Idiotypic Analysis of Antibodies to Hen Egg‐White Lysozyme (HEL): III. Further Studies on the Idiotypic Cross‐Reactivity among Anti‐HEL Antibodies in Mice

Abstract

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-

Suppression of Hen Egg‐White Lysozyme (HEL)‐Specific Delayed‐Type Hypersensitivity Responses in Mice by Suppressor T Cells after Neonatal Administration of Anti‐Idiotypic Antibodies

“…Mice were immunized by intraperitoneal (i.p.) inoculation of HEL (500 p,g per injection, in 0.02 ml of PBS emulsified with 0.18 ml of adjuvant), first in incomplete Freund's adjuvant (IFA) and thereafter in complete Freund's adjuvant (CFA), to produce large amounts of antibodies in individual mice as previously described (27,28,33). Two weeks elapsed between the first and second injections, and thereafter the mice were inoculated weekly.…”

“…The immunoadsorbent was prepared by a slight modification of the method of March et al (16) using Sepharose CL-4B and cyanogen bromide. For a l-ml volume of Sepharose CL-4B, 250 mg of cyanogen bromide was found to be necessary for satisfactory activation (27). The bound antibody was eluted with 3 M KSCN in BBSh-EDTA and further chromatographed on a Sephadex G-200 column.…”

“…Idiotypes shared by anti-HEL antibody (C3H, No.2) were detected by a double-antibody radioimmunoassay as described previously (21,(26)(27)(28). Briefly, 20 p,l of 125I-anti-HEL antibody (C3H, No.2) (20 ng), 200 p'!…”

“…A solid-phase radioimmunoassay on polyvinyl microtiter plates (model 220-24; Cooke Laboratory Products Div., Dynatech Lab. Inc., Alexandria, Va., U.S.A.) was performed as described previously (27,28). Antibodies bound to HEL-coated wells were detected by 125 1_ labeled rabbit antibody to mouse F(ab'h prepared as described by Semma et al (28).…”

“…Anti-HEL antibody from a single C3H mouse (No.2) was isolated by an immunoadsorbent on which HEL had been coupled to Sepharose CL-4B as described previously (27,28). The immunoadsorbent was prepared by a slight modification of the method of March et al (16) using Sepharose CL-4B and cyanogen bromide.…”