Fig. 1. RIA IAA procedure 1. Serum samples (75 µL) were poured into two series of 1.7 mL Eppendorf conical tubes (Costar 3207, Corning). A total of 75 µL of the 0.23М incubation buffer solution (IBS), pH 7.4 with the following composition were added to each A series tube: NaH₂PO₄ (Sigma-Aldrich, REF S-0751) 0.014М; NaH₂PO₄ (Panreac, REF 141677) 0.067М; NaCl (Sigma-Aldrich, REF S-9625) 0.15М; bovine serum albumin (CDH, REF TC1546) 0.05%; Twin-20 (Panreac, REF 162312) 0.05%. A total of 75 µL of the IBS with the recombinant human insulin (Insulin Reference Standard, Eli Lilly, USA) at a concentration of 9 × 10^–3 U/mL were added to each B series tube. 2. Test tubes of both series were vortexed with a vortex mixer and incubated for 30 min on the на ELMI-ST3 orbital shaker (Elmi, Latvia) with the platform rotation speed 250 rpm at room temperature. IAA bound to insulin during incubation. 3. A total of 100 µL of IBS with the recombinant human insulin labeled with the ¹²⁵I (¹²⁵I-insulin) at a concentration of 7.5 × 10^–6 U/mL were added to each tube of both series. Furthermore, 100 µL of IBS with the ¹²⁵I-insulin were added to each of two tubes to calculate total radioactivity. The ¹²⁵I-insulin preparation was produced in the Institute of Bioorganic Chemistry by monoiodination of insulin based on tyrosine A14 using chloramine-T as an oxidizing agent, purified by gel filtration of the column with Sephadex G-15. Iodination involved the use of sodium iodide (¹²⁵I) (ISOTOPE, RF). Finally, we obtained a stabilized ¹²⁵I-insulin preparation with the following radiochemical charateristics: total activity — 352 kBq, specific activity — 58 TBq/mmol, radiochemical purity — 92%. 4. All the test tubes were sealed and incubated for 7 days in a refrigerator at +4 °С. During incubation the serum IAA bound to insulin and ¹²⁵I-insulin, and the equilibrium between IAA binding with the labeled and non-labeled ligands established. 5. A total of 500 µL of the buffer solution (pH 8.6) containing polyethyleneglycol (BS-PEG) with the following composition added to all tubes, except those for total activity calculation : Tris 0.05М (Sigma-Aldrich REF 7-9 Tris base T13,78); PEG-8000 (Polyethylenglycol 8000 BioChemica AppliChem REF A2204.0500) 14%. BS-PEG was previously cooled to 0 °С. 6, 7. The tubes were vortexed with a vortex mixer and centriguged in the Beckman G-2-21 centrifuge at 2000 g for 30 min at +4 °С. The supernatant was removed with an aspirator. As a result, a precipitate was obtained containing the IAA complexes with the labeled and unlabeled insulin, as well as IAA that did not bind to insulin. 8, 9. A total of 1000 µL of BS-PEG with the 11% PEG-8000 previously cooled to 0 °С were added to all tubes, except those for TR calculation. The tubes were vortexed with a vortex mixer and centriguged in the Beckman G-2-21 centrifuge at 2000 g for 30 min at +4 °С. The supernatant was removed with an aspirator. As a result, a precipitate was obtained containing the IAA complexes with the labeled and unlabeled insulin. 10. Radioactivity was measured in all the tubes (including those for total activity calculation) with the Wizard γ-spectrometer (PerkinElmer, USA) at a measuring time of 1 min

Fig. 2. Receiver Operating Curve of the IAA test. Dashed curve — the line bounding the AUC of 0.5. Solid curve — the test receiver operating characteristic curve. Dotted curves — borders of the 95% confidence interval (95%CI_AUC) for the operating characteristic curve. P_AUC — probability of significance of the null hypothesis about the lack of difference between AUC 0.5 and AUC of the test