THE EPITOPIC FINGERPRINT OF THYROID PEROXIDASE-SPECIFIC FAB ISOLATED FROM A PATIENTS THYROID-GLAND BY THE COMBINATORIAL LIBRARY APPROACH RESEMBLES THAT OF AUTOANTIBODIES IN THE DONORS 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 oligonucle otide primer. The KM1 L chain is encoded by a mutated B3 gene (V kappa IV family). Another mutated B3 L chain had keen 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 pa nel of V kappa family-specific primers. Both KM1 and WR1.223 bind TPO with high affinity (similar to 1 x 10(-9) M) and interact with an epit ope 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 recognized the TPO A domain and none us ed a B3-like L chain. Remarkably, for both patients, Fab isolated from L chains generated with the promiscuous V kappa primer had epitopic p rofiles similar to autoantibodies in the donor's serum (RM-B domain; W R-A domain). Our data indicate that the promiscuous primer preferentia lly amplifies the dominant L chain present in vivo. However, to obtain a relatively rare Fab (such as the B domain Fab from WR), family-spec ific kappa primers are required. These findings provide insight into t he relationship between TPO autoantibody gene usage, epitopic recognit ion, and the effectiveness of the combinatorial library approach. (C) 1997 Academic Press.