Development and validation of a synthetic peptide-based field deployable latex slide agglutination test for detection of Tilapia tilapinevirus

Tilapia significantly contributes to global food security and is an affordable protein source for most developing nations. Tilapia tilapinevirus (TiLV) poses a significant economic threat to the global tilapia industry. This study aimed to develop a rapid and accurate detection method for TiLV by synthesizing a monoclonal antibody (MAb) against it. A novel peptide, KLH-CQ, derived from the TiLV sequence, was designed considering physicochemical properties like net cationic charge, amphipathicity, helicity, and hydrophobicity. The KLH-CQ (50 µg) was used to immunize Balb/c mice with Freund's complete adjuvant. The presence of specific antibodies in the mice serum was confirmed by ELISA, which showed a high antibody titre of 2.67:0.12 (mean OD of treated Vs control sera). The mouse with the strongest immune response was used for spleen donor in hybridoma production. Epitope mapping via ELISA screening identified five positive clones (TiLV-MAb 1-5), with the most reactive clone selected for further analysis. Using Classen's method, a cutoff OD value of 1.24 ± 0.45 was determined for virus detection. The selected TiLV-MAb was then used as a probing antibody to develop a latex slide agglutination assay (TiLV-LAT) using passive adsorption method. Validation of the assay with tissue and mucus samples revealed a specificity of 88.37% and a sensitivity of 82.37% for TiLV detection. The overall accuracy of the assay was 83.51%, with positive and negative likelihood ratios of 7.06 and 0.2, respectively. The TiLV-LAT successfully detected TiLV in various tissues, showing variable sensitivity: liver (77.35%), mucus (73.53%), brain (67.92%), and kidney (62.26%). TiLV-LAT developed here has minimized the tedious steps involved in nucleic acid-based detection assays, with the recorded sensitivity and specificity; it can be used as a presumptive diagnosis for testing and point of care/farm site. Moreover, non-lethal sampling and virus testing in mucus samples would be useful for fish health monitoring.