Designing of immunodiagnostic assay using polyclonal antibodies for detection of Enteropathogenic Escherichia coli strains

Enteropathogenic Escherichia coli (EPEC) is a significant bacterial pathogen that causes infantile diarrhea, particularly in low- and middle-income countries. The lack of a reliable diagnostic method greatly contributes to the increased occurrence and severity of the disease. This study aimed at developing of a cost-effective, rapid, and efficient immunodiagnostic assay for detecting EPEC infection. Lipopolysaccharide (LPS) was extracted from overnight EPEC cultures and combined with alum adjuvant, and then injected into mice for three rounds of immunizations. Subsequently, sera were collected after each immunization and utilized in agglutination assays conducted on glass slides. Both the LPS and colonies of the EPEC isolate used for LPS preparation were employed in these agglutination assays. To evaluate the assay's performance, a total of 34 bacteria, which comprise pathogenic, non-diarrheic E. coli and non-E. coli pathogenic bacteria were used. The developed assay detected EPEC, which yielded positive reactions within 6 minutes on average for both purified LPS and bacterial isolates. The assay exhibited 100% sensitivity and a 95.83% specificity for the detection of EPEC local isolates. Moreover, the assay also detected a low number of bacteria forming units (104X 104 CFU/ml) in spiked fecal samples. This study conclusively confirms that the developed immunodiagnostic assay possesses multiple favorable characteristics, including user-friendliness, high sensitivity, high specificity, cost-effectiveness, and time-efficiency. Hence, this assay can be used as ideal diagnostic assay, which is highly suitable for the detection and screening of EPEC infection in both humans and cattle in one health perspective of resource-limited laboratories.