Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries.
Thyroid lymphocyte RNA from a Hashimoto patient with high serum levels of autoantibodies to thyroid peroxidase (TPO) was used to construct a phage display antibody library in the phagemid vector pComb3. The library (100,000cfu) encoded IgG1 heavy chains together with kappa light chains. Selection of the phages displaying TPO antibody on TPO-coated ELISA plates yielded a phage population enriched for surface expression of TPO antibody Fabs. 3 different Fabs specific for TPO were subsequently isolated with affinities in the region of 10(9) molar-1. 2 of the Fabs recognised the same, or closely related, epitopes on TPO whereas the third Fab recognised a different epitope. These 2 epitopes were recognised by TPO autoantibodies in the serum of the lymphocyte donor and a series of 10 patient sera. Available sequence data showed that several non-self antibodies and non-thyroid autoantibodies use the same V kappa and VH germline genes as TPO autoantibodies. There appeared to be no clear relationship between gene sequence or gene family usage by TPO autoantibodies of the same or similar epitope specificity.
Hexham
J M
JM
Krebs Institute, Department of Molecular Biology, University of Sheffield, UK.
Partridge
L J
LJ
Furmaniak
J
J
Petersen
V B
VB
Colls
J C
JC
Pegg
C
C
Rees Smith
B
B
Burton
D R
DR
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000096:biosynthesis / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D015698:Genomic Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007453:Iodide Peroxidase / Q000276:immunology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger
D001323:Autoantibodies; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D012333:RNA, Messenger; D007453:Iodide Peroxidase