Abstract: Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90×130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 μg (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation 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.

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Istex/Checkpoint

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{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    Istex
   |étape=   Checkpoint
   |type=    RBID
   |clé=     ISTEX:C7833F77814D3441F6B65AF955DC4908B86719AF
   |texte=   In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen
}}