Studies on the 52 kDa antigen of Salmonella typhi: physicochemical stability, purification by affinity chromatography and immunochemical specificity.
The 52 kDa specific protein antigen of Salmonella typhi, as identified by monoclonal antibodies (Ekpo et al. 1990) has been studied with respect to its physicochemical stability, purification by affinity chromatography and immunochemical specificity. It was found that the 52 kDa protein was degraded into smaller antigenic fragments of MW 30-51 kDa when treated with acetone, ethanol, sodium thiocyanate, 0.3M sodium chloride and Veronal and Tris buffers. The exact chemical nature of the degradation of the protein under these conditions is not known but digestion by conventional proteases and dissociation of the non-covalent subunit type have been ruled out. It is proposed that the degradation may be the result of yet unidentified enzyme(s) which become activated by various physical or chemical treatments. Affinity chromatography using a specific monoclonal antibody has been carried out in an attempt to purify the 52 kDa protein. The binding of S. typhi protein to the column was saturable at 65.6 microgram protein/ml gel. The amount of S. typhi protein adsorbed on the column was 0.51% of the total sonicated cell protein. SDS-PAGE of the immunoadsorbent purified protein revealed bands at Mr 15-58 kDa, indicating that the protein obtained had been severely degraded. However, Western blot of the purified protein stained with a specific monoclonal antibody and with rabbit polyclonal antibody against S. typhi showed striking similarity, indicating that the protein obtained was close to immunochemical purity. The 52 kDa protein purified by affinity adsorbent was used as an antigen for the detection of specific IgM in sera of patients. It was shown that sera of patients infected with S. typhi as well as those infected with other bacteria, contained specific IgM against the 52 kDa protein. Thus, it appears that the 52 kDa protein contains species specific as well as cross-reacting epitopes. The possible development of specific diagnosis of S. typhi based on the present experimental results in discussed.
Anuntagool
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N
Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Sarasombath
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S
Ratanabanangkoon
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K
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003429:Cross Reactions / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques; D012485:Salmonella typhi / Q000276:immunology*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014435:Typhoid Fever / Q000097:blood / Q000453:epidemiology
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes