Abstract
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.
Highlights
We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT
Hybridomas producing anti-human LCAT antibody were established by fusing mouse spleen cells that had high antibody titers to the immunized purified LCAT or peptide-keyhole limpet hemocyanin (KLH) with myeloma cells
Where the start methionine of the amino acid sequence predicted by the human LCAT mRNA is residue number 1, monoclonal antibody 36487 reacts with the peptide corresponding to human LCAT amino acid residues 159–179 (RDETVRAAPYDWRLEPGQQEE), 36442 reacts with the peptide corresponding to human LCAT amino acid residues 258–273 (MSSIKLKEEQRITTTS), and 36486 reacts with the peptide corresponding to human LCAT amino acid residues 415–440 (NAILLGAYRQGP PASPTASPEPPPPE)
Summary
We established five monoclonal antibodies that reacted with human LCAT and recognized different epitopes on LCAT. Specific monoclonal antibodies will be useful in the study of the molecular pathology of LCAT 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. Abnormality of plasma lipids and lipoprotein profiles adversely affects these assays Such assays may reflect the physiological esterification rate, they do not readily distinguish influences of the enzyme from those of the substrate, cofactors, and products [4]. FED is caused by missense mutations only; these mutations affect either LDL or HDL activity in Class 3 defects, and LCAT mass is reduced.
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