Generation and characterization of monoclonal antibodies against human interferon-lambda1
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Background: Members belonging to the interferon-lambda (IFN-λ) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-λ biology, such as endocytosis of IFN- λ, we produced monoclonal antibodies (Abs) against human IFN-λ and examined their usefulness. Methods: We purified recombinant human IFN-λ1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-λ1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-λ1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-λ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-λ1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-λ1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-λ1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-λ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-λ1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-λ.
Abstract
Background: Members belonging to the interferon-lambda (IFN-λ) family exert protective action against viral infection; however, the mechanisms of their action have remained elusive. To study IFN-λ biology, such as endocytosis of IFN- λ, we produced monoclonal antibodies (Abs) against human IFN-λ and examined their usefulness. Methods: We purified recombinant human IFN-λ1 expressed in Escherichia coli by using affinity columns. Then, we generated hybridoma cells by fusing myeloma cells with splenocytes from IFN-λ1-immunized mice. For evaluating the neutralizing activity of the monoclonal Abs against IFN-λ1, we performed RT-PCR for the MxA transcript. In order to study the binding activity of IFN-λ and the monoclonal Ab complex on HepG2 cells, we labeled the monoclonal Ab with rhodamine and determined the fluorescence intensity. Results: Four hybridoma clones secreting Abs specific to IFN-λ1 were generated and designated as HL1, HL2, HL3, and HL4. All the Abs reacted with IFN-λ1 in the denatured form as well as in the native form. Abs produced by HL1, HL3, and HL4 did not neutralize the induction of the MxA gene by IFN-λ1. We also demonstrated the binding of the HL1 monoclonal anbitody and IFN-λ complex on HepG2 cells. Conclusion: Monoclonal Abs against IFN-λ1 were produced. These Abs can be used to study the cellular binding and internalization of IFN-λ.
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