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In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2000 Jan 13; Vol. 233 (1-2), pp. 95-105. - Publication Year :
- 2000
-
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 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.
- Subjects :
- Amino Acid Sequence
Animals
Antigens genetics
Cell Line
Cellulose
Chromatography, High Pressure Liquid
Immunodominant Epitopes genetics
Mice
Mice, Inbred C3H
Molecular Sequence Data
Nucleoproteins genetics
Nucleoproteins immunology
Peptide Library
Rabies virus genetics
Rabies virus immunology
Viral Proteins genetics
Viral Proteins immunology
Antigens administration & dosage
Hybridomas immunology
T-Lymphocytes, Helper-Inducer immunology
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 233
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 10648860
- Full Text :
- https://doi.org/10.1016/s0022-1759(99)00194-5