A natural carrier effect and the generation of specific antibodies to biologically active peptides

doi:10.1016/j.ab.2006.03.018

Analytical Biochemistry

Volume 353, Issue 2, 15 June 2006, Pages 174-180

https://doi.org/10.1016/j.ab.2006.03.018 Get rights and content

Abstract

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In this work, the production of antibodies to BPPs by use of an efficient immunization protocol in mice genetically modified for the high antibody responsiveness (H_III line) is described. Although it was possible to induce antibody production by single-dose administration of free BPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated to this carrier. Interestingly, both mouse groups had a higher titer of IgG₁ than IgG_2a isotypes, regardless of prior immunization with the carrier protein. However, a lower titer of IgG_2a was observed in unprimed mice. A single band of about 27 kDa corresponding to the BPP precursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca venom gland. This work proposes an efficient immunization protocol based on classic studies described for the hapten–carrier effect for generating specific antibodies against biologically active peptides.

Section snippets

Peptide synthesis

The peptides pBPP5a (<EKWAP), BPP5a (QKWAP), pBPP10c (<ENWPHPQIPP), and BPP10c (QNWPHPQIPP) were synthesized by solid-phase synthesis using the Fmoc strategy as previously described [29]. “<E” represents the pyroglutamic acid at the N terminus. Peptides were purified by HPLC (Shimadzu Corporation, Kyoto, Japan), and the structure was confirmed with Ettan matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Amersham Biosciences UK Limited, Buckinghamshire,

Production of specific antibodies to peptides by immunization with free peptides

In preliminary experiments, we used a conventional immunization protocol to obtain antibodies against the peptides. H_III mice were immunized subcutaneously with 50 μg of BPP5a conjugated to the carrier KLH, and high antibody titers were obtained with a single immunization (Fig. 1). However, for the booster, to obtain more specific sera against the peptides, only free peptides were administered to the animals. Surprisingly, the antibody titer remained high after more than 150 days of this

Discussion

The present work demonstrates that specific antibodies to biologically active peptides can be successfully generated even by immunization with the peptides in the free form. Although the anti-BPP titers obtained by immunization with the peptides in their free form were lower than those obtained with the peptides conjugated to the carrier protein, satisfactory antibody levels were generated in H_III mice immunized with either peptide: BPP5a, pBPP5a, or pBPP10c (Fig. 2). It is well known that the

Acknowledgments

This work was supported by funds from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) through the CAT/CEPID program. L.R. Tsuruta is a recipient of a FAPESP fellowship. D.V. Tambourgi, A.C.M. Camargo, and O.A. Sant’Anna are researchers of CNPq-Brazil. The technical assistance of Ms. Maria Aparecida Siqueira and Aparecida das Dores Coelho is gratefully acknowledged. We thank Mr. Valdir José Germano and Dr. Otávio A.V. Marques from the Laboratory of Herpetology for their providing

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Cited by (2)

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain - Potential BPP-like precursor protein?
2015, Biochemical Pharmacology
Citation Excerpt :
The use of specific antibodies for this purpose is classically employed [58,59], although the high content of proline residues characteristic of the snake bradykinin-potentiating peptides (BPPs) may represent the principal limitation for this approach due to the risk of eventual immune cross-reactivity. In fact, previous attempts to raise specific antibodies against snake BPPs demonstrated the challenging limitations imposed by using short proline-rich peptides (PRPs) as antigen [60]. The employment of several innovative strategies culminated in the production of anti-BPPs antiserum able to recognize a single band corresponding to the BPP precursor protein in the cytosol of the B. jararaca venom gland [56], but which was not able to specifically recognize proteins in any other snake or rat tissue homogenates (data not shown).
Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.
A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating Lupus aggravation in mice
2011, PLoS ONE

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A natural carrier effect and the generation of specific antibodies to biologically active peptides

Abstract

Section snippets

Peptide synthesis

Production of specific antibodies to peptides by immunization with free peptides

Discussion

Acknowledgments

Virology

Mol. Immunol.

Mol. Immunol.

Mol. Immunol.

FEMS Immunol. Med. Microbiol.

Biochimie

Immunopharmacology

J. Allergy Clin. Immunol.

Mol. Immunol.

Lancet

Toxicon

Peptides

J. Immunol. Methods

Immunochemistry

J. Biol. Chem.

Vaccine

Mol. Immunol.

Cell cooperation in antibody production: a perspective with questions

Adv. Exp. Med. Biol.

Chemically synthesized peptides predicted from the nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive with the native envelope protein of Dane particles

Proc. Natl. Acad. Sci. USA

Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides

Nature

Localization, synthesis, and activity of an antigenic site on influenza virus hemagglutinin

Proc. Natl. Acad. Sci. USA

Induction of antibodies against a peptide hapten does not require covalent linkage between the hapten and a class II presentable T helper peptide

Eur. J. Immunol.

Immunogenicity and vaccine efficacy of synthetic peptides containing Semliki Forest virus B and T cell epitopes

J. Gen. Virol.

A novel retro-inverso gonadotropin-releasing hormone (GnRH) immunogen elicits antibodies that neutralize the activity of native GnRH

Endocrinology