Mapping epitope specificities of monoclonal antibodies to thyroid peroxidase using recombinant antigen preparations.
Five separate monoclonal antibodies (MoAbs) to human thyroid peroxidase (hTPO) were raised by immunising Balb/c mice with hTPO purified from detergent solubilised thyroid microsomes by high performance liquid chromatography (HPLC). The epitope specificities of these MoAbs were determined by assessing their ability to bind to purified recombinant fusion protein fragments of human TPO (TPO(r)) generated in E. coli. A total of seven small overlapping fragments (averaging 104 amino acid residues) of hTPO, encompassing over 90% of the extracellular region of the molecule, were generated as glutathione S-transferase (GST) fusion proteins. The sequential epitopes on TPO(r) recognised by these MoAbs were analysed by both immunoblotting and enzyme linked immunosorbent assay (ELISA). Two different MoAbs (A4 and A5) recognised sequential epitopes within the TPO(r) preparation termed R1a + b (residues 1-160) and more specifically, in the case of MoAb A4, within the subfragment R1b (residues 70-160). The inability of the other MoAbs (A1-A3) to recognise recombinant fragments, suggests they either recognise conformational determinants on the TPO molecule or epitopes that are present on the small regions of the TPO molecule which have not been expressed as recombinant proteins.
Ewins
D L
DL
Department of Medicine, Kings College School of Medicine and Dentistry, London.
Barnett
P S
PS
Tomlinson
R W
RW
McGregor
A M
AM
Banga
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001324:Autoantigens / Q000276:immunology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006111:Graves Disease / Q000201:enzymology; D006801:Humans; D007453:Iodide Peroxidase / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008861:Microsomes / Q000201:enzymology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D007453:Iodide Peroxidase