A plaque assay for cells making antibody against HLA antigens

Abstract

Plaque assays that can detect single antibody-forming cells can be highly informative. A need was identified for a plaque assay which uses nucleated cells as targets, instead of the traditional erythrocyte targets. The objective of this thesis was to develop such an assay and to try it on some simple cellular problems in the field of monoclonal antibody technology. -- The principle of the plaque assay was to use carboxyfluorescein diacetate to stain living, EBV-transformed target cells and a red fluorescent dye [propidium iodide) to stain the dead cells, in a plaque, that had been lysed by antibody and complement. A fluorescence microscope was then used to count the plaques. The final method involved the use of microscope slides for the assay and resulted in permanent preparations that could be stored and examined at leisure. -- Trials with this technique using a mouse hybridoma, Nfld.M2, secreting cytotoxic antibody of anti-HLA-A2 and A28 specificity and of IgG2a subclass showed that less than 22% of these recently cloned cells formed plaques and that the majority of the cells were not secreting antibody, at least at levels detectable in this plaque assay. Efforts to increase the number of plaques by the use of a second antibody revealed a prozone effect. Another mouse hybridoma , Nfld.M1, secreting non-cytotoxic antibody of anti-DR4 specificity and of IgG1 subclass was also used in the plaque assay. No plaques were obtained even with the addition of developing antiserum. -- A human hybridoma, Nfld.H1, secreting cytotoxic antibody of IgM class was also used in the plaque assay. No plaques were observed and this was attributed to the low rate of antibody secretion by the hybridoma cells. When human EBV-transformed cells were used in the plaque assay as a source of antibody-secreting cells, clear and distinct plaques were obtained. -- The experiments described here show that the plaque assay is suitable for use in analysing problems in cellular immunology in both mouse and human systems. The results have revealed at least one unexpected finding and this is the low proportion of antibody secreting cells found in a clone and ways are suggested for further research on these. Finally, the method for producing permanent preparations for immunofluorescence may find other applications for teaching or research.