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

Abstract

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.