In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen
Identifieur interne : 000F56 ( Istex/Checkpoint ); précédent : 000F55; suivant : 000F57In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen
Auteurs : Laszlo Otvos Jr. [États-Unis] ; Anne Marie Pease [États-Unis] ; Krisztina Bokonyi [États-Unis] ; Wynetta Giles-Davis [États-Unis] ; Mark E. Rogers [États-Unis] ; Paul A. Hintz [États-Unis] ; Ralf Hoffmann [États-Unis] ; Hildegund C. J Ertl [États-Unis]Source :
- Journal of Immunological Methods [ 0022-1759 ] ; 2000.
English descriptors
- Teeft :
- Accessory cells, Amino, Amino acid concentration, Amino acid derivatives, Amino acid solutions, Amino acids, Antigenic potency, Assay, Assay medium, Assay plate, Binding peptide mixtures, Carrier cellulose membrane, Cellulose membrane, Cellulose membranes, Contact sites, Degradation rate, Dependent epitopes, Deprotection, Deprotection steps, Different batches, Eagles medium, Elsevier science, Entire length, Epitope, Error bars, Ertl, Febs lett, Final volume, Helper cell activity, Helper cell epitopes, Helper cell hybridoma, Helper cells, High performance, Human serum, Hxthymidine incorporation, Hybridoma, Immunodominant epitope, Immunological, Immunological methods, Immunosorbent assay, Lowest amounts, Lymphokine release, Major histocompatibility, Mass spectra, Mass spectrometry, Membrane, Negative control, Negative control peptide, Next step, Nitrocellulose immunoblots, Nmole amounts, Nucleoprotein, Open triangles, Otvos, Paper dots, Peptide, Peptide amount, Peptide array, Peptide dots, Peptide fragments, Peptide libraries, Peptide spot, Peptide spots, Peptide synthesis, Rabies, Rabies virus, Rabies virus nucleoprotein, Same medium, Simultaneous synthesis, Solid carrier, Solid line, Solid phase, Solution controls, Solution techniques, Splenocyte culture, Splenocytes, Spot size, Standard assay, Standard deviation, Stimulation assay, Stimulation assays, Stimulatory activity, Synthetic peptides, Target peptides, Test samples, Trifluoroacetic acid, Triplicate assay data, True reaction, Tryptic fragments.
Abstract
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.
Url:
DOI: 10.1016/S0022-1759(99)00194-5
Affiliations:
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<front><div type="abstract" xml:lang="en">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.</div>
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