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

Abstract 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” Vκ oligonucleotide primer. The KM1 L chain is encoded by a mutated B3 gene (VκIV 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 Vκ family-specific primers. Both KM1 and WR1.223 bind TPO with high affinity (∼1 × 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 Vκ primer recognized 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 Vκ 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 κ 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.