DOT ENZYME IMMUNOSORBENT ASSAY FOR THE SERODIAGNOSIS OF TYPHOID FEVER Asma Ismail, Zainoodin SA Kader and Ong Kok- Hai Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Abstract. A nitrocellulose membrane strip Clotted with a specific 50 kDa outer membrane protein of Salmonella typhi was applied for the serodiagnosis of typhoid fever. Using horseradish peroxidase conju- gated IgM and IgG antibodies with 4-chloronaphthol as substrate, antibodies in typhoid patients were clearly visualised as bluish purple dots while sera fiom patients with non-typhoid fevers gave negative results. The detection of specific IgM and IgG antibodies in typhoid patients suggest either recent or current infection. Combined with the high specificity, reliability and rapidity of the test, the dot EIA technique provides a simple and useful method for the serodiagnosis of typhoid using a single serum specimen. INTRODUCTION Enzyme immunoassays (EIA) using micro titer plates have been used in the diagnosis of typhoid fever (Barrett et ai, 1982; Beasley and Weiss, 1981). Although the microtiter plate assays have the added advantage of processing many samples at a time with high sensitivity, they do have some limitations. Microtiter plate assays require expen- sive and special equipment which restricts its use to the larger hospitals and laboratories. Thus there is a need for a simple and rapid method to diagnose typhoid fever. Dot EIA, which detects antibodies in patient sera to antigen dotted on nitrocellulose membrane has been applied in the diagnosis of microbial diseases (ltoh and Sato 1990; Oprandy et ai, 1988). With this simple yet sensitive method, laboratory results can be interpreted without the use of spe- cial equipment. The outer membrane proteins (OMPs) on the surfece of gram-negative bacteria have been con- sidered as important antigens to induce host immune responses (Ortiz et ai, 1989). Since cross- reactions among gram-negative OMPs can occur, purifications of antigenic components specific to Salmonella typhi must be made. We have pre- viously determined that the 50 kDa OMP of S. typhi was specific for this bacterial species since it only reacted immunologically with typhoid sera (Ismail et ai, 1991). In this study, we attempted to test the specificity of the dot EIA as a diagnostic tool, to detect spe- cific IgM and IgG antibodies in typhoid patients, using the 50 kDa OMP as the test antigen. MATERIALS AND METHODS Bacteria and antigen preparations S. typhi USMI was isolated from a patient with typhoid fever and has been maintained in our laboratory since 1987. Partially purified OMPs of S. typhi were obtained following the methodology as described by Schnaitman (1971). The concen- trations of OMPs were determined using a color- imetric microassay procedure as recommended by Bio-Rad (Richmond, CA, USA) with bovine serum albumin as a standard. The 50 kDa OMP of S. typhi was isolated via preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and e1ectroelu- tion. SDS-PAGE was performed under reducing conditions using the discontinuous buffer systems (Laemmli, 1970) with a vertical slab electrophoresis unit (Bio-Rad). The stacking and separating gels contained 4.5% and 9% acrylamide respectively. Vol 22 No 4 December 1991 563