Monoclonal antibodies distinguishing α and β forms of calcitonin gene-related peptide

Andrew, David P.; Bidgood, T.D.; Bose, C.; Brown, Derek; Galfrè, G.; Sherwood, Margaret E.

doi:10.1016/0022-1759(90)90115-c

Cited by 20 publications

(15 citation statements)

References 18 publications

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“…As a possible alternative strategy, the use of a panel of monoclonal antibodies (MAbs) which bind and possibly inhibit specific functional regions of a peptide may yield information regarding the active site of the molecule. In this study the modification of the vasodilator effect of CGRP by a panel of MAbs (Andrew et al, 1990) was investigated in the pig coronary artery ring preparation.…”

Section: Introductionmentioning

confidence: 99%

The effect of monoclonal antibodies to calcitonin gene‐related peptide (CGRP) on CGRP‐induced vasodilatation in pig coronary artery rings

Shaw

Foulkes

Andrew

et al. 1992

British J Pharmacology

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1 The modification of the vasodilator effect of calcitonin gene-related peptide (CGRP) by a panel of monoclonal antibodies (MAbs), which map to discrete epitopes on the CGRP molecule, was investigated in pig coronary artery rings (PCA). The preparations were pre-constricted with acetylcholine (3 x 10-7M) and concentration-response curves to CGRP (2 x 10-'0-2.56 x 10-8M) were obtained in the presence or absence of each MAb. 2 CGRP caused a concentration-dependent relaxation of PCAs which reached a maximum (98.2 ± 4.8%, n = 25) at 1.28 x 10-8M and gave an EC50 of 3.8 ± 0.8 x 10-9M. 3 Two MAbs which map to the N-terminal, CNl and CRA3, did not affect the CGRP response whilst a third, CRA5, significantly inhibited its effect. 4 The C-terminal MAb, CRA2, did not modify the CGRP response whilst, in contrast, CB3 (Cterminal) potentiated its effect. A similar augmentation of the CGRP-induced vasodilatation was seen in the presence of the middle-region MAb, CRA8. 5 These results suggest that regional specific MAbs can modify the vasodilator effect of CGRP causing either inhibition (CRA5, N-terminal) or potentiation (CB3, C-terminal; CRA8, middle region).

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Section: Introductionmentioning

confidence: 99%

The effect of monoclonal antibodies to calcitonin gene‐related peptide (CGRP) on CGRP‐induced vasodilatation in pig coronary artery rings

Shaw

Foulkes

Andrew

et al. 1992

British J Pharmacology

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“…Additional residues also interact strongly with the CDl antibody, most notably those near the N-terminus of the peptide. Thus, in both cases the epitope, as defined by NMR, corresponds broadly to serological typing (Andrew et al, 1990), although NMR has also identified residues involved in the interaction with the FAB in addition to those seen in the serological typing.…”

Section: Discussionmentioning

confidence: 87%

“…High-affinity monoclonal antibodies to epitopes on CGRP were obtained as described previously (Andrew et al, 1990). Tissue cultures from Sp/20 myeloma cell lines producing CB3 or CDI, which are both mouse IgG isotype I, were concentrated 10-fold and stored frozen at -20 "C in 100 mL batches.…”

Section: Preparation Of Monoclonal Fab Fragmentsmentioning

confidence: 99%

“…Eighteen monoclonal antibodies have been raised to CGRP using either the whole peptide or peptide fragments as the immunogen (Andrew et al, 1990). The smaller peptides produce antibodies of lower affinity than those produced by the whole molecule, and are not able to function well in two-site enzyme-linked immunoassays.…”

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confidence: 99%

“…In this paper we describe the use of this approach to examine the interaction of CGRP with two monoclonal antibodies (CB3 and CDI). Both these antibodies have high affinities for CGRP (lo8 M" (Andrew et al, 1990)) although serological studies indicate that different residues are involved in the interactions between the two molecules.…”

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confidence: 99%

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Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR

Hubbard¹,

Raleigh

Bonnerjea³

et al. 1997

Protein Science

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The interactions between calcitonin gene-related peptide and FAB fragments prepared from two different high-affinity anti-CGRP monoclonal antibodies (CB3 and CDI) have been studied at physiological pH using the ability of 'H NMR to detect selectively regions of dynamic flexibility. The 37-residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However, the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C-terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CDI, the conformationally restricted residues appear to lie predominantly within the N-terminal half of the CGRP molecule, particularly residues 4-16, although several residues in the middle section of the sequence (22-31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.Keywords: antibody peptide interactions; calcitonin gene-related peptide; epitope mapping; NMR Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide with potent vasodilatory and cardioexcitatory properties whose effects are receptor mediated (Abello et al., Raynaud et al., 1994). Little is known about the active conformation of the peptide. The N-terminal region, which is constrained by a disulphide bond, is required for activity, although N-terminal fragments by themselves have only low activity (Maggi et al., 1990) Abbreviarions: CGRP, calcitonin gene-related peptide; CD, circular dichroism spectroscopy; CPMG, Cam Purcell Meiboom Gill; COSY, correlated spectroscopy: DQF, double quantum filtered: ELISA, enzyme linked immunoassay; FAB, fast atom bombardment mass spectrometry; HOHAHA, homonuclear Hartmann-Hahn: NMR, nuclear magnetic resonance: NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser spectroscopy; RELAY COSY, two-dimensional relayed coherence transfer spectroscopy: TOCSY. total correlation spectroscopy: lD, one-dimensional: 2D, two-dimensional. ture of the peptide is flexible and disordered in solution, although an amphiphilic helix is formed in the middle of the peptide between residues 8 and 18 in trifluoroethanol/H20 mixtures (Breeze et al., Hubbard et al., 1991). There are some indications of a preference for a turn-type conformation in the C-terminal half of the peptide in the TFE-induced conformation and also in C-terminal fragments of CGRP (Sagoo et al., 1991). The requirement for activity, if any, for these regions of secondary structure remains to be determined.Eighteen monoclonal antibodies have been...

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KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA‐1‐ and CR3‐dependent adhesion

et al. 1993

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Unactivated peripheral blood leukocytes show little tendency to bind to other cells or matrix components, whilst, in the presence of inflammatory mediators, adhesive interactions can rapidly increase. The Leu-CAM (beta 2 integrin) family of adhesion molecules have been shown to mediate a variety of these induced adhesion events. Here we describe a monoclonal antibody against CD18, KIM185, which stimulates JY cell homotypic aggregation by a CD11 a pathway as well as inducing the adherence of neutrophils to protein-coated plastic by a CD11b-dependent mechanism. The antibody recognizes an epitope distinct from the previously described KIM127 antibody and evidence is presented that the binding of KIM185 can cause a change in the conformation of the CD18 molecule.

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Monoclonal antibodies distinguishing α and β forms of calcitonin gene-related peptide

Cited by 20 publications

References 18 publications

The effect of monoclonal antibodies to calcitonin gene‐related peptide (CGRP) on CGRP‐induced vasodilatation in pig coronary artery rings

The effect of monoclonal antibodies to calcitonin gene‐related peptide (CGRP) on CGRP‐induced vasodilatation in pig coronary artery rings

Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR

KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA‐1‐ and CR3‐dependent adhesion

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