In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen.
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 90x130 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 microgram (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.
Otvos
L
L
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, USA. otvos@wistar.upenn.edu
Pease
A M
AM
Bokonyi
K
K
Giles-Davis
W
W
Rogers
M E
ME
Hintz
P A
PA
Hoffmann
R
R
Ertl
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000008:administration & dosage* / Q000235:genetics; D002460:Cell Line; D002482:Cellulose; D002851:Chromatography, High Pressure Liquid; D006825:Hybridomas / Q000276:immunology*; D016056:Immunodominant Epitopes / Q000235:genetics; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000235:genetics / Q000276:immunology; D019151:Peptide Library; D011820:Rabies virus / Q000235:genetics / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology
D000941:Antigens; D016056:Immunodominant Epitopes; D009698:Nucleoproteins; D019151:Peptide Library; D014764:Viral Proteins; D002482:Cellulose