Isolation of hybridomas expressing a specific heavy chain variable region gene segment by using a screening technique that detects mRNA sequences in whole cell lysates.

Abstract

A technique is described that allows single hybridoma cell colonies to be assayed for the productive rearrangement of a single immunoglobulin variable region (V) gene segment by utilizing expression of V mRNA for analysis. Hybridomas growing in microwell tissue culture plates are lysed in situ, cellular RNA is directly transferred to nitrocellulose by filtration, and specific immunoglobulin mRNA is detected by hybridization of the filter with a DNA probe. The method is simple and sensitive. A single species of mRNA can be detected in a lysate of 1000 cells; 5000 hybridoma colonies can be easily screened per day. The technique has been successfully used to isolate cell lines from nonimmune mice expressing a particular heavy chain variable region (VH) gene segment.