Monoclonal antibodies distinguish between storage and secreted forms of eosinophil cationic protein

Abstract

The toxic effects of eosinophils on parasites and cells are due largely to the secretion of various granule proteins, following stimulation. In order to study this secretory process (degranulation) further, we have raised mouse monoclonal antibodies against both human eosinophil granule extracts and secretion products. From immunocytochemical studies it appears that one antibody, EG1 , recognized both the storage and secreted forms of eosinophilcationic protein (ECP), whereas antibody EG2 only bound to ECP during secretion (and extraction). This antibody also bound to eosinophil protein-X (EP-X). As both antibodies stained eosinophils in formalin-fixed tissues, they were used to demonstrate sites of eosinophil activation and secretion in chronic urticaria. The capacity of monoclonal antibodies to detect differences between storage and secreted forms of proteins is an important property of these reagents with many potential applications in cell biology.

Reactivity of monoclonal antibodies EG1 and EG2 with eosinophils and their granule proteins

“…3), EG1 reacted with ECP and EG2 reacted with ECP and EDN, consistent with the RIA findings. Overall, these results confirm the prior findings of Tai and colleagues [12] on the reactivity of EG1 and EG2 with ECP and EDN. Rosenberg and Tiffany reported that EG1 and polyclonal anti-ECP yielded three ECP bands on Western analyses, whereas EG2 detected only one ECP band at 18 kDa [34].…”

“…The monoclonal antibodies EG1 and EG2 were developed to further understand this degranulation, or secretory, process and to identify activated eosinophils in tissues. EG1 was thought to react with both stored and secreted forms of ECP, whereas EG2 was thought to react with EDN and only secreted ECP [12]. In addition, unactivated normal blood eosinophils reacted only with EG1, but activated eosinophils reacted with both EG1 and EG2, suggesting that EG2 selectively identified secreting activated eosinophils [12].…”

“…EG1 was thought to react with both stored and secreted forms of ECP, whereas EG2 was thought to react with EDN and only secreted ECP [12]. In addition, unactivated normal blood eosinophils reacted only with EG1, but activated eosinophils reacted with both EG1 and EG2, suggesting that EG2 selectively identified secreting activated eosinophils [12]. Demonstration of EG2-reactive eosinophils in tissue specimens from patients with a variety of allergic and inflammatory diseases [reviewed in ref.…”

“…EG1 was derived from a mouse injected with extracts of unstimulated eosinophils, and EG2 was derived from a mouse injected with secreted proteins from zymosan-C3b-stimulated eosinophils [12]. EG1 was thought to recognize both stored and secreted forms of eosinophil cationic protein (ECP), whereas EG2 was thought to recognize only secreted ECP and eosinophil-derived neurotoxin (EDN) [12]; amino acid sequencing and cloning of ECP and EDN [13][14][15][16][17] showed that these molecules are homologous and are members of the RNase superfamily. This report [12] further suggested that EG2 could be used to identify activated eosinophils in tissues, and a later report [18] confirmed the apparent ability of EG2 to identify activated eosinophils.…”

“…To study eosinophil activation and secretion, two murine monoclonal antibodies, EG1 and EG2, were developed [12] using eosinophils from patients with the hypereosinophilic syndrome (HES). EG1 was derived from a mouse injected with extracts of unstimulated eosinophils, and EG2 was derived from a mouse injected with secreted proteins from zymosan-C3b-stimulated eosinophils [12].…”