Analysis of the repertoire of anti‐(4‐hydroxy‐3‐nitro‐phenyl)acetyl (NP) antibodies in C 57 BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti‐idiotope antibodies

Abstract

CBA and SJL mice were immunized against the hybrid cell antibody Bl‐8 (μ,λ 1) which we have previously isolated from the primary response of C57BL/6 mice against the hapten (4‐hydroxy‐3‐nitro‐phenyl)acetyl (NP) (Eur. J. Immunol. 1978. 8: 393). Cell lines secreting monoclonal antibodies against B1–8 idiotopes were isolated, and the anti‐idiotope antibodies were used to characterize the variable portion of B1–8 and of other anti‐NP antibodies all belonging to an antibody family which carries a major idiotypic marker, namely the NP b idiotype. Three groups of idiotopes were defined by the monoclonal anti‐idiotope antibodies on the B1–8 protein; a first, in the NP binding site so that the corresponding anti‐ idiotopes compete with the NP hapten for binding to B1–8 (group 1); a second, presumably further apart from the binding site so that there is competition of the corresponding anti‐idiotopes with a polyvalent NP‐protein conjugate but not the free hapten for binding to B1–8 (group 2); and a third (group 3), specific for a determinant constructed by B1–8 μ chains and MOPC 21 χ chains which are still present in the B1–8 preparation as the product of the X63‐Ag8 parent cell line. Group 1 idiotopes were expressed on 1–10% of χ 1‐bearing primary anti‐NP antibodies of C57BL/6 origin and group 2 idiotopes on 10–100% of these antibodies. Group 3 idiotopes were not detected in anti‐NP antisera. The analysis of a set of monoclonal hybrid cell anti‐NP antibodies from C57BL/6 confirmed that group 2 idiotopes are more abundantly expressed in the primary anti‐NP response than any given group 1 idiotope. Group 2 idiotopes can thus be expressed independently of group 1 idiotopes. In addition, group 1 idiotopes appear to exhibit a higher degree of diversity than idiotopes of group 2. The present analysis dissects the NP b idiotype into a large set of idiotopes which can be grouped in terms of localization on the antibody molecule and of variability and which are combined in various ways on a large number of χ chain‐bearing antibody species. At least some of the idiotopes recognized by the monoclonal antibodies are under the control of the heavy chain linkage group, as shown by the idiotope analysis of serum antibodies from Igh‐congenic mice. In the course of the fusion experiments, also cell lines secreting antibodies of SJL origin with specificity for the constant region of χ 1 chains were isolated, and we also describe the preparation of an Fv fragment from B1–8, an IgM antibody.