采用热水(80℃)提取和75%乙醇沉淀、Sepharose CL-4B 凝胶过滤及 DEAE Sepharose CL-4B 阴离子交换等方法分离纯化大杯伞(Clitocybe maxima )柄粗多糖,获得伞柄多糖纯品,制备多糖抗体；采用热水法提取大杯伞菌丝体粗多糖。用碳二亚胺法将纯化伞柄多糖与牛血清白蛋白进行共价化学偶联,并将偶联产物(拟糖蛋白)免疫兔子以制备抗血清,以酶联免疫法检测抗体滴度。依据该抗体与来自相同菌株的菌丝体粗多糖免疫反应差异对这两种多糖进行免疫识别,结果表明：经过亲和层析纯化后,第六次免疫抗血清对伞柄多糖的抗体滴度可达64 K,而且对菌丝体粗多糖反应呈阴性,显示该抗体可识别这两种多糖之间的结构差异。%Crude Clitocybe maxima stipe polysaccharide (CMSP)and mycelium polysaccharide were isolated by hot water (80 ℃)extraction and alcohol (75%)precipitation.CMSP was purified by Sepharose CL-4B gel filtration and DEAE Sepharose CL-4B anion exchange chromatography, and antibody to the purified polysaccharide was obtained by coupling to bovine serum albumin using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC)and injection of the resultant neoglycoprotein complex into rabbits.IgG serum antibody titers were measured by the ELISA (Enzyme Linked Immunosorbent Assay)method.Antiserum obtained after six injections was able to recognize stipe polysaccharide at a 1∶64000-fold dilution while a negative reaction was recorded with crude mycelium polysaccharide.Our data demonstrated that the purified CMSP antiserum differentiated between the stipe polysaccharide and crude mycelium polysaccharide prepared from the same fungal strain.
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