Antigen-Based Immunofluorescence Analysis of B-Cell Targeting: Advanced Technology for the Generation of Novel Monoclonal Antibodies with High Efficiency and Selectivity

Abstract

On the basis of immunofluorescence analysis, an entire pathway of B-cell targeting was successfully identified, which can drive selective production of monoclonal antibodies with high efficiency and selectivity. The technique comprises three critical steps, antigen-based preselection of B lymphocytes, formation of antigenselected B lymphocyte and myeloma cell complexes, and selective fusion of B-cell-myeloma cell complexes with electrical pulses. Intriguingly, expression of surface immunoglobulin receptors on B lymphocytes was recognized even after immunization in vitro. The number of the antigen-selected B lymphocytes after in vitro immunization was in fact higher than that obtained after in vivo immunization, suggesting that such shortterm immunization is applicable for B-cell targeting. Immunofluorescence analysis revealed the targeting technique to demonstrate fivefold to tenfold higher efficiency for formation of hybridoma cells than a polyethylene glycol (PEG)-mediated method. This efficiency is in good agreement with production of hybridoma cells secreting desired monoclonal antibodies determined by an enzyme-linked immunosorbent assay (ELISA). The addition of a low concentration of PEG brought about enhanced fusion efficiency and reduced cell damage at even electric intensities as high as 4.0 kV/cm and 5.0 kV/cm. Here we demonstrate that immunofluorescence analysis can successfully clarify an entire pathway of B-cell targeting and provide advantages over the PEG-mediated method. This advanced technology may be applicable for rapid production of monoclonal antibodies based on in vitro immunization.

Hybridoma technology: is it still useful?

“…The BCT technique demonstrated five-to-ten times greater efficiency in the formation of hybridoma cells secreting the antibodies of interest, in comparison with the PEG-mediated method. However, based on the reported data, such fusion efficiency does not seem to go far beyond 20% ( Tomita et al., 2006 ), and the BCT protocol is more complex than the original hybridoma one. Another point to note is that the electrofusion yields are low when the fusion partner cells have different sizes, although this is a limitation that can be overcome with the use of nanosecond pulse electroporation ( Rems et al., 2013 ).…”

Highly Accurate Profiling of Exosome Phenotypes Using Super-resolution Tricolor Fluorescence Co-localization

“…Then, the fluorescent antibodies are used as probes to detect corresponding antigens (or antibodies) in tissues, cells, or exosomes, forming antigen–antibody complexes. The antibodies can produce fluorescence signals under proper excitation, and the specimen thus can be visualized. − Furthermore, qualitative and quantitative analysis of the targeting proteins are usually realized through the changes of fluorescence signals. , Fluorescent immunolabeling methods can be applied to the detection of various biological molecules owing to some advantages such as no radioactivity, simple operation, high sensitivity, and good selectivity. − However, it is widely known that nonspecific adsorption has long been the most worrisome phenomenon in immunoassays. , Previously, we proposed a strategy in which the nonspecific binding sites can be excluded simply and directly by visualizing the immune recognition process, which can reduce the false positive rate of immunoassays effectively . Due to the small size of exosomes and Abbe’s diffraction limit, traditional optical microscopes with spatial resolutions reaching only 200–300 nm are unsuitable for observing exosomes. − So, the specific and nonspecific binding points of exosomes cannot be observed through traditional optical microscopes.…”