Synthetic peptide-based latex agglutination test for the detection of Bacillus anthracis spores from soil samples

Abstract

The environmental persistence of Bacillus anthracis spores continues to challenge rapid detection and surveillance of anthrax, necessitating simple, field-deployable analytical tools. This study aimed to develop a peptide-directed IgY-based latex agglutination test (LAT) as a rapid, cost-effective analytical method for the selective detection of B. anthracis spores. Four species-specific peptides derived from protective antigen (PA) and S-layer (EA1) proteins were identified through bioinformatic screening and used to generate polyclonal IgY antibodies. Latex beads (1.25%) were functionalized with optimized antibody concentrations to develop the LAT. Analytical performance was evaluated in terms of sensitivity, specificity, and cross-reactivity using inactivated spores of B. anthracis and related Bacillus spp., with validation by indirect ELISA. Applicability in complex matrices was assessed through spiking studies in soil and meat meal samples, followed by evaluation using 257 field samples from anthrax-endemic regions, benchmarked against WOAH-recommended pag gene PCR. The optimized LAT (200 µg ml⁻¹ IgY) achieved a detection limit of 10⁵ spores per ml, with high specificity for peptides PA-1 and EA-1 and minimal cross-reactivity. ELISA corroborated the specificity of peptide-derived IgY antibodies. In spiked matrices, recovery efficiency ranged from 50–75%, with a practical detection limit of 10⁶ spores per g. Field validation demonstrated 72.7% sensitivity and 100% specificity (κ = 0.83) relative to PCR. This study presents a novel peptide-based immunoanalytical platform for on-site biosurveillance and resource-limited analytical settings that integrates specificity, rapid response, and low cost, offering a practical alternative to conventional methods for environmental monitoring of B. anthracis.