In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen

Many enzyme-linked immunosorbent assays take advantage of immobilized antig ens for the identification of antibody binding sites. Generally, the analys is of cellulose membrane-bound B-cell epitopes is currently considered of h igh utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides correspon ding to the entire rabies virus nucleoprotein were synthesized in duplicate s on a single sheet of 90 x 130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted am ino groups. After completion of the synthesis, the side-chain protecting gr oups were removed, and the membrane was thoroughly cleaned of all organic a nd inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antig ens. From all the 40 peptide spots only peptide 31D stimulated the prolifer ation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 mu g (approximately 200 pmole) of pep tide could be detected. According to mass spectrometry the T-cell stimulati on proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium-splenocyte mixture. Additionally , tryptic digestion of the cellulose membrane released the expected peptide fragments. (C) 2000 Elsevier Science B.V. All rights reserved.