Surface plasmon resonance-based immunoassay for human C-reactive protein.

A rapid and highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) has been developed and validated for detecting human C-reactive protein (CRP), a specific biomarker for inflammatory and metabolic disorders, and infections. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A/G (Pr A/G) was diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), dispensed on a KOH-treated gold (Au)-coated SPR chip, and incubated for 30 min. The Pr A/G functionalized Au SPR chip was then bound to anti-human CRP capture antibody (Ab), blocked with bovine serum albumin, and subsequently used for the detection of CRP. The highly-simplified oriented Ab immobilization strategy enabled the leach-proof binding of capture Ab in 5-fold shorter time than conventional procedures. The developed IA detected 1.2-80 ng mL(-1) of CRP with a limit of detection (LOD) and a limit of quantification (LOQ) of 1.2 ng mL(-1) and 4.6 ng mL(-1), respectively. It detected CRP spiked in diluted human whole blood, serum and plasma as well as the CRP levels in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients with the same precision as the clinically-accredited analyzer-based IA and conventional CRP sandwich ELISA. The Ab-bound SPR chips stored at 4 °C retained their functional activity for 10 weeks, resulting in significant reduction in the overall analysis time.

Surface plasmon resonance-based immunoassay for human fetuin A.

This article describes a highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The assay is based on a novel immobilization procedure that simply involves the dilution of an anti-HFA capture antibody (Ab) in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), followed by its dispensing on a KOH-treated gold (Au)-coated SPR chip and incubation for 30 min. The developed SPR IA detected 0.3-20 ng mL(-1) of HFA with a limit of detection and sensitivity of 0.7 ng mL(-1) and 1 ng mL(-1), respectively. The highly-simplified Ab immobilization procedure is also 5-fold more rapid than conventional procedures. It leads to the leach-proof binding of the capture Ab, which means that the developed SPR IA is highly cost-effective, as the Ab-bound SPR chip could be reused for many repeated HFA IAs after regeneration with 10 mM glycine-HCl, pH 2.0. The Ab-bound SPR chip, stored at 4 °C, lost only 18% of its original activity after 4 months. For the detection of HFA spiked in diluted human whole blood and plasma, the results obtained by the developed SPR IA agreed well with the commercial HFA sandwich ELISA.

Bioluminescence assay for the highly sensitive detection of botulinum neurotoxin A activity.

This article describes a novel bioluminescence assay for detecting the proteolytic activity of Botulinum NeuroToxins (BoNT) in complex matrices. The assay is capable of detecting traces of BoNT in blood samples as well as in food drinks. The assay was responsive to BoNT/A subtypes 1 to 5, and serotype E3 in buffered solutions. It was responsive to filtered Clostridium botulinum supernatants and BoNT/A1 in complex with neurotoxin associated proteins in bouillon and milk (3.8% fat) down to 400 fM after 4 h RT incubation and in bouillon at concentrations down to 120 fM after 21 h RT incubation. In combination with an immunocapture/enrichment step it could detect BoNT/A1 in citrated plasma at concentrations down to 30 fM (1.2 mouse LD50 per mL). The simplicity of the assay, combined with a demonstrated ability to lyophilize the reagents, demonstrates its usefulness for detection of BoNT in non-specialised analytical laboratories.