Recombinant human interferons (rhIFNs) are broadly used as effective therapeutic agents with antiviral, antitumor, and immune-modulating properties. Advances in protein biochip technology have benefited the medical community greatly, making true parallelism, miniaturization, and high throughput possible. In this study, 5 rhIFN proteins (IFN-α1b, IFN-α2a, IFN-α2b, IFN-β, and IFN-γ) were immobilized onto an N-hydroxysuccinimide (NHS)-modified gold-based biochip. The protein biochip was incubated with 6 specific mouse IgG antibodies (AK1, AK2, AK3, AK4, BK1, and CK1) against the human IFNs and then with Cy3-conjugated goat anti-mouse IgG antibody. The results showed that monoclonal antibody AK1 presented a unique binding characteristic to IFN-α1b. AK2 reacted in immunoassays equally with IFN-α2a and IFN-α2b. AK3 detected IFN-α1b, IFN-α2a, and IFN-α2b. AK4 had positive immunological responses directed to both IFN-α1b and IFN-α2b. Monoclonal antibodies BK1 and CK1 recognized epitope of IFN-β and IFN-γ, specifically. The assay specificity of the biochip was further confirmed by enzyme-linked immunosorbent assay (ELISA) and western blotting. Finally, 88 serum samples from patients treated with rhIFN-α2b were simultaneously tested on a single biochip. The result demonstrated that 6.8% (6 of 88 cases) presented positive reactions to anti-IFN-α2b antibodies, indicating that the patients under rhIFN-α2b therapy produced neutralized antibody against the IFN. The biochip format would offer a competitive alternative tool not only for facilitating characterization of IFN subtypes but also potentially for enabling clinical serum detection of corresponding antibodies directed against IFNs.
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