Yutaka Takagaki

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme.

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of Nα-[14C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of Nα-[14C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 107 to 2.6 × 108m−1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.