AN ISOTYPE-SPECIFIC SPOT-ELISA FOR THE ENUMERATION OF ANTIBODY-SECRETING HYBRIDOMAS AND THE DETERMINATION OF ISOTYPE SWITCH VARIANTS

A solid-phase spot enzyme-linked immunosorbent assay (spot-ELISA) using rat monoclonal antibodies (MAbs) and an image-processing system is described. This isotype-specific spot-ELISA permits the enumeration of antibody-secreting cells irrespective of the specificity of the secreted antibodies. When used in combination with an ELISA, the antibody production per cell can also be evaluated. In addition, isotype switch variants, which arise spontaneously in antibody-producing cell lines, can be determined. This study compared four assays: three antigen-specific spot-ELISAs, using enzyme-conjugated polyclonal antibodies as well as rat MAbs; and an isotype-specific spot-ELISA using rat MAbs. There were no significant differences between these four spot-ELISA systems. For one tested cell line (alpha-huIgA1/gamma-1), the number of antibody-secreting cells fluctuated between 60% and 95% during several passages. For the other tested cell line (alpha-huIgA1/gamma-2b), the number of antibody-secreting cells decreased from 90% to 70% after several passages. The results of the spot-ELISA were in agreement with flow cytometric (FC) analysis of cytoplasmic IgG. This indicates that for these two cell lines, the synthesized IgG was also secreted into the culture fluid. Using the isotype-specific spot-ELISA, the switch frequency of five murine hybridomas (alpha-huIgA1/gamma-1, alpha-huIgA1/gamma-2b, alpha-HRP, RIV6, MN12) was determined. The switch frequencies varied from 1/82,000 for the alpha-HRP cell line to 1/660,000 for the alpha-huIgA1/gamma-2b cell line.