Apolipoprotein D and cross-reacting human plasma apolipoproteins identified using monoclonal antibodies.
We have produced five hybridomas which secreted monoclonal antibodies that reacted with human plasma apolipoprotein D. On analysis by polyacrylamide gel electrophoresis (PAGE) high density lipoproteins and lecithin:cholesterol acyltransferase (EC 2.3.1.43)-enriched fractions of plasma contained many protein bands that reacted with the antibodies. Purified apolipoprotein D had the lowest Mr (29,000), the lowest pI (4.8-5.2), and the greatest migration on alkaline urea-PAGE of all the immunoreactive bands. These characteristics agreed with those described for apolipoprotein D in the literature. The other immunoreactive proteins had apparent Mr from about 39,000 to 98,000, they migrated more slowly than apolipoprotein D on alkaline urea-PAGE, and there were 10 polymorphs on isoelectric focusing. These cross-reacting proteins were present in the high density lipoproteins of each of four individuals sampled on several occasions and in pooled plasma. All of the monoclonal antibodies reacted both with apo-D and the higher Mr cross-reacting proteins. Each of our five monoclonal antibodies bound to one of two distinct antigenic sites on apo-D, determined by antibody competition immunoassays. Neither of these two sites was composed of carbohydrate, but expression of both sites seemed to be influenced by thiol-reducing agents: site 5G10 gained but 4E11 either lost immunoreactivity or was unchanged by reduction according to the conditions. We conclude that apolipoprotein D is only one of several plasma proteins, which contain two homologous polypeptide antigenic sites, recognized by monoclonal antibodies and also by a specific goat antiserum. Apolipoprotein D had the least Mr of these proteins.
P K
PK
Weech
R
R
Camato
R W
RW
Milne
Y L
YL
Marcel
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001053:Apolipoproteins / Q000097:blood* / Q000302:isolation & purification; D053399:Apolipoproteins D; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D011863:Radioimmunoassay
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001053:Apolipoproteins; D053399:Apolipoproteins D