Competitive insulin binding curves of IAAs. (A) Competition of an IAA-positive serum against [¹²⁵I] insulin labeled at Tyr¹⁴A (circles), Tyr¹⁹A (triangles), and Tyr¹⁶B (diamonds) with increasing concentrations of unlabeled human insulin. Binding curves were similar with the [¹²⁵I] insulin labeled at residues Tyr¹⁴A, Tyr¹⁹A, or Tyr¹⁶B, and calculated IAA affinities did not significantly differ among Tyr¹⁴A [¹²⁵I] insulin (2.1 × 10¹¹ l/mol), Tyr¹⁹A [¹²⁵I] insulin (2.3 × 10¹¹ l/mol), and Tyr¹⁶B [¹²⁵I] insulin (5.7 × 10¹¹ l/mol). Insulin labeled at position Tyr¹⁶B was associated with considerably higher nonspecific binding than insulin labeled at Tyr¹⁴A or Tyr¹⁹A. (B) Scatchard analysis performed for the competition curve obtained against Tyr¹⁴A radiolabeled insulin. (C) Binding curves of a serum mix containing a serum with high-affinity IAAs (1.7 × 10¹¹ l/mol) and a serum with low-affinity IAAs (2 × 10⁵ l/mol) (squares) and a serum mix containing a serum with high-affinity IAAs (1.7 × 10¹¹ l/mol) and a serum with moderate-affinity IAAs (6.3 × 10⁷ l/mol) (circles). Both curves fit a 2-site binding model. (D) Binding curves obtained for the first IAA-positive serum from 56 children in the BABYDIAB study. IAA binding in 1 serum conforms to a 2-site binding model (dotted line), whereas the remaining sera conform to a 1-site binding model. Curves that are shifted to the right indicate lower-affinity IAAs.