Immunochemical analysis of Lewis rat antisera to the synthetic encephalitogenic peptide S49

Abstract

Discrete populations of anti-S49 antibodies were found in the antisera of Lewis rats recovered from S49-induced experimental allergic encephalomyelitis (EAE). A potent inducer of EAE in Lewis rats, S49 is a synthetic peptide representing residues 69–84 of bovine myelin basic protein but with deletions at Gly-77 and His-78 to form an analogue of guinea pig or rat 69–84, GSLPQKAQRPQDENG. Each population within a given antiserum, as identified by Scatchard and Sipsian window analysis, was found to exhibit reactivity for a different S49 determinant, and the affinities of each population were relatively restricted and discontinuous. The high affinity populations (10⁷–10⁸ M⁻¹) were cross-reactive with YS8 (YGSLPQKAQGHRPQDENG) in equilibrium competitive inhibition reactions whereas the low affinity populations (10⁵–10⁶ M⁻¹) were reactive only with S49 and YS49 among a panel of peptide analogues. Of the YS8 cross-reactive antibodies the highest affinity (10⁸ M⁻¹) were also cross reactive with S81 (YGSLPQKAQGHRPQDEG) but not S49 (69-84-Gly), thus emphasizing the need for Tyr-68 for format stability of the determinant involved. The other YS8 cross-reactive population (10⁷ M⁻¹) was completely reactive with S49 but totally unreactive with S81 in equilibrium reactions, thus emphasizing the requirement for Asn-84 but not Tyr-68 for the determinant's topographic stability. Peptides shorter than S49 from the N-terminal end, but retaining the sequences AQRPQDEN or SQRSQDEN (suspected residence of minimal encephalitogenic determinants), reacted only under conditions of two-step non-equilibrium competitive inhibition assays. Such reactions would occur only at very low affinity (<10⁵ M⁻¹) with the anti-S49 antibodies. It was hypothesized that the encephalitogenic T-cell determinant for Lewis rats, although permitting B-cell responses at very low affinity, may exclude high affinity responses in susceptible animals.