Phage-displayed recombinant single-chain antibody fragments with high affinity for cholesteryl ester transfer protein (CETP): cDNA cloning, characterization and CETP quantification.
Cholesteryl ester transfer protein (CETP) greatly affects the metabolism of all lipoprotein classes including low-density lipoprotein (LDL) and high-density lipoprotein (HDL), both known to constitute powerful risk factors for coronary artery disease (CAD). We now report the successful first cloning and characterization of single-chain antibody fragments specific for CETP. A recombinant phage display library was generated using spleen mRNA isolated from BALB/c mice that had been immunized with highly purified CETP. Screening of the library yielded two single-chain antibody fragments with high affinity for CETP, termed 1CL8 and 1CL10, displaying respective KD values of 4.36 x 10(-9) M and 4.64 x 10(-9) M as determined by affinity sensor technology. Amino acid sequence comparison indicated the complementarity-determining regions of the respective heavy chains to be responsible for CETP high affinity binding. Fragment 1CL8 was successfully employed in clinical chemical quantification systems that uncovered an association in humans between plasma CETP concentration and total body fat mass (r=0.50, p<0.002). Because of the demonstrated superb CETP capturing capacity, combined with high binding affinity to CETP, ready access and unlimited supply, 1CL8 and 1CL10 are expected to prove powerful tools for studies on the role of CETP in atherogenesis.
Ritsch
Andreas
A
Department of Medicine, University of Innsbruck, Innsbruck, Austria. andreas.ritsch@uibk.ac.at
Ebenbichler
Christoph
C
Naschberger
Elisabeth
E
Schgoer
Wilfried
W
Stanzl
Ursula
U
Dietrich
Hermann
H
Heinrich
Peter C
PC
Saito
Kazunori
K
Patsch
Josef R
JR
D000818:Animals; D000906:Antibodies / Q000097:blood; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D001823:Body Composition / Q000502:physiology; D002352:Carrier Proteins / Q000097:blood / Q000276:immunology*; D002784:Cholesterol / Q000097:blood; D053480:Cholesterol Ester Transfer Proteins; D003090:Coliphages / Q000235:genetics; D022801:Complementarity Determining Regions / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D003627:Data Interpretation, Statistical; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000097:blood / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008074:Lipoproteins / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D019151:Peptide Library; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D013154:Spleen / Q000737:chemistry; D014280:Triglycerides / Q000097:blood; D014611:Vaccination
D000906:Antibodies; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D022801:Complementarity Determining Regions; D018076:DNA, Complementary; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008074:Lipoproteins; D019151:Peptide Library; D011994:Recombinant Proteins; D014280:Triglycerides; D012313:RNA; D002784:Cholesterol