A natural carrier effect and the generation of specific antibodies to 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. HIII 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 HIII 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 PharmacologyCitation 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).