these monoclonal antibodies is the first step to successfully managing a portfolio of critical reagents.  Additional assays will require additional reagents, advancing the lead drug molecule will require long-term storage and retesting these reagents. More molecules in the pipeline further complicate things.² 

Labcorp is pleased to offer a solution beyond the generation of these antibodies.  Plugging a B-cell cloning project into our Critical Reagent Management services provides a single solution from generation through long-term storage – whether for one or one hundred biotherapeutics in your portfolio.^1,2

We’ll take care of it: supply, maintenance and storage

The expression plasmids may be used for transient transfections to replenish the supply of needed antibody reagents.  Alternatively, a stable cell line may be created for longer-term needs, as appropriate.^1,2 

Reproducibility is key

For more than two decades, biotherapeutic drug molecules have been expressed using a variety of expression systems thanks, in part, to their reproducible nature. Older technologies, such as hybridoma cell lines, suffer from a variety of shortcomings when planning for unlimited, consistent supply of monoclonal antibodies. These technologies were limited to therapeutic generation due to cost and timeline constraints, as well as low availability of the equipment and technologies.² 

Recent advancements allow the same technologies and techniques to now be applied to antibody reagents.  Those same benefits of unlimited supply, greater consistency and reproducibility and increased characterization are now possible, working with partners like Labcorp, for your critical antibody programs.

Box 1: Advantages of B-cell cloning^1,3

Utilizing B-cell based platforms for monoclonal antibody discovery is an important technique with clear advantages, used throughout the industry.

In addition to the critical reproducibility, other key advantages of B-cell cloning include:

• Accelerated screening time of around 3 weeks in comparison to over 12 weeks for hybridoma and 6 weeks for phage display
• >25% more antigen positive clones with B-cell cloning vs standard technologies 
• Variety of host species (unlike hybridoma technology)
• Production of expression plasmids with sequenced heavy and light chain genes 

The main barrier to use is the associated cost

Are you interested in capitalizing on these advantages and working with a reliable supplier of critical antibody reagents that utilizes B-cell cloning and offers supply chain and maintenance support? Contact Us to learn more about how we can work together to achieve your goals or visit our webpage.