Journal of Lipid Research, Vol 28, 818-827, Copyright © 1987 by Lipid Research, Inc.
Expression of a monoclonal antibody-defined aminoterminal epitope of human apoC-I on native and reconstituted lipoproteins
ES Krul, K Oida and G Schonfeld
Nine distinct mouse monoclonal antibodies were produced in two fusions
using holo-human very low density lipoprotein (VLDL) as antigen. On
immunoblotting first with human VLDL and then with isolated human apoC- I,
seven of the antibodies, representing three isotypes, manifested
specificity for apoC-I. Two antibodies were directed against apoB. To
assess whether the seven anti-apoC-I antibodies were directed against the
same or distinctively different epitopes, cross-competition assays were
performed wherein 125I-labeled monoclonal antibodies were made to compete
with unlabeled antibodies for occupancy on immobilized VLDL- associated
apoC-I. All antibodies cross-competed to varying extents implying that they
were directed against closely spaced epitopes, but based on these
experiments three different epitopes were defined. On immunoblotting with
CNBr fragments, all of the epitopes were assigned to the CNBr I fragment of
human apoC-I (amino acids 1-38) suggesting that the NH2-terminal region of
apoC-I is more immunogenic in mice than other parts of the molecule when
apoC-I is associated with VLDL. A competitive solid-phase radioimmunoassay
(RIA) was developed employing one of the anti-apoC-I antibodies (A3-4).
VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the
antibody was reacted with the adsorbed VLDL. The amount of monoclonal
antibody that bound to the immobilized VLDL-apoC-I was determined with a
125I-labeled goat anti-mouse IgG antibody. The addition of competitor
apoC-I complexed with lipids resulted in reduced binding of the anti-apoC-I
antibody to the immobilized VLDL-apoC-I. Competitor complexes consisted of
an artificial lipid emulsion (Intralipid) incubated with apoC-I at
phospholipid/apoC-I ratios of 1:1 to 60:1 (w/w). As the lipid/protein
ratios were increased, the competitive displacement curves produced by the
complexes become progressively steeper, while isolated lipid-free apoC-I
produced curves with very shallow slopes, suggesting that a
conformation-dependent epitope was being probed. Other apoproteins (C- II,
C-III, A-I, A-II, and E) whether lipid-free or complexed with lipids did
not compete. Fractionation of the 30:1 apoC-I-Intralipid complex by gel
permeation chromatography suggested that apoC-I bound to phospholipids was
the most effective competitor. This was confirmed by testing of apoC-I-DMPC
complexes, which yielded curves that paralleled those produced by
apoC-I-Intralipid.(ABSTRACT TRUNCATED AT 400 WORDS)
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