International Journal of Nanomedicine (Dec 2019)
In-vitro Detection of Phytopathogenic Fungal Cell Wall by Polyclonal Sera Raised Against Trimethyl Chitosan Nanoparticles
Abstract
Hemant Joshi,1,* Anshu Malik,1,* Soumya Aggarwal,2 Manoj Munde,2 Subhrangsu Sundar Maitra,1 Nidhi Adlakha,3 Rakesh Bhatnagar1,4 1Molecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India; 2School of Physical Science, Jawaharlal Nehru University, New Delhi 110067, India; 3Regional Centre for Biotechnology, NCR Biotech Cluster, Faridabad 121001, India; 4Banaras Hindu University, Banaras, Uttar Pradesh 221005, India*These authors contributed equally to this workCorrespondence: Rakesh BhatnagarMolecular Biology and Genetic Engineering Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, IndiaEmail [email protected] AdlakhaRegional Centre for Biotechnology, NCR Biotech Cluster, Faridabad 121001, IndiaEmail [email protected]: 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.Methods: 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).Results: 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.Conclusion: 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.Keywords: polyclonal sera, chitosan, chitin, fungus, cell-wall, detection
