Anuntagool N, Sarasombath S, Ratanabanangkoon K
Department of Microbiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Southeast Asian J Trop Med Public Health. 1991 Sep;22(3):362-71.
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.
伤寒沙门氏菌52kDa特异性蛋白抗原已通过单克隆抗体鉴定（埃克波等人，1990年），并对其物理化学稳定性、亲和层析纯化及免疫化学特异性进行了研究。结果发现，用丙酮、乙醇、硫氰酸钠、0.3M氯化钠以及巴比妥缓冲液和Tris缓冲液处理时，52kDa蛋白会降解为分子量30 - 51kDa的较小抗原片段。在这些条件下蛋白质降解的确切化学性质尚不清楚，但已排除了传统蛋白酶的消化作用以及非共价亚基类型的解离。有人提出，这种降解可能是由各种物理或化学处理激活的尚未鉴定的酶所致。已尝试使用特异性单克隆抗体进行亲和层析来纯化52kDa蛋白。伤寒沙门氏菌蛋白与柱的结合在65.6微克蛋白/毫升凝胶时达到饱和。吸附在柱上的伤寒沙门氏菌蛋白量占总超声破碎细胞蛋白的0.51%。免疫吸附纯化蛋白的SDS - PAGE显示在Mr 15 - 58kDa处有条带，表明所获得的蛋白已严重降解。然而，用特异性单克隆抗体和抗伤寒沙门氏菌兔多克隆抗体对纯化蛋白进行的Western印迹显示出惊人的相似性，表明所获得的蛋白接近免疫化学纯度。用亲和吸附剂纯化的52kDa蛋白用作抗原检测患者血清中的特异性IgM。结果表明，感染伤寒沙门氏菌的患者血清以及感染其他细菌的患者血清中均含有针对52kDa蛋白的特异性IgM。因此，52kDa蛋白似乎既含有种属特异性表位，也含有交叉反应表位。基于目前实验结果讨论了伤寒沙门氏菌特异性诊断的可能发展。
