Idiotypic cross-reactivity of low-affinity anti-fluorescyl monoclonal antibodies

Abstract

Previous studies concerning structure-function relationships of anti-fluorescyl hybridoma proteins utilized primarily high-affinity proteins ( K a > 5.0 × 10 7 M −1) possessing distinct idiotypes. Low-affinity anti-fluorescyl monoclonal antibodies, predominantly IgG1 or IgG2a possessing κ light chains were analyzed. Two fusions produced 18 monoclonals, 13 binding fluorescein with a low affinity (≤3.0 × 10 6 M −1) and five possessing high affinities (≥5.3 × 10 8 M −1). Solid-phase idiotype assays, utilizing rabbit anti-idiotype reagents against two low-affinity proteins (3–13 and 3–17), showed that all the low-affinity clones (except 2–9 and 2–21) were capable of inhibiting (40–100%) these two idiotype-anti-idiotype interactions while no high-affinity proteins inhibited them. The interactions with 3–13 and 3–17 were inhibited 100 and 88%, respectively, by free fluorescein. When these idiotype-anti-idiotype interactions were inhibited with increasing concns of heterologous hybridoma proteins, three clones inhibited both interactions as effectively as the homologous proteins at all concns tested and inhibition reached 100%. These three clones appeared to possess all the idiotopes that the anti-3–13 and anti-3–17 reagents detected on 3–13 and 3–17. Screening of eight high-affinity anti-fluorescyl proteins previously produced [Kranz and Voss, Molec. Immun. 18, 889–898 (1981 a)] identified a single clone [20-4-4 ( K a = 5.0 × 10 7 M −1)] significantly inhibiting the 3–13 and 3–17 interactions (71.0 and 63.6%, respectively). In addition, recombination experiments utilizing H- and L-chains derived from three low-affinity and three high-affinity antibodies resulted in reformation of active sites in all six heterologous combinations when both chains were derived from low-affinity antibodies, and in only one of six combinations when both chains were derived from high-affinity molecules. Thus, the apparent lack of private idiotopes on clones 3–13 and 3–17 and the presence of these idiotopes (or cross-reactive ones) on 11 of 13 low-affinity antibodies and on one of 13 high-affinity antibodies may indicate that clones 3–13 and 3–17 are encoded by germline genes. The H- and L-chain recombination experiments indicated that the idiotype and affinity of parental molecules may be involved in H- and L-chain association.