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.

人血浆中（1→3）-β-D-葡聚糖的存在是真菌感染的标志。目前，Li基于血细胞裂解物（LAL）的测定法广泛用于定量血浆（1→3）-β-D-葡聚糖。但是，它在临床使用中有局限性，例如自然资源的不稳定供应，复杂的制造过程以及试剂的低通量。已经开发出利用针对（1→3）-β-D-葡聚糖的特异性抗体的替代检测方法来克服这些挑战。但是，这些方法灵敏度低，并且与基于LAL的测定所获得的数据关联性较差。这项研究的目的是开发一种新型的酶免疫测定法，与基于LAL的测定法相比，该方法在测定血浆（1→3）-β-D-葡聚糖水平方面既灵敏又准确。我们生成了针对（1→3）-β-D-葡聚糖的特异性单克隆抗体，该抗体可识别具有（1→3）-β-D-连锁的四单元葡萄糖寡聚体，并使用以下方法构建了夹心酶联免疫吸附测定（ELISA）这些抗体。新开发的ELISA显示吸光度随添加的（1→3）-β-D-葡聚糖的体积成比例增加。该检测的检测限为4 pg / ml血浆（1→3）-β-D-葡聚糖，相当于基于LAL的检测，工作范围为4–500 pg / ml。使用三种不同浓度的血浆样品，测定内变异系数为2.2–5.4％。我们观察到强相关性（新开发的ELISA显示吸光度随添加的（1→3）-β-D-葡聚糖的体积成比例增加。该检测的检测限为4 pg / ml血浆（1→3）-β-D-葡聚糖，相当于基于LAL的检测，工作范围为4–500 pg / ml。使用三种不同浓度的血浆样品，测定内变异系数为2.2–5.4％。我们观察到强相关性（新开发的ELISA显示吸光度随添加的（1→3）-β-D-葡聚糖的体积成比例增加。该检测的检测限为4 pg / ml血浆（1→3）-β-D-葡聚糖，相当于基于LAL的检测，工作范围为4–500 pg / ml。使用三种不同浓度的血浆样品，测定内变异系数为2.2–5.4％。我们观察到强相关性（ 我们的ELISA和Fungitec G测试ES Nissui（一种常用的基于LAL的分析方法）使用26种血浆样品得到的测量值之间的差R = 0.941，斜率= 0.986）。这可以归因于抗体的表位。两种抗体均可以抑制基于LAL的测定，表明该抗体识别基于β-D-葡聚糖的相同区域，从而在基于LAL的测定中失活因子G（凝血级联反应的起始酶原）。因此，这项研究中开发的ELISA可以在临床环境中检测真菌感染，其效率与基于LAL的检测方法相似。该测定法的特点是性能好，材料稳定，制造工艺简单，更适合于真菌感染的高通量诊断。