[Hybridoma cloning by the end-point dilution method].

Area of Science:

• Immunology
• Virology
• Cell Biology

Background:

• Hybridomas are crucial for producing monoclonal antibodies (mAbs).
• Efficient cloning of hybridomas is essential for obtaining stable, high-producing cell lines.
• Monoclonal antibodies are vital diagnostic and therapeutic tools.

Purpose of the Study:

• To optimize the end-point dilution method for cloning hybridomas producing mAbs against specific viruses.
• To assess the impact of cell seeding density on cloning efficiency and hybridoma productivity.
• To evaluate the effect of repeated cloning on hybridoma stability and immunoglobulin secretion.

Main Methods:

• Hybridomas producing mAbs against tick-borne encephalitis, Venezuelan equine encephalomyelitis, and vaccinia viruses were used.
• End-point dilution cloning was performed in 96-well plates with a cell feeder layer.
• Cell seeding doses were systematically varied to determine optimal conditions.
• Titers of secreted immunoglobulins and cell growth characteristics were analyzed post-cloning.

Main Results:

• Decreasing cell seeding dose reduced the average number of clones per well but increased cloning effectiveness.
• Introducing an average of one cell per well yielded single clones in 5.4% to 37.5% of wells.
• The first cloning round significantly increased immunoglobulin titers and improved hybridoma growth.
• Subsequent cloning rounds generally did not alter productivity but were necessary for line stability.

Conclusions:

• The end-point dilution method, with optimized cell seeding, is effective for cloning hybridomas.
• Initial cloning enhances mAb production and hybridoma cell characteristics.
• Repeated cloning is important for ensuring hybridoma cell line stability without compromising productivity.