BALB/c mice were sensitized/challenged as described in Methods. Twenty-four hours after allergen challenge, the mice were sacrificed and blood collected in heparinized tubes. Plasma was obtained by centrifugation (2000 r.p.m., 10 min). Three hundred μl immune plasma were transferred to each naïve BALB/c recipient by i.p. injection. In selected experiments, plasma was submitted to different treatments to deplete total antibody or to inactivate IgE before transfer. For antibody depletion, protein A sepharose (Sigma Ref. P-3391) was rehydrated in protein-free RPMI 1640 medium, under constant stirring, for 1 h 30 min at 4°C, washed three time and brought up to 10% until use. Absorption was carried out by mixing heparinized plasma (either naïve or immune) with pelleted protein A sepharose beads (10% beads final v v−1), and incubating for 1 h at 4°C, with constant stirring. After centrifugation at 2500 r.p.m. for 5 min, the supernatant was submitted to two further cycles of absorption under identical conditions. To eliminate residual immunoglobulins (Ig) from classes that fail to bind directly to protein A sepharose, one final cycle of absorption was carried out after adding 50 μl of goat polyclonal antibody to mouse IgG with specificity for both heavy and light chains (H+L, Immunotech, Marseille, France, Ref. 0815) to each ml of the preabsorbed plasma. The mixture was incubated for 30 min at room temperature, and for 1 h more at 4°C, with stirring. The supernatant was collected and 300 μl were injected i.p. in each recipient mouse. The efficiency of these procedures in removing antibody was monitored as follows: (a) binding of Igs to the beads was monitored by SDS-polyacrylamide gel electrophoresis of the proteins that could be extracted from the protein A sepharose beads pelleted after each cycle, by boiling for 3 min in sample buffer; (b) complete absorption of IgG, including IgGl, was confirmed by immunoblotting (see below); (c) complete removal of specific antibody activity was directly demonstrated by ELISA (see below). For immunoblots, the following samples were separated by SDS – PAGE on a 7.5% minigel, for 1 h at 150 V: plasma from ovalbumin-sensitized and challenged mice (before and after absorption of Ig as described above); the eluate from the last cycle and the goat anti-mouse IgG (H+L) polyclonal antibody used for the last cycle (see above); and monoclonal mouse IgG (Ref. 0107 – 01, Southern Biotechnology Associates, Birmingham, AL, U.S.A.), used as a positive control for immune reactivity, as well as a molecular weight marker. The minigel was soaked for 30 min in transfer buffer (Tris 20 mM, glycine 100 mM), before transfer at 100 V 500 mA−1, for 1 h. Complete transfer from the gel was demonstrated by Ponceau red staining of both the blot and the gel. The blot was washed in distilled water and quenching was carried out overnight with PBS – BSA 2%, at 4°C, before washing four times with PBS containing BSA 0.5% and tween 20 0.05%, for 15 min. Blots were incubated with anti-mouse IgG (Fab specific) peroxidase conjugate (Sigma, A-3682) at 1 : 4000, for 90 min washed four times (20 min each) as above and developed with diaminobenzidine solution (50 mg DAB, 100 μl de H2O2 30% in 100 ml PBS) for 5 min. Image processing was carried out with the help of the Kodak Digital Science 1 D Software, with inverted brightness-contrast for optimal visualization. For ELISA, ovalbumin (10 μg ml−1 in 0.1 M sodium phosphate buffer, pH 8), was used to coat 96-well plates (NUNC MaxiSorp™ Surface, 50 μl per well) overnight, at room temperature, with constant stirring, before washing with PBS-tween 20 (0.1%) and quenching with PBS – BSA 1% (200 μl per well), for 2 h. One hundred μl aliquots of immune plasma, naïve plasma or immune plasma absorbed on protein A sepharose, diluted in PBS – BSA, were added for 1 h, before washing and addition of goat anti-mouse IgG (γ-chain specific) alkaline phosphatase conjugate (Ref. A-1047, Sigma, 100 μl per well at 1 : 1000 dilution) for 2 h. Each well was washed and incubated with 100 μl p-nitrophenyl phosphate disodium (1 mg ml−1 in MgCl2 3 mM, Tris 100 mM) in the dark, for 15 min before reading OD405. To control for the effects of the absorption procedure, as well as the effects of carryover of goat antimouse Ig antibody into the absorbed plasma samples, naïve plasma was treated under identical conditions and injected into naïve recipients (see Results). In order to selectively eliminate IgE, immune plasma was incubated for 30 min at 56°C before injecting 300 μl i.p. in each naïve recipient. In all cases, 24 h later, recipient mice were sacrificed and used as bone-marrow donors for liquid culture.