Three lysozymes (DLs-1, -2, and -3) were purified from Peking-duck egg white by adsorption on CM-Sephadex C-25 resin, followed by CM-Sephadex C-25 and Sephadex G-50 column chromatographies. The three enzymes each moved as a single band, but showed different electrophoretic mobilities, on disc-polyacrylamide gel electrophoresis at pH 4.1. Final yields of DL-1, DL-2, DL-3 were 18.2%, 22.0%, and 6.0%, respectively, from the crude material adsorbed on CM-Sephadex resin. The enzymatic activities of DL-1, DL-2, and DL-3 were 1.53, 1.52, and 1.34 times that of hen egg white lysozyme, respectively, using Micrococcus lysodeikticus cell wall as a substrate at pH 6.2 and 37 degrees C. All the DLs lacked histidine and their amino acid compositions differed from each other by a few amino acid exchanges. The amino acid sequences of DL-2 and DL-3 differed from that of DL-1 by two displacements (Ser-37 to Gly and Gly-71 to Arg) and three displacements (Pro-79 to Arg in addition to the same substitutions), respectively. In comparison with Duck II and Duck III lysozymes from Kaki-duck (Hermann and Jollès (1970) Biochim. Biophys. Acta 200, 178-179; Hermann et al. (1971) Eur. J. Biochem. 24, 12-17) as regards amino acid sequences, Duck II is identical to DL-1 except for one displacement of Gln-57 in DL-1 to Glu in Duck II, while Duck III is rather different from our three lysozymes. All DLs gave single precipitin lines with any rabbit antisera against DLs and each precipitin line fused completely with that of any of the DLs in Ouchterlony double diffusion tests. However, the radiobinding inhibition assay showed that some anti-DL antisera clearly discriminated fine differences among the three DLs. The displacement at residue 79 (Pro in equilibrium Arg) gave the clearest immunological difference among the three kinds of displacements found in DLs.

Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.

The affinity of antihapten antibody was evaluated as a function of the length of the immunization period. Purified antibody in a yield of 7 5 4 0 % was prepared from rabbit antisera, induced with a large haptenic group (formula wt 540), covering a range of intervals between primary and secondary injections of 2-10 weeks. Association constants were determined by equilibrium dialysis with a tritiated hapten (formula wt 297) allowing concentration measurements to 10-8 M. This hapten (amino dye) constituted the terminal portion of the haptenic group. The association constant for this hapten increased over the period studied from 1.2 X 106 to 3.4 X lo7 M-' without an apparent

Twenty monoclonal antibodies (MAbs) were obtained by immunizing Balb/c mice with recombinant p17 (rp17) of HIV-1. Epitope specificity of each MAb was determined using six peptides that cover the entire region of p17. We found that each MAb reacts with only one of the peptides, residues 12-29, 30-52, 53-87, and 87-115 (P12-29, P30-52, P53-87, P87-115) of p17 with the exception of one MAb. Three kinds of MAbs that recognize P30-52, P87-115, and a conformational epitope, suppressed the infectivity of HIV-1 (JMH-1) when they added in the culture of MT-4 cells infected by HIV-1 within 24 h of the infection.

An isolated antibody preparation directed to the native hen egg-white lysozyme (HEL) from a single AI] mouse (~a-II), termed Id~r~, was inoculated into rabbits to produce anti-idiotypic sera (anti-Id). The antisera were extensively absorbed with normal AIJ Ig to render them idiotype specific. Radioimmunoassay utilizing 125I-Id~r~and the anti-Id sera (R103 and R104) was performed to examine the idiotypic cross-reactivity of the humoral immune response to HEL in various mouse strains and other animal species. Idiotypes shared by Id~f~were detected in the sera of five mouse strains tested, but not in any of the examined sera of other animal species such as rats, goats, guinea pigs, or sheep, indicating the occurrence of species-specific cross-reactive idiotypes (CRI) of antibodies to HEL. Our experiments also suggested the presence of intrastrain CRT. The present experiments, along with our earlier results (IS), suggest that idiotypic cross-reactivity among murine antibodies to HEL appears to be weak. Thus when R103 and R104 were the anti-Id sera used, the frequency of occurrence of CRI shared by Id~r~in 5 ftg of anti-HEL antibody in strain A mice was 74 ng and III ng; in other strains it was 25-44 ng and 60-98 ng, respectively.Idiotypic (variable-region) antigenic determinants (Id) of immunoglobulins (Ig) have been widely used for probing Ig diversity and the regulation of Ig pro-

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The antigenic reactivity of the region at residues 38-54 of hen egg white lysozyme (lysozyme) was investigated by using various rabbit, goat, and sheep anti-lysozyme antisera by the charcoal-dextran method. This continuous antigenic region was completely independent in its specificity from other discontinuous antigenic regions of lysozyme. The antibodies to this region were shown to be reactive with native lysozyme as well as thermolytic nondisulfide peptides (residues 38-54 and 38-57) and synthetic peptides (sequences 38-48 and 34-54) by the following evidence. (1) The antigen binding activity could be inhibited by the addition of native lysozyme.(2) The antibodies to this region could be absorbed with lysozyme immunoadsorbent. (3) The specific antibodies prepared by the synthetic peptide (sequence 38-48) immunoad-

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