Preparation of monoclonal antibody and development of test strips enabling on-field detection of Botrytis cinerea infection on herbal medicinal plant Fritillaria thunbergii Miq

摘要

As one of the most frequently used components of traditional Chinese herbal medicine, the economic plant Fritillaria thunbergii Miq is widely grown in several provinces of China. The so-called "gray mold disease" of the plant that may lead to fatal economic loss is caused by the fungal pathogen Botrytis cinerea. Sensitive and rapid detection of fungal infections is highly desired as a preventive step to fight against the disease. In this study, we prepared a monoclonal antibody (McAb) against a pathogenic fungus using classic hybridoma technology. After analyzing the titer, sensitivity, specificity, type, and binding protein of the prepared McAbs, we developed a colloidal carbon competitive immunochromatographic test strip with the McAbs. Results showed that we successfully obtained two highly specific McAbs, IgG3 and IgG2a, against B. cinerea from hybridoma cell lines BcA4 and BcE4, respectively. The McAbs from BcA4 could bind a 25 kDa protein, while the antibodies from BcE4 could bind two proteins (53 and 340 kDa). The test strip prepared with antibodies against BcE4 reached a detection sensitivity of 6.25 mu g/mL. The strips could be conveniently stored in a dry environment ( 6 months without any noticeable decrease in performance. The design was user-friendly, and all the steps required to perform the detection were sample preparation and sample loading. The results could be read directly within 15 min. The use of test strips enabled the detection of disease at a stage 4 days earlier than the traditional method.