Establishment of enzyme-linked immunosorbent assays for lipoprotein lipase with newly developed antibodies.
We developed eight new antibodies against lipoprotein lipase (LPL), which included polyclonal antibodies raised against recombinant human LPL produced by transformant cells and two synthetic peptides corresponding to either amino (N)- or carboxy (C)-terminus of human LPL. With these antibodies, we established three effective sandwich enzyme-linked immunosorbent assays (ELISAs) for LPL, which enabled us to examine LPL mass not only in the postheparin plasma from human, rat, mouse, and guinea pig but also in the media and lysates of cultured cells. All of the developed antibodies showed high affinities for LPL, but their binding to LPL did not always influence the lipolytic activity of the enzyme. Interestingly, although the anti-C-terminus antibody should bind to a common epitope of human and mouse LPL, its binding selectively suppressed only human LPL activity. Because amino acid sequence surrounding the epitope is common to both LPLs, difference in the sequence outside the epitope will contribute to the selective suppression of LPL activity by the antibody. Our results also suggested that both termini of LPL would be exposed on the surface of the molecule because they were fully accessible to antibodies and that the N-terminus of LPL would be functionally less important because binding of the anti-N-terminus antibody did not affect human LPL activity. The ELISAs were further utilized to demonstrate the presence of C-terminus truncated LPL protein in the postheparin plasma of an LPL-deficient patient, to map an epitope of the anti-C-terminus antibody within residues 433-436, and to gain insight into the structure-function relationship of the LPL molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
Kawamura
M
M
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Gotoda
T
T
Mori
N
N
Shimano
H
H
Kozaki
K
K
Harada
K
K
Shimada
M
M
Inaba
T
T
Watanabe
Y
Y
Yazaki
Y
Y
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D000939:Epitopes; D006168:Guinea Pigs; D006801:Humans; D008071:Lipoprotein Lipase / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D012680:Sensitivity and Specificity
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D008071:Lipoprotein Lipase