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Titolo:
NEUTRALIZING MURINE MONOCLONAL-ANTIBODIES TO HUMAN IL-5 ISOLATED FROMHYBRIDOMAS AND A FILAMENTOUS PHAGE FAB DISPLAY LIBRARY
Autore:
AMES RS; TORNETTA MA; MCMILLAN LJ; KAISER KF; HOLMES SD; APPELBAUM E; CUSIMANO DM; THEISEN TW; GROSS MS; JONES CS; SILVERMAN C; PORTER TG; COOK RM; BENNETT D; CHAIKEN IM;
Indirizzi:
SMITHKLINE BEECHAM PHARMACEUT,DEPT MOLEC IMMUNOL UE0548,709 SWEDELANDAVE KING OF PRUSSIA PA 19406 SMITHKLINE BEECHAM PHARMACEUT,DEPT MOLEC IMMUNOL KING OF PRUSSIA PA 19406 SMITHKLINE BEECHAM PHARMACEUT,DEPT GENE EXPRESS SCI KING OF PRUSSIA PA 19406 SMITHKLINE BEECHAM PHARMACEUT,DEPT PROT BIOCHEM KING OF PRUSSIA PA 19406 SMITHKLINE BEECHAM PHARMACEUT,DEPT MACROMOLEC SCI KING OF PRUSSIA PA 19406 SMITHKLINE BEECHAM PHARMACEUT,DEPT FERMENTAT & CELL SCI EPSOM KT18 5XQ SURREY ENGLAND
Titolo Testata:
The Journal of immunology
fascicolo: 12, volume: 154, anno: 1995,
pagine: 6355 - 6364
SICI:
0022-1767(1995)154:12<6355:NMMTHI>2.0.ZU;2-Y
Fonte:
ISI
Lingua:
ENG
Soggetto:
AFFINITY MATURATION; INTERLEUKIN-5; MICE; EOSINOPHILIA; GENERATION; SELECTION; ANTIGEN; CELLS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
38
Recensione:
Indirizzi per estratti:
Citazione:
R.S. Ames et al., "NEUTRALIZING MURINE MONOCLONAL-ANTIBODIES TO HUMAN IL-5 ISOLATED FROMHYBRIDOMAS AND A FILAMENTOUS PHAGE FAB DISPLAY LIBRARY", The Journal of immunology, 154(12), 1995, pp. 6355-6364

Abstract

Conventional hybridomas and combinatorial Ab libraries were used to develop neutralizing murine mAbs to human IL-5. Mice were immunized with rlL-5. Spleens from two mice were used to generate hybridomas. Spleens from an additional three mice were used to construct a combinatorial library. In both instances, Abs were identified and selected by ELISA using 96-well plates coated with rlL-5. These Abs were tested for the ability to block binding of iodinated rIL-5 to the ac-chain of the human IL-5 receptor (IL-5R alpha) and to inhibit proliferation of IL-5-dependent cells. By hybridoma technology, 16 mAbs were obtained, 11 ofwhich blocked binding to IL-5R alpha, including three that inhibited proliferation. Quantitative binding assays and sequence analysis revealed that these latter three mAbs were closely related. Combinatorial cloning and selection by phage display was used to isolate 24 bacterialcolonies secreting Fabs that bound to I-125-rIL-5 and to rIL-5-coatedplates. Sequencing of 10 of the Fabs indicated that four unique Abs were obtained, comprising one predominant V-H paired with one of two different V-L. The sequence of the Fabs was distinct from the sequences of the neutralizing mAbs. In contrast to the mAbs, none of the Fabs blocked binding of I-125-IL-5 to IL-5R alpha or neutralized the biologicactivity of IL-5. The inability to identify neutralizing Fabs was shown not to result from their monovalency, because a Fab derived from one of the neutralizing mAbs, by cloning and expression of its Fd and kappa light chains, retained neutralizing activity. By chain shuffling, pairing of the Fd fragment of the heavy chain of one of the neutralizing mAbs (2B6), with the light chain library derived from the IL-5-immunized mice, neutralizing Fabs were obtained. These Fabs contained light chain sequences closely related to the original light chain of 2B6. Hence, chain shuffling allowed detection of a light chain sequence that was not evident upon two-chain combinatorial selection. The results reveal differences in the Abs obtained from a combinatorial library vshybridomas and demonstrate how these approaches can be used in concert to select mAbs with neutralizing activity.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 15/07/20 alle ore 21:11:39