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.
Anhui Academic Institute of Biology, Anhui, People's Republic of China.
Zhenshan
Z
Xu
Weidong
W
Du
Ping
P
Zhang
Xuan
X
Wang
Xueling
X
Ma
Liqin
L
Shi
Lihua
L
Song
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