E. Ferguson et al., GENERATION AND INITIAL CHARACTERIZATION OF A NOVEL POLYCLONAL ANTIBODY-DIRECTED AGAINST HOMOCYSTEINE THIOLACTONE-MODIFIED LOW-DENSITY-LIPOPROTEIN, Journal of lipid research, 39(4), 1998, pp. 925-933
Elevated plasma homocysteine (homocysteinemia) are presumed to be resp
onsible for the development of coronary artery disease, however, the p
recise etiology is unclear. We examined the possibility that the adduc
t formed from the reaction between homocysteine thiolactone, a metabol
ic product of homocysteine, and apolipoprotein B-100 lysyl residues of
low density lipoprotein (LDL) was immunogenic. New Zealand White rabb
its were immunized with this adduct at 6-week intervals. Antisera coll
ected following the 3rd immunization was assayed for antibody titers u
sing solid phase ELISA techniques. Titers (defined as the inverse of t
he greatest serum dilution in which there was a significant difference
(P < 0.05) between the percentage antibody bound from the antiserum a
nd the pre-immune serum) were approximately 10(5). In competition-base
d ELISAs, homocysteine thiolactone-treated LDL competed for binding wi
th the antiserum, as the 50% inhibitory concentration was approximatel
y 10 mu g/ml. Neither homocysteine, homocystine (homocysteine disulfid
e), nor Cu2+-oxidized LDL competed for binding. LDL in which lysyl res
idues were derivatized by acetylation or methylation were not recogniz
ed by the antiserum. Homocysteine thiolactone-treated plasma competed
for binding to the antiserum, whereas native plasma did not. All lipop
rotein fractions from the homocysteine thiolactone-treated plasma comp
eted for binding to the antiserum. We conclude that homocysteine thiol
actone-modified LDL is highly immunogenic and specific for homocystein
e thiolactone-modified lysines. The potential for using this antibody
as a diagnostic tool for measuring plasma homocysteine concentrations
and the implications for understanding diseases induced by homocystein
emia are discussed.