Preparation of synthetic polypeptide domains of carcinoembryonic antigen and their use in epitope mapping.
Genes encoding the four principal polypeptide domains (N, A1-B1, A2-B2, and A3-B3) of carcinoembryonic antigen (CEA) were synthesized and expressed in Escherichia coli as fusion products with bacterial CMP-KDO synthetase (CKS). The four synthetic fusion proteins were purified in high yield and used as targets in Western blots for 11 anti-CEA MAbs and to compete with immobilized CEA for binding to four of these MAbs. Each of the MAbs showed strong binding to one or more of the fusion proteins. In Western blots, MAbs H19C91 and 4230 bound only to CKS-N. MAbs H8C2 and H11C35 bound only CKS-A1-B1, and MAbs T84.66, H46C136, and H21C83 appeared to be specific for CKS-A3-B3. None of the MAbs tested bound only to CKS-A2-B2. However, two MAbs bound both CKS-A1-B1 and CKS-A3-B3 and one MAb (3519) bound to all three of the repeated domains. Since these three domains exhibit over 90% amino acid sequence homology, the latter results were not surprising. The competition studies largely confirmed the results of Western blots but did show some MAb-fusion protein interactions not observed in Western blots. These competition studies also allowed estimation of the relative affinities of the MAbs for the synthetic domains and for native CEA. These studies demonstrated that epitopes in CEA recognized by the MAbs in this study are peptide in nature and that the fusion proteins are of utility in the localization of the epitopes on the polypeptide chain of CEA.
Hass
G M
GM
Abbott Laboratories, Abbott Diagnostics Division, Abbott Park, Illinois 60064.
Bolling
T J
TJ
Kinders
R J
RJ
Henslee
J G
JG
Mandecki
W
W
Dorwin
S A
SA
Shively
J E
JE
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001667:Binding, Competitive; D002272:Carcinoembryonic Antigen / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002874:Chromosome Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005802:Genes, MHC Class II; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000235:genetics / Q000276:immunology; D010957:Plasmids / Q000235:genetics
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase