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OD ratio33031530011060105432102/1 4/3 6/5 8/7 10/9 2/1 4/3 6/5 8/7 10/9peptidesCCP+ serahealthy seraFig. 3. Indirect ELISA with biotinylatedpeptides, pre-coated with NeutrAvidin.specific secondary antibodies (Figure 2).Comparing the short sequences offilaggrin and vimentin, we have found thatthe 5-mer filaggrin-peptide was recognizedby the RA sera with sensitivity andspecificity comparable with the currentlyused tests.We detected lower sensitivity andspecificity than in our first test. However,OD ratios of all peptides have shown asignificant correlation with the MCV(mutated citrullinated vimentin) titer, whichis associated with activity of disease. Onthe other hand, OD ratios did not showcorrelation with the CCP3 titer. These datasuggest that the short citrulline containingfilaggrin peptide (Filaggrin 311-315 XX)might be a useful biomarker for RA.Selected peptides (Table 1) weresynthesised manually by SPPS, accordingto Fmoc/ t Bu strategy using Rink-Amide MBHA or MBHA resin. For the coupling Fmocaminoacid : HOBt : DIC = 2 : 2 : 2 activation was used. C- or N-terminally biotinylatedforms were made using biotin, biotinyl-6-aminohexanoic acid and 4,7,10-trioxa-1,13-tridecanediamino succinic acid linker (Ttds) [3]. The ratio of the coupling reagents wasbiotin : HOBt : PyBOP : DIEA = 3 : 3 : 3 : 6. We coupled the Fmoc-Ttds as a protectedamino acid. The crude products were purified by reversed-phase chromatography. Thestructure of the peptides was proved by electron spray ionization (ESI) mass spectrometry.We have used these peptides in an indirect ELISA, on NeutrAvidin pre-coated plates, thereaction was detected by anti IgG-HRP (Figure 3).The CCP3+ serum samples specifically recognized the C terminally biotinylated5-mer filaggrin peptide, while showed no reaction with the N-terminally biotinylated ones.Table 1. Structures of studied peptides1 biotin-6-amino-hexanoyl- Filaggrin 311-315 RR -NH 22 biotin-6-amino-hexanoyl- Filaggrin 311-315 XR -NH 23 biotinyl- Filaggrin 311-315 RR -NH 24 biotinyl- Filaggrin 311-315 XR -NH 25 Ac- Filaggrin 311-315 RR -K(biotin-6-amino-hexanoyl)-NH 26 Ac- Filaggrin 311-315 XR -K(biotin-6-amino-hexanoyl)-NH 27 Ac- Filaggrin 311-315 RR -K(Ttds-biotin)-NH 28 Ac- Filaggrin 311-315 XR -K(Ttds-biotin)-NH 29 Ac- Filaggrin 311-315 RR -Ttds-K(biotin-6-amino-hexanoyl)-NH 210 Ac- Filaggrin 311-315 XR -Ttds-K(biotin-6-amino-hexanoyl)-NH 2As a conclusionwe can say that theshort Filaggrin 311-315XX peptide andMultipin ELISA maybe a useful addition tothe array of diagnostictools of early RA.Our results werevalidated by conventionalindirect ELISA,also showing thatonly the C-terminalbiotinylation can beapplied for the 5-merfilaggrin-peptide.AcknowledgmentsWe thank to EPS for travel grant. This work was supported by National Office for Research andTechnology (RET-06/2006, GVOP-3.1.1.-2004-05-0183/3.0, OTKA-NKTH CK_80689), Hungary.References1. Vossenaar, E.R., Despres, N., Lapointe, E., van der Heijden, A., Lora, M., Senshu, T., VanVenrooij, W.J., Menard, H.A. Arthritis Res. Ther. 6(2), 86-89 (2004).2. Schellekens, G.A., de Jong, B.A.W., van den Hoogen, F.H.J., van de Putte, L.B.A., Van Venrooij,W.J. J. Clin. Invest. 101, 273-281 (1998).3. Bartos, Á., Uray, K., Hudecz, F. Peptide Science 92, 110-115 (2009).463