201509010009). or in vivo. (infected sheep and goats or their products [1C3]. The 31C34?kDa outer membrane protein (OMP) or (Omp31) is a major membrane protein paederoside of except for [4]. Omp31 plays an important role in cellular and humoral immune protective responses against contamination [4C11]. Previously we fully evaluated Omp31 epitopes in specific T-cell response in sheep vaccinated with attenuated vaccine [12]. However, the B-cell epitopes have not yet been extensively investigated. To date, only few epitopes recognized by antibodies to Omp31, such as monoclonal antibody A59/10F09/G10 realizing amino acid 48C83 of M16 and presenting protective activity were reported [4, 13, 14]. In this study, we generated and characterized 22 novel murine monoclonal antibodies (mAbs) binding native Omp31 of or to detect other species beyond proteins; TC IFS, Lentivirus-mediated Omp31, transduced 293Tcells detected by immunofluorescent staining; ICS, strain detected by immunochemical staining; L, linear; SC, semi-conformational; C, conformational. The figures show the S/CO values in the EIA; the reactivity levels are indicated by ++ (strongly reactive), + (reactive), (indeterminate) or C (non-reactive) in the Western-blot, IFS and ICS. (+), indicate false positive for the unfavorable control of cells. Five IgG and three IgM clones in strong are associates of high capacity of mAbs reacting with native Omp31 antigens of in various assays. Classification of Omp31 epitopes by mAbs acknowledgement In order to classify the Omp31 antigenic epitopes, all mAbs were tested for reactivity with 27 16mer overlapping peptides derived from full-length amino acid (aa) sequence, denatured or non-denatured protein forms of Omp31 in various immunoassays. Thirteen mAbs were reactive with 7 linear peptides in Peptide-ELISA (Fig.?(Fig.1a).1a). Twenty mAbs were reactive to the denatured rOmp31 and 14 mAbs to the denatured native membrane protein extract (NMP) by Western blot (Fig. ?(Fig.1b),1b), respectively. The mAbs reactivity was also tested against the non-denatured native antigens in ELISA using the NMP or the supernatant of sonicated proteins (SSP) from M5C90) by Western-blot. (C) Reactivity of mAbs to NMP and SSP (supernatant of sonicated proteins of M5C90) by ELISA. NS3, an HCV NS3 peptide, recombinant protein and an un-related mAb to HCV NS3 were used as negative-controls, respectively. The dotted collection indicates the level of cut off defined as mean?+?2SD of OD value to negative controls According to the nature of Omp31 antigens recognized by 22 mAbs, the epitopes were stratified into three groups of linear (L), semi-conformational (SC) and conformational (C) forms. Among these 22 mAbs, 13 reacted with the linear epitopes, 7 reacted with the semi-conformational and 2 reacted with the conformational epitopes offered in either rOmp31 or native Omp31 antigens of (Table ?(Table11). Linear epitope mapping of Omp31 by mAbs Among seven reactive linear paederoside peptides (Fig. ?(Fig.1a),1a), the epitope shared by peptides P05 and P06 was reactive with mAbs 1H2, 2D2, 2G9 and 7A3. However, due to the stronger reactivity with P05 than P06, the minimal aa common sequence of Omp31 was designated as epitope Ep5 (39SWTGGYIGINA49) (Fig. ?(Fig.2).2). Similarly, epitope Ep20 (168GDDASALHT176) overlapped by peptides P19 and P20 reacted with mAbs 2C1, 2E7, 4E9, 4H10 and 8F11. Epitope Ep21 (183AGWTLGAGAE192) reacted with both mAbs 2A8 and paederoside 6D8. Epitopes Ep11 (87QAGYNWQLDNGVVLGA102) and Ep24 (204EYLYTDLGKRNLVDVD219) were recognized only by mAb 5B1 or 5B3, respectively (Fig. ?(Fig.2).2). Alignment of Omp31 aa sequences showed that these five epitopes were completely conserved among and except for a single aa mutation (S172P) within Rabbit Polyclonal to RPC5 Ep20 of strains (Fig. ?(Fig.33). Open in a separate windows Fig. 2 Mapping for linear epitopes of Omp31 recognized by mAbs. The amino acid (aa) sequences of 16mer peptides reactive to the mAbs are offered, of which the epitopes (Ep) are designated on the top of underlined aa sequences. Aa position of Omp31 is usually indicated at the beginning and the end of the peptide sequence. MAbs are indicated below the epitopes they recognize Open in a separate windows Fig. 3 Alignment of Omp31 sequences from four species of strains. The aa sequences of Omp31 from (B.m), paederoside (B.o), (B.s) and (B.c) strains were retrieved from Genbank database. The accession figures are “type”:”entrez-protein”,”attrs”:”text”:”ADZ88512.1″,”term_id”:”326553873″,”term_text”:”ADZ88512.1″ADZ88512.1 (M5C90/China), “type”:”entrez-protein”,”attrs”:”text”:”ADZ67646.1″,”term_id”:”326410582″,”term_text”:”ADZ67646.1″ADZ67646.1 (M28/China), “type”:”entrez-protein”,”attrs”:”text”:”P0A3U4″,”term_id”:”61229373″,”term_text”:”P0A3U4″P0A3U4.1 (M16/US), “type”:”entrez-protein”,”attrs”:”text”:”ACQ84164.1″,”term_id”:”229620416″,”term_text”:”ACQ84164.1″ACQ84164.1 (293/Malaysia), “type”:”entrez-protein”,”attrs”:”text”:”AAL27294.1″,”term_id”:”16611675″,”term_text”:”AAL27294.1″AAL27294.1 (BCCN 91.264/ Argentina), “type”:”entrez-protein”,”attrs”:”text”:”AAL27292.1″,”term_id”:”16611669″,”term_text”:”AAL27292.1″AAL27292.1 (Reo 198/US), “type”:”entrez-protein”,”attrs”:”text”:”AAL27290.1″,”term_id”:”16611663″,”term_text”:”AAL27290.1″AAL27290.1 (513/Former USSR), “type”:”entrez-protein”,”attrs”:”text”:”AAL27287.1″,”term_id”:”16611654″,”term_text”:”AAL27287.1″AAL27287.1 (Thomsen/Denmark), “type”:”entrez-protein”,”attrs”:”text”:”AAL27296.1″,”term_id”:”16611681″,”term_text”:”AAL27296.1″AAL27296.1 (BCCN R18/US). The recognized mAbs acknowledgement epitopes are underlined on top of aa sequences Acknowledgement of Omp31-lentivirus transduced cells To detect Omp31 intracellularly, 293FT cells were transduced by recombinant Omp31-lentivirus (LV-HAGE-Omp31) for mimicking contamination in human or animal cells. By using IFS, one IgM mAb (2D2) and 16 IgG mAbs were reactive to the expressed rOmp31 in transduced 293FT cells (Fig. ?(Fig.44). Open in a separate windows Fig. 4 Acknowledgement of lentivirus-mediated Omp31 expressing cells by mAbs in IFS. The lentivirus (LV-HAGE-Omp31) transduced 293FT cells were stained by.