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Development of specific and quantitative real-time detection PCR and immunoassays for λ3-interferon

Authors


Professor Masashi Mizokami, The Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, 1-7-1 Kohnodai, Ichikawa 272-8516, Japan. Email: mmizokami@hospk.ncgm.go.jp

Abstract

Aim:  Single nucleotide polymorphisms (SNP) around interferon (IFN)-λ3 have been associated with the response to pegylated IFN-α treatment for chronic hepatitis C. Specific quantification methods for IFN-λ3 are required to facilitate clinical and basic study.

Methods:  Gene-specific primers and probes for IFN-λ1, 2 and 3 were designed for real-time detection PCR (RTD–PCR). Dynamic range and specificity were examined using specific cDNA clones. Total RNA from hematopoietic and hepatocellular carcinoma cell lines was prepared for RTD–PCR. Monoclonal antibodies were developed for the IFN-λ3-specific immunoassays. The immunoassays were assessed by measuring IFN-λ3 in serum and plasma.

Results:  The RTD–PCR had a broad detection range (10–107 copies/assay) with high specificity (∼107-fold specificity). Distinct expression profiles were observed in several cell lines. Hematopoietic cell lines expressed high levels of IFN-λ compared with hepatocellular carcinoma cells, and Sendai virus infection induced strong expression of IFN-λ. The developed chemiluminescence enzyme immunoassays (CLEIA) detected 0.1 pg/mL of IFN-λ3 and showed a wide detection range of 0.1–10 000 pg/mL with little or no cross-reactivity to IFN-λ1 or IFN-λ2. IFN-λ3 could be detected in all the serum and plasma samples by CLEIA, with median concentrations of 0.92 and 0.86 pg/mL, respectively.

Conclusion:  Our newly developed RTD–PCR and CLEIA assays will be valuable tools for investigating the distribution and functions of IFN-λ3, which is predicted to be a marker for predicting outcome of therapy for hepatitis C or other virus diseases.

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