Sensitive array-based assay for determination of serological protein kinase A autoantibody levels based on its antigen protein activation

초록

Objectives: We investigated the effect of cPKA alpha conformational states during protein immobilization on an array platform for cPKA autoantibody assays for sensitive and high-throughput profiling of protein kinase A (PICA) autoantibody levels in human sera. Design and methods: We prepared activated human cPKA alpha protein arrays by addition of cofactors including ATP, MgCl2, and Triton X-100 to incubation buffer. Anti-human cPKA alpha antibody or PICA autoantibody levels in human sera were analyzed using activated human cPKA alpha protein arrays. Results: Activation of cPKA alpha with ATP, Mg2+, and Triton X-100 enhanced the sensitivity of the assay by increasing the signal/noise ratio and lowering the limit of detection. cPKA alpha activation also enhanced the sensitivity of cPKA autoantibody detection in human sera. We successfully applied this assay to determine cPKA autoantibody levels in human sera from normal individuals (n = 30) and hepatic cancer patients (n = 30). Conclusions: Our results demonstrate that cPKA alpha activation enhanced the sensitivity of array-based WA autoantibody assays, and that this assay is suitable for high-throughput analyses of cPKA autoantibodies in human sera. (C) 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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