Degranulation of mast cells and basophils, with release of agents of the allergic response, ensues when multivalent antigens bind to and cross-link the cells receptor-bound IgE antibodies. tightly to high-affinity receptors (FcRI) on mast cells in surface tissues, or on basophils in the blood, to sequester the parasite antigens or toxins and evoke an immediate and potent immune response. A central feature of this mechanism is cross-linking of the receptor by attachment of a multivalent antigen to two or more receptor-bound IgEs, thereby delivering the signal for mast cell or basophil activation2, 3. The cost of the evolution of this protective mechanism in mammals is the risk of sensitization to otherwise harmless antigens (allergens) with the development of allergic disease. IgE binding to the receptors in the absence of antigen should not induce cell activation, which would serve no purpose in adaptive immunity, and could indeed pose a severe danger. The discovery of several monoclonal IgEs that stimulate mast cell and basophil activation without the need for an antigen was therefore perplexing4. At the time of this discovery, it was already known that IgE at extremely high concentrations (5?g/ml), some two orders of magnitude higher than that required for antigen-dependent cell activation, could up-regulate IgE receptor expression on mast cells5C7. Kitaura et al. further established that some monoclonal IgEs could induce cell activation in the absence of antigen. Different monoclonal IgEs could 1187595-84-1 manufacture be classified according to their antigen-independent activities, for which they coined the term, cytokinergic activity4. The higher this cytokinergic activity of the IgE, the greater the variety and potency 1187595-84-1 manufacture of the activities it evoked. Monoclonal IgEs were grouped according 1187595-84-1 manufacture to whether they were highly cytokinergic (HC), moderately (MC), or poorly (PC) so. Only IgEs in the first (HC) category could induce mast cell degranulation in the absence of allergen. The most highly cytokinergic IgE was the mouse NS1 hybridoma SPE-7 IgE, an anti-dinitrophenyl (DNP) antibody8, 9. At 5?g/ml the SPE-7 IgE alone activates mast cells and basophils to nearly the same extent as it does at 0.02?g/ml in the presence of antigen, and by the same receptor cross-linking mechanism normally initiated by the antigen4. In some cases, sufferers from allergic disease were found by Bennich and Johansson10 to have concentrations of circulating IgE of up to 5?g/ml and it was therefore suggested that antigen-independent (cytokinergic) IgE action may play a part in the pathophysiology of allergic diseases4, 11, 12. The inhibition of cytokinergic activity by free DNP, and more recently by recombinant SPE-7 IgE Fab, pointed to the Fv domain as the seat of the cytokinergic activity of the SPE-7 IgE antibody4, 13, 14. In the present study, with the aid of ATF3 higher resolution from a new size-exclusion column matrix, we show that SPE-7 IgE monomers are devoid of cytokinergic activity when isolated from all contaminants. This new technology has also enabled us to demonstrate the presence of a previously unrecognized component in SPE-7 IgE preparations: an SPE-7 IgE trimer that drives the cytokinergic activity. We thus resolve a long-standing issue concerning the nature of the cytokinergic phenomenon. Results To determine the origins of the exceptional cytokinergic action of SPE-7 IgE (Sigma), we isolated the pure monomeric IgE from the crude commercial product. For comparison we purified the equivalent monomeric IgE from the NS1 hybridoma (kindly provided by Dr. Zelig Eshhar) and recombinant SPE-7 produced in HEK cells using the sequence derived from the hybridoma. The recombinant SPE-7 was expressed both as wild-type mouse protein (mSPE-7) and as a mouse-human chimaeric form, comprising mouse SPE-7 IgE heavy- and light-chain variable regions and human constant regions (chSPE-7; Supplementary Figure?1). The monomers were isolated by size-exclusion chromatography on a Superdex 200 column and pooled fractions were assayed for antigen-independent activity in a rat basophil cell line expressing both the human and mouse high-affinity IgE receptors (RBL-SX38 cells). By contrast with the potent degranulating activity of the crude commercial product, none of our three purified proteins showed any detectable degranulating action (Fig.?1). At the same time, the four proteins became comparably active on addition of antigen 1187595-84-1 manufacture (DNP coupled to human serum albumin, DNP-HSA). These results imply that the highly cytokinergic activity of SPE-7 may be due.