Lateral Flow Immunochromatographic Assay for Detection of Antibodies Against a Recombinant Human Adenovirus Type 3 Hexon Fragment Using Gold Nanoparticles and Quantum Dots.

Background/Objectives: Human adenoviruses (HAdVs) are widely used as vectors for vaccines and gene therapy; however, pre-existing immunity can reduce their efficacy. Therefore, rapid and accessible serological methods are required to assess antibody levels against adenoviruses. Lateral flow immunochromatographic assay (LFIA) sensitivity depends on the label and antigen. In this study, we aimed to develop and evaluate LFIA systems based on gold nanoparticles (GNPs) and quantum dots (QDs) using a recombinant HAdV hexon protein. Methods: A recombinant HAdV hexon protein fragment (rhHAdV, 35 kDa; amino acids A120-R316) was expressed in Escherichia coli and purified using Ni2+ affinity chromatography. Protein identity was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and liquid chromatography-tandem mass spectrometry analyses. Two LFIA formats were developed using Protein G-conjugated GNPs (GNP-G) and QDs (QD-G). Agreement with the reference ELISA was evaluated using 90 human serum samples. Results: The rhHAdV antigen demonstrated reactivity with anti-adenovirus-positive human sera in ELISA. Antibody detection was achieved at serum dilutions of up to 1:300 and 1:1000 for the GNP- and QD-based LFIA, respectively. The GNP-G and QD-G LFIA formats demonstrated positive percent agreements of 96.9% and 100%, respectively, while both assays showed a negative percent agreement of 98.2% compared with the reference ELISA. Conclusions: The findings demonstrate the feasibility of a QD-based LFIA for qualitative detection of antibodies reactive with the recombinant HAdV-B3 hexon fragment.