BIOMAC-2030; No. of Pages 7 International Journal of Biological Macromolecules xxx (2006) xxx–xxx Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies Marija Brgles b , Krunoslav Mirosavljevi´ c a , Vesna Noethig-Laslo a,∗ , Ruˇ za Frkanec b , Jelka Tomaˇ si´ c b a “Rudjer Boˇ skovi´ c” Institute, Bijeniˇ cka cesta 54, 10002 Zagreb, Croatia b Institute of Immunology Inc., P.O. Box 266, 10001 Zagreb, Croatia Received 5 July 2006; received in revised form 18 August 2006; accepted 22 August 2006 Abstract Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine- 1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG. © 2006 Elsevier B.V. All rights reserved. Keywords: Spin-labelled ovalbumin; Multilamellar liposomes; Anti-ovalbumin antibodies 1. Introduction Liposomes are microscopic vesicles consisting of one or more lipid bilayers surrounding aqueous compartments. The physico- chemical properties of liposomes depend mainly on the lipid composition that determines the liposomal charge as well as the fluidity of the liposomal membrane. A wide variety of dif- ferent compounds can be encapsulated in liposomes and the resulting liposomal formulations are often more convenient for experimental and therapeutic purposes [1]. Liposomes have also been considered and tested as carriers of antigens, vaccines and immunomodulatory compounds and tested in different experi- mental models [2,3]. In numerous studies of immune reactions and effects of immunostimulators ovalbumin (OVA) has been used as a model antigen of low immunogenicity according to previously pub- lished recommendations [4]. OVA is a glycoprotein from avian egg white. It has a well-defined structure [5] and belongs ∗ Corresponding author. Tel.: +385 1 4561 136; fax: +385 1 4680 245. E-mail address: laslo@irb.hr (V. Noethig-Laslo). structurally to the serpin (serine protease inhibitor) super- family but shows no protease inhibitor activity [6]. In our previous studies on immunostimulating properties of pepti- doglycan monomer, GlcNAc-MurNAc-l-Ala-d-isoGln-meso- DAP(-NH 2 )-d-Ala-d-Ala (PGM) and adamantyltripeptides, d- (adamant-2-il)-Gly-l-Ala-d-isoGln (AdTP1) and l-(adamant- 2-il)-Gly-l-Ala-d-isoGln (AdTP-2) [7,8], OVA was used as an antigen. The encapsulation of PGM and AdTPs into liposomes [9] and possible interactions of these compounds with lipids in liposomal membrane were studied using ESR [10], but the pos- sible interaction of OVA and lipids was not addressed. However, our recent results [11] on immune reaction to OVA encapsulated into liposomes demonstrated that the direction of immune reac- tion was shifted towards a specific T-helper phenotype (Th1) and this finding prompted us to study the interaction of OVA with liposomes using ESR. Only two reports published previously concern the spin- labelling of OVA. In one report [12], the label different from TEMPO-maleimide was mixed with OVA and the analysis of ESR spectra of not covalently labelled OVA described. In the other [13] the spin-label was covalently linked to OVA and used for immunizations of rabbits with the aim of eliciting specific 0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.08.012 Please cite this article as: Marija Brgles et al., Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies, International Journal of Biological Macromolecules (2006), doi:10.1016/j.ijbiomac.2006.08.012