The advantages of enzyme-linked immunosorbent assay(ELISA) are its sensitivity and simplicity in detecting IgG, IgM and IgA antibody. To apply ELISA to diagnosis of typhoid fever, antigen such as lip polysaccharide of Salmonella typhi or killed whole cell must be coated on solid phase. It is easy to coat lip polysaccharide on ELISA plate but troublesome to purify it. As it is easy to obtain the killed whole cells, the development of the appropriate method by which those.: antigens of S. typhi are´ optimally coated on solid phase is needed.
To establish the appropriate method, carbonate buffer, methanol or poly-L-lysine was applied as binding substance on polystyrene or polyvinylchloride plate as solid phase when the killed whole cell antigens - of . S. typhi varided as follows : 106, 107, 108 and 109 cell/ ml.
The criteria of the optimal method were determined as follows: 1. The optical density of positive sera is above 1.0(0.6 in IgM) at 1:10 serum dilution and is 0.3(0.2 in IgM) higher than that of negative sera: 2. The O.D. of sera is flat or lowering according to serum dilution: 3. It must be that the O.D. of negative sera is lower than 0.2 at the point of serum dilution where the O.D. of positive sera is higher than 1.0 (0.5 in IgM). The results obtained were summarized as follows
1. The methods which fitted the above criteria were to use poly-L-lysine as binding substance, polyvinylchloride plate as solid phase and 107 cell/ml as antigen concentration of S typhi (poly-L- lysine/ polyvinylchloride/107 and poly-L-lysine/polyvinylchloride/108 in detecting IgG antibody, methanol/polystyrene/109, poly-L-lysine /polyvinylchloride/108 and poly-L-lysine /poolyvinylchloride /108 in IgM and carbonate buffer/polystyrene/108 carbonate buffer/polystyrene/109 methanol/polystyrene/108 methanol/ polyvinylchloride/108 methanol/polyvinylchloride/109 poly-L-lysine/polyvinylchloride/108 and poly-L-lysine/polyvinylchloride/109 in IgA.
2. The coating method using poly-L-lysine, polyvinylchloride plate and 108 cell/ml was best to assay IgG, IgM and IgA antibody all in one. By this method, to assay the each immunoglobulin class with an appropriate fixed serum dilution, 1 : 320 dilution was best.
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