Hepatology research : the official journal of the Japan Society of Hepatology · 2012
Development of specific and quantitative real-time detection PCR and immunoassays for λ3-interferon.
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Sugiyama Masaya, Kimura Tatsuji, Naito Shigeko, Mukaide Motokazu, Shinauchi Takanori, Ueno Masakatsu, Ito Kiyoaki, Murata Kazumoto, Mizokami Masashi
Аннотация
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 (10-10(7) copies/assay) with high specificity (∼10(7) -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.
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 (10-10(7) copies/assay) with high specificity (∼10(7) -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.