Preparation and characterization of anti‐framework antibodies to the heavy chain variable region (V_H) of mouse immunoglobulins

Abstract

The heavy chain variable portion of mouse myeloma protein MOPC-3 15 (a, h z ) was obtained from its Fv fragment (J. Hochman, D. Inbar and D. Givol, Biochemistry 1973. 1 2 : 11 30.) and was used t o immunize rabbits. Rabbit anti-V, antibodies precipitated V,, Fv and the intact protein 315. The V, region specificity of these antibodies was evident from the line of identity in agar gel diffusion between Fv and M315, and from the lack of precipitation with another (11 chain of XRPC-25 or with light (L) chains. Radioimmunoassay binding and inhibition studies demonstrated that anti-VH 31 5 antibodies cross-react with heavy (H) chains derived from various mouse myeloma proteins (MOPC-460, XRPC-25, MOPC-I 04E, UPC-10, TEPC-15, HOPC-8, MOPC-167) as well as with H chains derived from mouse IgG. These H chains can inhibit 90-100 % of the binding of VH3 15 by anti-VH indicating extensive sharing of antigenic determinants by various VH regions. The relative affinity of anti-V, for various H chains was 20-1 000-fold weaker than its affinity for the MOPC-3 1 5 H chain. The cross-reaction of the various H chains was independent of antibody specificity, subclass, subgroup or considered as anti-V framework antibody.Anti-VH315 cross-reacts with isolated H chains but not with intact molecules and can therefore be used t o detect VH determinants only in the absence of chains.allotype. This suggests that anti-VH may be

Antigen-specific T lymphocyte clones. III. Papain splits purified T suppressor molecules into two functional domains.

“…This was because of binding to the heavy (H) chain fragments as judged by direct binding to purified MOPC-315 H-chains and lack of reactivity to constant (C) and to light (L) chains. It should be noted, however, that the reagent does not precisely correspond to that reported by Ben-Neriah et al (13) since only a restricted number of myeloma proteins were bound by this antibody, in contrast to the considerably wider reactivity of the reagent described by Ben-Neriah et al (13).…”

“…Antisera with specificity for framework regions of VH chains of Ig were prepared in rabbits after immunization with the myeloma protein MOPC-315, as described previously (13). The MOPC-315 affinity-purified Ig fraction of this antiserum reacted with myeloma proteins MOPC-315, MOPC-460, and, to a lesser extent, T15 and S107.…”

“…After washing with PBS four times, proteins were eluted from the columns with 3 ml glycine-HCl (pH 2.4) and applied to SDS-PAGE slab gels. A control slot was dried and exposed to detect the position of the eluted proteins, and several fractions were sliced out and extracted from the gel, as described (13). These fractions were tested (1:10 dilution) for suppression of 105 SRBC-immune Ly-1+2-T helper cells, 10 6 B cells, or mixtures of both.…”

Two separate genes regulate self-Ia and carrier recognition in H-2-restricted helper factors secreted by hybridoma cells.

“…Anti-Vn-, anti-variable region of the h light chain (Va), and antivariable region of the x light chain (V,) specific affinity-purified antibodies were prepared by immunizing rabbits with the appropriate myeloma protein fragments, as previously described (13)(14)(15). Previous experiments have suggested that anti-Vn-Ma15 have a selectively lower affinity to AKR VH than to the VH region of C57BL/6 or other mouse strains.…”

“…Anti-Vn.315 recognizes allotypelike Igh-l-linked determinants in VH and reacts weakly or fails to react with AKR heavy chains or AKR T cell receptors (10,13,16,19; see also Materials and Methods). This characteristic of anti-Vn was used to investigate the presence of BW-5147 (AKR)-type VH products in the helper factors secreted by clone T77-146-C3.…”