Development and characterization of novel monoclonal antibodies against tartrate-resistant acid phosphatase 5.
Serum band 5 tartrate-resistant acid phosphatase (TRACP 5; EC 3.1.3.2) is a glycoprotein that exists as two very similar isoforms, TRACP 5a and TRACP 5b. The similarity of these two isoforms has made it difficult to establish monoclonal antibodies specific for either isoform. We report here the development of a monoclonal antibody with high specificity for TRACP 5b. We prepared TRACP 5b antigens from four sources: TRACP 5b purified from human bone, recombinant TRACP 5 from Escherichia coli, recombinant TRACP 5 from insect cells, and a synthetic TRACP 5b peptide. Thirty-seven mice were each immunized with 1 of the 4 different TRACP antigens to generate 473 antibody-producing clones. Three of these clones, Trk27, Trk49, and Trk62, reacted with TRACP 5b. These three clones were all established from mice exposed to native bone TRACP 5b antigen. In fact, none of the other antigens were able to generate anti-TRACP 5b monoclonal antibodies in mice. Furthermore, Trk62 interacted more strongly with TRACP 5b than with TRACP 5a. These results suggested that although recombinant proteins can be effective antigens, the native TRACP 5 protein might be more effective at generating monoclonal antibodies of greater specificity due to its more faithful representation of the native three-dimensional structure of the protein.
Ohashi
Tatsuya
T
Biochemical Laboratory, Nitto Boseki Co., Ltd., Fukushima, Japan.
Miura
Toshihide
T
Igarashi
Yoshihiko
Y
Kiyokawa
Iwao
I
Sato
Yasuhito
Y
Sasagawa
Kumiko
K
Katagiri
Ken
K
Mochizuki
Yoshiko
Y
Tomonaga
Takeshi
T
Nomura
Fumio
F
Kojima
Ryo
R
Katayama
Katsuhiro
K
D000135:Acid Phosphatase / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001842:Bone and Bones / Q000201:enzymology; D002460:Cell Line; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D018411:Spodoptera; D000071681:Tartrate-Resistant Acid Phosphatase
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; C000605510:ACP5 protein, human; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase