Antibody-derived biologics have become a major class of modern medicine, particularly in the fight against cancer and autoimmune diseases.
Highly efficacious immunoglobulin-based drugs have been naturally developed by antibody-producing B cells from within the mammalian immune system. Despite this, finding rare cells with the right characteristics remains a challenge.
Mouse B cells have traditionally been immortalized through hybridoma fusion. However, there are numerous problems because B cells die quickly in culture, and many do not survive the fusion process.
As a result, the rare B cell of interest may be lost throughout the hybridoma formation process. The development of hybridoma fusions is time-consuming, costly, and inefficient, as it may not result in the identification and immortalization of the rare antigen-specific antibody secreting B cells of interest.
To screen and isolate rare antigen-specific producing cells, semi-automated technologies like cell sorting, colony picking, and cell-in-well imagers are now employed in tandem.
The Cyto-Mine® Single Cell Analysis and Monoclonality Assurance System is the first completely integrated biopharma platform that inspects millions of primary B cells or hybridomas for secreted immunoglobulin, recognizes and sorts antigen-specific candidates, and gently dispenses them into 96- or 384-well microtiter plates with visual proof of cell number via high-quality imaging (Figure 1).
The current research demonstrates how Cyto-Mine® uses an antigen-specific test to analyze a heterogeneous B cell or hybridoma population in order to recognize target-specific variations in a high-throughput way.
Figure 1. Cyto-Mine® technology finds and isolates cells secreting antigen-specific antibodies from complex cell populations. Image Credit: Sphere Fluidics
Aims and objectives
This article will outline how Cyto-Mine® can:
- Precisely identify and isolate “hit” cells from a large hybridoma population using an antigen-specific assay
- Evaluate different concentrations of antigen-specific antibodies
- Use a single completely integrated instrument to substantially simplify the Antibody Discovery workflow
Single cell encapsulation
Cells from a hybridoma population were diluted to a concentration that maximizes the number of picodroplets containing only a single cell using Poisson distribution statistics. A dilution can be made for exceptionally large populations to allow for the encapsulation of pools of cells in a single picodroplet (Figure 2).
Figure 2. These images show the encapsulation of single cells or multiple cells per picodroplet. A) Cells were diluted to a concentration of 1 × 106 cells/mL to obtain 1 cell per picodroplet. B) A large population of cells (greater than 1 million) were diluted to a concentration of 1 × 108 cells/mL in medium resulting in multiple cells per picodroplet. Image Credit: Sphere Fluidics
The culture medium was added with OptiPrep™ Density Gradient Medium (Sigma Aldrich Merck) and a set of detection probes, a fluorescently conjugated antigen donor probe and an immunoglobulin-specific acceptor probe, prior to cell suspension (see Box 1).
After that, the diluted mixture was pipetted into a Cyto-Cartridge® and loaded into Cyto-Mine®. The method begins with Cyto-Mine® encapsulating the cells in 300 pL picodroplets.
The number of cells per picodroplet can be adjusted to meet the needs of the user, for example, one cell per picodroplet if single-cell cloning is required, or more than one cell per picodroplet if the population size exceeds 1 million cells, with the latter resulting in the collection of enriched mini-pools of cells rather than single cells (Figure 2).
The cells were incubated in situ in Antigen-Specific Secretion Assay Next Cyto-Mine® so that the secreted immunoglobulins concentrated inside the picodroplets and could be identified by the antigen-specific detection reagent present in the medium (Box 1).
The miniaturized format provides an ultrasensitive, quick assay (usually with an incubation duration of 0.5 to 2 hours) depending on the cell population’s production rate.
The fluorescent antigen-positive hits are sorted according to their fluorescence intensity. Figure 3 depicts the integrated steps of the Cyto-Mine® antigen-specific assay process.
Figure 3. The Cyto-Mine® workflow integrates the screening, sorting, isolation, and verification of antigen-specific clones into a fully automated process. Image Credit: Sphere Fluidics
Prior to dispensing, as the picodroplet travels through the final dispensing microfluidic channel, the encapsulated cells are imaged multiple times to provide verification of clonality.
Box 1. Antigen-specific secretion assay
A key enabling component of Cyto-Mine® is its ability to analyze the secreted immunoglobulins of millions of cells for antigen specificity while maintaining the cells in a highly viable state with no prior modification.
Figure 4 depicts the procedure for reporting positives. The detection probe pair (fluorescently conjugated antigen and an immunoglobulin-specific acceptor probe) binds to the released antigen-specific immunoglobulin, causing a fluorescence shift via FRET.
The fluorescent signal created is subsequently measured and converted into a quantitative output by Cyto-Mine®. The use of an acceptor probe allows for the simultaneous detection of antigen specificity and immunoglobulin isotype.
Figure 4. The Cyto-Mine® picodroplet-based antigen-specific assay. This model shows the standard assay to screen for antigen-specific IgG. Antigen-specific IgG secreted from the encapsulated cell during incubation is recognized by both the antigen and the IgG-specific acceptor probe forming a 3-body FRET complex that induces a fluorescent signal. Image Credit: Sphere Fluidics
Figure 5. Cyto-Mine® Scatter Plot. Large numbers of individual picodroplets were loaded with the indicated concentrations of anti-human TNF-alpha IgG and then resolved using the Cyto-Mine® antigen-specific assay and analysis. Image Credit: Sphere Fluidics
Figure 6. Representative titration curve generated using the Cyto-Mine® antigen-specific assay. In this example, the control IgG confirms specificity of the assay for human TNF-alpha. Image Credit: Sphere Fluidics
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About Sphere Fluidics
Our philosophy is simple. We combine our knowledge and resources to help you find rare and valuable biological variants, while helping you to save time, reduce costs and stay a step ahead of the competition.
Our novel single cell analysis systems offer the rapid screening and characterization of single cells. These systems are underpinned by our patented picodroplet technology, specifically designed to increase your chances of finding that rare ‘one-in-a-billion’ molecule or cell that could be an industry blockbuster.
We understand that time is of the essence. That’s why our technologies boost throughput and assay sensitivity across a range of applications. Most importantly, our flexible systems evolve alongside your changing research needs, providing an adaptable platform that helps you to meet your goals.
Founded in 2010, Sphere Fluidics is an established Life Sciences company, originally spun out from the University of Cambridge. We initially developed 25 patented products – biochips and specialist chemicals – which currently assist hundreds of customers globally with their research.
We initially focused on producing novel biochip systems and providing R&D services. We have since extended our expertise and are developing a technology platform that enables discovery in a range of growing markets through single cell analysis. Our systems make the development of new biopharmaceuticals faster and more cost-effective, improve monoclonal antibody screening, cell line development, and overall research efficiency in a number of other applications including synthetic biology, single cell diagnostics, prognostics and single cell genome editing.
The Cyto-Mine® Single Cell Analysis System is our flagship product – the first integrated, benchtop system to automatically analyse, sort and dispense millions of individual cells in just a single day.
We value and are always open to discussing new collaborative, successful and innovative academic and industry partnerships to further develop and improve our single cell technologies.
Our Technology Access Programmes and Collaborative Services exist to enable academic researchers and companies alike to tap into our application-specific expertise through direct partnerships.
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