Analysis of isotype switch variants of a Qa-5 specific hybridoma.

Qa-5 is a Class I MHC Ag expressed on mature T cells and natural killer (NK) cells of mice, and is defined by specific reactivity with an IgM secreting hybridoma (B16-167). The availability of IgG antibodies with Qa-5 specificity would greatly facilitate in vivo studies of Qa-5 positive NK cells. Using a sequential subline technique in conjunction with ELISA screening, several isotype variants of the B16-167 parent line were selected and cloned. An IgG2a, an IgG2b, and two IgG1 switch variants were selected, and it was demonstrated that one of these cloned lines produced both IgM and IgG1 simultaneously. Binding of the variant antibodies to Qa-5+ cells was not detected by flow cytometry analysis, nor by the ability to deplete NK activity from Qa-5+ large granular lymphocytes (LGL). However, all of the variant lines appeared to use the same VH region genes by Southern blot analysis. Furthermore, IgG2a from the switch variants inhibited binding of the parental IgM anti-Qa-5 when used in excess, and reduction of the parental IgM anti-Qa-5 to monomeric form abolished its binding capacity. These data together indicate that the weak binding by the IgG variants may be due to loss of an innate low affinity/high avidity binding of the parent IgM. Therefore, the isotype switch detection procedure is a useful technique for selection of antibodies with various effector functions, provided the initial antibody is an antibody with sufficient binding affinity.