A novel enzyme immunoassay for the measurement of plasma (1 → 3)-β-D-glucan levels

Abstract

The presence of (1 → 3)-β-D-glucan in human plasma is a marker for fungal infections. Currently, the Limulus amebocyte lysate (LAL)-based assay is widely used for the quantification of plasma (1 → 3)-β-D-glucan. However, it has limitations in clinical use, such as an unstable supply of natural resources, complicated manufacturing process, and low-throughput of the reagents. Alternative assays exploiting specific antibodies against (1 → 3)-β-D-glucan have been developed to overcome these challenges. However, these methods are associated with low sensitivity and poorly correlate with the data obtained by the LAL-based assay. The aim of this study is to develop a novel enzyme immunoassay that is as sensitive and accurate in determining plasma (1 → 3)-β-D-glucan levels as compared to that obtained with the LAL-based assay. We generated specific monoclonal antibodies against (1 → 3)-β-D-glucan that recognizes four-unit glucose oligomers with (1 → 3)-β-D-linkages, and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) using these antibodies. The newly developed ELISA showed proportional increase in absorbance with the volume of (1 → 3)-β-D-glucan added. The limit of detection of the assay was 4 pg/ml of plasma (1 → 3)-β-D-glucan that was equivalent to the LAL-based assay and the working range was 4-500 pg/ml. The intra-assay coefficient of variation was 2.2-5.4% using three different concentrations of plasma samples. We observed strong correlation (R = 0.941, slope = 0.986) between the measurements obtained by our ELISA and Fungitec G test ES Nissui, a commonly used LAL-based assay, using 26 types of plasma samples. This could be attributed to the epitopes of the antibodies. Both antibodies could inhibit the LAL-based assay, suggesting that the antibodies recognize the identical regions in β-D-glucan, thereby inactivating factor G, an initiation zymogen for coagulation cascade, in the LAL-based assay. Thus, the ELISA developed in this study can detect fungal infections in clinical settings with similar efficiency as the LAL-based assay. This assay is characterized by good performance, stable supply of materials, and simple manufacturing process and is more suitable for the high-throughput diagnosis of fungal infections.

Keywords: (1 → 3)-β-D-glucan; Monoclonal antibody; Sandwich enzyme immunoassay.