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过去广泛应用的过碘酸钠法(简称原法)需事先将辣根过氧化物酶(HRP)在pH8.0溶液中经二硝基氟苯处理、封闭氨基,防止与醛化的HRP发生自身交联。Wilson和Nakane(1978)报道了过碘酸钠改进法(简称改进法),我们对这二种方法进行了比较。原法按文献报道进行。改进法将4mg HRP溶解后加入新制备的NaIO_4,用醋酸钠缓冲液(pH4.4)透析,4℃过夜,用碳酸钠缓冲液将HRP醛基溶液调至pH9.0～9.5,然后加入8mg IgG,再加入新配制的硼氢化钠。酶结合物混合液过SephadexG-200柱层析,收集第一峰前半部的酶-IgG结合物,除去未能结合的IgG和HRP。收集物分管测0.D_(280nm)和0.D_(403nm),计算IgG含量和酶含量。
In the past, the sodium periodate method widely used in the past (referred to as the original method) required before horseradish peroxidase (HRP) in pH8.0 solution by dinitrofluorobenzene treatment, blocking the amino group to prevent the formation of HRP and hydroformylation Self-crosslinking. Wilson and Nakane (1978) reported the sodium periodate improvement method (referred to as improved method), we have compared these two methods. The original method according to the literature. Improved method 4mg HRP was dissolved in freshly prepared NaIO_4, dialyzed with sodium acetate buffer (pH4.4) overnight at 4 ° C, the HRP aldehyde solution was adjusted to pH9.0 ~ 9.5 with sodium carbonate buffer, then add 8mg IgG, then add freshly prepared sodium borohydride. The enzyme conjugate mixture was subjected to Sephadex G-200 column chromatography to collect the enzyme-IgG conjugate in the first half of the first peak to remove unbound IgG and HRP. Collected in charge of measuring 0.D_ (280nm) and 0. D_ (403nm), calculate IgG content and enzyme content.