There are various techniques developed to isolate monoclonal antibodies (mAbs). Hybridoma technology has a relatively low efficiency, which compromises sampling the diversity of immune repertoire. Antibody phage display is widely adopted for mAb isolation, but they are prone to generate biased repertoires and lose information of natural pairing. View more about "mAb technology".
However, in antibody development, it is thought to be important to maintain the original VH and VL pairing as exists in human B cells. Single B cell technology is an efficient strategy based on the direct amplification of VH and VL region encoding genes from single human B cells. It's a powerful technique to generate monoclonal antibodies from humans and immunized animals.
The single B cell antibody approaches harbor the potential to isolate functional mAbs reactive against conformational determinants that are present predominantly in vivo and difficult to emulate in vitro. Single B cell technologies have evolved rapidly in recent years for the straightforwardness, requirement of relatively few cell numbers and the high efficiency in obtaining specific mAbs in a rapid way.
Sino Biological leverages single B cell sorting platform to provide single B cell antibody service worldwide, involving single B cell isolation, sequencing, cloning and screening of single B cell antibodies.
Single B cell isolation can be performed either in a random way or in an antigen-selective manner from peripheral blood or from lymphoid tissues. For random B cell isolation, micromanipulation, laser capture microdissection and fluorescence-activated cell sorting (FACS) can be applied. Alternatively, selection of antigen-specific B cells can be performed using antigen coated magnetic beads, fluorochrome-labeled antigens via multi-parameter FACS, the hemolytic plaque assay and a fluorescent foci method.
With FACS technology, cells can be sorted and clearly distinguished based upon the expression patterns of specific cell surface markers. For efficient recovery of specific mAbs, the donor's immune response must be assessed in this method, for example, by determining the frequency of antibody-secreting cells (ASCs) in peripheral blood using an enzyme-linked immunospot technique (ELISPOT) before single cell isolation.
Additionally, before single B cell cloning, high-throughput methods such as microengraving can be used to lead to the efficient identification and desired specificity of B cells. These techniques simplify the screening of various antigens to allow the selection of different clones with distinct specificities.
Single cell cDNA synthesis is usually performed in the 96-well plate. Full-length Ig gene transcripts are amplified by nested or semi-nested RT-PCR. Typically, forward primer mixes complementary to the corresponding IgH and IgL V gene leader sequences and a single reverse primer specific to the constant region sequence are used. If necessary, for example, when isotype independent cell sorting is performed, mixed reverse primers can be used for the amplification of IgH chains with different constant regions. In the second round, nested primer or primer mixes are used to increase sensitivity and specificity. During this step, restriction sites for subsequent cloning steps can be incorporated into the amplicons. Linear expression cassettes can be directly transfected into mammalian cells for in vitro expression of monoclonal antibodies.
To determine the reactivity profile and biophysical characteristics of antibodies or fragments, the proteins have to be expressed, purified and tested in various assays. Conveniently, in the cell-based microarray systems this reactivity screening can be performed before Ig gene cloning. To analyze the corresponding antibodies in more detail, however, molecular Ig gene cloning steps are indispensable, as large-scale amounts of protein are needed. The most common choices for expression systems are bacterial systems (e.g. Escherichia coli) or transient and/or stable mammalian cell systems (e.g. HEK 293, CHO cells). In E. coli., the cloned Ig genes are typically expressed as antigen-binding fragments (Fabs), whereas in mammalian cells expression can be in complete IgG format.
|Single cell sorting||Variable region gene amplification|
|• Enrich target cells before sorting
• Adjustment of instrument compensation
• Perform assay to determine optimal antibody usage
• Add 7-AAD and other cell reactive dyes to remove interference from dead cells
• Properly reduce the sorting speed to ensure higher sorting efficiency
|• Ensure RNase removal
• Lysis condition optimization
• Design effective reverse transcription primer
• Variable region amplification
1. Tiller, T. (2011). Single B cell antibody technologies. New biotechnology, 28(5), 453-457.
2. Lei, L., Tran, K., Wang, Y., Steinhardt, J. J., Xiao, Y., Chiang, C. I., ... & Li, Y. (2019). Antigen-Specific Single B Cell Sorting and Monoclonal Antibody Cloning in Guinea Pigs. Frontiers in microbiology, 10, 672.