Preparation and characterization of monoclonal antibodies against human myeloperoxidase.
We established 11 hybridomas producing monoclonal antibodies (MoAbs), designated AM, against human myeloperoxidase (MPO), by immunizing mice with the three forms of MPO (I, II, and III) purified from healthy human polymorphonuclear leukocytes (PMN) and characterized the specificity of the AM MoAbs. Ten of the AM MoAbs reacted similarly to each of the three forms using an enzyme-linked immunosorbent assay. When a cetyltrimethylammonium bromide (CETAB) extract of human PMN was electrophoresed in a CETAB polyacrylamide gel and transferred to a nitrocellulose filter, IgG1 class AM MoAbs immunostained only the MPO band of the proteins of the extract. In addition, the AM MoAbs reacted to two radioactive bands of 94 and 92 kDa in a HL-60 cell lysate labeled with [35S]methionine for 1 h. After a chase period of 24 h, these bands were replaced by four radioactive bands of 64.5, 43, 16.7, and 13.4 kDa, demonstrating that the MoAbs recognize not only mature MPO but also the MPO precursors of 94 and 92 kDa. The data also indicated that the two major bands of 64.5 and 13.4 kDa corresponded to heavy and light chains of mature MPO, respectively, and the additive intermediate bands of 43 and 16.7 kDa were MPO-related proteins. Moreover, AM MoAbs reacted to a similar extent to the deglycosylated form of MPO III with endo-beta-N-acetylglucosaminidase H (Endo-H). Thus, IgG1 class AM MoAbs recognized MPO with high specificity and reacted to the structure which is commonly conserved in the three mature forms of MPO (I, II, and III), MPO precursors, and deglycosylated MPO with Endo-H. AM MoAbs also specifically reacted to PMN and/or monocytes but did not react to lymphocytes when the cell staining method was used.
Homma
T
T
Mitsubishi Yuka Bio-Clinical Laboratories, Inc., Tokyo Japan.
Suzuki
K
K
Kudo
Y
Y
Inagawa
M
M
Mizuno
S
S
Yamaguchi
K
K
Tagawa
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D009504:Neutrophils / Q000201:enzymology*; D009195:Peroxidase / Q000032:analysis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D009195:Peroxidase