Thiols enhance the sensitivity of luminescent assays for non‐cross‐linked and covalently cross‐linked aminophthalhydrazides

Abstract

Transglutaminases catalyse an acyl-transfer reaction between the gamma-carboxamide of a protein or peptide-bound glutamine (P-CH2-gammaCH2-CO-1NH2) and the primary amino group of mono- or polyamines (R-2NH2), covalently cross-linking the reactants by an isopeptide bond: P-CH2-gammaCH2-CO-1NH2 + R-2NH2 --> P-CH2-gammaCH2-CO-2NH-R + 1NH3. We reported that N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was a chemiluminescent (CL) amine substrate for transglutaminases. We now identified N-(6-aminohexyl)-N-ethylisoluminol (AHEI) as a second, less reactive, transglutaminase substrate. A structure-based explanation is offered for the lower reactivity of AHEI. Optimum CL from non-cross-linked or cross-linked ABEI or AHEI was elicited in the presence of 10 mmol/L dithiothreitol, by oxidizing with a mixture of 20 mmol/L potassium ferricyanide and 10 mmol/L hydrogen peroxide in 100 mmol/L NaOH. The limits of detection and quantitation for non-cross-linked aminophthalhydrazides obtained in this system were: 20 fmol and 60 fmol for ABEI and 10 fmol and 30 fmol for AHEI. These values represented a 500-800-fold improved sensitivity. Delayed peak CL and CL decay in the presence of dithiothreitol contributed to improving the sensitivity. The data could be useful for improving the immunoassays for aminophthalhydrazides and facilitate the development of high throughput assays for transglutaminases.

Design Considerations for High Throughput Screening and In Vitro Diagnostic Assays

“…This amount is known as the limit of quantitation or LOQ [33][34][35][36]. LOQ is sometimes defined as:…”

“…In the HTS environment, it is important to distinguish between Instrument Detection Limit (IDL) which is simply 3 of baseline "noise" from instrument using a "pure" sample vs Method Detection Limit (MDL) which is much less sensitive and uses "real world" samples for testing the analyte [33][34][35][36]. Likewise, in the IVD testing situations, it is important to distinguish Analytical Sensitivity (AS) which refers to the detection of an analyte under purified, laboratory test conditions and Clinical Sensitivity (CS), where the same analyte is detected/quantized under operationally relevant conditions such as patient specimens of throat swab, nasal swab, nasal aspirate, sputum, tears, sweat, saliva, urine, blood, feces, semen, or other similar complex milieu.…”

ZstatFlu®‐II test: a chemiluminescent neuraminidase assay for influenza viral diagnostics

“…Light emission from the reaction mixtures was also measured using an MGM Optocomp II Luminometer (MGM Instruments, Hamden, CT). Chemiluminescence measurements, light emission kinetics and signal-noise ratio calculations were conducted as described in detail previously (19,(22)(23)(24). Light emission measured in the luminometer is expressed as relative light units (RLU).…”