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Development of an enzyme linked immunosorbent assay for detection of cyathane diterpenoids

机译：酶联免疫吸附测定法测定胞苷二萜的开发

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Background So-called cyathane type diterpenoids are produced as secondary metabolites by basidiomycetes. Based on their antibacterial, fungicidal, and cytotoxic properties, cyathane type terpenoids represent interesting target compounds in fungal biotechnology. Results An indirect competitive enzyme linked immunosorbent assay has been developed for detection of cyathane type diterpenoids. Rabbit polyclonal antibodies were raised against a mixture of striatal A and B conjugated to bovine serum albumin. The conditions for direct attachment of the hapten striatal B to a solid phase by passive adsorption were optimized. The cross reactivities of the striatals A, C and D, of the striatins A and B, and of the erinacines C and P to striatal B were determined. The validation study showed that the ELISA was precise and sensitive. The average IC50 of striatal B was 36.0 ng mL?1 with an inter-assay coefficient of variation (CV) of 13.2% (n = 5). Recoveries from striatal B spiked samples in the assay were in the range of 97.3 – 125.9%. A good correlation between the striatal B concentration measured by the ELISA and by HPLC-DAD (y = 1.1122× – 0.1585, R2?=?0.9942) was obtained from linear regression analysis. The suitability of the ELISA for detection of cyathane type diterpenoids in submerged cultures and fruiting bodies of H. erinaceus was studied. It showed cross reactivity with supernatants from submerged cultures and extracts thereof, but did not show cross reactivity with extracts from fruiting bodies. Conclusions The developed method is appropriate for qualitative and quantitative detection of cyathane diterpenoids in complex mixtures. Due to its high sensitivity and specificity, it represents an ideal screening method for discovering new cyathane diterpenoids and new potential producers of them.
机译：背景技术所谓的胞嘧啶型二萜类化合物是担子菌产生的次级代谢产物。基于它们的抗菌，杀真菌和细胞毒性特性，鞘型萜类化合物代表了真菌生物技术中令人关注的目标化合物。结果已开发出一种间接竞争性酶联免疫吸附测定法，用于检测胞嘧啶型二萜。产生针对与牛血清白蛋白偶联的纹状体A和B的混合物的兔多克隆抗体。优化了通过被动吸附将半抗原纹状体B直接附着到固相的条件。测定了纹状体A，C和D，纹状体A和B，以及芥兰碱C和P与纹状体B的交叉反应性。验证研究表明ELISA准确且灵敏。纹状体B的平均IC 50 为36.0 ng mL ？1 ，批间变异系数（CV）为13.2％（n = 5）。在测定中从纹状体B加标样品中的回收率在97.3 – 125.9％的范围内。通过线性回归分析，通过ELISA和HPLC-DAD测得的纹状体B浓度之间具有良好的相关性（y = 1.1122×– 0.1585，R 2 Δ=？0.9942）。研究了酶联免疫吸附测定（ELISA）在海蓝藻浸没培养物和子实体中检测胞嘧啶型二萜类化合物的适用性。它显示出与来自浸没培养物的上清液及其提取物的交叉反应性，但没有表现出与来自子实体的提取物的交叉反应性。结论该方法适用于定性和定量检测复杂混合物中的胱氨酸二萜类化合物。由于其高灵敏度和特异性，它是发现新的胞嘧啶二萜及其新潜在生产者的理想筛选方法。

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