Analytical Sciences

Comparison of Candidate Pairs of Hydrolytic Enzymes for Spectrophotometric-dual-enzyme-simultaneous-assay

Hongbo LIU,* Mei YUAN,* Xiaolan YANG,* Xiaolei HU,* Juan LIAO,* Jizheng DANG,* Yanling XIE,*,** Jun PU,* Yuanli LI,* Chang-Guo ZHAN,*** and Fei LIAO*

*Unit for Analytical Probes and Protein Biotechnology, Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Yuzhong Dist., Chongqing 400016, P. R. China
**Chongqing Nurse School, No. 249, Yuqing Road, Banan Dist., Chongqing 401320, P. R. China
***Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA

Spectrophotometric-dual-enzyme-simultaneous-assay (SDESA) for enzyme-linked-immunosorbent-assay (ELISA) of two components in one well is a patented platform when a special pair of labels is accessible. With microplate readers, alkaline phosphatase on 4-nitro-1-naphthylphosphate (4NNPP) served as label A; Pseudomonas aeruginosa arylsulfatase (PAAS) and acetylcholinesterase (AChE) on their substrates derived from 4-nitrophenol/analogue served as candidate label B, and were compared for SDESA with an engineered alkaline phosphatase of Eschrichia coli (ECAP). For SDESA, the interference from overlapped absorbance was corrected based on linear additivity of absorbance to derive initial rates reflected by absorbance change at 450 nm for ECAP and at 405 nm for PAAS or AChE, after the correction of spontaneous hydrolysis. For SDESA with ECAP, AChE already had sufficient activity in an optimized buffer; PAAS was more favorable for substrate stability and product absorbance except for lower activity. Therefore, PAAS engineered for sufficient activity plus alkaline phosphatase is absorbing for ELISA via SDESA.

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