A monoclonal antibody and an enzyme immunoassay for human Ala-IL-8(77).
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-8(77)) and a second containing 72 amino acids (Ser-IL-8(72)), which comprise more than 90% of IL-8 protein in cell cultures. IL-8(77) was reported to be produced predominantly by endothelial cells and is known as "endothelial" IL-8. IL-8(72) 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-8(77) and the development of a corresponding immunoassay. Various immunization protocols were investigated. Immunization with conjugates of a peptide from the N-terminus of IL-8(77) (NTP(77)) resulted in the production of an IgG1 mAb (N11) that recognizes human IL-8(77) and neutralizes its chemotactic activity. A sensitive ELISA specific for IL-8(77) was developed using N11 for capture and a biotinylated mAb to IL-8(72) for detection. Using this immunoassay it was shown that the only form of IL-8 secreted in cell culture was IL-8(77) and that the IL-8(72) present was the result of proteolysis of IL-8(77). IL-8(77) was detected in plasma and cerebrospinal fluid (CSF) from patients with sepsis and meningitis.
Nashkevich
Natalia N
NN
Laboratory of Cellular and Molecular Immunology, Institute of Hematology and Blood Transfusion, Dolginovsky Tract 160, Minsk, Belarus.
Akalovich
Svetlana
S
Louneva
Natalia
N
Heavner
George A
GA
Voitenok
Nikolai N
NN
D000409:Alanine; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D002633:Chemotaxis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007074:Immunoglobulin G; D016209:Interleukin-8 / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009500:Neutralization Tests; D010455:Peptides / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013194:Staining and Labeling / Q000379:methods
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D016209:Interleukin-8; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000409:Alanine