Synthetic peptide-based latex agglutination test for the detection of <i>Bacillus anthracis</i> spores from soil samples.

Abstract

The environmental persistence of <i>Bacillus anthracis</i> 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 <i>B. anthracis</i> 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 <i>B. anthracis</i> and related <i>Bacillus</i> 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 <i>pag</i> 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 (<i>κ</i> = 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 <i>B. anthracis</i>.