Use of bacterial expression cloning to localize the epitopes for a series of monoclonal antibodies against apolipoprotein B100.
Bacterial expression of apolipoprotein (apo) B cDNA constructs has been used to map a series of monoclonal antibodies (mAbs) to apoB by immunoblotting. In some cases assignments have been confirmed and refined by (i) semipurification of expressed protein, CNBr digestion, and assignment of the immunoreactive fragments; (ii) controlled digestion of the cDNA with the exonuclease Bal31 and bacterial expression of the truncated proteins that result; or (iii) expression of specific segments of cDNA amplified by the polymerase chain reaction. Forty mAbs were mapped to a minimum of 17 separate determinants on apoB. Tryptic fragments have been used to confirm the epitope assignments. In addition, this approach in conjunction with immunoassay, enables some deductions to be made about the trypsin-accessible regions in low density lipoprotein (LDL). The cleavage pattern obtained predicts retention of structure in the cysteine-rich domain of the amino terminus and also in the LDL receptor binding region. Trypsinized LDL was shown to bind to the LDL receptor by an authentic process, using monoclonal antibodies as competing ligands. In conjunction with the previous paper (Milne, R. W., Theolis, R., Maurice, R., Pease, R. J., Weech, P. K., Rassart, E., Fruchart, J.-C., Scott, J., and Marcel, Y. L. (1989) J. Biol. Chem. 265, 19754-19760) the mapped mAbs have been used to define the receptor-binding domain of apoB100 in LDL.
Division of Molecular Medicine, Medical Research Council Clinical Research Centre, Harrow, United Kingdom.
R J
RJ
Pease
R W
RW
Milne
W K
WK
Jessup
A
A
Law
P
P
Provost
J C
JC
Fruchart
R T
RT
Dean
Y L
YL
Marcel
J
J
Scott
D000911:Antibodies, Monoclonal / Q000276:immunology*; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D008077:Lipoproteins, LDL / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011973:Receptors, LDL / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D000939:Epitopes; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D004247:DNA; D003488:Cyanogen Bromide