The epitopic "fingerprint" of thyroid peroxidase-specific Fab isolated from a patient's thyroid gland by the combinatorial library approach resembles that of autoantibodies in the donor's serum.
A new thyroid peroxidase (TPO)-specific Fab (KM1) was obtained from an immunoglobulin gene combinatorial library of patient KM containing L chain genes amplified with a single "promiscuous" V kappa oligonucleotide primer. The KM1 L chain is encoded by a mutated B3 gene (V kappa IV family). Another mutated B3 L chain had been identified previously in a TPO-specific Fab (WR1.223) isolated from a different patient (WR). In contrast to patient KM, the WR L chains were amplified with a panel of V kappa family-specific primers. Both KM1 and WR1.223 bind TPO with high affinity (approximately 1 x 10(-9) M) and interact with an epitope in the B domain of the TPO immunodominant region. TPO-specific Fab previously isolated from a WR combinatorial library constructed with the promiscuous V kappa primer recognised the TPO A domain and none used a B3-like L chain. Remarkably, for both patients, Fab isolated from L chains generated with the promiscuous V kappa primer had epitopic profiles similar to autoantibodies in the donor's serum (KM-B domain; WR-A domain). Our data indicate that the promiscuous primer preferentially amplifies the dominant L chain present in vivo. However, to obtain a relatively rare Fab (such as the B domain Fab from WR), family-specific kappa primers are required. These findings provide insight into the relationship between TPO autoantibody gene usage, epitopic recognition, and the effectiveness of the combinatorial library approach.
Jaume
J C
JC
Thyroid Molecular Biology Unit, Veterans' Administration Medical Center, San Francisco, California, USA.
Guo
J
J
Kakinuma
A
A
Rapoport
B
B
McLachlan
S M
SM
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000302:isolation & purification; D007453:Iodide Peroxidase / Q000276:immunology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013961:Thyroid Gland / Q000737:chemistry*; D013967:Thyroiditis, Autoimmune / Q000235:genetics*
D001323:Autoantibodies; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D007453:Iodide Peroxidase