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Journal of Lipid Research, Vol. 43, 325-334, February 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase

Correspondence to: Kiichiro Kobori, To whom correspondence should be addressed., kobori{at}daiichichem.co.jp (E-mail)

We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. These are mouse anti-human LCAT monoclonal antibodies designated 36487, 36454, 36442, 36405, and 36486, which react with the peptides corresponding to human LCAT amino acid residues R159-E179, M258-S273, S274-S294, D352-S376, and N415-E440, respectively. We also successfully used two of these antibodies to develop an ELISA, which uses a solid phase monoclonal antibody, 36486, that reacts with the C-terminus of LCAT, and a detection monoclonal antibody, 36487, that reacts with an epitope located in the center of the LCAT primary structure. We observed a significant positive correlation between the values of LCAT protein determined with ELISA and LCAT activity determined with liposome substrate (r = 0.871, P < 0.001) or the endogenous self-substrate method (r = 0.864, P < 0.001), and we obtained inter- and intra-assay coefficients of variation less than 6.1%, minimum detection limit of 0.1 µg/ml. Highly specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT. Therefore, this precise and sensitive LCAT assay will help clarify the role of this enzyme in the metabolism of HDLs, and can be used for diagnostic purposes in investigating liver function.

We obtained five monoclonal antibodies that recognized different epitopes on LCAT and developed a sandwich-type ELISA. Highly specific monoclonal antibodies provide a sensitive and specific analytical system for measurements of LCAT protein. — Kobori, K., K. Saito, S. Ito, K. Kotani, M. Manabe, and T. Kanno. A new enzyme-linked immunosorbent assay with two monoclonal antibodies to specific epitopes measures human lecithin-cholesterol acyltransferase. J. Lipid Res. 2002. 43: 325–334.

Supplementary key words: ELISA, LCAT, epitope

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