BIOMEDICAL AND ENVIRONMENTAL SCIENCES 21, 137-143 (2008) www.besjournal.com Enrichment-ELISA for Detection of Salmonella typhi From Food and Water Samples S. KUMAR * , K. BALAKRISHNA, AND HV. BATRA Division of Microbiology, Defence R & D Establishment, Jhansi Road, Gwalior 474002, India Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay (sELISA) for rapid detection of Salmonella enterica serovar typhi (S. typhi) from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin (H=d) antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water (BPW) and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW (Enrichment-ELISA) was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food (30) and water (35) samples collected from field were also tested by Enrichment-ELISA and culture method. Results Out of four specific clones to H=d antigen, one clone (# 2/56, IgG2a isotype) was used in sELISA. The sELISA had the detection limit of 10 4 -10 5 cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10 2 S. typhi cfu/mL within 10 h from various food rinses (meat, vegetable) and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples (water) gave false positive result by Enrichment-ELISA. Conclusion In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation. Key words: Enrichment-ELISA; Food; Monoclonal antibody; Salmonella; Sandwich ELISA; Water INTRODUCTION Salmonella enterica serovar typhi (S. typhi) is the causative agent of typhoid fever. The disease continues to be a major health problem in many parts of the world particularly the developing countries. The incidence of typhoid fever has been estimated to be about 16 million cases annually with 600 000 associated deaths [1] . S. typhi is an obligate human pathogen and causes infection by fecal-oral route. Typhoid fever is typically acquired by ingesting food or water that has been contaminated by the feces of typhoid infected individual. However, unlike ubiquitous serovars such as S. typhimurium, S. typhi is generally excluded from the group of foodborne salmonellas because it is not a pre-harvest food safety issue. Probably, for this reason, the rapid methods for detection of S. typhi from environmental sources and food matrices are almost nonexistent. However, many typhoid fever outbreaks have been reported to be caused either by consumption of contaminated water [2] or food [3] . The laboratory procedures for detection and identification of Salmonella by conventional methods are laborious and time consuming taking 3-5 days. Various rapid methods for the detection of foodborne salmonellas have been reported e.g. selective media, bacterial agglutination, fluorescent antibody technique, enzyme linked immunosorbant assay (ELISA), immunomagnetic assays and PCR [4-6] . Several ELISAs for detection of Salmonella in food samples take one to two days to deliver results [7-8] . The detection limit of most of these ELISAs ranges between 10 4 to 10 7 cfu/mL in enrichment broth. Almost all studies on improving the detection methods for S. typhi have been directed towards * Correspondence should be addressed to S. Kumar, Tel: 91-751- 2340245/354. Fax: 91-751-2341148. E-mail: subodh_kumar@email.com Biographical note of the first author: S. Kumar, scientist, majoring in development of rapid methods for detection of typhoid bacteria from non-clinical and clinical samples as well as newer strategies for vaccine development. 0895-3988/2008 CN 11-2816/Q Copyright © 2008 by China CDC 137