Anti-rhodopsin monoclonal antibodies of defined specificity

Characterization and application

Grazyna Adamus, Z. Suzanne Zam, Anatol Arendt, Krzysztof Palczewski, J. Hugh McDowell, Paul A. Hargrave

Research output: Contribution to journalArticle

158 Citations (Scopus)

Abstract

A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbant assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 188-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 108-1010M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similary, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

Original languageEnglish (US)
Pages (from-to)17-31
Number of pages15
JournalVision Research
Volume31
Issue number1
DOIs
StatePublished - 1991
Externally publishedYes

Fingerprint

Rhodopsin
Antibody Specificity
Monoclonal Antibodies
Antibodies
Epitopes
Phosphorylation
Peptides
Membranes
Hybridomas

Keywords

  • Antibody epitope determination
  • Enzyme-linked immunosorbant assay
  • Membrane topography
  • Monoclonal antibody
  • Protein phosphorylation
  • Rhodopsin

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Anti-rhodopsin monoclonal antibodies of defined specificity : Characterization and application. / Adamus, Grazyna; Zam, Z. Suzanne; Arendt, Anatol; Palczewski, Krzysztof; McDowell, J. Hugh; Hargrave, Paul A.

In: Vision Research, Vol. 31, No. 1, 1991, p. 17-31.

Research output: Contribution to journalArticle

Adamus, Grazyna ; Zam, Z. Suzanne ; Arendt, Anatol ; Palczewski, Krzysztof ; McDowell, J. Hugh ; Hargrave, Paul A. / Anti-rhodopsin monoclonal antibodies of defined specificity : Characterization and application. In: Vision Research. 1991 ; Vol. 31, No. 1. pp. 17-31.
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AU - McDowell, J. Hugh

AU - Hargrave, Paul A.

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N2 - A panel of anti-bovine rhodopsin monoclonal antibodies (MAbs) of defined site-specificity has been prepared and used for functional and topographic studies of rhodopsins. In order to select these antibodies, hybridoma supernatants that contained anti-rhodopsin antibodies have been screened by enzyme-linked immunosorbant assay (ELISA) in the presence of synthetic peptides from rhodopsin's cytoplasmic regions. We selected for antibodies against predominantly linear determinants (as distinct from complex assembled determinants) and have isolated antibodies that recognize rhodopsin's amino terminus, its carboxyl terminus, as well as the hydrophilic helix-connecting regions 61-75, 96-115, 188-203, 230-252 and 310-321. Detailed specificities have been further determined by using a series of overlapping peptides and chemically modified rhodopsins as competitors. A group of seven antibodies with epitopes clustered within the amino terminal region of rhodopsin and a group of 15 antibodies with epitopes within the carboxyl terminal region are described. These MAbs have high affinities for rhodopsin with Kas in the range of 108-1010M-1. Some MAbs specific for the carboxyl and amino terminal regions were used to compare these bovine rhodopsin sequences to those of different vertebrates. The MAbs cross-reacted with the different species tested to different extents indicating that there is some similarity in the sequences of these regions. However, some differences in the sequences were indicated by a reduced or absent cross-reactivity with some MAbs. In membrane topographic studies the MAbs showed both the presence and the accessibility of rhodopsin sequences 330-348, 310-321 and 230-252 on the cytoplasmic surface of the disk membrane. Similarly, sequences 1-20 and 188-203 were shown to reside on the lumenal surface of the disk and to be accessible to a macromolecular (antibody) probe. Antibodies directed against rhodopsin's carboxyl terminal sequence did not bind well to highly phosphorylated rhodopsin. Similary, these antibodies as well as those against the V-VI loop inhibited phosphorylation of rhodopsin. Antibody A11-82P, specific for phosphorylated rhodopsin, recognized rhodopsin containing two or more phosphates and inhibited its further phosphorylation.

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