ATR-FTIR Spectroscopy for Detection of Anti-interferon-Gamma Autoantibodies

<b>Introduction:</b> Adult-onset immunodeficiency (AOID) unrelated to HIV is commonly linked to anti-interferon-gamma autoantibodies (AIGAs), predisposing patients to life-threatening opportunistic infections. Current AIGA detection methods are time-consuming and resource intensive. This study aimed to develop and validate an attenuated total reflectance-Fourier transform infrared (ATR-FTIR) technique for detecting and differentiating AIGA-positive patients. <b>Methods:</b> We recruited individuals into three groups: AIGA-positive patients (<i>n</i> = 45), AIGA-negative patients (<i>n</i> = 15), and healthy controls (<i>n</i> = 15). Heparinized plasma or serum samples underwent ATR-FTIR spectroscopy. Spectral profiles were examined using principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA). The reference standard for AIGA concentration was determined by the inhibitory enzyme-linked immunosorbent assay (ELISA). <b>Results:</b> ATR-FTIR spectra revealed significant differences, especially in the amide and fingerprint regions, particularly at key wavenumbers 1632 cm^-1 and 1535 cm^-1. The Amide I/Amide I + II + III ratio is significant between AIGA-positive and others and a secondary structure protein conformational change α-helix to β-sheet. PCA and PLS-DA enabled effective separation of patient groups; PLS-DA and k-NN achieved 95% accuracy, 93.3% sensitivity, and 100% specificity for differentiating AIGA-positive from negative/healthy individuals (NPV 83.3%; PPV 100%). The amide region (1700-1500 cm^‑1) delivered the best prediction accuracy. <b>Conclusion:</b> ATR-FTIR spectroscopy demonstrates significant potential as a rapid and effective diagnostic tool for differentiating AIGA-positive patients from healthy individuals, providing a valuable addition to clinical diagnostic practices. Further validation in larger, diverse populations is warranted.