Development of a protein biochip to identify 6 monoclonal antibodies against subtypes of recombinant human interferons.
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-alpha1b, IFN-alpha2a, IFN-alpha2b, IFN-beta, and IFN-gamma) 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-alpha1b. AK2 reacted in immunoassays equally with IFN-alpha2a and IFN-alpha2b. AK3 detected IFN-alpha1b, IFN-alpha2a, and IFN-alpha2b. AK4 had positive immunological responses directed to both IFN-alpha1b and IFN-alpha2b. Monoclonal antibodies BK1 and CK1 recognized epitope of IFN-beta and IFN-gamma, 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-alpha2b were simultaneously tested on a single biochip. The result demonstrated that 6.8% (6 of 88 cases) presented positive reactions to anti-IFN-alpha2b antibodies, indicating that the patients under rhIFN-alpha2b 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.
Xu
Zhenshan
Z
Anhui Academic Institute of Biology, Anhui, People's Republic of China.
Du
Weidong
W
Zhang
Ping
P
Wang
Xuan
X
Ma
Xueling
X
Shi
Liqin
L
Song
Lihua
L
D000911:Antibodies, Monoclonal / Q000032:analysis*; D000941:Antigens / Q000737:chemistry; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007120:Immunochemistry; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000097:blood / Q000627:therapeutic use; D007372:Interferons / Q000032:analysis / Q000145:classification / Q000276:immunology*; D040081:Protein Array Analysis / Q000295:instrumentation*; D011994:Recombinant Proteins; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D000941:Antigens; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D007372:Interferons