Allotype-defined mRNA for rabbit immunoglobulin H and L chains isolated from rabbit-mouse hybridomas

Improved procedures have expedited the formation and propagation of stable rabbit-mouse hybridomas (RMH) that secrete rabbit immunoglobulin (Ig) chains and that serve as sources for allotype-defined mRNA, specifying both constant and variable regions of rabbit Ig. The Ig-secreting hybridomas were stabilized by multiple recloning steps and eventually attained a 90% frequency of cells secreting rabbit Ig chains. Stabilized RMH were propagated in vivo in nude (athymic) and in some instances in conventional BALB/c mice. Rabbit Ig products secreted by these RMH cell lines were isolated and were shown to have amino acid sequence characteristics of rabbit Ig chains and that are distinct from those of the mouse. Preparative amounts of polyadenylated RNA-(poly(A) RNA) encoding allotype-defined rabbit Ig chains were isolated from RMH grown as solid tumors. Poly(A) RNA encoding a b4 L chain from one RMH (12F2) and an a3 H chain from another (7D2) were purified 10-fold by sucrose density gradient centrifugation and were characterized in an in vitro translation system. The mRNA encoding the b4 allotype had an S value of 12 and directed the synthesis of a rabbit precursor L chain with m.w. 27,500. Radiochemical amino acid sequence analysis of the L chain precursor has shown it to include a 22 residue leader sequence with a leucine profile similar to that of murine L chain precursors. Analysis of the mRNA encoding the a3 H chain revealed it has an S value of 16 and directed the synthesis of an Ig chain with a m.w. identical to that of the rabbit H chain secreted by the RMH.