Screening for Positive Hybridoma Cells by Flow Cytometry
The production of hybridomas is a way to develop mAbs for various targets used for research, diagnosis, or therapeutic purposes. In the production of the monoclonal antibody, hybridoma screening is a key step, which requires the rapid identification of positive clones from hundreds to thousands of hybridoma cell lines against the desired target. Here, Creative Biolabs has established a time-saving and labor-saving Hybridoma platform to quickly and effectively provide global clients convenient and high-quality hapten-specific hybridoma screening services.
Since discovered by Georges Kohler and Cesar Milstein in 1975, hybridoma technology has made indelible merit for antibody production and development. Generally, combining the short-lived antibody-producing B lymphocyte with an immortal myeloma cell forms an antibody-secreting hybridoma, which features with both antibody-producing ability and limitless reproductivity. So now comes the question, how to ensure that the hybridomas you choose after cell fusion are capable of continually secreting antibodies. Therefore, a simple and rapid method for efficient hybridoma screening is needed to select positive hybridomas without labor-intensive and time-consuming.
Over the years, several approaches have been developed for screening positive hybridoma against the desired target antigen. With the improvement of biotechnology, hybridoma screening tends to be more high-throughput, efficient, and specific.
- Microplate-based enzyme-linked immunosorbent assay (ELISA)
- Fluorescence-activated cell sorting (FACS) or flow cytometry (FC)
- High-throughput cell-based Celigo Image Cytometer
- Droplet-based microfluidics
Hapten-Specific Hybridoma Screening by Flow Cytometry
Different from macromolecular antigens which directly stimulate the immune system and induce an immune response, haptens usually do not cause an immune response and should be linked to a carrier protein for immunization. Despite this fact, the principle of hapten-specific hybridoma screening is identical to that of macromolecular antigen. Generally, the anti-hapten membrane antibody on the hybridoma surface shares the same binding characteristics as its secreted form, so the anti-immunoglobulin antibodies or fluorescence-labeled antigens bound to the membrane immunoglobulin can be used for FACS/FC-based positive hybridoma screening. The hapten specificity can be detected by adding a hapten-peroxidase conjugate together with a fluorophore-labeled anti-peroxidase antibody to hybridoma culture, and the fluorophore-labeled anti-immunoglobulin antibody can be used for screening the antibody-expressing hybridoma by confocal laser scanning microscopy of FASC/FC.
Fig.1 Hapten-specific hybridoma screening by flow cytometry and ELISA. (Dippong, 2017)
Compared with conventional screening methods, this FASC/FC-based screening technology is efficient enough to select the antibody-secreting or hapten-specific hybridoma in a high-yielding and time-saving manner. More importantly, this screening method can also be applied for hapten-specific isolation of B lymphocytes as well as single-cell fusion.
Fig.2 The general principle of hybridoma screening by flow cytometry. (Uribe-Benninghoff, 2014)
Creative Biolabs' flow cytometry service can quickly and efficiently screen positive hybridoma cells to speed up the production of mAbs. Based on an advanced experimental platform, powerful analysis software, and abundant experience, our experts can provide you with customized solutions and professional data analysis. Please contact us for more hybridoma cell screening services.
- Dippong, M.; et al. Hapten-Specific Single-Cell Selection of Hybridoma Clones by Fluorescence-Activated Cell Sorting for the Generation of Monoclonal Antibodies. Analytical Chemistry. 2017, 89(7): 4007-4012.
- Uribe-Benninghoff, A.; et al. Screening hybridomas for cell surface antigens by high-throughput homogeneous assay and flow cytometry. Methods in Molecular Biology. 2014, Chapter 6, 1131:81-103.