NEW MONOCLONAL-ANTIBODIES TO THE T-CELL ANTIGENS CD4 AND CD8 - PRODUCTION AND CHARACTERIZATION IN FORMALIN-FIXED PARAFFIN-EMBEDDED TISSUE

We have generated a recombinant protein representing part of the CD4 m olecule and a peptide representing an epitope of predicted high antige nicity on the CD8 molecule and employed these to generate mouse monocl onal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mR NA from peripheral blood lymphocytes followed by polymerase chain reac tion. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escheric hia coli, Purified fusion protein was used to immunize mice. The CDS m onoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic doma in. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly ef fective on paraffin-embedded tissue. In normal lymphoid tissue, the ex pected distribution of CD4 and CD8 lymphocytes was observed. In a seri es of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in fr ozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterizati on of T cell disorders.