B-318 Development of Non-competitive Immunoassay System for Cyclosporine a Using Alpaca Naïve Library-derived VHH

Abstract Background Cyclosporine A is a potent immunosuppressive drug. It has a narrow therapeutic index, and therefore the measurement of cyclosporine’s blood concentration is essential to obtain optimal therapy. Cyclosporine A, a small cyclic polypeptide, is generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. In this study, we report a novel sandwich immunoassay for cyclosporine A using an alpaca naïve library-derived variable domain of heavy chain of heavy chain antibody (VHH) developed against an immunocomplex of cyclosporine A and anti-cyclosporine A mouse monoclonal antibody. Methods We generated a VHH against an anti-cyclosporine A mouse monoclonal antibody complexed with cyclosporine A by alpaca naïve VHH phage display library. A bivalent tandem VHH was generated using the established VHH, and a new sandwich immunoassay for measuring cyclosporine A was constructed using the tandem VHH conjugated with alkaline phosphatase. A prototype fully automated sandwich immunoassay was developed on a fully automated chemiluminescence analyzer using magnetic particles immobilized with the mouse monoclonal antibody and the tandem VHH conjugated with alkaline phosphatase. The prototype assay was further developed by implementing an automated whole blood pretreatment process with strong denaturing agents. The performances of the prototype immunoassays were investigated. Results The established VHH showed a highly specific reaction to the complex of cyclosporine A and the anti-cyclosporine A mouse monoclonal antibody, and a sandwich immunoassay for cyclosporine A was successfully developed using the VHH and the mouse monoclonal antibody. The tandem bivalent VHH exhibited higher reactivity to the complex than the monomeric VHH. The fully automated reagent prototype achieved a measurement range of 30 to 1500 ng/mL. Measurement of cyclosporine A in whole blood samples by the prototype reagent showed a correlation coefficient of 0.96 with mass spectrometry, and a correlation coefficient of 0.95 with a commercial IVD reagent. Conclusion We successfully developed a sandwich reagent prototype for cyclosporine A using VHH established by alpaca naïve VHH phage display library. This is the first report of a fully automated reagent for cyclosporine A measurement including an automated whole blood pretreatment process.