Production of monoclonal antibodies against antigens of the human erythrocyte

McNicholas, Susan (1981) Production of monoclonal antibodies against antigens of the human erythrocyte. Masters thesis, Memorial University of Newfoundland.

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Abstract

The objectives of this program were to establish, in Newfoundland, the recently described method for making monoclonal antibodies by cell fusion, to explore various aspects of the technique and to attempt to produce lymphocyte hybridomas secreting monoclonal antibodies against human erythrocyte antigens. -- Fusion, with the aid of polyethylene glycol, of spleen cells from an immunized mouse with mouse plasmacytoma cells, may result in formation of hybrid cells. Such hybrids, which contain a B lymphocyte nucleus from the spleen cells, may make monoclonal antibody. -- Tests showed the plasmacytoma cells grew well in medium RPMI 1640 with 10% fetal calf serum from a concentration of 2.5 x 10⁴ to 4 x 10⁵ cells per ml. but viability declined rapidly if cultured longer. Polyethylene glycol was found to be toxic to these cells but a suitable concentration was found which allowed both cell fusion and survival of the resulting hybrids. Preliminary testing of the mouse response to red cells (RBC) of various species showed that the standard Jerne plaque technique gave good results with sheep RBC but no plaques with human RBC, even though serum antibody titres were satisfactory. Techniques were later established for detection, in culture fluid, of mouse immunoglobulin (inhibition of the antiglobulin test) and of specific anti-RBC antibody (agglutination). -- After 35 fusions and various modifications, the method began to produce hybridomas. Altogether in a series of seven successful fusions 152 colonies were produced with 82 colonies secreting mouse immunoglobulin and 4 of those secreting anti-human RBC antibody. -- Of nine species of red cells tested, the only ones to be agglutinated by the four culture supernatants were human RBC. Red cell samples from 87 unrelated humans were screened and all were agglutinated by each of the four culture supernatants. Using 2-mercaptoethanol all four antibodies behaved as if they were Ig M. It was concluded that these monoclonal antibodies were species specific and not detecting alleles. -- It is evident from these and other published studies that monoclonal antibodies with allelic specificity may be less easy to obtain than expected.

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