The production of equine monoclonal immunoglobulins by horse-mouse heterohybridomas.

The research investigates the best conditions for producing horse monoclonal antibodies, specifically against a strain of equine influenza virus. It involved combining certain horse and mouse cells and specifically mutating the resulting hybrid cells to increase the continued production of the antibodies.

Research Context and Objectives

• The study sought to figure out the optimal conditions required for the production of equine monoclonal antibodies (MAbs), which are antibodies derived from one cell line used in detecting or combating particular foreign substances or toxins.
• The focus of this research was on creating MAbs against the influenza A equine 2 virus of the strain A/Equine/Newmarket/79 (H3N8).

Methodology

• Lymphocytes, a type of white blood cell, were taken from ponies that had been immunised against the H3N8 influenza strain.
• These pony lymphocytes were fused with mouse myeloma cells, cancerous cells that are widely used in immunology research due to their ability to produce a significant amount of antibodies.
• The resulting hybrid cells, called heterohybridomas, were then treated with 8-azaguanine, a substance often used in lab settings to select specific cell types, to make them sensitive to aminopterin, an agent which inhibits cell growth and reproduction.

Observations and Findings

• The research found that while all fusions initially resulted in heterohybridoma colonies secreting equine immunoglobulin, many could not maintain this secretion for more than a few weeks.
• By increasing the period between immunisation and the booster injection of the H3N8 virus, adding Freund’s incomplete adjuvant – an agent that boosts immune response, and by using an aminopterin-sensitive primary heterohybridoma as the fusion partner, the production of antibody-secreting heterohybridomas was significantly improved.
• After running this improved process twice, eight stable cell lines were established which could maintain secretion of anti-H3N8 antibodies for more than a year.

Additional Benefits and Applications

• The study demonstrated that Fluorescence-Activated Cell Sorting (FACS) analysis, which is a method to sort and classify cells, of these cell lines could predict potential breakdowns of monoclonal antibody secretion.
• With this predictive capability, the researchers could carry out a necessary re-cloning process early, thereby assuring a continuous antibody production.