A monoclonal antibody and an enzyme immunoassay for human Ala-IL-877

• Published in: Journal of immunological methods Vol. 270; no. 1; pp. 37 - 51
• Main Authors: Nashkevich, Natalia N, Akalovich, Svetlana, Louneva, Natalia, Heavner, George A, Voitenok, Nikolai N
• Format: Journal Article
• Language: English
• Published: 01.12.2002
• ISSN: 0022-1759
• DOI: 10.1016/S0022-1759(02)00279-X

Interleukin-8 (IL-8) plays a central role in neutrophil chemotaxis and exerts a wide range of effects on various cells, ranging from tumor angiogenesis to impairment of neuronal signaling. Two main forms of IL-8 exist, one containing 77 amino acids (Ala-IL-877) and a second containing 72 amino acids (Ser-IL-872), which comprise more than 90% of IL-8 protein in cell cultures. IL-877 was reported to be produced predominantly by endothelial cells and is known as 'endothelial' IL-8. IL-872 predominates in monocyte cultures and is known as 'leukocyte' IL-8. While both forms have equal chemotactic activity in vivo, recent data suggest that their biological activities might be different. Here we describe the generation of a mouse monoclonal antibody (mAb) specific for IL-877 and the development of a corresponding immunoassay. Various immunization protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-877 (NTP77) resulted in the production of an IgG1 mAb (N11) that recognizes human IL-877 and neutralizes its chemotactic activity. A sensitive ELISA specific for IL-877 was developed using N11 for capture and a biotinylated mAb to IL-872 for detection. Using this immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-877 and that the IL-872 present was the result of proteolysis of IL-877. IL-877 was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and meningitis.