Cloning of A Human Autoimmune Response: Preparation and Sequencing of A Human Anti-Thyroglobulin Autoantibody Using A Combinatorial Approach

Abstract

Thyroid lymphocyte RNA from a Hashimoto patient exhibiting high titre serum IgG autoantibodies against thyroglobulin (Tg) has been used to construct a Fab library in phage lambda. Screening of this library with radioiodinated Tg has permitted the cloning of an anti-Tg antibody (MH52) with an affinity of 4.5x10' molar¹ as determined by inhibition ELISA. Sequence analysis showed MH52 to be an authentic antibody of the IgGl/K isotype with variable region genes from the VHI and VKIII families in combination with the JH3, DK4 and JK2 gene segments.

The MH52 light chain gene showed high sequence homology (93%) with the germline gene used by several rheumatoid factors and some DNA autoantibodies. Greater divergence from the germline was observed in the case of the MH52 heavy chain gene which showed 86% homology with a germline heavy chain gene isolated from human liver. Overall the similarity between the genes coding for MH52 and the genes coding for some other autoantibodies of non-related specificity might suggest that similar regulatory processes control the formation of these different autoantibodies.

Key Words:

• Hashimoto's disease
• thyroid autoantibodies
• immune repertoires
• combinatorial libraries
• thyroglobulin
• Tg
• thyroglobulin
• TPO
• thyroid peroxidase
• dsDNA
• double stranded DNA
• BSA
• bovine serum albumin
• PBS
• phosphate buffered saline
• ELISA enzyme-linked immunosorbent assay
• HC
• heavy chain
• LC
• light chain
• PCR
• polymerase chain reaction