12 Dec 2019 00:00
In-vitro Detection of Phytopathogenic Fungal Cell Wall by Polyclonal Sera Raised Against Trimethyl Chitosan Nanoparticles
The technique of generating this antibody repertoire against chitosan utilizing its methylated derivative nanoparticles is a novel attempt in the direction of development of a detection tool for fungal contamination. The downstream translation of this technology could be applied for many conditions. This polyclonal antibody has the ability to be advanced into a quality control assay, an infection detection assay, or a tool to analyze the structure of fungal cell walls. The technique could also be utilized for the detection of human fungal pathogens involved in the different fungal infections. For example, there are a number of fungi that could cause the onychomycosis, which is a nail fungal infection in human. A similar approach could be explored for other topical or dermal infections as well. The antibodies could also be conjugated with fungal inhibitors to further promote their application. In the future, the ability to target specific fungi could lead to applications in crop protection, and the containment of fungal disease outbreaks.
The objective of this research was to generate a tool for the first-line detection of fungal infection in plants. Chitin is one of the unique fungal cell wall polysaccharide which is naturally deacetylated to chitosan upon infection. It is said to be involved in the fungal cell wall modulation and plant-pathogen communication. Therefore, detection of chitosan could be potentially helpful in the detection of fungal contamination. Five different phytopathogenic fungi strains were used for the study. Polyclonal sera were raised in the mice against Trimethylchitosan nanoparticles to generate an enhanced humoral immune response and generate a rich and heterogeneous repertoire of antibodies. The binding affinity of the sera with fungal cell wall was analyzed by ELISA, Langmuir isotherm, confocal microscopy and ITC (Isothermal Calorimetry). The raised polyclonal sera could detect chitosan in the fungal cell wall, as analyzed with the different techniques. However, the detection specificity varied among the strains in proportion to the chitin content of their cell wall. Fusarium oxysporum was detected with the highest affinity while Trichoderma reesei was detected with the least affinity by ELISA. Adsorption isotherm, as well as ITC, revealed the specific and high binding capacity. Confocal microscopy also confirmed the detection of all strains used in the study. This novel technique employing TMC nanoparticulate system could be potentially used as a source to raise sera against chitosan in an inexpensive and less laborious manner. Rapid detection of fungal contamination by the polyclonal antibodies could help in devising a quick solution. The polyclonal sera are expected to detect a span of epitopes and provide precise detection. The detection system could be advanced for future applications such as food quality control, crop protection, and human fungal infection detection and treatment.